RNA-seq data of phenotypic pools of lax.a.. Laxa_RNASeq

The barley mutant laxatum.a (lax.a) displays a set of pleiotropic phenotypic characteristics compared to the wild type: (i) increased spike length due to extended rachis internodes, (ii) awns with a very wide base, (iii) thin and exposed grains, (iv) as well as a homeotic conversion of the lodicules into two additional anthers. High resolution mapping in ~2000 F2 plants delimited the gene locus to a 0,2 cM target interval in a region which is characterized by reduced recombination. Thus, a large physical distance that comprises up to 200 genes required further mapping effort. On the basis of advanced genomic resources (IBSC, 2012) our aim was to test an adapted version of cloning-by-sequencing method in barley. By combining whole-genome-shotgun sequencing (WGS) data of the parental genotypes, RNA-seq data of phenotypic pools as well as exome capture (Mascher et al. 2013) re-sequencing of genetically closely linked lax.a recombinant plants a single candidate gene could be identified for lax.a. These results proof the feasibility of adapting high-throughput sequencing methodology for gene isolation in barley.

与野生型相比，大麦laxatum.a（lax.a）突变体呈现出一系列多效性表型特征：(i) 因穗轴节间伸长导致穗长增加；(ii) 芒的基部极度宽大；(iii) 籽粒瘦小且外露；(iv) 浆片发生同源异型转化，形成两枚额外的花药。研究人员在约2000株F2群体中开展高分辨率定位，将该基因座位界定至重组率降低区域内一个0.2 cM的目标区间。由于该区间物理跨度较大，包含多达200个基因，因此需要进一步开展定位工作。依托IBSC（国际大麦基因组测序联盟）2012年公布的先进基因组学资源，本研究旨在验证适配于大麦的测序克隆法可行性。通过整合亲本基因型的全基因组鸟枪测序（WGS）数据、表型混池的RNA测序数据，以及对遗传紧密连锁的lax.a重组植株开展的外显子组捕获重测序（Mascher等，2013），最终成功鉴定出lax.a的唯一候选基因。上述结果证实，将高通量测序技术应用于大麦基因克隆具有可行性。