Despite the large number of genomic and transcriptomic resources in maize, there is still much to learn about the function of genes in developmental and biochemical processes. Some maize mutants that were generated by gamma-irradiation showed clear segregation for the kernel phenotypes in B73 × Mo17 F2 ears. To better understand the functional genomics of kernel development, we developed a mapping and gene identification pipeline, bulked segregant exome sequencing (BSEx-seq), to map mutants with kernel phenotypes including opaque endosperm and reduced kernel size. BSEx-seq generates and compares the sequence of the exon fraction from mutant and normal plant F2 DNA pools. The comparison can derive mapping peaks, identify deletions within the mapping peak, and suggest candidate genes within the deleted regions. We then used the public kernel-specific expression data to narrow down the list of candidate genes/mutations and identified deletions ranging from several kb to more than 1 Mb. A full deletion allele of the Opaque-2 gene was identified in mutant 531, which occurs within a ~200-kb deletion. Opaque mutant 1486 has a 6248-bp deletion in the mapping interval containing two candidate genes encoding the RNA-directed DNA methylation 4 (RdDM4) and AMP-binding protein, respectively. This study demonstrates the efficiency and cost-effectiveness of BSEx-seq for causal mutation mapping and candidate gene selection, providing a new option in mapping-by-sequencing for maize functional genomics studies.
尽管有很多玉米研究的基因组和转录组资源，但是对于发育和生化过程中基因功能，我们的理解还不够，仍然有很多方面需要进行研究。本研究中，我们首先用伽马射线创造了一些玉米突变体，当我们将它们与玉米Mo17株系杂交得到F1代，在F1代自交获得的F2代中，发现其玉米籽粒有非常明显的性状分离。为了更好地理解玉米籽粒发育中的功能基因组学，我们开发出一个基因定位和鉴定的流程，叫作bulked segregant exome sequencing (BSEx-seq)，在不透明籽粒和小籽粒表型的玉米突变体中，成功进行了定位分析。BSEx-seq在性状分离为突变体表型和正常表型的F2代植株中，提取叶子中的基因组DNA，开展外显子测序，并进行比较分析。这种比较分析可以得到定位峰，进而在定位峰中找到缺失突变，最后在缺失区域中确定候选基因。然后，我们利用已发表的玉米籽粒中特异表达的基因数据，来缩小候选基因/变异的数量，同时也识别出长度从几个kb到大于1Mb的缺失。我们在籽粒不透明突变体531中，发现Opaque-2基因完全缺失了，这个缺失长度大约为200kb。在籽粒不透明突变体1486中，我们在定位区间中发现了一个6248bp的缺失，这个缺失涉及到两个基因，分别编码RNA-directed DNA methylation 4 (RdDM4)和AMP-binding protein。本研究中，BSEx-seq可以有效地定位突变变异位点和突变基因，其成本也很低，这为玉米功能基因组学研究，提供了新的测序定位方法。





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