Abstract
Genotyping by sequencing (GBS) enables genotyping of multiple loci at low cost. However, the single nucleotide polymorphisms (SNPs) revealed by GBS tend to be randomly distributed between individuals, limiting their direct comparisons without applying the various filter options to obtain a comparable dataset of SNPs. Here, we developed a panel of a multiplex targeted sequencing method, genotyping-in-thousands by sequencing (GT-seq), to genotype SNPs in Capsicum spp. Previously developed Fluidigm® SNP markers were converted to GT-seq markers and combined with new GT-seq markers developed using SNP information obtained through GBS. We then optimized multiplex PCR conditions: we obtained the highest genotyping rate when the first PCR consisted of 25 cycles. In addition, we determined that 101 primer pairs performed best when amplifying target sequences of 79 bp. We minimized interference of multiplex PCR by primer dimer formation using the PrimerPooler program. Using our GT-seq pipeline on Illumina Miseq and Nextseq platforms, we genotyped up to 1,500 (Miseq) and 1,300 (Nextseq) samples for the optimum panel size of 100 loci. To allow the genotyping of Capsicum species, we designed 332 informative GT-seq markers from Fluidigm SNP markers and GBS-derived SNPs. This study illustrates the first application of GT-seq in crop plants. The GT-seq marker set developed here will be a useful tool for molecular breeding of peppers in the future.
Highlights
Backcross breeding introduces a desired trait from a donor parent to an elite line lacking this trait, followed by multiple rounds of backcrosses to restore as much of the genetic background of the elite line in the new germplasm (Allard, 1999)
To test the efficacy of these single nucleotide polymorphisms (SNPs) markers to genotype a genomic segment introgressed to C. annuum from C. baccatum, we genotyped a 80 backcross population derived from a cross between (C. annuum “CM334” × C. baccatum “PBC81”) and “CM334,” designated CPIL BC1F1, by Genotyping by sequencing (GBS) (Supplementary Table 2)
We demonstrate that genotyping-in-thousands by sequencing (GT-seq) can be applied to plants, in this case using various chili pepper species
Summary
Backcross breeding introduces a desired trait from a donor parent to an elite line lacking this trait, followed by multiple rounds of backcrosses to restore as much of the genetic background of the elite line in the new germplasm (Allard, 1999). In traditional backcross breeding, backcrossing is repeated for at least five generations to sufficiently dilute the genetic information of the donor parent, outside of the trait of interest. Marker-assisted backcrossing (MABC) aims to speed up selection by combining foreground selection to select useful traits and background selection to evaluate the residual genetic contribution of the donor parent to the elite line genome (Charcosset, 1997). MABC, makes it possible to develop elite lines harboring novel useful traits in only two backcross generations (Visscher et al, 1996; Frisch et al, 1999; Collard and Mackill, 2008). By utilizing the MABC marker set, individuals with 96% of the recurrent parental genome could be obtained in the second backcross (Jeong et al, 2015)
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