Abstract
High-density single nucleotide polymorphism (SNP) markers are crucial to improve the resolution and accuracy of genome-wide association study (GWAS) and genomic selection (GS). Numerous approaches, including whole genome sequencing, genome sampling sequencing, and SNP chips are able to discover or genotype markers at different densities and costs. Achieving an optimal balance between sequencing resolution and budgets, especially in large-scale population genetics research, constitutes a major challenge. Here, we performed improved double-enzyme digestion genotyping by sequencing (ddGBS) on chicken. We evaluated eight double-enzyme digestion combinations, and EcoR I- Mse I was chosen as the optimal combination for the chicken genome. We firstly proposed that two parameters, optimal read-count point (ORP) and saturated read-count point (SRP), could be utilized to determine the optimal sequencing volume. A total of 291,772 high-density SNPs from 824 animals were identified. By validation using the SNP chip, we found that the consistency between ddGBS data and the SNP chip is over 99%. The approach that we developed in chickens, which is high-quality, high-density, cost-effective (300 K, $30/sample), and time-saving (within 48 h), will have broad applications in animal breeding programs.
Highlights
Genetic markers, as material for genetic research, have evolved from early restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs) to currently widely-used single nucleotide polymorphism (SNP) markers
A nine-generation advanced intercross population was established from two divergent chicken lines, High Quality chicken Line A (HQLA), a broiler line bred by Guangdong Wiz Agricultural Science and Technology, Co. (Guangzhou, China), and Huiyang Beard chicken (HB), a native Chinese meat-type breed
We evaluated each double-enzyme digestion strategy based on the enzyme digestion fragment size, the fragment consistency index (FCI), the coefficient of variation of sequencing depth across three samples (CVdepth), the number of SNPs, and the distribution uniformity of SNPs across the chromosomes
Summary
As material for genetic research, have evolved from early restriction fragment length polymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs), and simple sequence repeats (SSRs) to currently widely-used SNP markers. Numerous related methods are proposed, including restriction-site-associated DNA sequencing (RAD-seq) [9], genotyping by sequencing (GBS) [10], reduced-representation libraries (RRLs) [11], complexity reduction of polymorphic sequences (CRoPS) [12], their improved versions [13,14,15,16], etc. These RRGS methods are widely applied in animal, plant, and microorganism research [17,18,19]
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