marker-assisted selection in wheat - ictsd

Chapter 6 – Targeted introgression of cotton fibre quality quantitative trait loci using molecular markers 71planted in two replications, each plot (onerow) measuring 5 m. The next BC 3 andBC 4 generations were grown under field(411 BC 3 in 2002) or greenhouse (450 BC 4in 2003) conditions in Montpellier. Everyplant in each BC 1-4 generation was used forDNA extraction from young fresh leavesusing different methods described elsewhere(Lacape et al., 2003; Nguyen et al.,2004). In each BC 3 and BC 4 generation, anearly genotyping was conducted (beforeflowering of BC 3 plants and at the seedlingstage for BC 4 plants), to reduce the numberof plants to be manipulated and raised toflowering for selfing and backcrossing.From each generation (75 BC 1 , 200 BC 2 ,400 BC 2 S 1 , 43 selected BC 3 and 37 selectedBC 4 ), the cotton seed harvest was ginned(separation of the fibre from the seed) on alaboratory roller gin and the fibre was sampledfor analyses at the Fibre TechnologyLaboratory of the French AgriculturalResearch Centre for International Development(CIRAD).Fibre analysesAll fibre quality measurements (11 traits)were conducted at CIRAD, Montpellier,on a high volume instrument line (ZellwegerUster 900, Uster Technologies, Switzerland).These included length, uniformity, strength,elongation and colour. A FMT3 maturimeter(Shirley Dev Ltd., UK) was used to determinemicronaire value, maturity and fineness.Molecular analysesThe different types of markers displayingpolymorphism between G. hirsutum andG. barbadense included restriction fragmentlength polymorphisms (RFLPs) (used onlyin the BC 1 generation), simple sequencerepeats (SSRs) and amplified fragment lengthpolymorphisms (AFLPs). Details of themarkers and protocols used are providedin Lacape et al. (2003) and Nguyen et al.(2004). The AFLP markers were all derivedfrom combinations of EcoRI/MseI primerpairs (64 pairs in the BC 1 , 45 in the BC 2 and30 in the BC 3 and BC 4 generations). Thecotton microsatellites were derived essentiallyfrom two public libraries, BrookhavenNational Laboratory (BNL) and CIRAD(CIR). In the BC 1 generation, the microsatellitesused included 188 polymorphic BNLmarkers out of the 216 available (Lacapeet al., 2003) and 204 CIR markers out of392 developed (Nguyen et al., 2004). Fromthe results of the combined QTL analysesof the BC 1 /BC 2 /BC 2 S 1 generations (Lacapeet al., 2005), QTL-rich regions were identifiedon “carrier” chromosomes, and SSRloci present within or in the vicinity ofthese regions were assembled for constitutinggroups of three SSRs (one group perregion) to be tested as multiplexes, takinginto account both annealing temperatureand compatibility of sizes of amplified fragments.A subset of 60 SSR (20 region-specifictriplexes) was used for early genotyping ofall 411 BC 3 and 450 BC 4 plants (see examplesin Figure 2). The individual plants selectedfrom BC 3 and BC 4 (43 and 37 plants respectively)were further analysed using knownAFLPs to provide broad genome coverage.In the context of our marker-assisted introgressionprogramme, the SSR markerstarget the QTL-rich regions, i.e. those lociof the “foreground genome” expected tohave been introgressed, while the AFLPmarkers essentially serve to cover the rest ofthe genome, i.e. the “background genome”,aimed at returning to the recipient genomecomposition.Construction of genetic mapThe BC 1 (75 individuals) and BC 2 (200individuals) maps were constructed separatelyusing the MapMaker 3.0 software