Discovery of polymorphisms in starch-related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing
Discovery of polymorphisms in starch-related genes in rice germplasm by amplification of pooled DNA and deeply parallel sequencing
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DOI：
10.1111/j.1467-7652.2011.00629.x
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发表时间：
2011-12-01
影响因子：
13.8
通讯作者：
Henry, Robert J.
中科院分区：
文献类型：
--
作者：
Kharabian-Masouleh, Ardashir;Waters, Daniel L. E.;Henry, Robert J.
聚合DNA的高通量测序应用于淀粉合成相关候选基因的多态性发现。该方法采用半远程PCR（LR-PCR），然后是下一代测序技术。利用半远程PCR技术从233个水稻基因型中扩增出17个水稻淀粉合成基因，它们分别编码ADP-葡萄糖焦磷酸化酶（AGP-glucose pyrophosphorylase，AGP-glucose pyrophosphorylase，AGP-glucose）、颗粒淀粉合成酶（granule starch synthase，GBSS）、可溶性淀粉合成酶（soluble starch synthase，SS）、淀粉分支酶（starch branching enzyme，BE）、淀粉脱分支酶（starch debranching enzyme，DBE）、淀粉磷酸化酶（starch phosphorylase，SPHOL）和磷酸转运蛋白（phosphate translocator，GPT 1）等7类酶。将扩增产物等摩尔地合并并使用大规模平行测序技术（MPS）测序。通过检测基因编码区和非编码区的单核苷酸多态性（SNP/Indel），我们确定了淀粉候选基因的遗传差异，并描述了SNP/Indel变异和分布模式。产生了大约6090万个读段，其中5480万个（90%）映射到参考序列。SSIIa和SSIIb的平均覆盖率分别为12 708 ~ 38 300次。在116 403 bp的总组装长度中分析了SNP和单/多碱基Indel。在17个淀粉相关基因座中共检测到501个SNP和113个Indel。同义与非同义SNP的比率（Ka/Ks）测试表明GBSSI和异淀粉酶1（ISA 1）是最不多样化（最纯化）和保守的基因，因为所研究的群体已经通过选择循环。这份报告展示了一个有用的策略筛选种质MPS发现变异的特定目标群体的基因。
High-throughput sequencing of pooled DNA was applied to polymorphism discovery in candidate genes involved in starch synthesis. This approach employed semi-to long-range PCR (LR-PCR) followed by next-generation sequencing technology. A total of 17 rice starch synthesis genes encoding seven classes of enzymes, including ADP-glucose pyrophosphorylase (AGPase), granule starch synthase (GBSS), soluble starch synthase (SS), starch branching enzyme (BE), starch debranching enzyme (DBE) and starch phosphorylase (SPHOL) and phosphate translocator (GPT1) from 233 genotypes were PCR amplified using semi-to long-range PCR. The amplification products were equimolarly pooled and sequenced using massively parallel sequencing technology (MPS). By detecting single nucleotide polymorphism (SNP)/Indels in both coding and noncoding areas of the genes, we identified genetic differences and characterized the SNP/Indel variation and distribution patterns among individual starch candidate genes. Approximately, 60.9 million reads were generated, of which 54.8 million (90%) mapped to the reference sequences. The average coverage rate ranged from 12 708 to 38 300 times for SSIIa and SSIIIb, respectively. SNPs and single/multiple-base Indels were analysed in a total assembled length of 116 403 bp. In total, 501 SNPs and 113 Indels were detected across the 17 starch-related loci. The ratio of synonymous to nonsynonymous SNPs (Ka/Ks) test indicated GBSSI and isoamylase 1 (ISA1) as the least diversified (most purified) and conservative genes as the studied populations have been through cycles of selection. This report demonstrates a useful strategy for screening germplasm by MPS to discover variants in a specific target group of genes.