Translational Genomics for Crop Breeding (Biotic Stress) || Enabling Tools for Modern Breeding of Cowpea for Biotic Stress Resistance

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<ul><li><p>Chapter 10</p><p>Enabling Tools for Modern Breeding ofCowpea for Biotic Stress ResistanceBao-Lam Huynh, Jeffrey D. Ehlers, Timothy J. Close, Ndiaga Cisse, Issa Drabo, OusmaneBoukar, Mitchell R. Lucas, Steve Wanamaker, Marti Pottorff, and Philip A. Roberts</p><p>Abstract</p><p>This chapter summarizes recent advances in genomic resources, technologies, breeding platforms,and molecular markers that are being used to expedite delivery of cowpea cultivars with resistanceto important biotic stresses. The genomic resources available in cowpea include high-throughputsingle nucleotide polymorphism (SNP) genotyping platforms, a high-density consensus genetic mapwith more than 1,100 markers, a whole genome sequence assembly, bacterial articial chromosomes(BACs), and a physical map anchored to the genetic map.Markers linked to important biotic resistancetraits, including resistance to foliar and ower thrips, Fusarium wilt, root-knot nematode, bacterialblight, ashy stem blight (Macrophomina), Striga, and viruses are described, together with toolsdeveloped to integrate SNP genotypes, genetic maps, and trait information to guide breeding. Agenetic transformation system has been developed for cowpea and breeding lines developed withresistance to cowpea weevil and Maruca pod borer. Some examples of initial work in evaluating andoptimizing marker-assisted backcross and marker-assisted pedigree breeding are described. Majorchallenges that must be overcome to allow for the adoption of modern marker-assisted breeding(MAB) in cowpea include human and precision phenotyping capacity issues. Further, as essentiallyall cowpea breeding is being conducted by the public sector with modest resources, efcient strategiesare needed to minimize running costs.</p><p>Introduction</p><p>Cowpea (Vigna unguiculata L. Walp.) is perhapsthemost widely adapted, versatile, and nutritiousgrain and fodder legume for warm and dry agro-ecologies of the tropics and subtropics. World-wide there aremore than 14million ha of cowpea</p><p>being cultivated, of which about three-fourthsof the area and two-thirds of the productionoccur in the semi-arid Sudan savanna and Sahe-lian zones of the African continent running fromwest to east, south of the Saharan desert, and inmore dispersed but extensive pockets of similaragro-ecologies in eastern and southern Africa.</p><p>Translational Genomics for Crop Breeding, Volume I: Biotic Stress, First Edition. Edited by Rajeev K. Varshney and Roberto Tuberosa.C 2013 John Wiley &amp; Sons, Inc. Published 2013 by John Wiley &amp; Sons, Inc.</p><p>183</p></li><li><p>184 TRANSLATIONAL GENOMICS FOR CROP BREEDING</p><p>Substantial quantities of cowpea also are pro-duced in similar agro-ecologies in South Amer-ica, with Brazil being the second-largest produc-ing country in the world after Nigeria. Althoughof less importance today than in the recentpast, cowpea is also grown in the southern andwestern parts of North America and is a tra-ditional crop of several indigenous tribes oflower-elevation central Mexico. A form of cow-pea (subspecies sesquipedialis) known as longbean or asparagus bean is cultivated as a veg-etable crop throughout East Asia for its longeshy green pods that can be onemeter in length.Long bean is fully fertile with other cultivatedand some wild subspecies of cowpea and com-parisons of genetic maps show essential iden-tity (Xu et al. 2010). Cowpea was documentedfrom before Roman times in Europe and thepresence of signicant phenotypic and genotypicdiversity exists among landrace germplasm fromItaly (Tosti and Negri 2002) and other southernEuropean countries, suggesting a long history ofuse in this region. As a general rule, cowpeasare grown in hotter low-elevation equatorial andsubtropical areas, often being replaced by com-mon bean (Phaseolus vulgaris L.) at altitudesabove 1,300 m, although cowpeas are grownat altitudes up to 1,500 m in Kenya. Commer-cial production of the crop extends as far northas 40 latitude in California, and experimen-tal plantings of early maturing breeding lineshave been successful as far north as Minnesota(45 N latitude) in the United States (Daviset al. 1986).</p><p>While cowpea is both responsive to favorablegrowing conditions and tolerant to drought, hightemperatures, and poor soils (Hall 2004; Fery1990), biotic stresses from pests and diseasesinict heavy losses and are key factors under-lying why on-farm cowpea yields of traditionalvarieties in Africa are 3- to 5-fold lower thanpotential yields (Ehlers and Hall 1997).</p><p>Development of cowpea cultivars that resistor tolerate biotic stresses would result in dra-matic yield improvements. Breeding resistant</p><p>cultivars is a particularly desirable strategy forthis crop because it is grown mostly by resource-poor farmers, many of whom are women wholack access to capital for application equipment,pesticides, and protective wear, as well as toexpertise in the efcacious and safe use of theseproducts.</p><p>Breeding for resistance to biotic stresses hasbeen undertaken by the International Institutefor Tropical Agriculture (IITA) and by AfricanNational Research Systems (NARS), in somecases supported by the USAID CollaborativeResearch Support Program (CRSP) and otherdonors. These breeding programs have achieveda number of successes in the last 20 years,having bred cultivars resistant to some of thekey pests such as cowpea aphid, cowpea wee-vil, the parasitic weed Striga gesneroides, bac-terial blight, root-knot nematodes, and cowpeaaphid-borne mosaic virus (CABMV) (Hall et al.1997; Hall et al. 2003; Timko et al. 2007) andthereby contributing to raised on-farm yields.These breeding efforts employed wholly con-ventional breeding approaches and typically tookmore than ten years from concept and crossing torelease.</p><p>Similar to breeding programs in other crops,the need to employ sequential and repeated phe-notypic evaluations and performance trials isthe most resource-intensive and time-consumingaspect of the process. In many cases, these eval-uations require complex, specialized conditions,techniques and expertise to assess phenotypesfor selection. These evaluation resources are dif-cult to assemble and costly to operate, and inmany cases it is not possible to conduct a com-plete breeding program consisting of teams ofscientists of all necessary disciplines because ofbudget and manpower limitations for orphancrops (Delmer 2005) such as cowpea. Selec-tion based on molecular markers linked to keybiotic resistance traits or quantitative trait loci(QTL) can be used to reduce the phenotyp-ing burden typically required through conven-tional breeding efforts and thereby help speed the</p></li><li><p>ENABLING TOOLS FOR MODERN BREEDING OF COWPEA 185</p><p>delivery of improved varieties. Neutral, geneticbackground molecular markers that are welldistributed through the genome are also partof molecular marker-assisted selection (MAS)schemes such as marker-assisted backcrossing(MABC), where the goal is to reassemble thegenetic background of the recurrent parent withthe addition of one to a few target traits. Accord-ingly, MAS provides a powerful and potentiallycost-saving avenue for increasing the rates ofgenetic gain in plant breeding programs (Xu andCrouch 2008).</p><p>In the recent past, cost and technology limi-tations meant that MAS was restricted to majorcrops and to one or a very few high priority traits,where phenotypingwas costly or otherwise prob-lematic. The development of relatively low costhigh-throughput SNP genotyping platforms inmany crops and the availability of high-densitygenetic linkagemaps have greatly enhancedwhatis now possible with MAS. These capabilitiesmean new breeding strategies can now be con-sidered that utilize molecular marker informa-tion at tens to thousands of points in the genome,encompassing selection for multiple traits and/ormultigenic traits simultaneously. Selection tar-gets include easy-to-phenotype agronomic andgrain quality traits such as grain size, texture,and color, in addition to multiple biotic stresstolerance and resistance traits that are more dif-cult to phenotype.</p><p>The effectiveness of MAS breeding schemesdepends on high quality phenotyping and preci-sion genotyping for assembly of robust marker-trait associations and QTL estimates, closenessof marker-trait determinant linkages, and rel-ative cost of genotypic selection compared totraditional selection protocols based on phe-notypic selection. MAS can increase the ef-ciency of breeding in several other ways, throughthe selection of desirable progeny for crossingbefore owering, providing year-round, environ-mentally independent selection capability, andthrough simultaneous selection of multiple traitsand for a particular genetic background. In addi-</p><p>tion, it is possible to deduce useful marker-trait associations and marker effects in earlygenerations of breeding cycles that can thenbe used for selection in later generations ifgenotypic information is available at the laterstage.</p><p>Pioneering the Use of SSRMarkers for Introgressionof Striga Resistance</p><p>To date only limited deployment of molecu-lar markers in cowpea breeding programs hasoccurred. MAS using simple sequence repeat(SSR, or microsatellite) markers to develop cow-pea varieties resistant to the parasitic weed Strigagesnerioides (Striga) was initiated in 2006 bythe national research organizations of Senegal,Burkina Faso, Nigeria, and Mali, and at IITAin Nigeria in collaboration with the Universityof Virginia (Timko et al. 2007). These projectshave focused on the use of a limited numberof SSR markers with the goal of introgress-ing Striga resistance into improved or local sus-ceptible varieties. This approach eliminates theneed for phenotypic evaluation of Striga resis-tance at each stage of the breeding process.Also, by incorporating resistance genes effectiveagainst multiple races of Striga prevalent acrossnational boundaries, MAS eliminates the needfor meeting quarantine requirements associatedwith the movement of Striga seeds of differentraces across national boundaries that would berequired for centralized phenotypic evaluationsof resistance. However, the approach uses onlytrait-linked foreground markers, meaning thatrecovery of the recurrent parent backgroundgenotype will occur at nearly the same rateas with conventional backcross breeding. SSRgenotyping with numerous background markerson multiple individuals to facilitate recovery ofthe recurrent parent would be cost prohibitive.Another major drawback of MAS using SSRmarkers in backcross breeding is that high qual-ity gels are difcult to produce consistently, thus</p></li><li><p>186 TRANSLATIONAL GENOMICS FOR CROP BREEDING</p><p>progress can be hampered by misclassied indi-viduals. This is in contrast to single nucleotidepolymorphisms, or SNPs, which show very highdelity of allele calling in repeated rounds ofgenotyping.</p><p>SNPs in Cowpea Breeding forBiotic Stress Resistance</p><p>Genotyping platforms based on SNPs are pre-ferred over other marker types in plant breed-ing applications because they are amenableto high-throughput yet highly precise assays(Rafalski 2002). SNPs derived from expressedsequence tags (ESTs) are especially usefulbecause they are derived ultimately from mRNAtranscripts and thereby reect allelic differ-ences between individuals and present oppor-tunities for cloning genes through comparisonswith annotated genome sequences of other plantspecies. SNP genotyping is highly developedfrom a technical and equipment perspectivebecause of the large investments made in thisarea in human and animal genetics that are beingleveraged for crop improvement. The privatecrop breeding sector was quick to grasp thisopportunity and develop these systems for majorcrop plants such as maize and soybean, hav-ing employed them in breeding programs formore than a decade (Eathington et al. 2007). Itappears that the private sector has found marker-assisted breeding (MAB) with SNPs to be aneffective approach, as evidenced by the factthat SNP genotyping platforms continue to bedeveloped for more and more crops bred in thissector. In cowpea, a 1536-Illumina GoldenGateSNP genotyping platformwas developed in 2009(Muchero et al. 2009) and is described brieybelow.</p><p>High-Throughput SNP GenotypingPlatform for Cowpea BreedingSeveral key resources are needed for the com-prehensive implementation of modern cowpea</p><p>breeding. First among these is high-throughputgenotyping capability, for rapid and dense n-gerprinting of parents and breeding progenies ata reasonable cost. This multiplexing approach isa critical advance over low-throughput systemswhere genotyping is conducted on a marker-by-marker, individual-by-individual basis. Forcowpea, a high-throughput genotyping platformwas developed between 2007 and 2009 as animportant outcome of a Tropical Legumes Iproject based at the University of California,Riverside, and funded by the ConsultativeGroup on International Agricultural Research(CGIAR) Generation Challenge Programme(GCP). The genotyping platform was developedas an Illumina GoldenGate Assay for 1536 SNPloci. This platform can genotype simultaneously96 DNA samples at 1,536 SNP loci. Thegenotypic information is provided to users withspecialized Illumina GenomeStudio software,which allows rapid visual inspection and datasummarization. The development and technicaldetails of this platform were presented in detailin the work of Muchero and colleagues (2009).Briey, this platform consists of 1,536 EST-derived SNPs chosen from a selected subset ofabout 10,000 SNPs identied from comparisonsof 183,000 EST sequences from 13 cowpeagenotypes. Of these, 1,375 SNPs performedtechnically well in the assay. Among the severaltechnical considerations in choosing the 1,536SNP subset, high polymorphic informationcontent among African germplasm was primary,in order to ensure that the chosen SNPs werehighly polymorphic in African breeding mate-rials. Examples of the numbers of polymorphicmarkers observed in crosses between breedinglines both within and between several Africanbreeding programs following genotyping withthis platform are shown in Table 10.1. Theseresults show a high average level of polymorphicmarkers between any two parents, ranging from134 to 391 and indicating that the SNPs selectedfor inclusion on the current 1,536 IlluminaGoldenGate platform represent an effective setfor MAS in West African breeding programs.</p></li><li><p>ENABLING TOOLS FOR MODERN BREEDING OF COWPEA 187</p><p>Table 10.1. Examples of the number of SNP markers that are polymorphic between two parental lines of potentialcombinations for intra- (e.g., IITA x IITA) and inter- (e.g., IITA x INERA) breeding program crosses.</p><p>Parent 1 Parent 2 Cross Type No. Polymorphic markers</p><p>IT84S-2246 IT93K-503-1 IITA x IITA 134IT89KD-288 Suvita-2 IITA x INERA 245KVx 525 KVx 396-4-5-20 INERA x INERA 257KVx 442-3 KVx 396-4-5-20 INERA x INERA 261Suvita-2 Melakh INERA x ISRA 274IT97K-499-35 Mouride IITA x ISRA 289Suvita-2 Calif. Blackeye No. 27 INERA x UCR 291IT84S-2246 IT00K-1263 IITA x IITA 294IT97K-499-35 IT00K-1263 IITA x IITA 309KVx 525 Bambey 21 INERA x ISRA 313Bambey 21 Melakh ISRA x ISRA 325...</p></li></ul>


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