A Next-Generation Sequencing Method for Genotyping-by-Sequencing of Highly Heterozygous Autotetraploid Potato

Assessment of genomic DNA sequence variation and genotype calling in autotetraploids implies the ability to distinguish among five possible alternative allele copy number states. This study demonstrates the accuracy of genotyping-by-sequencing (GBS) of a large collection of autotetraploid potato cultivars using next-generation sequencing. It is still costly to reach sufficient read depths on a genome wide scale, across the cultivated gene pool. Therefore, we enriched cultivar-specific DNA sequencing libraries using an in-solution hybridisation method (SureSelect). This complexity reduction allowed to confine our study to 807 target genes distributed across the genomes of 83 tetraploid cultivars and one reference (DM 1–3 511). Indexed sequencing libraries were paired-end sequenced in 7 pools of 12 samples using Illumina HiSeq2000. After filtering and processing the raw sequence data, 12.4 Gigabases of high-quality sequence data was obtained, which mapped to 2.1 Mb of the potato reference genome, with a median average read depth of 63× per cultivar. We detected 129,156 sequence variants and genotyped the allele copy number of each variant for every cultivar. In this cultivar panel a variant density of 1 SNP/24 bp in exons and 1 SNP/15 bp in introns was obtained. The average minor allele frequency (MAF) of a variant was 0.14. Potato germplasm displayed a large number of relatively rare variants and/or haplotypes, with 61% of the variants having a MAF below 0.05. A very high average nucleotide diversity (π = 0.0107) was observed. Nucleotide diversity varied among potato chromosomes. Several genes under selection were identified. Genotyping-by-sequencing results, with allele copy number estimates, were validated with a KASP genotyping assay. This validation showed that read depths of ∼60–80× can be used as a lower boundary for reliable assessment of allele copy number of sequence variants in autotetraploids. Genotypic data were associated with traits, and alleles strongly influencing maturity and flesh colour were identified.

对同源四倍体（autotetraploid）的基因组DNA序列变异与基因型分型进行评估，意味着需具备区分五种可能的等位基因拷贝数状态的能力。本研究采用下一代测序（next-generation sequencing）技术，针对大规模同源四倍体马铃薯栽培品种群体开展测序分型（genotyping-by-sequencing, GBS），并验证了该方法的准确性。不过，在栽培种质的全基因组范围内获取足够的测序深度仍需较高成本。为此，本研究采用液相杂交法（SureSelect）对品种特异性DNA测序文库进行富集。通过该复杂度降低策略，本研究的分析范围限定为83个四倍体马铃薯栽培品种及1个参考品种（DM 1–3 511）的全基因组分布的807个靶基因。我们将带索引的测序文库按12个样本为1组，共分为7组，采用Illumina HiSeq2000平台进行双端测序。对原始测序数据进行过滤与处理后，共获得12.4吉碱基对的高质量测序数据，这些数据可比对到马铃薯参考基因组的2.1兆碱基对区域，每个栽培品种的平均测序深度中位数为63×。本研究共检测到129156个序列变异，并为每个栽培品种的每一个变异位点完成了等位基因拷贝数分型。在该栽培品种群体中，外显子区域的变异密度为1个单核苷酸多态性（Single Nucleotide Polymorphism, SNP）/24 bp，内含子区域则为1个SNP/15 bp。每个变异位点的次要等位基因频率（minor allele frequency, MAF）平均值为0.14。马铃薯种质资源存在大量相对稀有变异和/或单倍型，其中61%的变异位点的MAF低于0.05。本研究检测到极高的平均核苷酸多样性（nucleotide diversity, π=0.0107）。不同马铃薯染色体的核苷酸多样性存在差异，同时鉴定出多个受选择的基因。本研究通过KASP基因分型实验，对带有等位基因拷贝数估算结果的测序分型结果进行了验证。该验证结果表明，约60~80×的测序深度可作为同源四倍体物种序列变异等位基因拷贝数可靠评估的下限阈值。本研究将基因型数据与性状进行关联分析，鉴定出对成熟期和果肉颜色具有显著影响的等位基因。