Establishment And Application Of BSR-Seq For Gene Mapping In Wheat And Sequence Analysis Of Aegilops Tauschii Chromosome Arm 3DS

Mapping and cloning of genes regulating agronomically important traits is the key to genetically dissect and improve these traits. In wheat, however, because of its complex genome and unavailable reference sequence, the processes to map and clone agronomically important genes are labor intensive and time-consuming. The arm of this study is to develop a rapid and efficient gene mapping method in wheat by combining bulked segregant analysis with RNA-Seq (Bulked segregant RNA-Seq, BSR-Seq) to effectively identify the closely linked transcripts or variations for a target gene without the help of reference sequence. Meanwhile, the reference sequence of chromosome arm 3DS of Aegilops tauschii, the D genome progenitor of common wheat, was obtained via physical map based B AC pools sequencing. The main results are shown below:1. Based on comparative genenomic analysis and premliminary genetic linkage map of powdery mildew resistance gene Pm5e, a high efficiency and rapid gene mapping method using bulked segregant analysis and RNA-Seq (BSR-Seq) was established in wheat. The BSR-Seq data analysis pipeline identified canidates SNPs located in the same genetic interval of the Pm5e, suggesting the power of BSR-Seq analysis in hexploid wheat. To optimize the parameters used in the BSR-Seq analysis pipeline, more sequencing data was generated and different experimental designs were conducted. The results indicated that parental transcriptome data and biological replicates have no significant influence on BSR-Seq gene mapping results, and sequencing depth played certain roles, but phenotyping contaiminations in sequencing pools construction are critical for BSR-Seq analysis.2. The optimized BSR-Seq data analysis pipeline was successfully applied to map a leaf rust resistance gene (Lr42),4 powdery mildew resistance genes (Pm5e, PmTm4, MlHLT and MIH962),5 stripe rust resistance genes(YrHuaiyangl, YrMengmai58, YrZhengmai103, YrZhoumai22, YrZhongyu1152), a glossy gene(W1), a dwarf gene (Rht2BL), and a spot blotch resistance gene (Sb3). Candidate SNPs for most of these genes can be physically mapped in small intervarls ranging from 7 Mb to 36 Mb, thereafter validated by routine genetic mapping of SNP markers in segregating populations, indicating the power and efficiency of the BSR-Seq data analysis pipeline using different mapping populations in mapping wheat genes responsible for different traits. The identified high-quality SNPs in mapping intervals are valuable for fine mapping and cloning these important genes. Moreover, the results provide additional imformation regarding to the application of BSR-Seq in wheat, such as trait type, polymorphism level, population size, pooling strategy, pool size, sequencing depth and sequencing read length etc.3. A combinational approach of BAC pooling and next-generation sequencing technology was employed to sequence the minimal tiling path (MTP) of 3,337 BAC clones for At3DS. The final assembly of 135 super-scaffolds with a N50 of 4.3 Mb was used to build a 247-Mb pseudomolecule with an estimated coverage of 90% of the At3DS sequence. Evidence based annotation predicts that At3DS contains 2,222 protein-coding genes, with 81% transposable elements (TEs). For comparative analysis with orthologous regions in rice, Brachypodium, and sorghum, a stringent gene filtration process was applied to identify core gene sets in all the species and found that At3DS contains 38% more genes with a core gene set of 1,929. We estimate that 48% of the At3DS non-syntenic genes are resulted from inter-chromosomal gene duplications, while intra-chromosomal gene duplications also contribute to this high number of non-syntenic genes (21%) in the Ae. tauschii region. Compared to the common wheat cv. Chinese Spring chromosome 3B, the corresponding region (Ta3BS) is-100 Mb larger than At3DS with 879 more genes. The larger size of Ta3BS is primarily due to the expansion of the non-centromeric region while the centromeric region is of similar size, suggesting that the transposable element (TE) burst in 3B that occurred approximately 1.5 million years ago likely occurred in the non-centromeric region. The increase of genes in 3B appears to be proportional to its increase in size. In addition, we found about 0.36% gene loss in the chromosome 3D of the hexaploid wheat (Ta3DS) when compared with At3DS, indicating that major gene loss did not occur after polyploidization. The significant sequence divergences in the D genome were mostly reflected by large amount of SNPs and InDels between At3DS and Ta3DS.