A next-generation sequencing method for genotyping-by-sequencing of highly heterozygous autotetraploid potato.
A next-generation sequencing method for genotyping-by-sequencing of highly heterozygous autotetraploid potato.
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DOI：
10.1371/journal.pone.0062355
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
van Eck HJ
中科院分区：
文献类型：
--
作者：
Uitdewilligen JG;Wolters AM;D'hoop BB;Borm TJ;Visser RG;van Eck HJ
同源四倍体中基因组DNA序列变异和基因型识别的评估意味着区分五种可能的替代等位基因拷贝数状态的能力。本研究证明了使用下一代测序技术对大量同源四倍体马铃薯品种进行测序基因分型（GBS）的准确性。在整个培养的基因库中，在基因组范围内达到足够的读取深度仍然是昂贵的。因此，我们使用溶液中杂交方法（SureSelect）来富集培养物特异性DNA测序文库。这种复杂性的降低使我们的研究限制在807个目标基因分布在83个四倍体品种和一个参考（DM 1-3 511）的基因组。使用Illumina HiSeq 2000在12个样品的7个池中对索引的测序文库进行末端配对测序。在过滤和处理原始序列数据后，获得了12.4千兆字节的高质量序列数据，其映射到2.1 Mb的马铃薯参考基因组，每个品种的平均读取深度中值为63×。我们检测了129，156个序列变异，并对每个品种的每个变异的等位基因拷贝数进行了基因分型。在该栽培品种组中，获得外显子中1个SNP/24 bp和内含子中1个SNP/15 bp的变异密度。变异体的平均次要等位基因频率（MAF）为0.14。马铃薯种质显示出大量相对罕见的变体和/或单倍型，其中61%的变体具有低于0.05的MAF。观察到非常高的平均核苷酸多样性（π =0.0107）。 核苷酸多样性在马铃薯染色体之间存在差异。鉴定了几个选择中的基因。基因分型测序结果，等位基因拷贝数估计值，与KASP基因分型试验进行了验证。该验证表明，60-80×的读取深度可用作可靠评估同源四倍体中序列变体的等位基因拷贝数的下限。基因型数据与性状，和等位基因强烈影响成熟度和肉色进行了鉴定。
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.
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影响因子：
48
作者：
Craig, David W.;Pearson, John V.;Szelinger, Szabolcs;Sekar, Aswin;Redman, Margot;Corneveaux, Jason J.;Pawlowski, Traci L.;Laub, Trisha;Nunn, Gary;Stephan, Dietrich A.;Homer, Nils;Huentelman, Matthew J.
通讯作者：
Huentelman, Matthew J.
影响因子：
2.9
作者：
Ching A;Caldwell KS;Jung M;Dolan M;Smith OS;Tingey S;Morgante M;Rafalski AJ
通讯作者：
Rafalski AJ
影响因子：
1.2
作者：
Cingolani, Pablo;Platts, Adrian;Ruden, Douglas M.
通讯作者：
Ruden, Douglas M.
影响因子：
5.3
作者：
Grattapaglia D;Silva-Junior OB;Kirst M;de Lima BM;Faria DA;Pappas GJ Jr
通讯作者：
Pappas GJ Jr
影响因子：
7.2
作者：
Gonzalez-Schain, Nahuel D.;Diaz-Mendoza, Mercedes;Suarez-Lopez, Paula
通讯作者：
Suarez-Lopez, Paula