Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Eleven years of breeding efforts to combat cassava brown streak disease

Eleven years of breeding efforts to combat cassava brown streak disease Breeding Science 66: 560–571 (2016) doi:10.1270/jsbbs.16005 Research Paper 1) 1) 1) 1) 1) Robert Sezi Kawuki* , Tadeo Kaweesi , Williams Esuma , Anthony Pariyo , Ismail Siraj Kayondo , 1) 1) 1) 1) 1) Alfred Ozimati , Vincent Kyaligonza , Alex Abaca , Joseph Orone , Robooni Tumuhimbise , 1) 1) 1) 1) 1) Ephraim Nuwamanya , Philip Abidrabo , Teddy Amuge , Emmanuel Ogwok , Geoffrey Okao , 1) 1) 1) 1) 1) 2) Henry Wagaba , Gerald Adiga , Titus Alicai , Christopher Omongo , Anton Bua , Morag Ferguson , 3) 1) Edward Kanju and Yona Baguma 1) National Crops Resources Research Institute, 9 km Gayaza-Zirobwe Road, P.O. Box 7084, Kampala, Uganda 2) International Institute of Tropical Agriculture (IITA), C/o International Livestock Research Institute (ILRI), P.O. Box 30709, Nairobi 00100, Kenya 3) International Institute of Tropical Agriculture (IITA), P.O. Box 34441, Dar es Salaam, Tanzania Cassava (Manihot esculenta Crantz) production is currently under threat from cassava brown streak disease (CBSD), a disease that is among the seven most serious obstacles to world’s food security. Three issues are of significance for CBSD. Firstly, the virus associated with CBSD, has co-evolved with cassava outside its center of origin for at least 90 years. Secondly, that for the last 74 years, CBSD was only limited to the low lands. Thirdly, that most research has largely focused on CBSD epidemiology and virus diversity. Accordingly, this paper focuses on CBSD genetics and/or breeding and hence, presents empirical data generated in the past 11 years of cassava breeding in Uganda. Specifically, this paper provides: 1) empirical data on CBSD resis- tance screening efforts to identify sources of resistance and/or tolerance; 2) an update on CBSD resistance population development comprising of full-sibs, half-sibs and S families and their respective field perfor - mances; and 3) insights into chromosomal regions and genes involved in CBSD resistance based on genome wide association analysis. It is expected that this information will provide a foundation for harmonizing on-going CBSD breeding efforts and consequently, inform the future breeding interventions aimed at combat- ing CBSD. Key Words: BLUPs, CBSD genetics, incidence, resistance genes, root necrosis, severity. Introduction (CBSVs) could be associated with CBSD (Ndunguru et al. 2015). Cassava (Manihot esculenta Crantz) production, which cur- Historically, CBSD was first reported from northern rently supports livelihoods of more than 800 million people Tanzania in the 1930s (Jennings 1957). Since then, the dis- worldwide, is under a threat of cassava brown streak disease ease has been reported in coastal areas of Kenya, northern (CBSD). This disease has been identified among the seven Mozambique, Zanzibar and areas close to the shores of most serious threats to world’s food security (Pennisi 2010). Lake Malawi (Hillocks et al. 2002). Recently, reports have CBSD was thought to be caused by two distinct virus spe- underlined the presence of CBSD especially that caused by cies; Uganda cassava brown streak virus (UCBSV) and UCBSV, in the Democratic Republic of Congo, Western Cassava brown streak virus (CBSV), both (+) ssRNA Kenya, Burundi and Lake Victoria region of Tanzania viruses belonging to genus Ipomovirus, family Potyviridae (Bigirimana et al. 2011, Legg et al. 2011). In Uganda, (Mbanzibwa et al. 2009, Winter et al. 2010). However, re- CBSD was first observed in the 1940s on cassava genotypes cent findings based on analysis of 470 symptomatic leaf introduced from Tanzania. The disease was, however, eradi- samples collected from Tanzania seem to suggest that up to cated through implementation of phytosanitary measures four different species of cassava brown streak viruses (Jameson 1964), but re-emerged in 2005 and attained epi- demic status (Alicai et al. 2007). Thus, without concerted efforts to control CBSD, it is likely to continue spreading to Communicated by J.M. Bonman all major cassava growing regions. Received January 14, 2016. Accepted May 7, 2016. Symptomatically, CBSD causes leaf chlorosis that ap- First Published Online in J-STAGE on August 5, 2016. *Corresponding author (e-mail: kawukisezi@yahoo.com) pears in a feathery pattern along the minor veins. These 560 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS symptoms can appear as early as three months after planting resistance screening efforts to identify sources of resistance (Ndunguru et al. 2015). In some genotypes, the leaf symp- and/or tolerance to CBSD; 2) an update on population de- toms are coupled with purple/brown lesions on stems, velopment comprising field performances of full-sibs, half- which in severe cases cause death of nodes, internodes and sibs and S families; and 3) insights into chromosomal re- the axillary buds, resulting into dieback (Hillocks and gions and genes potentially involved in CBSD resistance Thresh 2000). On the roots, the disease causes yellow and/ based on genome-wide association analysis. It is hoped that or brown, corky necrosis within the starch bearing tissues this information will provide direction to ongoing and fu- along with black streaks rendering roots unusable and thus ture efforts in combating CBSD in all breeding programs in causing up to 70–100% yield loss in susceptible genotypes the CBSD endemic regions, and help prepare regions cur- (Hillocks and Thresh 2000). Transient symptom expressions rently not affected by the disease. have also been observed (Mohammed et al. 2012). Starch quality notably quantities of amylose and amylopectin are Materials and Methods respectively reduced by 30 and 50 percent by CBSD (Nuwamanya et al. 2015). In this paper, we present empiri- Screening for sources of resistance and tolerance to CBSD cal CBSD field data generated from over 250 clones across Two major CBSD resistance and/or tolerance screening four propagation cycles. However, we limit detailed geno- efforts have been undertaken at a CBSD and CMD hot spot mic studies to storage root symptoms, as the storage roots area, Namulonge located in central Uganda (Abaca et al. are the most economically important part of the cassava 2012a, 2012b, Legg and Fauquet 2004). This has been (Tumuhimbise et al. 2015). undertaken in the last decade (2004 and 2014). Firstly, The pioneering formal cassava breeding program in Manihot esculenta germplasm introduced as botanical seeds Africa, which also coincided with first attempts to breed from Tanzania (referred to as germplasm-1). Secondly, wild for CBSD and cassava mosaic disease (CMD) resistance relatives and cassava F families sourced from Brazil and was initiated at Amani Research Station, Tanzania in the introduced as seeds (referred to as germplasm 2). 1930s (Jennings 1957, Nichols 1947). During that time, due to a lack of virus resistance in cultivated cassava, wild Germplasm 1: Tanzanian open-pollinated seed relatives that included Manihot glaziovii Muell-Arg. (Ceare At the onset of the CBSD epidemic in Uganda, we intro- Rubber), Manihot dichotoma Ule (Jaquie Manicoba duced germplasm from Tanzania in the form of open polli- Rubber), Manihot catingea Ule), Manihot saxicola Lang nated (OP) seeds (~5,000 OP seeds). These were derived and Manihot melanobasis Muell-Arg were reported to ex- from a polycross that had at least 10 CBSD tolerant cassava hibit high levels of resistance to CBSD (Jennings 1957, clones. In April 2005, a seedling nursery was established at 1959). One of the outstanding CBSD resistant inter-specific Namulonge for CBSD resistance screening. Both seedlings hybrid generated from these crosses was Namikonga (also and subsequent surviving clones were established at spacing referred to as clone No. 46106/27 or Kaleso). For CMD of 1 m × 1 m. We employed the independent culling selec- resistance, the outstanding clone was No. 58308, which be- tion method targeting CBSD for seven consecutive years came the main source of CMD resistance used in the breed- (between 2005 and 2011). Thus, for each annual selection ing program started in the early 1970s at IITA in Nigeria. event, only clones with <10% CBSD root incidence and Results of CBSD were not as successful as those for CMD. <5% foliar incidence were advanced; the rest were discard- Namikonga and/or its improved versions were not exten- ed. During each evaluation trial, spreader rows of CBSD sively used as a CBSD resistance progenitor. susceptible variety TME 204 were established to augment Based on the information available, the following is ap- CBSD pressure; single rows of TME 204 were also includ- parent for CBSD and its associated CBSVs. First, the virus ed in the evaluation trials to act as susceptible checks. has co-evolved with cassava in eastern and southern Africa CBSD was scored using the 1–5 severity scale as described (outside its center of origin) for at least 90 years. Second, in previous studies (Kaweesi et al. 2014). for the last 74 years (between 1930 and 2004), the disease Briefly, for this scale 1 = no root necrosis; 2 = mild root was only limited to the low lands i.e., less than 500 m.a.s.l. necrotic lesions (1–10%); 3 = pronounced root necrotic le- It is only after 2005, that the rapid spread of the disease has sions (11–25%); 4 = severe root necrotic lesions (26–50%) been observed at altitudes >500 m.a.s.l (Alicai et al. 2007, combined with mild root constriction; and 5 = very severe Ndunguru et al. 2015), which is likely to continue if not root necrotic lesion (>50%) coupled with severe constric- controlled. Third, most studies have focused on CBSD epi- tion. This seven-year culling exercise was undertaken at demiology and virus diversity, with limited published work Namulonge using single-row plots of 10 plants/row. In on breeding for CSBD resistance except perhaps the excel- 2012, the surviving 16 clones were established in replicated lent work done in 1930s (Jennings 1957) and more recently plots for final CBSD evaluation. In addition, open pollinat - in Mozambique (Zacarias and Labuschagne 2010), Kenya ed seeds were also generated from these surviving 16 clones. (Munga 2008) and Tanzania (Kulembeka et al. 2012). The generated progeny were established in the field at This paper, therefore, responds to the information gaps Namulonge for CBSD evaluation as described earlier; this highlighted. Specifically, it provides: 1) insights into CBSD was done in 2013. Thus, for the Tanzanian material, both 561 Breeding Science BS Vol. 66 No. 4 Kawuki, Kaweesi, Esuma, Pariyo, Kayondo, Ozimati, Kyaligonza, Abaca, Orone, Tumuhimbise et al. the introduced germplasm and its progeny (generated by Full-sibs from Namikonga inter-mating the surviving 16 clones) were evaluated. Namikonga which is believed to be a progeny from wild cassava (Hillocks and Jennings 2003) with high levels Germplasm 2: Wild relatives and cassava families from CBSD resistance (Kaweesi et al. 2014, Maruthi et al. 2014), Brazil was extensively used as a progenitor. Thus four Namikonga For the wild relatives, open pollinated seeds were derived F families were generated and evaluated for CBSD sourced from Brazil and also screened for CBSD field re- resistance for a period of four years (2011 to 2014). These sistance at Namulonge in 2013. The introduced seeds were families included: 1) NASE 14 × Namikonga (referred to as sourced from eight wild relatives: Manihot anomala Pohl. CS1 crosses), 2) TME 14 × Namikonga (referred to as CS2 Manihot caerulescens Pohl., Manihot carthaginensis ssp. crosses), 3) NASE 12 × Namikonga (referred to as CS3 glaziovii Müll.Arg., Manihot dichotoma Ule., Manihot crosses) and 4) NASE 13 × Namikonga referred to as CS4 esculenta ssp. flabellifolia Pohl., Manihot glaziovii Muel. crosses. Arg., Manihot irwinii D.J. Rogers & Appan., and Manihot With exception of Namikonga, all other selected clones peruviana Müll. Arg. For the cassava families, F seeds were highly resistant to CMD. For CBSD, the clones had were generated by crossing elite cassava clones in Brazil varying reactions basing on data collected between 2009 (BGM1332 × Fécula Branca, BGM1428 × Fécula Branca, and 2013: Namikonga with mean root severity of 1.03 and BGM1584 × Fécula Branca and BGM1662 × Fécula root incidence of 10%, NASE 14 with mean root severity of Branca). The generated F seeds were introduced to Uganda 3.3 and root incidence of 38.2%, TME 14 with mean root and evaluated for both CMD and CBSD resistance. The germ- severity of 4.2 and root incidence of 57.9, NASE 12 with plasm from Brazil was kindly provided by Dr. Eder Jorge de mean root severity of 4.3 and root incidence of 97.9% and Oliveira, of Empresa Brasileira de Pesquisa Agropecuária NASE 13 with mean root severity of 5 and root incidence of (Embrapa). 98%. Thus, with exception of CS1 crosses, all other crosses In both cases (wild relatives and full-sib cassava fami- were between a resistant clone Namikonga, and a suscepti- lies), all seedlings were germinated in nurseries and there- ble clone. after transplanted to the field for resistance screening follow- The F seedling evaluation trial was established in 2011, ing established procedures (IITA 1990). Field evaluations while the respective clonal replicated trials (2 and/or 3 were done during the period May 2013 to November 2014 replicates) were established in 2012 and 2013. The clonal at Namulonge. For CMD, plants were assigned a severity evaluations undertaken in 2014 was only for clones that score according to the standard five point scoring scale consistently had no or few CBSD root symptoms (i.e., root (IITA 1990) at six months after planting (MAP). At harvest severity score ≤2). All these evaluations were undertaken (12 MAP), surviving seedlings were uprooted and scored at Namulonge and for each trial, spreader rows of a CBSD- for CBSD root necrosis as described earlier. susceptible variety TME 204 were included to augment The generated datasets were separately subjected to sta- CBSD pressure. In addition, single rows of TME 204 were tistical analysis to generate family and/or individual clone always included in the evaluation trials to act as susceptible means using R statistical program (R Development Core checks. In all trials, CBSD data was collected as described Team 2010). For “germplasm 1” datasets that were largely earlier. unbalanced owing to varying number of clones or replicates established and/or evaluated, data were subjected to mixed S cassava families model analysis and Best Linear Unbiased Predictions The procedures for generation and agronomic evaluation (BLUPs) computed for each clone as a basis for comparison of S was adopted from previous studies (Kawuki et al. (Bernardo 2010). 2011). The five selected S parental lines (Namikonga, I00142, I30040, 0040, and Tz 130), had mean CBSD root Populations developed and their response to CBSD severity of scores of <3 and root incidences of <35%. A var- Following the initial CBSD resistance screening efforts iable number of S progeny were generated (44 to 141) and that were undertaken during 2004–2010, promising clones subjected to CBSD resistance screening beginning with the were selected and consequently used as progenitors for de- seedling evaluation in 2011. Thereafter, only surviving velopment of populations that could be used for long-term clones (lower CBSD severities and able to raise reasonable CBSD breeding. Accordingly, three populations were gen- planting materials) were re-evaluated in clonal trials at erated and screened for CBSD field resistance/tolerance Namulonge following established procedures for three con- during the period 2011 to 2014. These included: 1) full-sibs secutive years (2012, 2013 and 2014). having Namikonga as progenitor; 2) S cassava clones from five selected genotypes; and 3) full-sibs and/or half-sib fam- Full-and half-sib families from diverse origin ilies generated from other introduced and/or local outstand- Other full-sibs and/or half-sibs were generated from 49 ing clones. different progenitors. These progenitors were per se charac- terized by agronomically important traits that included: CBSD and CMD resistance and/or tolerance, fresh root yield 562 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS and dry matter content. Approximately 3,500 resulting F lelic frequency (MAF) of 10% (145,391) were used in the seedlings were established in a seedling trial at Namulonge genome-wide association analysis. in 2011. Culling for CBSD was done and ~13% of seedlings Marker-trait association was implemented in Trait by associated with low CBSD severities (i.e., root severity aSSociation, Evolution and Linkage (TASSEL) software score ≤2), were cloned for further screening in 2012. The version 5.2.3, using a mixed linear model (MLM) where clonal trial (having 455 clones) was established in 2012 in kinship and population structure were included as random single-row unreplicated plots comprising of 10 plants/plot. and fixed effects respectively. For population structure, the In 2013, the clones (300 to 450) were re-evaluated in first three principal components were used. Kinship matrix replicated plots of single-rows comprising of 10 plants at was generated using filtered SNPs using scaled identity by two CBSD hot spots, Namulonge and Kasese. In all these state (IBS) method. As opposed to the pairwise IBS, the trials: seedling trials (2011), unreplicated clonal trial at sin- scaled IBS method produces a kinship matrix that is scaled gle site (2012) and replicated clonal trials at two sites to give a reasonable estimate of additive variance. The four (2013), entries were evaluated for CBSD root necrosis as traits (mean root severity, root necrosis index, maximum described by Kaweesi et al. (2014). root severity and root necrosis incidence) were separately The datasets generated from the three populations (full- analyzed in the GWAS analysis. Q-Q plots were used to sibs from Namikonga; S cassava families; and full-sibs and evaluate the best trait for association analysis. After identi- half-sib families) were each analyzed separately using R fying the significant SNPs through association analysis, the statistical program (R Development Core Team 2010) to sequence that flanked the significant SNPs was obtained as enable family and/or clone comparisons. In addition, regres- an open reading frame using Artemis (Rutherford et al. sions analyses were undertaken to compare CBSD foliar 2000). A BLAST search was performed against the entire and root severity data for both seedling and/or clonal trials; National Center for Biotechnology Information (NCBI) non- this was done for the S and Namikonga-derived F families. redundant protein database and Phytozome 10.3 (Goodstein 1 1 For the unbalanced dataset (full-sibs and/or half-sibs popu- et al. 2012), to establish whether or not these sequence en- lation), BLUPs were computed to enable clone comparisons coded proteins with known functions. Best hits were used as (Bernardo 2010). reference for interpretation of putative biological functions of the sequence from which the SNPs were obtained. CBSD genome-wide association studies We assembled a diverse panel of cassava breeding lines Results and subjected them to CBSD evaluation at Namulonge for a period of five years (2009–2013). These clones were either Screening for sources of resistance and tolerance to CBSD at second and/or or third round of recombination. From this Data on performance of the surviving 16 clones (out of dataset, it was possible to select 190 genotypes that would the introduced 5000 seeds from Tanzania) and their respec- be classified as resistant (with maximum severity score for tive progeny at seedling stage is presented in Table 1. After the root of 1 and 2 and very low root incidence i.e., < 20%), eight years of CBSV virus exposure, lowest CBSD root in- moderately susceptible (with root severity score 3 and inci- cidences were registered on clone Tz-80 with respective dence ranging between 20–50%) and highly susceptible BLUP and mean incidences of –32.1 and 12.2% (Table 1). (root severity score of 4 or 5 and root incidence >50) to Clones Tz-65, Tz-90 and Tz-177, also had BLUP values that constitute the panel. These genotypes were re-evaluated in ranged between –26.5 to –24.6, with mean root incidences 2014, which coincided with the fifth year of virus exposure of <20%. However, other clones like Tz-61, Tz-64, and Tz- and/or evaluation. Since cassava roots are the most econom- 100 had mean incidences of >70% with root severity scores ic part of the plant, CBSD root necrosis data was considered of ≥3 (Table 1). It is also evident that each clone had proge- for this study, and thus, data collected on four root-related ny that were classified as resistant with root severity scores traits: mean root severity, root necrosis index, maximum of 1 and/or 2 (Table 1). This finding confirm presence of root severity and root necrosis incidence, as described by CBSD resistance/tolerance genes in the introduced Tanzanian Kaweesi et al. (2014). germplasm. DNA was extracted from leaf samples following es- Data on performance of wild relatives is presented in tablished procedures (Dellaporta et al. 1983). Genotyping Table 2. A total of 239 seedlings established successfully by was done at Cornell University, using a genotyping-by- one MAP. However these reduced to 210 and 173 respec- sequencing (GBS) approach as described by Elshire et al. tively at three and six MAP. At six MAP, CMD severity (2011) using ApeKI enzymes. The bioinformatics procedure ranged from 1 for M. caerulescens Pohl, to 4.1 for in TASSEL-GBS pipeline (Glaubitz et al. 2014) was used to M. peruviana Müll. Arg. M. esculenta individuals had aver- process that raw data, and SNP calls were based on cassava age CMD severity of 3.8, indicating higher levels of CMD genome sequence v6.0. To clean up the raw dataset, indels susceptibility. At Namulonge, the most prevalent strains of were removed and imputation done using Beagle software cassava mosaic germiniviruses are East African Cassava v4.0 as described by Swarts et al. (2014). A total of 162,951 Mosaic Virus-Ug (EACMV-Ug) and the African Cassava SNPs were identified and only filtered SNPs with minor al- Mosaic Virus (ACMV), with EACMV-Ug strain being the 563 Breeding Science BS Vol. 66 No. 4 Kawuki, Kaweesi, Esuma, Pariyo, Kayondo, Ozimati, Kyaligonza, Abaca, Orone, Tumuhimbise et al. Table 1. Best Linear Unbiased Predictions (BLUPs) of CBSD root necrosis of cassava parental lines and their respective progeny evaluated at NaCRRI, Uganda Performance of parents F seedling evaluation trials Parental line a b CBSDRi CBSDRs Score 1 & 2 Score 3 Score 4 & 5 No. F s CBSDRs CBSDRi Tz_100 37.5 (81.9) 1.25 (3.06) 6 0 6 12 0.03 (1.71) 0.74 (27.2) Tz_110 0.19 (44.5) –0.05 (1.76) 27 1 6 34 –0.02 (1.64) –1.57 (24.4) Tz_146 12.4 (56.8) –0.01 (1.80) 7 1 0 8 –0.03 (1.63) –1.27 (24.8) Tz_163 –23.6 (20.7) –0.54 (1.26) 17 1 2 20 –0.04 (1.62) –1.22 (24.7) Tz_175 –1.66 (42.7) –0.13 (1.67) 16 1 6 23 0.06 (1.74) 2.17 (28.8) Tz_177 –24.6 (19.7) –0.58 (1.22) 21 3 3 27 –0.01 (1.66) –0.97 (25.2) Tz_61 31.5 (75.9) 0.76 (2.57) 16 3 7 26 0.06 (1.74) 2.49 (29.5) Tz_62 –14.8 (29.5) –0.36 (1.45) 20 2 11 33 0.04 (1.71) 1.26 (27.7) Tz_64 35.7 (80.1) 1.34 (3.16) 21 1 5 27 –0.02 (1.65) –0.84 (25.2) Tz_65 –26.5 (17.8) –0.54 (1.26) 29 2 8 39 –0.01 (1.66) –1.40 (24.5) Tz_66 19.2 (63.6) 0.35 (2.17) 38 3 13 54 0.06 (1.74) 2.28 (29.1) Tz_69 5.5 (49.9) –0.01 (1.80) 31 3 5 39 –0.05 (1.60) –2.93 (22.9) Tz_73 –12.0 (32.3) –0.38 (1.43) 15 0 1 16 –0.03 (1.63) 0.43 (26.8) Tz_80 –32.1 (12.2) –0.67 (1.14) 29 6 6 41 0.02 (1.70) 1.71 (28.2) Tz_88 8.5 (52.9) 0.12 (1.94) 41 2 6 49 –0.06 (1.60) –1.03 (25.1) Tz_90 –25.5 (18.8) –0.58 (1.22) 20 1 7 28 0.01 (1.69) 0.17 (26.5) P-values 0.026 0.007 0.51 0.406 2c H 0.60 0.70 0.10 0.14 a b c Cassava brown streak disease root incidence assessed at 12 MAP; Cassava brown streak disease root severity assessed at 12 MAP; Broad- sense heritability. CBSD root necrosis was assessed on a scale of 1 to 5, where 1 = no necrosis, 2 = mild necrotic lesions (1–10%), 3 = pro- nounced necrotic lesion (11–25%), 4 = severe necrotic lesion (26–50%) combined with mild root constriction and 5 = very severe necrotic lesion (>50%) coupled with severe constriction. Data in parentheses are least-square (LS) means for parents and family progeny means, respectively. Table 2. Field response of selected Manihot species to CMD and scores >3 (Table 2). Seven individuals from Manihot CBSD assessed at NaCRRI, Uganda during 2013–2014 carthaginensis ssp. glaziovii and Manihot glaziovii had no a b c Manihot Species Number CMDs CBSDs root necrosis (Table 2). Individuals belonging to the M. esculenta F families registered the highest CBSD root Manihot caerulescens 22 1.0 1.5 1 Manihot dichotoma 21 1.5 1.1 incidence (85%) and highest CBSD root severity (3.5). In Manihot esculenta ssp. flabellifolia 12 3.3 1.2 all these evaluations, the susceptible check (TME 204) reg- Manihot sp. 4 1.5 1.0 istered mean CBSD root severities scores >4 and incidences Manihot carthaginensis ssp. glaziovii 8 1.6 1.1 Manihot irwinii 11 1.0 1.0 of >80%. M. esculenta F 74 3.8 1.4 Manihot glaziovii 2 2.5 1.0 Populations developed and their response to CBSD Manihot peruviana 19 4.1 1.0 Three populations were generated and their respective d e CBSDRi CBSDRs progeny evaluated for CBSD resistance and/or tolerance: 1) Manihot peruviana ssp. glaziovii 4 0.0 1.0 full-sibs having Namikonga as progenitor, 2) S cassava fam- M. esculenta F 23 85.4 3.5 Manihot glaziovii 3 0.0 1.0 ilies, and 3) full-sibs and/or half-sib families derived from a b 49 progenitors. Data on the performance of Namikonga- Number of genotypes evaluated; Mean severity of cassava mosaic derived crosses across four years of screening is presented disease assessed at six MAP; Mean severity of cassava brown streak disease assessed on the foliar parts at six MAP; Cassava brown streak in Table 3. It is evident from these datasets that: 1) progeni- disease root incidence assessed at 12 MAP; Cassava brown streak tor Namikonga provided CBSD resistance alleles to the disease root severity assessed at 12 MAP using the 1–5 scale. Data progeny as reflected by the number of individuals (18 to 32) rd presented are family means. that had no and/or few CBSD root symptoms after the 3 th and 4 year of evaluation; 2) there is a persistent poor corre- lation between CBSD foliar and root symptoms i.e., R val- most virulent (Legg and Fauquet 2004). Thus, all individu- ues <22%; and 3) it requires at least three seasons of CBSD als that had scores 1 or 2 can be classified as truly resistant evaluation in a hot spot to reliably quantify the CBSD field and thus sources of CMD resistance genes. response, as reflected by the sharp contrast of clones with At harvest, a variable number of roots were obtained. For scores 1 and 2 in 2011 (214 clones) and those observed in instance, 143 seedlings had no roots purportedly due to the 2013 (only 46 clones) or 2014 (24). CMD pressure, 16 seedlings had one root and two seedlings Data on the CBSD reaction of the S families is present- had five roots. Thus, CBSD root necrosis was only assessed ed in Table 4. Though a drastic reduction (~66%) was ob- on 30 seedlings. Up to 20 individuals all of which are served in number of progeny evaluated between seedling M. esculenta F had average CBSD root necrosis severity (2011 trial) and first clonal trial (2012 trial), each family had 564 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS Table 3. Field reaction of Namikonga-derived progeny to cassava brown streak disease at NaCRRI, Uganda evaluated across three propagation cycles F s used for scoring CBSD a b c d 2 e Year Family CBSDs CBSDRi Rootless Total R Score 1 & 2 Score 3 Score 4 & 5 2011 CS1 1.85 21.6 138 6 31 148 323 0.013 CS2 2.10 32.6 42 3 13 32 90 0.031 CS3 1.50 13.5 28 2 2 30 62 0.113 CS4 2.54 29.3 6 1 4 21 32 0.148 2012 CS1 3.02 33.2 59 24 60 0 143 0.069* CS2 3.77 56.1 7 8 21 0 36 0.052 CS3 2.64 31.1 12 6 7 0 25 0.218* CS4 3.77 58.6 2 1 6 0 9 0.398* 2013 CS1 2.79 40.3 32 23 39 8 102 0.016 CS2 1.59 16.18 7 1 1 5 14 0.045 CS3 1.20 5.13 7 – – 2 9 0.208 CS4 2014 CS1 1.37 23.5 18 1 – – 19 0.0001 CS2 1.10 5.4 4 – – – 4 CS3 1.2 10.0 2 – – – 2 a b c d Cassava brown streak disease root severity; Cassava brown streak disease root incidence; Number of F s without roots at evaluation; Total number of F s per family. Regression coefficient of CBSD root severity regressed on CBSD foliar severity: no regression was done for data for some families of 2014 owing to the small sample size i.e., <10 observations. CBSD root necrosis was scored on a scale of 1 to 5, where 1 = no necrosis; 2 = mild necrotic lesions (1–10%); 3 = pronounced necrotic lesion (11–25%); 4 = severe necrotic lesion (26–50%) combined with mild root constriction; and 5 = very severe necrotic lesion (>50%) coupled with severe constriction. CS1 = NASE 14 × Namikonga; CS2 = TME 14 × Namikonga; CS3 = NASE 12 × Namikonga; and CS4 = NASE 13 × Namikonga referred to as CS4 crosses. Check variety TME 204 had CBSDRs > 4 and CBSDRi > 80%. Data presented are family means. Table 4. Field response of S cassava partial inbreds to CBSD over three propagation cycles at NaCRRI, Uganda F s used for scoring CBSD a b c d 2 e Year Family CBSDs CBSDRi Rootless Total R Score 1 & 2 Score 3 Score 4 & 5 2011 Namikonga 1.04 10.6 32 – 2 10 44 0.116* I00142 1.63 19.1 93 5 14 29 141 0.0003 I30040 1.23 16.9 92 3 6 – 101 0.01 0040 1.20 2.28 79 – 4 17 100 0.13* Tz130 1.87 22.6 55 5 12 16 88 0.0000 2012 Namikonga 3.57 49.2 4 1 9 0 14 0.38* I00142 4.00 46.8 3 2 11 0 16 0.001 I30040 3.22 36.2 15 5 16 0 36 0.01 0040 3.88 44.2 6 4 17 0 27 0.03 Tz130 3.54 36.6 16 7 30 0 53 0.11* 2013 Namikonga 1.45 18.4 4 0 0 0 4 – I00142 2.21 29.8 1 1 2 0 4 – I30040 1.68 17.7 11 0 1 0 12 – 0040 1.92 19.9 4 1 0 0 5 – Tz130 2.59 38.2 9 0 6 0 15 – 2014 Namikonga 1.12 6.25 4 0 0 0 4 – I30040 1.04 1.54 6 0 0 0 6 – 0040 1.0 0 2 0 0 0 2 – Tz130 1.55 23.9 7 – 1 0 7 – a b c d Cassava brown streak disease root severity; Cassava brown streak disease root incidence; Number of F s without roots at evaluation; Total number of F s per family. Regression coefficient of CBSD root severity regressed on CBSD foliar severity: no regression was done for data for some families owing to the small sample size i.e., <16 observations. CBSD root necrosis scored on a scale of 1 to 5, where 1 = no necrosis; 2 = mild necrotic lesions (1–10%); 3 = pronounced necrotic lesion (11–25%); 4 = severe necrotic lesion (26–50%) combined with mild root con- striction; and 5 = very severe necrotic lesion (>50%) coupled with severe constriction. Check variety TME 204 had CBSDRs > 4 and CBSDRi > 80%. Data presented are family means. individuals (3 to 16) that showed no and/or limited CBSD number of S clones that remained symptomless were: root symptoms (Table 4). It was also evident that correla- seven for Tz 130, six for I30040, four for Namikonga and tions between CBSD foliar and root severities were negligi- two for 0040. These S clones had mean harvest index of ble (i.e., most r values were <20%) for both seedling and 0.18 (ranging from 0.03 to 0.47) and mean root dry matter clonal trials (Table 4). By the fourth year (2014 trial), the content of 28.6% (ranging from 16.9%–38.8%). It is these 565 Breeding Science BS Vol. 66 No. 4 Kawuki, Kaweesi, Esuma, Pariyo, Kayondo, Ozimati, Kyaligonza, Abaca, Orone, Tumuhimbise et al. Table 5. Best Linear Unbiased Prediction (BLUP) of CBSD root necrosis of five-best and five-worst progeny from selected parental lines evalu - ated at NaCRRI, Uganda a b a b Clone Female Male BLUPs CBSDRs Clone Female Male BLUPs CBSDRs Top TMS 30572 Progeny Worst TMS 30572 Progeny Ug120001 TMS30572 NASE 12 –46.79 1.49 Ug120013 NASE 11 TMS30572 44.01 3.06 Ug120104 TMS30572 TMS30572 –46.70 1.67 Ug120042 NASE 11 TMS30572 46.34 3.72 Ug120058 TMS30572 MM96/0686 –39.97 1.83 Ug120111 TMS30572 MH04/236 54.83 4.02 Ug130126 TMS30572 –39.63 1.57 Ug120011 NASE 11 TMS30572 55.29 3.37 Ug130087 TMS30572 –30.90 1.68 Ug120061 TMS30572 NASE 4 57.76 3.52 Top TMS 60142 Progeny Worst TMS 60142 Progeny Ug120002 NASE 11 TMS 60142 –30.64 1.66 Ug120249 SE95/00036 TMS60142 21.83 3.18 Ug120303 TMS 60142 NASE 14 –34.65 1.66 Ug120005 SE95/00036 TMS 60142 21.87 3.22 Ug120251 TMS 60142 NASE 9 –22.70 1.74 Ug120009 NASE 11 TMS 60142 48.33 3.34 Ug120267 TMS 60142 TME 14 –28.69 1.80 Ug120250 SE95/00036 TMS60142 36.84 3.58 Ug120289 TMS 60142 NASE 11 –13.51 1.91 Ug120010 NASE 11 TMS 60142 17.39 3.70 Top NASE 14 Progeny Worst NASE 14 Progeny Ug120124 NASE 14 MH04/2767 –38.71 1.95 Ug120125 NASE 14 MH04/236 27.23 3.14 Ug120123 NASE 14 MH04/2767 –31.76 1.97 Ug120200 SE95/00036 NASE 14 34.76 3.33 Ug120135 NASE 14 MH04/2575 –30.36 1.80 Ug120121 NASE 14 MH02/0441 46.24 3.07 Ug120116 NASE 14 –20.87 1.94 Ug120202 SE95/00036 NASE 14 46.24 3.31 Ug120105 I92/0067 NASE 14 –19.49 2.53 Ug120201 SE95/00036 NASE 14 46.34 3.72 Top TME 14 Progeny Worst TME 14 Progeny Ug120095 TME 14 TME 14 –33.84 1.62 Ug120134 TME 14 26B/27 24.20 3.47 Ug120274 TME 14 –33.57 1.92 Ug120233 TME 14 Nyaraboke 24.20 3.47 Ug130005 TME 14 26B-27 –32.84 1.76 Ug120212 NASE 12 TME 14 39.20 3.50 Ug130009 TME 14 11B-91 –32.03 1.65 Ug120295 TME 14 40.55 3.55 Ug130110 TME 14 Nyaraboke –31.69 1.73 Ug120292 TME 14 47.35 3.21 Best linear unbiased predictions, based on CBSD root necrosis data collected from trials established during 2012 (unreplicated trials at NaCRRI) and 2013 (replicated trials at both NaCRRI and Kasese); Cassava brown streak root severity scored using the 1–5 severity scale. This dataset is based on evaluation of 300 to 450 clones that were established in single row plots of 10 plants/row. Parental lines SE95/00036, NASE 12, NASE 4, and NASE 11 are highly susceptible to CBSD, while parental lines TMS 30572, TMS 60142 and NASE 14 are classified as tolerant to CBSD. The best clone was Ug120198 (with BLUP value of –48.2) and the worst performing clone was Ug120278 (BLUP value of 65.8). few outstanding individuals (19 out of the original 474 S BLUP value of –48.2), Ug120024 (F of NASE 14 × 1 1 clones) that are of interest for CBSD breeding, as they Namikonga, with BLUP value of –48.1), Ug120190 (intro- demonstrate the benefit of inbreeding in cassava, particular - duction from Tanzania, with BLUP value of –48.1), ly when combined with stringent selection. Ug120022 (F of NASE 14 × Namikonga, with BLUP value Data on the performance of the best-five and worst-five of –47.7) and Ug120001 ((F of NASE 3 × NASE 12, with progeny generated from four of the 49 parental lines is pre- BLUP value of –46.7). The worst clone Ug120278 (an F of sented in Table 5. The selected parental clones, all of IITA NASE 10 × NASE 9) had BLUP value of 65.8 with respec- pedigree included: NASE 1 (TMS 60142), NASE 3 (TMS tive root mean severity of 3.64. 30572), NASE 14 (MM96/4271) and TME 14. The parental lines NASE 1, NASE 3 and NASE 14, have all been associ- CBSD genome-wide association studies ated with low CBSD symptoms. Progeny of TME 14 were Five SNPs all located on chromosome 11 had significant added for comparison purposes. It is evident from the data signals (Table 6, Fig. 1). However, these SNPs did not that each of the parental lines had outstanding progeny; reach genome-wide Bonferroni significance threshold (of –7 among the top-best, the most CBSD resistant progeny P = 3.44 × 10 ). Four of these SNPs were identified with (Ug120001 and Ug120104) were all derived from TMS mean root severity. Two SNPs (S11-22909579 and S11- 30572, as they had BLUP values of –46.7 (Table 5). The 19872319) were identified with both mean root severity and best (Ug120002) and worst (Ug120009) progeny of parental disease index data. SNP S11-23228224 was the only signifi- clone TMS 60142, were all derived from the same cross cant signal that was obtained using maximum root severity. combination (Table 5), an illustration of the heterozygosity These SNPs are physically located between 19872319 to challenge in cassava breeding. 23751929 bp, a segment which most likely harbors the gene However, when all progeny from different combinations locus conditioning resistance and/or tolerance to CBSD root were analyzed together (including the introductions from necrosis. On average the identified SNPs explained 14.6% Tanzania; data not shown), we observed that the top five of CBSD root necrosis phenotypic variation. clones were: Ug120198 (introduction from Tanzania, with Based on the BLASTx plant protein search, four different 566 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS Table 6. SNP markers significantly associated with CBSD root necrosis resistance and their respective P-values Candidate gene a 2 b Trait CHR Marker Position (Mb) P-value R Gene BLASTx plant protein d –6 Mean Sev 11 S11-19872319 19872319 4.27 × 10 0.14 Cassava4.1_019379m lysM domain containing protein –6 11 S11-23751929 23751929 4.42 × 10 0.14 Cassava4.1_028097m glycine-rich protein –6 11 S11-22909579 22909579 5.78 × 10 0.14 – Ankyrin-3-like protein –6 11 S11-22909532 22909532 7.65 × 10 0.16 – – e –6 Index 11 S11-22909579 22909579 5.43 × 10 0.13 – Ankyrin-3-like protein –6 11 S11-19872319 19872319 6.11 × 10 0.13 Cassava4.1_019379m lysM domain containing protein f –6 Max Sev 11 S11-23228224 23228224 3.15 × 10 0.17 Cassava4.1_00037m 3.5.2.9-5-oxoprolinase enzyme a b c d Chromosome; Proportion of genetic trait variation explained by SNPs; Obtained through BLAST search against Phytozome 10.3; Mean root e f severity; Disease index as described by Kaweesi et al. (2014); Maximum root severity. proteins were identified from this study. These included (Ndunguru et al. 2015) and/or virus load during the evalua- lysM domain containing protein, glycine-rich protein, tion periods. For example, if evaluations are conducted for ankyrin-3-like protein and 3.5.2.9-5-oxoprolinase enzyme three seasons/years, virus monitoring should be done for (Table 6). The Q-Q plots which were used to evaluate the each season; 10–20 plots/clones with severe disease symp- best trait for CBSD association tests are displayed in Fig. 2. toms and 10–20 plots/clones with transient and/or no symp- It’s evident that 99% of the SNPs had P-values greater than toms should be assayed. This is particularly relevant in early 0.001 for mean severity and disease index showing that the selection stages i.e., clonal trials which often involve evalua- bulk of distribution behaved the way it should, based on the tion of >150 clones. Fortunately, optimal sampling sched- no association hypothesis. However, for root incidence and ules (with details of plant growth stage and plant part) for maximum severity, there was an early deviation from the CBSVs have been described (Kaweesi et al. 2014, Ogwok perfect diagonal (which corresponds to the null hypothesis), et al. 2015). Thus, as cheaper and reliable methods to quan- which means that statistical distribution is not appropriate tify CBSV viral loads become available, each individual for association tests. plot and/or clone can be monitored and its respective viral load compared with the disease severity. Discussion It is also evident from the generated field datasets that entailed evaluation of >250 clones for minimum of three Since the first reports of CBSD breeding in the early 1930’s years, that no consistent relationship exists between CBSD (Jennings 1957, Nichols 1947), limited genetics and/or foliar and root symptoms i.e., most R values were <10% breeding information has been generated. This is evident in (Tables 3, 4). This could suggest that these are different the few published work between the 1930s and 2015, as cit- traits under different genetic and/or biochemical mecha- ed herein. Thus, this paper presents and discusses empirical nisms. Thus, in terms of measurement, both traits can be mea- CBSD data generated in the last 11 years. From these data- sured and final categorizations of CBSD response based on sets, important information relating to CBSD and/or CBSV both as proposed by Kaweesi et al. (2014). In fact, in absence evaluation, scoring methodologies and breeding strategies of immunity, foliar CBSD assessments will continue to be have been gained. This information will specifically be rele - critically important in early selection stages (for purposes of vant for on-going CBSD breeding efforts and consequently, culling) and for cassava seed certification. inform the future breeding interventions aimed at combat- In practice, methodologies for measuring root severity ing CBSD. and/or root incidence can be variable. From experience, we It is evidently clear that CBSD and/or CBSV evaluations observe that different genotypes exhibit varying frequencies be conducted in a truly CBSD hotspot for a minimum of of total harvested roots and/or root severity scores per plant, three years to reliably classify clone responses. The sharp a situation that complicates genotype categorization and/or contrast in number of clones with CBSD root severity comparison. It is commonplace for breeding programmes to scores of 1 and/or 2 (classified as resistant) in 2011, and in use a maximum score, on the scale of 1–5, for the entire 2014 for both F and S families (Tables 3, 4) is testimony plot, others may use a maximum score per plant and then 1 1 for this. This also provides an opportunity to assess degen- compute plot averages; clearly, the two do not equate. It’s eration purported to arise from increased viral load observed also evident that a root that is assigned a score of 1 is eco- during the clonal propagation cycles. It is preferable to nomically very different from a root that is assigned a score measure virus load using real-time PCR during CBSD eval- of either 3, 4 or 5. Thus, discussions are on-going to find uations. solutions to this by way of accounting for the variable num- However in situations where large-scale virus monitor- ber of roots sampled per genotype through the development ing is not possible for each individual plot and/or clone, of a CBSD root necrosis index. then representative plots and/or clones could be sampled Regarding CBSD resistance breeding it is encouraging to and monitored to get insights into virus species dynamics note that outstanding CBSD resistant and/or tolerant clones 567 Breeding Science BS Vol. 66 No. 4 Kawuki, Kaweesi, Esuma, Pariyo, Kayondo, Ozimati, Kyaligonza, Abaca, Orone, Tumuhimbise et al. Fig. 1. Manhattan plots for genome wide association analysis for CBSD root necrosis resistance based on mixed linear models. Mean severity of root necrosis (a); disease index of root necrosis (b); maximum root severity (c); and incidence of root necrosis (d). The redline represents the Bonferroni correction threshold that determines SNPs with genome wide significance signal. have been identified. Based on evaluations undertaken in Notable of these are progeny derived from Namikonga, the past decade, we have identified some clones with rea- NASE 1 (TMS 60142), NASE 3 (TMS 30572), NASE 14 and sonable resistance and/or tolerance to CBSD. These clones M. esculenta selections introduced from Tanzania. Namikonga come from half-sibs, full-sibs and/or S cassava families derived F s (particularly those involving NASE 14), and the 1 1 generated from CBSD tolerant and/or resistant genotypes. S partial inbreds were truly outstanding (Tables 3, 4). 568 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS evaluations were based on seedlings, it will be necessary to re-evaluate the promising wild seedlings in clonal trials where a larger number of roots can be assessed per clone. In addition, the identified CBSD resistance/tolerance genes particularly those identified within M. esculenta germplasm sourced from Tanzania, can be exploited. On the other hand, CMD datasets (generated from the wild relatives), demonstrated their genetic value. Some in- dividuals from M. caerulescens, M. dichotoma, M. irwinii and M. cartheginensis ssp. glaziovii, registered CMD severi- ty scores of 1 and/or 2 (Table 2), and thus qualifying them as potentially useful sources of resistance to cassava germini- viruses. These CMD resistance sources from the wild will certainly compliment the widely deployed resistance that was exploited from M. glaziovii in the 1930s (Jennings 1957). It also suffices to note that during this period cassava varieties NASE 14, NARO-CASS 1 (synonym Tz 130) and NARO- CASS 2 have been officially released for commercial pro- duction; NASE 14 and NARO-CASS 1, have also been shared with four other countries (Kenya, Tanzania, Malawi and Mozambique) that are equally challenged by CBSD. Within limits, the CBSD association genetic study identi- fied five significant SNPs, all physically located between 19872319–23751929 bp on chromosome 11. The consistent presence of more than one SNP in this region suggests that this chromosome region is one of the quantitative trait loci (QTL) for CBSD root necrosis resistance. In this study, Q-Q plots were used to evaluate the best phenotype to use for CBSD genetic association tests. It was evident that 99% of the SNPs had P-values greater than 0.001 for mean severity and disease index showing that the bulk of distribution behaved the way it should, based on the no association hypothesis. However, for CBSD incidence and maximum severity, there was an early deviation from the perfect diagonal (which corresponds to the null hypothesis), which limits the utility of the two traits (incidence and maximum severity) for CBSD association studies. Plants possess pattern recognition receptors (PRRs) for their defense against pathogens (Nicaise et al. 2009). In- deed, a number of membrane-bound or soluble PRRs with lectin domain, have been identified as frontiers for plant de- fense (Lannoo and Van Damme 2014, Van Damme et al. Fig. 2. Q-Q plots of SNPs at marker level (P-values). Mean root se- 2008). Based on the BLASTx plant protein search, four dif- verity (a); disease index of root necrosis (b); maximum root severity ferent proteins were identified: lysM domain containing (c); and incidence of root necrosis (d). Deviation from the identity line protein, glycine-rich protein, ankyrin-3-like protein and at different significance levels showed the amount of false positive 3.5.2.9-5-oxoprolinase enzyme (Table 6). LysM-domain tests resulted from the analysis of the data; most deviations were ob- lectins is one of the four lectin receptor kinases (LecRK) served for maximum severity and root incidence. that have been reported to act both upon biotic and abiotic stresses (Vaid et al. 2013). These identified clones have remained symptomless or have It can therefore be hypothesized that the LysM domain shown mild CBSD symptoms (maximum severity score 2) containing protein observed from this study, may have a with low foliar and/or root incidences (<15%) after five role in CBSD root necrosis. Lozano et al. (2015), observed years of evaluation. that cassava chromosome 11 has three TOLL/interleukin-1 Equally striking was the identification of some wild rela - receptor (TIR-NBS-LRR) and one coiled- coil N-terminal tives, notably from M. carthaginensis ssp. glaziovii and domain (CC-NBS-LRR). None of these known resistance M. glaziovii that showed no CBSD symptoms. Because gene orthologs were identified in this study. This concurs 569 Breeding Science BS Vol. 66 No. 4 Kawuki, Kaweesi, Esuma, Pariyo, Kayondo, Ozimati, Kyaligonza, Abaca, Orone, Tumuhimbise et al. with the finding of Maruthi et al. (2014) where none of the We also thank IITA and the Agricultural Research Insti- known resistance gene orthologs were uniquely over ex- tute (ARI) Kibaha, Tanzania for sharing with us the cassava pressed in CBSD-resistant genotype (Namikonga). Thus, germplasm. more studies are therefore needed to further explore this region and/or other genomic regions to get further insights Literature Cited into genes that contribute to resistance and/or tolerance to CBSD root necrosis. Currently, efforts are underway to un- Abaca, A., R. Kawuki, P. Tukamuhabwa, Y. Baguma, A. Pariyo, T. Alicai, dertake detailed CBSD genome wide analysis studies that C.A. Omongo and A. Bua (2012a) Progression of cassava brown aim to get further insights into genes and/or chromosomal streak disease (CBSD) in Infected cassava roots in Uganda. Uganda regions controlling CBSD resistance. J. Agri. Sci. 13: 45–51. So far, all CBSD genetic studies conducted confirm the Abaca, A., R. Kawuki, P. Tukamuhabwa, Y. Baguma, A. Pariyo, T. Alicai, preponderance of additive genetic effects (Kulembeka C.A. Omongo and A. Bua (2012b) Evaluation of local and elite cassava genotypes for resistance to cassava brown streak disease in 2010, Kulembeka et al. 2012, Munga 2008, Zacarias and Uganda. J. Agron. 11: 65–72. Labuschagne 2010) and significant genotype by environ- Alicai, T., C.A. Omongo, M.N. Maruthi, R.J. Hillocks, Y. Baguma, ment interactions (Pariyo et al. 2015, Tumuhimbise et al. R. Kawuki, A. Bua, G.W. Otim-Nape and J. Colvin (2007) Re- 2014b). A number of factors are likely to amplify the geno- emergence of cassava brown streak disease in Uganda. Plant Dis. type × environmental interaction for CBSD including geno- 91: 24–29. type susceptibility levels, predominant virus species in lo- Bernardo, R. (2010) Breeding for Quantitative Traits in Plants, 2nd cality and/or season, and climatic factors that either edn. Stemma Press, Woodbury, Minnesota. influence the abundance of whitefly vectors and/or the Bigirimana, S., P. Barumbanze, P. Ndayihanzamaso, R. Shirima and growth rate of the crop (Katono et al. 2015). The discovery J.P. Legg (2011) First report of cassava brown streak disease and of four distinct virus species (Ndunguru et al. 2015), is like- associated Ugandan cassava brown streak virus in Burundi. New ly to further complicate the extent of genotype by environ- Dis. Rep. 24: 26. Dellaporta, S., J. Wood and J. Hicks (1983) A plant DNA miniprepara- ment interaction, as CBSD symptom expression (pheno- tion: version II. Plant Mol. Biol. Rep. 1: 19–21. types) associated with virus species are likely to differ Elshire, R.J., J.C. Glaubitz, Q. Sun, J.A. Poland, K. Kawamoto, between environments. Therefore, future CBSD breeding E.S. Buckler and S.E. Mitchell (2011) A robust, simple genotyping- strategies have to be designed mindful of these factors. by-sequencing (GBS) approach for high diversity species. PLoS Accordingly, if selection for hybridization is to be based ONE 6: e19379. on phenotypes, then cycle time can be reduced by having Glaubitz, J.C., T.M. Casstevens, F. Lu, J. Harriman, R.J. Elshire, Q. Sun field nurseries that serve both as evaluation and hybridiza - and E.S. Buckler (2014) TASSEL-GBS: a high capacity genotyping tion plots. For instance, final CBSD phenotypes (foliar and by sequencing analysis pipeline. PLoS ONE 9: e90346. rd root) can be scored (during 3 year of evaluation) at seven Goodstein, D.M., S. Shu, R. Howson, R. Neupane, R.D. Hayes, J. Fazo, MAP and then clones with low severities (combined with T. Mitros, W. Dirks, U. Hellsten, N. Putnam et al. (2012) Phyto- desired agronomic traits) crossed to constitute next cycle for zome: a comparative platform for green plant genomics. Nucleic Acids Res. 40: 1178–1186. selection. This simplistic 3–4 year CBSD breeding cycle Hillocks, R.J. and J.M. Thresh (2000) Cassava mosaic and cassava can be explored to attain higher levels of resistance. On the brown streak virus diseases in Africa: A comparative guide to other hand, if selection is to be based on genotypic data as symptoms and aetiologies. Roots 7: 1–8. implemented for genomic selection (Oliviera et al. 2012), Hillocks, R.J., J.M. Thresh, J. Tomas, M. Botao, R. Macia and R. Zavier then training populations will initially be needed to develop (2002) Cassava brown streak disease in northern Mozambique. Int. prediction models. It is through this approach that SNP J. Pest Manag. 48: 178–181. markers associated with CBSD resistance genes can be giv- Hillocks, R.J. and D.L. Jennings (2003) Cassava brown streak disease: en more weights in the estimation of genomic estimated a review of present knowledge and research needs. Int. J. Pest breeding values that are used in parental selection. Manag. 49: 225–234. IITA (1990) Cassava in Tropical Africa: A Reference Manual. Inter- Acknowledgements national Institute of Tropical Agriculture, Ibadan, Nigeria. Jameson, J.D. (1964) Cassava mosaic disease in Uganda. East Afr. Agric. For. J. 29: 208–213. The authors thank all technicians and support staff of Root Jennings, D.L. (1957) Further studies in breeding cassava for virus Crops Programme, NaCRRI, who participated in CBSD resistance. East Afr. Agric. J. 22: 213–219. data collection for experiments conducted during the period Jennings, D.L. (1959) Manihot melanobasis Müll. Arg.—A useful 2004 to 2015. Several research projects supported CBSD parent for cassava breeding. Euphytica 8: 157–162. research in Uganda. Notable of these included: The Millen- Katono, K., T. Alicai, Y. Baguma, R. Edema, A. Bua and C.A. Omongo nium Science Initiative (MSI); the East African Agricultural (2015) Influence of host plant resistance and disease pressure on Productivity Project (EAAPP); Biotechnology Tools to spread of cassava brown streak disease in Uganda. Am. J. Exp. Combat CBSD; Africa-Brazil Market Place the Government Agric. 7: 284–293. of Uganda; and the Next Generation Cassava Breeding Pro- Kaweesi, T., R. Kawuki, V. Kyaligonza, Y. Baguma, G. Tusiime and M.E. ject. Ferguson (2014) Field evaluation of selected cassava genotypes for 570 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS e0139321. cassava brown streak disease based on symptom expression and Nicaise, V., M. Roux and C. Zipfel (2009) Recent advances in virus load. Virol. J. 11: 216. PAMP-triggered immunity against bacteria: pattern recognition re- Kawuki, R.S., A. Pariyo, T. Amuge, E. Nuwamanya, G. Ssemakula, ceptors watch over and raise the alarm. Plant Physiol. 150: 1638– S. Tumwesigye, A. Bua, Y. Baguma, C. Omongo, T. Alicai et al. (2011) A breeding scheme for local adoption of cassava (Manihot Nichols, R.F.W. (1947) Breeding cassava for virus resistance. East Afr. esculenta Crantz). J. Plant Breed. Crop Sci. 3: 120–130. Agric. J. 12: 184–194. Kulembeka, H.P.K. (2010) Genetic linkage mapping of field resistance Nuwamanya, E., Y. Baguma, E. Atwijukire, S. Acheng and T. Alicai to cassava Brown Streak disease in cassava (Manihot esculenta (2015) Effect of cassava brown streak disease (CBSD) on cassava Crantz) landraces from Tanzania. University of the Free State, (Manihot esculenta Crantz) root storage components, starch quan- Bloemfontein, South Africa. tities and starch quality properties. Int. J. Plant Physiol. Biochem. Kulembeka, H.P., M. Ferguson, L. Herselman, E. Kanju, G. Mkamilo, 7: 12–22. E. Masumba, M. Fregene and M.T. Labuschagne (2012) Diallel Ogwok, E., T. Alicai, M.E.C. Rey, G. Beyene and N.J. Taylor (2015) analysis of field resistance to brown streak disease in cassava Distribution and accumulation of cassava brown streak viruses (Manihot esculenta Crantz) landraces from Tanzania. Euphytica within infected cassava (Manihot esculenta) plants. Plant Pathol. 187: 277–288. 64: 1235–1246. Lannoo, N. and E.J.M. Van Damme (2014) Lectin domains at the fron- Pariyo, A., Y. Baguma, T. Alicai, R. Kawuki, E. Kanju, A. Bua, C. tiers of plant defense. Front. Plant Sci. 5: 397. Omongo, P. Gibson, D.S. Osiru, D. Mpairwe et al. (2015) Stability of Legg, J.P. and C. Fauquet (2004) Cassava mosaic geminiviruses in resistance to cassava brown streak disease in major agro-ecologies Africa. Plant Mol. Biol. 56: 585–599. of Uganda. J. Plant Breed. Crop Sci. 7: 67–78. Legg, J.P., S.C. Jeremiah, H.M. Obiero, M.N. Maruthi, I. Ndyetabula, Pennisi, E. (2010) Armed and dangerous. Science 327: 804–805. G. Okao-Okuja, H. Bouwmeester, S. Bigirimana, W. Tata-Hangy, Rutherford, K., J. Parkhill, J. Crook, T. Horsnell, P. Rice, M.A. G. Gashaka et al. (2011) Comparing the regional epidemiology of Rajandream and B. Barrell (2000) Artemis: sequence visualization the cassava mosaic and cassava brown streak virus pandemics in and annotation. Bioinformatics 16: 944–945. Africa. Virus Res. 159: 161–170. Swarts, K., H. Li, J.A. Romero Navarro, D. An, M.C. Romay, S. Hearne, Lozano, R., M.T. Hamblin, S. Prochnik and J.L. Jannink (2015) Identi- C. Acharya, J.C. Glaubitz, S. Mitchell, R.J. Elshire et al. (2014) fication and distribution of the NBS-LRR gene family in the cassa- Novel methods to optimize genotypic imputation for low-coverage, va genome. BMC Genomics 16: 360. next-generation sequence data in crop plants. Plant Genome 7: Maruthi, M.N., S. Bouvaine, H.A. Tufan, I.U. Mohammed and R.J. 1–12. Hillocks (2014) Transcriptional response of virus-infected cassava Tumuhimbise, R., R. Melis and P. Shanahan (2014a) Diallel analysis of and identification of putative sources of resistance for cassava early storage root yield and disease resistance traits in cassava brown streak disease. PLoS ONE 9: e96642. (Manihot esculenta Crantz). Field Crops Res. 167: 86–93. Mbanzibwa, D.R., Y.P. Tian, A.K. Tugume, S.B. Mukasa, F. Tairo, Tumuhimbise, R., R. Melis, P. Shanahan and R. Kawuki (2014b) Geno- S. Kyamanywa, A. Kullaya and J.P.T. Valkonen (2009) Genetically distinct strains of Cassava brown streak virus in the Lake Victoria type × environment interaction effects on early fresh storage root basin and the Indian Ocean coastal area of East Africa. Arch. Virol. yield and related traits in cassava. Crop J. 2: 329–337. 154: 353–359. Tumuhimbise, R., P. Shanahan, R. Melis and R. Kawuki (2015) Genetic Mohammed, I.U., M.M. Abarshi, B. Muli, R.J. Hillocks and M.N. variation and association among factors influencing storage root Maruthi (2012) The symptom and genetic diversity of cassava bulking in cassava. J. Agric. Sci. 153: 1267–1280. brown streak viruses infecting cassava in East Africa. Adv. Virol. Van Damme, E.J.M., N. Lannoo and W.J. Peumans (2008) Plant lectins. 2012: 795–697. Adv. Bot. Res. 48: 107–209. Munga, T.L. (2008) Breeding for Cassava Brown Streak Resistance in Winter, S., M. Koerbler, B. Stein, A. Pietruszka, M. Paape and A. Coastal Kenya. KwaZulu-Natal, South Africa. Butgereitt (2010) Analysis of cassava brown streak viruses reveals Ndunguru, J., P. Sseruwagi, F. Tairo, F. Stomeo, S. Maina, A. Djinkeng, the presence of distinct virus species causing cassava brown streak M. Kehoe and L.M. Boykin (2015) Analyses of twelve new whole disease in East Africa. J. Gen. Virol. 91: 1365–1372. genome sequences of cassava brown streak viruses and Ugandan Zacarias, A.M. and M.T. Labuschagne (2010) Diallel analysis of cassa- cassava brown streak viruses from East Africa: diversity, super- va brown streak disease, yield and yield related characteristics in computing and evidence for further speciation. PLoS ONE 10: Mozambique. Euphytica 176: 309–320. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Breeding Science Pubmed Central

Loading next page...
 
/lp/pubmed-central/eleven-years-of-breeding-efforts-to-combat-cassava-brown-streak-0RzCkEHbJr

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Pubmed Central
Copyright
Copyright © 2016 by JAPANESE SOCIETY OF BREEDING
ISSN
1344-7610
eISSN
1347-3735
DOI
10.1270/jsbbs.16005
Publisher site
See Article on Publisher Site

Abstract

Breeding Science 66: 560–571 (2016) doi:10.1270/jsbbs.16005 Research Paper 1) 1) 1) 1) 1) Robert Sezi Kawuki* , Tadeo Kaweesi , Williams Esuma , Anthony Pariyo , Ismail Siraj Kayondo , 1) 1) 1) 1) 1) Alfred Ozimati , Vincent Kyaligonza , Alex Abaca , Joseph Orone , Robooni Tumuhimbise , 1) 1) 1) 1) 1) Ephraim Nuwamanya , Philip Abidrabo , Teddy Amuge , Emmanuel Ogwok , Geoffrey Okao , 1) 1) 1) 1) 1) 2) Henry Wagaba , Gerald Adiga , Titus Alicai , Christopher Omongo , Anton Bua , Morag Ferguson , 3) 1) Edward Kanju and Yona Baguma 1) National Crops Resources Research Institute, 9 km Gayaza-Zirobwe Road, P.O. Box 7084, Kampala, Uganda 2) International Institute of Tropical Agriculture (IITA), C/o International Livestock Research Institute (ILRI), P.O. Box 30709, Nairobi 00100, Kenya 3) International Institute of Tropical Agriculture (IITA), P.O. Box 34441, Dar es Salaam, Tanzania Cassava (Manihot esculenta Crantz) production is currently under threat from cassava brown streak disease (CBSD), a disease that is among the seven most serious obstacles to world’s food security. Three issues are of significance for CBSD. Firstly, the virus associated with CBSD, has co-evolved with cassava outside its center of origin for at least 90 years. Secondly, that for the last 74 years, CBSD was only limited to the low lands. Thirdly, that most research has largely focused on CBSD epidemiology and virus diversity. Accordingly, this paper focuses on CBSD genetics and/or breeding and hence, presents empirical data generated in the past 11 years of cassava breeding in Uganda. Specifically, this paper provides: 1) empirical data on CBSD resis- tance screening efforts to identify sources of resistance and/or tolerance; 2) an update on CBSD resistance population development comprising of full-sibs, half-sibs and S families and their respective field perfor - mances; and 3) insights into chromosomal regions and genes involved in CBSD resistance based on genome wide association analysis. It is expected that this information will provide a foundation for harmonizing on-going CBSD breeding efforts and consequently, inform the future breeding interventions aimed at combat- ing CBSD. Key Words: BLUPs, CBSD genetics, incidence, resistance genes, root necrosis, severity. Introduction (CBSVs) could be associated with CBSD (Ndunguru et al. 2015). Cassava (Manihot esculenta Crantz) production, which cur- Historically, CBSD was first reported from northern rently supports livelihoods of more than 800 million people Tanzania in the 1930s (Jennings 1957). Since then, the dis- worldwide, is under a threat of cassava brown streak disease ease has been reported in coastal areas of Kenya, northern (CBSD). This disease has been identified among the seven Mozambique, Zanzibar and areas close to the shores of most serious threats to world’s food security (Pennisi 2010). Lake Malawi (Hillocks et al. 2002). Recently, reports have CBSD was thought to be caused by two distinct virus spe- underlined the presence of CBSD especially that caused by cies; Uganda cassava brown streak virus (UCBSV) and UCBSV, in the Democratic Republic of Congo, Western Cassava brown streak virus (CBSV), both (+) ssRNA Kenya, Burundi and Lake Victoria region of Tanzania viruses belonging to genus Ipomovirus, family Potyviridae (Bigirimana et al. 2011, Legg et al. 2011). In Uganda, (Mbanzibwa et al. 2009, Winter et al. 2010). However, re- CBSD was first observed in the 1940s on cassava genotypes cent findings based on analysis of 470 symptomatic leaf introduced from Tanzania. The disease was, however, eradi- samples collected from Tanzania seem to suggest that up to cated through implementation of phytosanitary measures four different species of cassava brown streak viruses (Jameson 1964), but re-emerged in 2005 and attained epi- demic status (Alicai et al. 2007). Thus, without concerted efforts to control CBSD, it is likely to continue spreading to Communicated by J.M. Bonman all major cassava growing regions. Received January 14, 2016. Accepted May 7, 2016. Symptomatically, CBSD causes leaf chlorosis that ap- First Published Online in J-STAGE on August 5, 2016. *Corresponding author (e-mail: kawukisezi@yahoo.com) pears in a feathery pattern along the minor veins. These 560 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS symptoms can appear as early as three months after planting resistance screening efforts to identify sources of resistance (Ndunguru et al. 2015). In some genotypes, the leaf symp- and/or tolerance to CBSD; 2) an update on population de- toms are coupled with purple/brown lesions on stems, velopment comprising field performances of full-sibs, half- which in severe cases cause death of nodes, internodes and sibs and S families; and 3) insights into chromosomal re- the axillary buds, resulting into dieback (Hillocks and gions and genes potentially involved in CBSD resistance Thresh 2000). On the roots, the disease causes yellow and/ based on genome-wide association analysis. It is hoped that or brown, corky necrosis within the starch bearing tissues this information will provide direction to ongoing and fu- along with black streaks rendering roots unusable and thus ture efforts in combating CBSD in all breeding programs in causing up to 70–100% yield loss in susceptible genotypes the CBSD endemic regions, and help prepare regions cur- (Hillocks and Thresh 2000). Transient symptom expressions rently not affected by the disease. have also been observed (Mohammed et al. 2012). Starch quality notably quantities of amylose and amylopectin are Materials and Methods respectively reduced by 30 and 50 percent by CBSD (Nuwamanya et al. 2015). In this paper, we present empiri- Screening for sources of resistance and tolerance to CBSD cal CBSD field data generated from over 250 clones across Two major CBSD resistance and/or tolerance screening four propagation cycles. However, we limit detailed geno- efforts have been undertaken at a CBSD and CMD hot spot mic studies to storage root symptoms, as the storage roots area, Namulonge located in central Uganda (Abaca et al. are the most economically important part of the cassava 2012a, 2012b, Legg and Fauquet 2004). This has been (Tumuhimbise et al. 2015). undertaken in the last decade (2004 and 2014). Firstly, The pioneering formal cassava breeding program in Manihot esculenta germplasm introduced as botanical seeds Africa, which also coincided with first attempts to breed from Tanzania (referred to as germplasm-1). Secondly, wild for CBSD and cassava mosaic disease (CMD) resistance relatives and cassava F families sourced from Brazil and was initiated at Amani Research Station, Tanzania in the introduced as seeds (referred to as germplasm 2). 1930s (Jennings 1957, Nichols 1947). During that time, due to a lack of virus resistance in cultivated cassava, wild Germplasm 1: Tanzanian open-pollinated seed relatives that included Manihot glaziovii Muell-Arg. (Ceare At the onset of the CBSD epidemic in Uganda, we intro- Rubber), Manihot dichotoma Ule (Jaquie Manicoba duced germplasm from Tanzania in the form of open polli- Rubber), Manihot catingea Ule), Manihot saxicola Lang nated (OP) seeds (~5,000 OP seeds). These were derived and Manihot melanobasis Muell-Arg were reported to ex- from a polycross that had at least 10 CBSD tolerant cassava hibit high levels of resistance to CBSD (Jennings 1957, clones. In April 2005, a seedling nursery was established at 1959). One of the outstanding CBSD resistant inter-specific Namulonge for CBSD resistance screening. Both seedlings hybrid generated from these crosses was Namikonga (also and subsequent surviving clones were established at spacing referred to as clone No. 46106/27 or Kaleso). For CMD of 1 m × 1 m. We employed the independent culling selec- resistance, the outstanding clone was No. 58308, which be- tion method targeting CBSD for seven consecutive years came the main source of CMD resistance used in the breed- (between 2005 and 2011). Thus, for each annual selection ing program started in the early 1970s at IITA in Nigeria. event, only clones with <10% CBSD root incidence and Results of CBSD were not as successful as those for CMD. <5% foliar incidence were advanced; the rest were discard- Namikonga and/or its improved versions were not exten- ed. During each evaluation trial, spreader rows of CBSD sively used as a CBSD resistance progenitor. susceptible variety TME 204 were established to augment Based on the information available, the following is ap- CBSD pressure; single rows of TME 204 were also includ- parent for CBSD and its associated CBSVs. First, the virus ed in the evaluation trials to act as susceptible checks. has co-evolved with cassava in eastern and southern Africa CBSD was scored using the 1–5 severity scale as described (outside its center of origin) for at least 90 years. Second, in previous studies (Kaweesi et al. 2014). for the last 74 years (between 1930 and 2004), the disease Briefly, for this scale 1 = no root necrosis; 2 = mild root was only limited to the low lands i.e., less than 500 m.a.s.l. necrotic lesions (1–10%); 3 = pronounced root necrotic le- It is only after 2005, that the rapid spread of the disease has sions (11–25%); 4 = severe root necrotic lesions (26–50%) been observed at altitudes >500 m.a.s.l (Alicai et al. 2007, combined with mild root constriction; and 5 = very severe Ndunguru et al. 2015), which is likely to continue if not root necrotic lesion (>50%) coupled with severe constric- controlled. Third, most studies have focused on CBSD epi- tion. This seven-year culling exercise was undertaken at demiology and virus diversity, with limited published work Namulonge using single-row plots of 10 plants/row. In on breeding for CSBD resistance except perhaps the excel- 2012, the surviving 16 clones were established in replicated lent work done in 1930s (Jennings 1957) and more recently plots for final CBSD evaluation. In addition, open pollinat - in Mozambique (Zacarias and Labuschagne 2010), Kenya ed seeds were also generated from these surviving 16 clones. (Munga 2008) and Tanzania (Kulembeka et al. 2012). The generated progeny were established in the field at This paper, therefore, responds to the information gaps Namulonge for CBSD evaluation as described earlier; this highlighted. Specifically, it provides: 1) insights into CBSD was done in 2013. Thus, for the Tanzanian material, both 561 Breeding Science BS Vol. 66 No. 4 Kawuki, Kaweesi, Esuma, Pariyo, Kayondo, Ozimati, Kyaligonza, Abaca, Orone, Tumuhimbise et al. the introduced germplasm and its progeny (generated by Full-sibs from Namikonga inter-mating the surviving 16 clones) were evaluated. Namikonga which is believed to be a progeny from wild cassava (Hillocks and Jennings 2003) with high levels Germplasm 2: Wild relatives and cassava families from CBSD resistance (Kaweesi et al. 2014, Maruthi et al. 2014), Brazil was extensively used as a progenitor. Thus four Namikonga For the wild relatives, open pollinated seeds were derived F families were generated and evaluated for CBSD sourced from Brazil and also screened for CBSD field re- resistance for a period of four years (2011 to 2014). These sistance at Namulonge in 2013. The introduced seeds were families included: 1) NASE 14 × Namikonga (referred to as sourced from eight wild relatives: Manihot anomala Pohl. CS1 crosses), 2) TME 14 × Namikonga (referred to as CS2 Manihot caerulescens Pohl., Manihot carthaginensis ssp. crosses), 3) NASE 12 × Namikonga (referred to as CS3 glaziovii Müll.Arg., Manihot dichotoma Ule., Manihot crosses) and 4) NASE 13 × Namikonga referred to as CS4 esculenta ssp. flabellifolia Pohl., Manihot glaziovii Muel. crosses. Arg., Manihot irwinii D.J. Rogers & Appan., and Manihot With exception of Namikonga, all other selected clones peruviana Müll. Arg. For the cassava families, F seeds were highly resistant to CMD. For CBSD, the clones had were generated by crossing elite cassava clones in Brazil varying reactions basing on data collected between 2009 (BGM1332 × Fécula Branca, BGM1428 × Fécula Branca, and 2013: Namikonga with mean root severity of 1.03 and BGM1584 × Fécula Branca and BGM1662 × Fécula root incidence of 10%, NASE 14 with mean root severity of Branca). The generated F seeds were introduced to Uganda 3.3 and root incidence of 38.2%, TME 14 with mean root and evaluated for both CMD and CBSD resistance. The germ- severity of 4.2 and root incidence of 57.9, NASE 12 with plasm from Brazil was kindly provided by Dr. Eder Jorge de mean root severity of 4.3 and root incidence of 97.9% and Oliveira, of Empresa Brasileira de Pesquisa Agropecuária NASE 13 with mean root severity of 5 and root incidence of (Embrapa). 98%. Thus, with exception of CS1 crosses, all other crosses In both cases (wild relatives and full-sib cassava fami- were between a resistant clone Namikonga, and a suscepti- lies), all seedlings were germinated in nurseries and there- ble clone. after transplanted to the field for resistance screening follow- The F seedling evaluation trial was established in 2011, ing established procedures (IITA 1990). Field evaluations while the respective clonal replicated trials (2 and/or 3 were done during the period May 2013 to November 2014 replicates) were established in 2012 and 2013. The clonal at Namulonge. For CMD, plants were assigned a severity evaluations undertaken in 2014 was only for clones that score according to the standard five point scoring scale consistently had no or few CBSD root symptoms (i.e., root (IITA 1990) at six months after planting (MAP). At harvest severity score ≤2). All these evaluations were undertaken (12 MAP), surviving seedlings were uprooted and scored at Namulonge and for each trial, spreader rows of a CBSD- for CBSD root necrosis as described earlier. susceptible variety TME 204 were included to augment The generated datasets were separately subjected to sta- CBSD pressure. In addition, single rows of TME 204 were tistical analysis to generate family and/or individual clone always included in the evaluation trials to act as susceptible means using R statistical program (R Development Core checks. In all trials, CBSD data was collected as described Team 2010). For “germplasm 1” datasets that were largely earlier. unbalanced owing to varying number of clones or replicates established and/or evaluated, data were subjected to mixed S cassava families model analysis and Best Linear Unbiased Predictions The procedures for generation and agronomic evaluation (BLUPs) computed for each clone as a basis for comparison of S was adopted from previous studies (Kawuki et al. (Bernardo 2010). 2011). The five selected S parental lines (Namikonga, I00142, I30040, 0040, and Tz 130), had mean CBSD root Populations developed and their response to CBSD severity of scores of <3 and root incidences of <35%. A var- Following the initial CBSD resistance screening efforts iable number of S progeny were generated (44 to 141) and that were undertaken during 2004–2010, promising clones subjected to CBSD resistance screening beginning with the were selected and consequently used as progenitors for de- seedling evaluation in 2011. Thereafter, only surviving velopment of populations that could be used for long-term clones (lower CBSD severities and able to raise reasonable CBSD breeding. Accordingly, three populations were gen- planting materials) were re-evaluated in clonal trials at erated and screened for CBSD field resistance/tolerance Namulonge following established procedures for three con- during the period 2011 to 2014. These included: 1) full-sibs secutive years (2012, 2013 and 2014). having Namikonga as progenitor; 2) S cassava clones from five selected genotypes; and 3) full-sibs and/or half-sib fam- Full-and half-sib families from diverse origin ilies generated from other introduced and/or local outstand- Other full-sibs and/or half-sibs were generated from 49 ing clones. different progenitors. These progenitors were per se charac- terized by agronomically important traits that included: CBSD and CMD resistance and/or tolerance, fresh root yield 562 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS and dry matter content. Approximately 3,500 resulting F lelic frequency (MAF) of 10% (145,391) were used in the seedlings were established in a seedling trial at Namulonge genome-wide association analysis. in 2011. Culling for CBSD was done and ~13% of seedlings Marker-trait association was implemented in Trait by associated with low CBSD severities (i.e., root severity aSSociation, Evolution and Linkage (TASSEL) software score ≤2), were cloned for further screening in 2012. The version 5.2.3, using a mixed linear model (MLM) where clonal trial (having 455 clones) was established in 2012 in kinship and population structure were included as random single-row unreplicated plots comprising of 10 plants/plot. and fixed effects respectively. For population structure, the In 2013, the clones (300 to 450) were re-evaluated in first three principal components were used. Kinship matrix replicated plots of single-rows comprising of 10 plants at was generated using filtered SNPs using scaled identity by two CBSD hot spots, Namulonge and Kasese. In all these state (IBS) method. As opposed to the pairwise IBS, the trials: seedling trials (2011), unreplicated clonal trial at sin- scaled IBS method produces a kinship matrix that is scaled gle site (2012) and replicated clonal trials at two sites to give a reasonable estimate of additive variance. The four (2013), entries were evaluated for CBSD root necrosis as traits (mean root severity, root necrosis index, maximum described by Kaweesi et al. (2014). root severity and root necrosis incidence) were separately The datasets generated from the three populations (full- analyzed in the GWAS analysis. Q-Q plots were used to sibs from Namikonga; S cassava families; and full-sibs and evaluate the best trait for association analysis. After identi- half-sib families) were each analyzed separately using R fying the significant SNPs through association analysis, the statistical program (R Development Core Team 2010) to sequence that flanked the significant SNPs was obtained as enable family and/or clone comparisons. In addition, regres- an open reading frame using Artemis (Rutherford et al. sions analyses were undertaken to compare CBSD foliar 2000). A BLAST search was performed against the entire and root severity data for both seedling and/or clonal trials; National Center for Biotechnology Information (NCBI) non- this was done for the S and Namikonga-derived F families. redundant protein database and Phytozome 10.3 (Goodstein 1 1 For the unbalanced dataset (full-sibs and/or half-sibs popu- et al. 2012), to establish whether or not these sequence en- lation), BLUPs were computed to enable clone comparisons coded proteins with known functions. Best hits were used as (Bernardo 2010). reference for interpretation of putative biological functions of the sequence from which the SNPs were obtained. CBSD genome-wide association studies We assembled a diverse panel of cassava breeding lines Results and subjected them to CBSD evaluation at Namulonge for a period of five years (2009–2013). These clones were either Screening for sources of resistance and tolerance to CBSD at second and/or or third round of recombination. From this Data on performance of the surviving 16 clones (out of dataset, it was possible to select 190 genotypes that would the introduced 5000 seeds from Tanzania) and their respec- be classified as resistant (with maximum severity score for tive progeny at seedling stage is presented in Table 1. After the root of 1 and 2 and very low root incidence i.e., < 20%), eight years of CBSV virus exposure, lowest CBSD root in- moderately susceptible (with root severity score 3 and inci- cidences were registered on clone Tz-80 with respective dence ranging between 20–50%) and highly susceptible BLUP and mean incidences of –32.1 and 12.2% (Table 1). (root severity score of 4 or 5 and root incidence >50) to Clones Tz-65, Tz-90 and Tz-177, also had BLUP values that constitute the panel. These genotypes were re-evaluated in ranged between –26.5 to –24.6, with mean root incidences 2014, which coincided with the fifth year of virus exposure of <20%. However, other clones like Tz-61, Tz-64, and Tz- and/or evaluation. Since cassava roots are the most econom- 100 had mean incidences of >70% with root severity scores ic part of the plant, CBSD root necrosis data was considered of ≥3 (Table 1). It is also evident that each clone had proge- for this study, and thus, data collected on four root-related ny that were classified as resistant with root severity scores traits: mean root severity, root necrosis index, maximum of 1 and/or 2 (Table 1). This finding confirm presence of root severity and root necrosis incidence, as described by CBSD resistance/tolerance genes in the introduced Tanzanian Kaweesi et al. (2014). germplasm. DNA was extracted from leaf samples following es- Data on performance of wild relatives is presented in tablished procedures (Dellaporta et al. 1983). Genotyping Table 2. A total of 239 seedlings established successfully by was done at Cornell University, using a genotyping-by- one MAP. However these reduced to 210 and 173 respec- sequencing (GBS) approach as described by Elshire et al. tively at three and six MAP. At six MAP, CMD severity (2011) using ApeKI enzymes. The bioinformatics procedure ranged from 1 for M. caerulescens Pohl, to 4.1 for in TASSEL-GBS pipeline (Glaubitz et al. 2014) was used to M. peruviana Müll. Arg. M. esculenta individuals had aver- process that raw data, and SNP calls were based on cassava age CMD severity of 3.8, indicating higher levels of CMD genome sequence v6.0. To clean up the raw dataset, indels susceptibility. At Namulonge, the most prevalent strains of were removed and imputation done using Beagle software cassava mosaic germiniviruses are East African Cassava v4.0 as described by Swarts et al. (2014). A total of 162,951 Mosaic Virus-Ug (EACMV-Ug) and the African Cassava SNPs were identified and only filtered SNPs with minor al- Mosaic Virus (ACMV), with EACMV-Ug strain being the 563 Breeding Science BS Vol. 66 No. 4 Kawuki, Kaweesi, Esuma, Pariyo, Kayondo, Ozimati, Kyaligonza, Abaca, Orone, Tumuhimbise et al. Table 1. Best Linear Unbiased Predictions (BLUPs) of CBSD root necrosis of cassava parental lines and their respective progeny evaluated at NaCRRI, Uganda Performance of parents F seedling evaluation trials Parental line a b CBSDRi CBSDRs Score 1 & 2 Score 3 Score 4 & 5 No. F s CBSDRs CBSDRi Tz_100 37.5 (81.9) 1.25 (3.06) 6 0 6 12 0.03 (1.71) 0.74 (27.2) Tz_110 0.19 (44.5) –0.05 (1.76) 27 1 6 34 –0.02 (1.64) –1.57 (24.4) Tz_146 12.4 (56.8) –0.01 (1.80) 7 1 0 8 –0.03 (1.63) –1.27 (24.8) Tz_163 –23.6 (20.7) –0.54 (1.26) 17 1 2 20 –0.04 (1.62) –1.22 (24.7) Tz_175 –1.66 (42.7) –0.13 (1.67) 16 1 6 23 0.06 (1.74) 2.17 (28.8) Tz_177 –24.6 (19.7) –0.58 (1.22) 21 3 3 27 –0.01 (1.66) –0.97 (25.2) Tz_61 31.5 (75.9) 0.76 (2.57) 16 3 7 26 0.06 (1.74) 2.49 (29.5) Tz_62 –14.8 (29.5) –0.36 (1.45) 20 2 11 33 0.04 (1.71) 1.26 (27.7) Tz_64 35.7 (80.1) 1.34 (3.16) 21 1 5 27 –0.02 (1.65) –0.84 (25.2) Tz_65 –26.5 (17.8) –0.54 (1.26) 29 2 8 39 –0.01 (1.66) –1.40 (24.5) Tz_66 19.2 (63.6) 0.35 (2.17) 38 3 13 54 0.06 (1.74) 2.28 (29.1) Tz_69 5.5 (49.9) –0.01 (1.80) 31 3 5 39 –0.05 (1.60) –2.93 (22.9) Tz_73 –12.0 (32.3) –0.38 (1.43) 15 0 1 16 –0.03 (1.63) 0.43 (26.8) Tz_80 –32.1 (12.2) –0.67 (1.14) 29 6 6 41 0.02 (1.70) 1.71 (28.2) Tz_88 8.5 (52.9) 0.12 (1.94) 41 2 6 49 –0.06 (1.60) –1.03 (25.1) Tz_90 –25.5 (18.8) –0.58 (1.22) 20 1 7 28 0.01 (1.69) 0.17 (26.5) P-values 0.026 0.007 0.51 0.406 2c H 0.60 0.70 0.10 0.14 a b c Cassava brown streak disease root incidence assessed at 12 MAP; Cassava brown streak disease root severity assessed at 12 MAP; Broad- sense heritability. CBSD root necrosis was assessed on a scale of 1 to 5, where 1 = no necrosis, 2 = mild necrotic lesions (1–10%), 3 = pro- nounced necrotic lesion (11–25%), 4 = severe necrotic lesion (26–50%) combined with mild root constriction and 5 = very severe necrotic lesion (>50%) coupled with severe constriction. Data in parentheses are least-square (LS) means for parents and family progeny means, respectively. Table 2. Field response of selected Manihot species to CMD and scores >3 (Table 2). Seven individuals from Manihot CBSD assessed at NaCRRI, Uganda during 2013–2014 carthaginensis ssp. glaziovii and Manihot glaziovii had no a b c Manihot Species Number CMDs CBSDs root necrosis (Table 2). Individuals belonging to the M. esculenta F families registered the highest CBSD root Manihot caerulescens 22 1.0 1.5 1 Manihot dichotoma 21 1.5 1.1 incidence (85%) and highest CBSD root severity (3.5). In Manihot esculenta ssp. flabellifolia 12 3.3 1.2 all these evaluations, the susceptible check (TME 204) reg- Manihot sp. 4 1.5 1.0 istered mean CBSD root severities scores >4 and incidences Manihot carthaginensis ssp. glaziovii 8 1.6 1.1 Manihot irwinii 11 1.0 1.0 of >80%. M. esculenta F 74 3.8 1.4 Manihot glaziovii 2 2.5 1.0 Populations developed and their response to CBSD Manihot peruviana 19 4.1 1.0 Three populations were generated and their respective d e CBSDRi CBSDRs progeny evaluated for CBSD resistance and/or tolerance: 1) Manihot peruviana ssp. glaziovii 4 0.0 1.0 full-sibs having Namikonga as progenitor, 2) S cassava fam- M. esculenta F 23 85.4 3.5 Manihot glaziovii 3 0.0 1.0 ilies, and 3) full-sibs and/or half-sib families derived from a b 49 progenitors. Data on the performance of Namikonga- Number of genotypes evaluated; Mean severity of cassava mosaic derived crosses across four years of screening is presented disease assessed at six MAP; Mean severity of cassava brown streak disease assessed on the foliar parts at six MAP; Cassava brown streak in Table 3. It is evident from these datasets that: 1) progeni- disease root incidence assessed at 12 MAP; Cassava brown streak tor Namikonga provided CBSD resistance alleles to the disease root severity assessed at 12 MAP using the 1–5 scale. Data progeny as reflected by the number of individuals (18 to 32) rd presented are family means. that had no and/or few CBSD root symptoms after the 3 th and 4 year of evaluation; 2) there is a persistent poor corre- lation between CBSD foliar and root symptoms i.e., R val- most virulent (Legg and Fauquet 2004). Thus, all individu- ues <22%; and 3) it requires at least three seasons of CBSD als that had scores 1 or 2 can be classified as truly resistant evaluation in a hot spot to reliably quantify the CBSD field and thus sources of CMD resistance genes. response, as reflected by the sharp contrast of clones with At harvest, a variable number of roots were obtained. For scores 1 and 2 in 2011 (214 clones) and those observed in instance, 143 seedlings had no roots purportedly due to the 2013 (only 46 clones) or 2014 (24). CMD pressure, 16 seedlings had one root and two seedlings Data on the CBSD reaction of the S families is present- had five roots. Thus, CBSD root necrosis was only assessed ed in Table 4. Though a drastic reduction (~66%) was ob- on 30 seedlings. Up to 20 individuals all of which are served in number of progeny evaluated between seedling M. esculenta F had average CBSD root necrosis severity (2011 trial) and first clonal trial (2012 trial), each family had 564 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS Table 3. Field reaction of Namikonga-derived progeny to cassava brown streak disease at NaCRRI, Uganda evaluated across three propagation cycles F s used for scoring CBSD a b c d 2 e Year Family CBSDs CBSDRi Rootless Total R Score 1 & 2 Score 3 Score 4 & 5 2011 CS1 1.85 21.6 138 6 31 148 323 0.013 CS2 2.10 32.6 42 3 13 32 90 0.031 CS3 1.50 13.5 28 2 2 30 62 0.113 CS4 2.54 29.3 6 1 4 21 32 0.148 2012 CS1 3.02 33.2 59 24 60 0 143 0.069* CS2 3.77 56.1 7 8 21 0 36 0.052 CS3 2.64 31.1 12 6 7 0 25 0.218* CS4 3.77 58.6 2 1 6 0 9 0.398* 2013 CS1 2.79 40.3 32 23 39 8 102 0.016 CS2 1.59 16.18 7 1 1 5 14 0.045 CS3 1.20 5.13 7 – – 2 9 0.208 CS4 2014 CS1 1.37 23.5 18 1 – – 19 0.0001 CS2 1.10 5.4 4 – – – 4 CS3 1.2 10.0 2 – – – 2 a b c d Cassava brown streak disease root severity; Cassava brown streak disease root incidence; Number of F s without roots at evaluation; Total number of F s per family. Regression coefficient of CBSD root severity regressed on CBSD foliar severity: no regression was done for data for some families of 2014 owing to the small sample size i.e., <10 observations. CBSD root necrosis was scored on a scale of 1 to 5, where 1 = no necrosis; 2 = mild necrotic lesions (1–10%); 3 = pronounced necrotic lesion (11–25%); 4 = severe necrotic lesion (26–50%) combined with mild root constriction; and 5 = very severe necrotic lesion (>50%) coupled with severe constriction. CS1 = NASE 14 × Namikonga; CS2 = TME 14 × Namikonga; CS3 = NASE 12 × Namikonga; and CS4 = NASE 13 × Namikonga referred to as CS4 crosses. Check variety TME 204 had CBSDRs > 4 and CBSDRi > 80%. Data presented are family means. Table 4. Field response of S cassava partial inbreds to CBSD over three propagation cycles at NaCRRI, Uganda F s used for scoring CBSD a b c d 2 e Year Family CBSDs CBSDRi Rootless Total R Score 1 & 2 Score 3 Score 4 & 5 2011 Namikonga 1.04 10.6 32 – 2 10 44 0.116* I00142 1.63 19.1 93 5 14 29 141 0.0003 I30040 1.23 16.9 92 3 6 – 101 0.01 0040 1.20 2.28 79 – 4 17 100 0.13* Tz130 1.87 22.6 55 5 12 16 88 0.0000 2012 Namikonga 3.57 49.2 4 1 9 0 14 0.38* I00142 4.00 46.8 3 2 11 0 16 0.001 I30040 3.22 36.2 15 5 16 0 36 0.01 0040 3.88 44.2 6 4 17 0 27 0.03 Tz130 3.54 36.6 16 7 30 0 53 0.11* 2013 Namikonga 1.45 18.4 4 0 0 0 4 – I00142 2.21 29.8 1 1 2 0 4 – I30040 1.68 17.7 11 0 1 0 12 – 0040 1.92 19.9 4 1 0 0 5 – Tz130 2.59 38.2 9 0 6 0 15 – 2014 Namikonga 1.12 6.25 4 0 0 0 4 – I30040 1.04 1.54 6 0 0 0 6 – 0040 1.0 0 2 0 0 0 2 – Tz130 1.55 23.9 7 – 1 0 7 – a b c d Cassava brown streak disease root severity; Cassava brown streak disease root incidence; Number of F s without roots at evaluation; Total number of F s per family. Regression coefficient of CBSD root severity regressed on CBSD foliar severity: no regression was done for data for some families owing to the small sample size i.e., <16 observations. CBSD root necrosis scored on a scale of 1 to 5, where 1 = no necrosis; 2 = mild necrotic lesions (1–10%); 3 = pronounced necrotic lesion (11–25%); 4 = severe necrotic lesion (26–50%) combined with mild root con- striction; and 5 = very severe necrotic lesion (>50%) coupled with severe constriction. Check variety TME 204 had CBSDRs > 4 and CBSDRi > 80%. Data presented are family means. individuals (3 to 16) that showed no and/or limited CBSD number of S clones that remained symptomless were: root symptoms (Table 4). It was also evident that correla- seven for Tz 130, six for I30040, four for Namikonga and tions between CBSD foliar and root severities were negligi- two for 0040. These S clones had mean harvest index of ble (i.e., most r values were <20%) for both seedling and 0.18 (ranging from 0.03 to 0.47) and mean root dry matter clonal trials (Table 4). By the fourth year (2014 trial), the content of 28.6% (ranging from 16.9%–38.8%). It is these 565 Breeding Science BS Vol. 66 No. 4 Kawuki, Kaweesi, Esuma, Pariyo, Kayondo, Ozimati, Kyaligonza, Abaca, Orone, Tumuhimbise et al. Table 5. Best Linear Unbiased Prediction (BLUP) of CBSD root necrosis of five-best and five-worst progeny from selected parental lines evalu - ated at NaCRRI, Uganda a b a b Clone Female Male BLUPs CBSDRs Clone Female Male BLUPs CBSDRs Top TMS 30572 Progeny Worst TMS 30572 Progeny Ug120001 TMS30572 NASE 12 –46.79 1.49 Ug120013 NASE 11 TMS30572 44.01 3.06 Ug120104 TMS30572 TMS30572 –46.70 1.67 Ug120042 NASE 11 TMS30572 46.34 3.72 Ug120058 TMS30572 MM96/0686 –39.97 1.83 Ug120111 TMS30572 MH04/236 54.83 4.02 Ug130126 TMS30572 –39.63 1.57 Ug120011 NASE 11 TMS30572 55.29 3.37 Ug130087 TMS30572 –30.90 1.68 Ug120061 TMS30572 NASE 4 57.76 3.52 Top TMS 60142 Progeny Worst TMS 60142 Progeny Ug120002 NASE 11 TMS 60142 –30.64 1.66 Ug120249 SE95/00036 TMS60142 21.83 3.18 Ug120303 TMS 60142 NASE 14 –34.65 1.66 Ug120005 SE95/00036 TMS 60142 21.87 3.22 Ug120251 TMS 60142 NASE 9 –22.70 1.74 Ug120009 NASE 11 TMS 60142 48.33 3.34 Ug120267 TMS 60142 TME 14 –28.69 1.80 Ug120250 SE95/00036 TMS60142 36.84 3.58 Ug120289 TMS 60142 NASE 11 –13.51 1.91 Ug120010 NASE 11 TMS 60142 17.39 3.70 Top NASE 14 Progeny Worst NASE 14 Progeny Ug120124 NASE 14 MH04/2767 –38.71 1.95 Ug120125 NASE 14 MH04/236 27.23 3.14 Ug120123 NASE 14 MH04/2767 –31.76 1.97 Ug120200 SE95/00036 NASE 14 34.76 3.33 Ug120135 NASE 14 MH04/2575 –30.36 1.80 Ug120121 NASE 14 MH02/0441 46.24 3.07 Ug120116 NASE 14 –20.87 1.94 Ug120202 SE95/00036 NASE 14 46.24 3.31 Ug120105 I92/0067 NASE 14 –19.49 2.53 Ug120201 SE95/00036 NASE 14 46.34 3.72 Top TME 14 Progeny Worst TME 14 Progeny Ug120095 TME 14 TME 14 –33.84 1.62 Ug120134 TME 14 26B/27 24.20 3.47 Ug120274 TME 14 –33.57 1.92 Ug120233 TME 14 Nyaraboke 24.20 3.47 Ug130005 TME 14 26B-27 –32.84 1.76 Ug120212 NASE 12 TME 14 39.20 3.50 Ug130009 TME 14 11B-91 –32.03 1.65 Ug120295 TME 14 40.55 3.55 Ug130110 TME 14 Nyaraboke –31.69 1.73 Ug120292 TME 14 47.35 3.21 Best linear unbiased predictions, based on CBSD root necrosis data collected from trials established during 2012 (unreplicated trials at NaCRRI) and 2013 (replicated trials at both NaCRRI and Kasese); Cassava brown streak root severity scored using the 1–5 severity scale. This dataset is based on evaluation of 300 to 450 clones that were established in single row plots of 10 plants/row. Parental lines SE95/00036, NASE 12, NASE 4, and NASE 11 are highly susceptible to CBSD, while parental lines TMS 30572, TMS 60142 and NASE 14 are classified as tolerant to CBSD. The best clone was Ug120198 (with BLUP value of –48.2) and the worst performing clone was Ug120278 (BLUP value of 65.8). few outstanding individuals (19 out of the original 474 S BLUP value of –48.2), Ug120024 (F of NASE 14 × 1 1 clones) that are of interest for CBSD breeding, as they Namikonga, with BLUP value of –48.1), Ug120190 (intro- demonstrate the benefit of inbreeding in cassava, particular - duction from Tanzania, with BLUP value of –48.1), ly when combined with stringent selection. Ug120022 (F of NASE 14 × Namikonga, with BLUP value Data on the performance of the best-five and worst-five of –47.7) and Ug120001 ((F of NASE 3 × NASE 12, with progeny generated from four of the 49 parental lines is pre- BLUP value of –46.7). The worst clone Ug120278 (an F of sented in Table 5. The selected parental clones, all of IITA NASE 10 × NASE 9) had BLUP value of 65.8 with respec- pedigree included: NASE 1 (TMS 60142), NASE 3 (TMS tive root mean severity of 3.64. 30572), NASE 14 (MM96/4271) and TME 14. The parental lines NASE 1, NASE 3 and NASE 14, have all been associ- CBSD genome-wide association studies ated with low CBSD symptoms. Progeny of TME 14 were Five SNPs all located on chromosome 11 had significant added for comparison purposes. It is evident from the data signals (Table 6, Fig. 1). However, these SNPs did not that each of the parental lines had outstanding progeny; reach genome-wide Bonferroni significance threshold (of –7 among the top-best, the most CBSD resistant progeny P = 3.44 × 10 ). Four of these SNPs were identified with (Ug120001 and Ug120104) were all derived from TMS mean root severity. Two SNPs (S11-22909579 and S11- 30572, as they had BLUP values of –46.7 (Table 5). The 19872319) were identified with both mean root severity and best (Ug120002) and worst (Ug120009) progeny of parental disease index data. SNP S11-23228224 was the only signifi- clone TMS 60142, were all derived from the same cross cant signal that was obtained using maximum root severity. combination (Table 5), an illustration of the heterozygosity These SNPs are physically located between 19872319 to challenge in cassava breeding. 23751929 bp, a segment which most likely harbors the gene However, when all progeny from different combinations locus conditioning resistance and/or tolerance to CBSD root were analyzed together (including the introductions from necrosis. On average the identified SNPs explained 14.6% Tanzania; data not shown), we observed that the top five of CBSD root necrosis phenotypic variation. clones were: Ug120198 (introduction from Tanzania, with Based on the BLASTx plant protein search, four different 566 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS Table 6. SNP markers significantly associated with CBSD root necrosis resistance and their respective P-values Candidate gene a 2 b Trait CHR Marker Position (Mb) P-value R Gene BLASTx plant protein d –6 Mean Sev 11 S11-19872319 19872319 4.27 × 10 0.14 Cassava4.1_019379m lysM domain containing protein –6 11 S11-23751929 23751929 4.42 × 10 0.14 Cassava4.1_028097m glycine-rich protein –6 11 S11-22909579 22909579 5.78 × 10 0.14 – Ankyrin-3-like protein –6 11 S11-22909532 22909532 7.65 × 10 0.16 – – e –6 Index 11 S11-22909579 22909579 5.43 × 10 0.13 – Ankyrin-3-like protein –6 11 S11-19872319 19872319 6.11 × 10 0.13 Cassava4.1_019379m lysM domain containing protein f –6 Max Sev 11 S11-23228224 23228224 3.15 × 10 0.17 Cassava4.1_00037m 3.5.2.9-5-oxoprolinase enzyme a b c d Chromosome; Proportion of genetic trait variation explained by SNPs; Obtained through BLAST search against Phytozome 10.3; Mean root e f severity; Disease index as described by Kaweesi et al. (2014); Maximum root severity. proteins were identified from this study. These included (Ndunguru et al. 2015) and/or virus load during the evalua- lysM domain containing protein, glycine-rich protein, tion periods. For example, if evaluations are conducted for ankyrin-3-like protein and 3.5.2.9-5-oxoprolinase enzyme three seasons/years, virus monitoring should be done for (Table 6). The Q-Q plots which were used to evaluate the each season; 10–20 plots/clones with severe disease symp- best trait for CBSD association tests are displayed in Fig. 2. toms and 10–20 plots/clones with transient and/or no symp- It’s evident that 99% of the SNPs had P-values greater than toms should be assayed. This is particularly relevant in early 0.001 for mean severity and disease index showing that the selection stages i.e., clonal trials which often involve evalua- bulk of distribution behaved the way it should, based on the tion of >150 clones. Fortunately, optimal sampling sched- no association hypothesis. However, for root incidence and ules (with details of plant growth stage and plant part) for maximum severity, there was an early deviation from the CBSVs have been described (Kaweesi et al. 2014, Ogwok perfect diagonal (which corresponds to the null hypothesis), et al. 2015). Thus, as cheaper and reliable methods to quan- which means that statistical distribution is not appropriate tify CBSV viral loads become available, each individual for association tests. plot and/or clone can be monitored and its respective viral load compared with the disease severity. Discussion It is also evident from the generated field datasets that entailed evaluation of >250 clones for minimum of three Since the first reports of CBSD breeding in the early 1930’s years, that no consistent relationship exists between CBSD (Jennings 1957, Nichols 1947), limited genetics and/or foliar and root symptoms i.e., most R values were <10% breeding information has been generated. This is evident in (Tables 3, 4). This could suggest that these are different the few published work between the 1930s and 2015, as cit- traits under different genetic and/or biochemical mecha- ed herein. Thus, this paper presents and discusses empirical nisms. Thus, in terms of measurement, both traits can be mea- CBSD data generated in the last 11 years. From these data- sured and final categorizations of CBSD response based on sets, important information relating to CBSD and/or CBSV both as proposed by Kaweesi et al. (2014). In fact, in absence evaluation, scoring methodologies and breeding strategies of immunity, foliar CBSD assessments will continue to be have been gained. This information will specifically be rele - critically important in early selection stages (for purposes of vant for on-going CBSD breeding efforts and consequently, culling) and for cassava seed certification. inform the future breeding interventions aimed at combat- In practice, methodologies for measuring root severity ing CBSD. and/or root incidence can be variable. From experience, we It is evidently clear that CBSD and/or CBSV evaluations observe that different genotypes exhibit varying frequencies be conducted in a truly CBSD hotspot for a minimum of of total harvested roots and/or root severity scores per plant, three years to reliably classify clone responses. The sharp a situation that complicates genotype categorization and/or contrast in number of clones with CBSD root severity comparison. It is commonplace for breeding programmes to scores of 1 and/or 2 (classified as resistant) in 2011, and in use a maximum score, on the scale of 1–5, for the entire 2014 for both F and S families (Tables 3, 4) is testimony plot, others may use a maximum score per plant and then 1 1 for this. This also provides an opportunity to assess degen- compute plot averages; clearly, the two do not equate. It’s eration purported to arise from increased viral load observed also evident that a root that is assigned a score of 1 is eco- during the clonal propagation cycles. It is preferable to nomically very different from a root that is assigned a score measure virus load using real-time PCR during CBSD eval- of either 3, 4 or 5. Thus, discussions are on-going to find uations. solutions to this by way of accounting for the variable num- However in situations where large-scale virus monitor- ber of roots sampled per genotype through the development ing is not possible for each individual plot and/or clone, of a CBSD root necrosis index. then representative plots and/or clones could be sampled Regarding CBSD resistance breeding it is encouraging to and monitored to get insights into virus species dynamics note that outstanding CBSD resistant and/or tolerant clones 567 Breeding Science BS Vol. 66 No. 4 Kawuki, Kaweesi, Esuma, Pariyo, Kayondo, Ozimati, Kyaligonza, Abaca, Orone, Tumuhimbise et al. Fig. 1. Manhattan plots for genome wide association analysis for CBSD root necrosis resistance based on mixed linear models. Mean severity of root necrosis (a); disease index of root necrosis (b); maximum root severity (c); and incidence of root necrosis (d). The redline represents the Bonferroni correction threshold that determines SNPs with genome wide significance signal. have been identified. Based on evaluations undertaken in Notable of these are progeny derived from Namikonga, the past decade, we have identified some clones with rea- NASE 1 (TMS 60142), NASE 3 (TMS 30572), NASE 14 and sonable resistance and/or tolerance to CBSD. These clones M. esculenta selections introduced from Tanzania. Namikonga come from half-sibs, full-sibs and/or S cassava families derived F s (particularly those involving NASE 14), and the 1 1 generated from CBSD tolerant and/or resistant genotypes. S partial inbreds were truly outstanding (Tables 3, 4). 568 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS evaluations were based on seedlings, it will be necessary to re-evaluate the promising wild seedlings in clonal trials where a larger number of roots can be assessed per clone. In addition, the identified CBSD resistance/tolerance genes particularly those identified within M. esculenta germplasm sourced from Tanzania, can be exploited. On the other hand, CMD datasets (generated from the wild relatives), demonstrated their genetic value. Some in- dividuals from M. caerulescens, M. dichotoma, M. irwinii and M. cartheginensis ssp. glaziovii, registered CMD severi- ty scores of 1 and/or 2 (Table 2), and thus qualifying them as potentially useful sources of resistance to cassava germini- viruses. These CMD resistance sources from the wild will certainly compliment the widely deployed resistance that was exploited from M. glaziovii in the 1930s (Jennings 1957). It also suffices to note that during this period cassava varieties NASE 14, NARO-CASS 1 (synonym Tz 130) and NARO- CASS 2 have been officially released for commercial pro- duction; NASE 14 and NARO-CASS 1, have also been shared with four other countries (Kenya, Tanzania, Malawi and Mozambique) that are equally challenged by CBSD. Within limits, the CBSD association genetic study identi- fied five significant SNPs, all physically located between 19872319–23751929 bp on chromosome 11. The consistent presence of more than one SNP in this region suggests that this chromosome region is one of the quantitative trait loci (QTL) for CBSD root necrosis resistance. In this study, Q-Q plots were used to evaluate the best phenotype to use for CBSD genetic association tests. It was evident that 99% of the SNPs had P-values greater than 0.001 for mean severity and disease index showing that the bulk of distribution behaved the way it should, based on the no association hypothesis. However, for CBSD incidence and maximum severity, there was an early deviation from the perfect diagonal (which corresponds to the null hypothesis), which limits the utility of the two traits (incidence and maximum severity) for CBSD association studies. Plants possess pattern recognition receptors (PRRs) for their defense against pathogens (Nicaise et al. 2009). In- deed, a number of membrane-bound or soluble PRRs with lectin domain, have been identified as frontiers for plant de- fense (Lannoo and Van Damme 2014, Van Damme et al. Fig. 2. Q-Q plots of SNPs at marker level (P-values). Mean root se- 2008). Based on the BLASTx plant protein search, four dif- verity (a); disease index of root necrosis (b); maximum root severity ferent proteins were identified: lysM domain containing (c); and incidence of root necrosis (d). Deviation from the identity line protein, glycine-rich protein, ankyrin-3-like protein and at different significance levels showed the amount of false positive 3.5.2.9-5-oxoprolinase enzyme (Table 6). LysM-domain tests resulted from the analysis of the data; most deviations were ob- lectins is one of the four lectin receptor kinases (LecRK) served for maximum severity and root incidence. that have been reported to act both upon biotic and abiotic stresses (Vaid et al. 2013). These identified clones have remained symptomless or have It can therefore be hypothesized that the LysM domain shown mild CBSD symptoms (maximum severity score 2) containing protein observed from this study, may have a with low foliar and/or root incidences (<15%) after five role in CBSD root necrosis. Lozano et al. (2015), observed years of evaluation. that cassava chromosome 11 has three TOLL/interleukin-1 Equally striking was the identification of some wild rela - receptor (TIR-NBS-LRR) and one coiled- coil N-terminal tives, notably from M. carthaginensis ssp. glaziovii and domain (CC-NBS-LRR). None of these known resistance M. glaziovii that showed no CBSD symptoms. Because gene orthologs were identified in this study. This concurs 569 Breeding Science BS Vol. 66 No. 4 Kawuki, Kaweesi, Esuma, Pariyo, Kayondo, Ozimati, Kyaligonza, Abaca, Orone, Tumuhimbise et al. with the finding of Maruthi et al. (2014) where none of the We also thank IITA and the Agricultural Research Insti- known resistance gene orthologs were uniquely over ex- tute (ARI) Kibaha, Tanzania for sharing with us the cassava pressed in CBSD-resistant genotype (Namikonga). Thus, germplasm. more studies are therefore needed to further explore this region and/or other genomic regions to get further insights Literature Cited into genes that contribute to resistance and/or tolerance to CBSD root necrosis. Currently, efforts are underway to un- Abaca, A., R. Kawuki, P. Tukamuhabwa, Y. Baguma, A. Pariyo, T. Alicai, dertake detailed CBSD genome wide analysis studies that C.A. Omongo and A. Bua (2012a) Progression of cassava brown aim to get further insights into genes and/or chromosomal streak disease (CBSD) in Infected cassava roots in Uganda. Uganda regions controlling CBSD resistance. J. Agri. Sci. 13: 45–51. So far, all CBSD genetic studies conducted confirm the Abaca, A., R. Kawuki, P. Tukamuhabwa, Y. Baguma, A. Pariyo, T. Alicai, preponderance of additive genetic effects (Kulembeka C.A. Omongo and A. Bua (2012b) Evaluation of local and elite cassava genotypes for resistance to cassava brown streak disease in 2010, Kulembeka et al. 2012, Munga 2008, Zacarias and Uganda. J. Agron. 11: 65–72. Labuschagne 2010) and significant genotype by environ- Alicai, T., C.A. Omongo, M.N. Maruthi, R.J. Hillocks, Y. Baguma, ment interactions (Pariyo et al. 2015, Tumuhimbise et al. R. Kawuki, A. Bua, G.W. Otim-Nape and J. Colvin (2007) Re- 2014b). A number of factors are likely to amplify the geno- emergence of cassava brown streak disease in Uganda. Plant Dis. type × environmental interaction for CBSD including geno- 91: 24–29. type susceptibility levels, predominant virus species in lo- Bernardo, R. (2010) Breeding for Quantitative Traits in Plants, 2nd cality and/or season, and climatic factors that either edn. Stemma Press, Woodbury, Minnesota. influence the abundance of whitefly vectors and/or the Bigirimana, S., P. Barumbanze, P. Ndayihanzamaso, R. Shirima and growth rate of the crop (Katono et al. 2015). The discovery J.P. Legg (2011) First report of cassava brown streak disease and of four distinct virus species (Ndunguru et al. 2015), is like- associated Ugandan cassava brown streak virus in Burundi. New ly to further complicate the extent of genotype by environ- Dis. Rep. 24: 26. Dellaporta, S., J. Wood and J. Hicks (1983) A plant DNA miniprepara- ment interaction, as CBSD symptom expression (pheno- tion: version II. Plant Mol. Biol. Rep. 1: 19–21. types) associated with virus species are likely to differ Elshire, R.J., J.C. Glaubitz, Q. Sun, J.A. Poland, K. Kawamoto, between environments. Therefore, future CBSD breeding E.S. Buckler and S.E. Mitchell (2011) A robust, simple genotyping- strategies have to be designed mindful of these factors. by-sequencing (GBS) approach for high diversity species. PLoS Accordingly, if selection for hybridization is to be based ONE 6: e19379. on phenotypes, then cycle time can be reduced by having Glaubitz, J.C., T.M. Casstevens, F. Lu, J. Harriman, R.J. Elshire, Q. Sun field nurseries that serve both as evaluation and hybridiza - and E.S. Buckler (2014) TASSEL-GBS: a high capacity genotyping tion plots. For instance, final CBSD phenotypes (foliar and by sequencing analysis pipeline. PLoS ONE 9: e90346. rd root) can be scored (during 3 year of evaluation) at seven Goodstein, D.M., S. Shu, R. Howson, R. Neupane, R.D. Hayes, J. Fazo, MAP and then clones with low severities (combined with T. Mitros, W. Dirks, U. Hellsten, N. Putnam et al. (2012) Phyto- desired agronomic traits) crossed to constitute next cycle for zome: a comparative platform for green plant genomics. Nucleic Acids Res. 40: 1178–1186. selection. This simplistic 3–4 year CBSD breeding cycle Hillocks, R.J. and J.M. Thresh (2000) Cassava mosaic and cassava can be explored to attain higher levels of resistance. On the brown streak virus diseases in Africa: A comparative guide to other hand, if selection is to be based on genotypic data as symptoms and aetiologies. Roots 7: 1–8. implemented for genomic selection (Oliviera et al. 2012), Hillocks, R.J., J.M. Thresh, J. Tomas, M. Botao, R. Macia and R. Zavier then training populations will initially be needed to develop (2002) Cassava brown streak disease in northern Mozambique. Int. prediction models. It is through this approach that SNP J. Pest Manag. 48: 178–181. markers associated with CBSD resistance genes can be giv- Hillocks, R.J. and D.L. Jennings (2003) Cassava brown streak disease: en more weights in the estimation of genomic estimated a review of present knowledge and research needs. Int. J. Pest breeding values that are used in parental selection. Manag. 49: 225–234. IITA (1990) Cassava in Tropical Africa: A Reference Manual. Inter- Acknowledgements national Institute of Tropical Agriculture, Ibadan, Nigeria. Jameson, J.D. (1964) Cassava mosaic disease in Uganda. East Afr. Agric. For. J. 29: 208–213. The authors thank all technicians and support staff of Root Jennings, D.L. (1957) Further studies in breeding cassava for virus Crops Programme, NaCRRI, who participated in CBSD resistance. East Afr. Agric. J. 22: 213–219. data collection for experiments conducted during the period Jennings, D.L. (1959) Manihot melanobasis Müll. Arg.—A useful 2004 to 2015. Several research projects supported CBSD parent for cassava breeding. Euphytica 8: 157–162. research in Uganda. Notable of these included: The Millen- Katono, K., T. Alicai, Y. Baguma, R. Edema, A. Bua and C.A. Omongo nium Science Initiative (MSI); the East African Agricultural (2015) Influence of host plant resistance and disease pressure on Productivity Project (EAAPP); Biotechnology Tools to spread of cassava brown streak disease in Uganda. Am. J. Exp. Combat CBSD; Africa-Brazil Market Place the Government Agric. 7: 284–293. of Uganda; and the Next Generation Cassava Breeding Pro- Kaweesi, T., R. Kawuki, V. Kyaligonza, Y. Baguma, G. Tusiime and M.E. ject. Ferguson (2014) Field evaluation of selected cassava genotypes for 570 Breeding Science Breeding for cassava brown streak disease resistance Vol. 66 No. 4 BS e0139321. cassava brown streak disease based on symptom expression and Nicaise, V., M. Roux and C. Zipfel (2009) Recent advances in virus load. Virol. J. 11: 216. PAMP-triggered immunity against bacteria: pattern recognition re- Kawuki, R.S., A. Pariyo, T. Amuge, E. Nuwamanya, G. Ssemakula, ceptors watch over and raise the alarm. Plant Physiol. 150: 1638– S. Tumwesigye, A. Bua, Y. Baguma, C. Omongo, T. Alicai et al. (2011) A breeding scheme for local adoption of cassava (Manihot Nichols, R.F.W. (1947) Breeding cassava for virus resistance. East Afr. esculenta Crantz). J. Plant Breed. Crop Sci. 3: 120–130. Agric. J. 12: 184–194. Kulembeka, H.P.K. (2010) Genetic linkage mapping of field resistance Nuwamanya, E., Y. Baguma, E. Atwijukire, S. Acheng and T. Alicai to cassava Brown Streak disease in cassava (Manihot esculenta (2015) Effect of cassava brown streak disease (CBSD) on cassava Crantz) landraces from Tanzania. University of the Free State, (Manihot esculenta Crantz) root storage components, starch quan- Bloemfontein, South Africa. tities and starch quality properties. Int. J. Plant Physiol. Biochem. Kulembeka, H.P., M. Ferguson, L. Herselman, E. Kanju, G. Mkamilo, 7: 12–22. E. Masumba, M. Fregene and M.T. Labuschagne (2012) Diallel Ogwok, E., T. Alicai, M.E.C. Rey, G. Beyene and N.J. Taylor (2015) analysis of field resistance to brown streak disease in cassava Distribution and accumulation of cassava brown streak viruses (Manihot esculenta Crantz) landraces from Tanzania. Euphytica within infected cassava (Manihot esculenta) plants. Plant Pathol. 187: 277–288. 64: 1235–1246. Lannoo, N. and E.J.M. Van Damme (2014) Lectin domains at the fron- Pariyo, A., Y. Baguma, T. Alicai, R. Kawuki, E. Kanju, A. Bua, C. tiers of plant defense. Front. Plant Sci. 5: 397. Omongo, P. Gibson, D.S. Osiru, D. Mpairwe et al. (2015) Stability of Legg, J.P. and C. Fauquet (2004) Cassava mosaic geminiviruses in resistance to cassava brown streak disease in major agro-ecologies Africa. Plant Mol. Biol. 56: 585–599. of Uganda. J. Plant Breed. Crop Sci. 7: 67–78. Legg, J.P., S.C. Jeremiah, H.M. Obiero, M.N. Maruthi, I. Ndyetabula, Pennisi, E. (2010) Armed and dangerous. Science 327: 804–805. G. Okao-Okuja, H. Bouwmeester, S. Bigirimana, W. Tata-Hangy, Rutherford, K., J. Parkhill, J. Crook, T. Horsnell, P. Rice, M.A. G. Gashaka et al. (2011) Comparing the regional epidemiology of Rajandream and B. Barrell (2000) Artemis: sequence visualization the cassava mosaic and cassava brown streak virus pandemics in and annotation. Bioinformatics 16: 944–945. Africa. Virus Res. 159: 161–170. Swarts, K., H. Li, J.A. Romero Navarro, D. An, M.C. Romay, S. Hearne, Lozano, R., M.T. Hamblin, S. Prochnik and J.L. Jannink (2015) Identi- C. Acharya, J.C. Glaubitz, S. Mitchell, R.J. Elshire et al. (2014) fication and distribution of the NBS-LRR gene family in the cassa- Novel methods to optimize genotypic imputation for low-coverage, va genome. BMC Genomics 16: 360. next-generation sequence data in crop plants. Plant Genome 7: Maruthi, M.N., S. Bouvaine, H.A. Tufan, I.U. Mohammed and R.J. 1–12. Hillocks (2014) Transcriptional response of virus-infected cassava Tumuhimbise, R., R. Melis and P. Shanahan (2014a) Diallel analysis of and identification of putative sources of resistance for cassava early storage root yield and disease resistance traits in cassava brown streak disease. PLoS ONE 9: e96642. (Manihot esculenta Crantz). Field Crops Res. 167: 86–93. Mbanzibwa, D.R., Y.P. Tian, A.K. Tugume, S.B. Mukasa, F. Tairo, Tumuhimbise, R., R. Melis, P. Shanahan and R. Kawuki (2014b) Geno- S. Kyamanywa, A. Kullaya and J.P.T. Valkonen (2009) Genetically distinct strains of Cassava brown streak virus in the Lake Victoria type × environment interaction effects on early fresh storage root basin and the Indian Ocean coastal area of East Africa. Arch. Virol. yield and related traits in cassava. Crop J. 2: 329–337. 154: 353–359. Tumuhimbise, R., P. Shanahan, R. Melis and R. Kawuki (2015) Genetic Mohammed, I.U., M.M. Abarshi, B. Muli, R.J. Hillocks and M.N. variation and association among factors influencing storage root Maruthi (2012) The symptom and genetic diversity of cassava bulking in cassava. J. Agric. Sci. 153: 1267–1280. brown streak viruses infecting cassava in East Africa. Adv. Virol. Van Damme, E.J.M., N. Lannoo and W.J. Peumans (2008) Plant lectins. 2012: 795–697. Adv. Bot. Res. 48: 107–209. Munga, T.L. (2008) Breeding for Cassava Brown Streak Resistance in Winter, S., M. Koerbler, B. Stein, A. Pietruszka, M. Paape and A. Coastal Kenya. KwaZulu-Natal, South Africa. Butgereitt (2010) Analysis of cassava brown streak viruses reveals Ndunguru, J., P. Sseruwagi, F. Tairo, F. Stomeo, S. Maina, A. Djinkeng, the presence of distinct virus species causing cassava brown streak M. Kehoe and L.M. Boykin (2015) Analyses of twelve new whole disease in East Africa. J. Gen. Virol. 91: 1365–1372. genome sequences of cassava brown streak viruses and Ugandan Zacarias, A.M. and M.T. Labuschagne (2010) Diallel analysis of cassa- cassava brown streak viruses from East Africa: diversity, super- va brown streak disease, yield and yield related characteristics in computing and evidence for further speciation. PLoS ONE 10: Mozambique. Euphytica 176: 309–320.

Journal

Breeding SciencePubmed Central

Published: Aug 5, 2016

References