Abstract
Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates trait-associated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads (⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had <10% missing data, and read depth had a median of 220–244×. Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy, flexibility, speed, high-throughput, low-cost and easily automated analysis.
Highlights
Marker-assisted selection (MAS) is commonly employed in perennial crop breeding programs to pursue the acceleration of cultivar development.[1,2,3] In particular, MAS has been shown to provide advantages for selection during the juvenile phase;[4,5] for pyramiding disease resistance genes;[6,7] and for replacing expensive, time-consuming or technically difficult traits.[8,9] inherited traits with Mendelian or near-Mendelian segregation patterns are major targets for MAS
The primer design pipeline and curation resulted in 54 amplicon sequencing (AmpSeq) markers physically located between the two flanking markers and genetically linked to the anchor markers, for further testing (Supplementary File 2)
The primer design procedure was facilitated by a decision support tool to predict which primers would perform appropriately in terms of predictability based on a pre-trained random forest model
Summary
Marker-assisted selection (MAS) is commonly employed in perennial crop breeding programs to pursue the acceleration of cultivar development.[1,2,3] In particular, MAS has been shown to provide advantages for selection during the juvenile phase;[4,5] for pyramiding disease resistance genes;[6,7] and for replacing expensive, time-consuming or technically difficult traits.[8,9] inherited traits with Mendelian or near-Mendelian segregation patterns are major targets for MAS. The development of molecular markers requires the detection of association between target traits and genotypes. Two approaches are often used to detect such associations: (a) QTL analysis with structured families, and (b) genome-wide association study, which takes advantage of linkage disequilibrium (LD) in diverse germplasm to capture the linkage between markers and causal genes.[16,17] for highly heterozygous and diverse crops, such as grape, genome-wide association study has limitations.[18,19,20] LD decays rapidly in species of Vitis, in which the square of the correlation coefficient (r2) declines to 0.1 within
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