Abstract
Conventional marker-based genotyping platforms are widely available, but not without their limitations. In this context, we developed Sequence-Based Genotyping (SBG), a technology for simultaneous marker discovery and co-dominant scoring, using next-generation sequencing. SBG offers users several advantages including a generic sample preparation method, a highly robust genome complexity reduction strategy to facilitate de novo marker discovery across entire genomes, and a uniform bioinformatics workflow strategy to achieve genotyping goals tailored to individual species, regardless of the availability of a reference sequence. The most distinguishing features of this technology are the ability to genotype any population structure, regardless whether parental data is included, and the ability to co-dominantly score SNP markers segregating in populations. To demonstrate the capabilities of SBG, we performed marker discovery and genotyping in Arabidopsis thaliana and lettuce, two plant species of diverse genetic complexity and backgrounds. Initially we obtained 1,409 SNPs for arabidopsis, and 5,583 SNPs for lettuce. Further filtering of the SNP dataset produced over 1,000 high quality SNP markers for each species. We obtained a genotyping rate of 201.2 genotypes/SNP and 58.3 genotypes/SNP for arabidopsis (n = 222 samples) and lettuce (n = 87 samples), respectively. Linkage mapping using these SNPs resulted in stable map configurations. We have therefore shown that the SBG approach presented provides users with the utmost flexibility in garnering high quality markers that can be directly used for genotyping and downstream applications. Until advances and costs will allow for routine whole-genome sequencing of populations, we expect that sequence-based genotyping technologies such as SBG will be essential for genotyping of model and non-model genomes alike.
Highlights
Marker assisted selection (MAS) is used to significantly accelerate the plant breeding process
The average number of reads obtained per lane was 25.3 M in arabidopsis and 127.6 M in lettuce, which is in agreement with the throughput of the sequencing platform used to sequence each crop (Illumina Genome Analyzer-II in arabidopsis; HiSeq2000 in lettuce)
The distribution of the number of reads per sample obtained in the arabidopsis and lettuce sequence datasets is indicated in Figures S1 and S2, respectively
Summary
Marker assisted selection (MAS) is used to significantly accelerate the plant breeding process. In MAS, molecular markers such as single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) are used to indirectly select for genetic determinant(s) of a trait of interest. The number of molecular markers has rapidly expanded with the development of high-throughput marker discovery and genotyping technologies, in some crop plants the number of markers remains surprisingly low [10]. In part this is a result of limitations specific to individual crops, such as those with polyploid or highly repetitive genomes, but technical and economic challenges prohibit the identification of large numbers of molecular markers
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