高粱无融合生殖基因的定位、分离与分析

Apomixis is the most active area of plant reproductive biology research as it allows fixing heterosis after only hybridization once. Sorghum is the model species of cereal apomixis research with its molecular mechanism is still barely understood. We are pioneering in the development of sorghum breeding line 2083 with high frequencies of apomixis and producing advanced experimental hybrids capable of fixing heterosis. This project is designed to fine mapping the apomictic loci using 120 evenly distributed SSR markers on 10 chromosomes and 2083 × E36B F2 segregating population and F5 isogenic inbred lines. Meanwhile, deep sequencing the transcriptome of ovule development by Solexa Hiseq will proceed and unveil the differentially expressed genes in magaspore mother cell, meiosis and mature embryo sac of apomictic ovule development stages by contrasting apomictic and non-apomictic lines.Differentially expressed apomictic candidate genes will be projected to above obtained apomixis chromosome regions based on the publicly available Sorghum near-whole Genome Sequence.The final apomictic gene sets will be catalogued and further analyzed by real-time reverse transcript PCR and compared to canonical apomictic genes such as Agonaute, Apostart and DDM1. The apomictic genes will be cloned into Gateway vectors and functionally characterized in transgenic plants by overexpression and ds-RNA suppression. Genomic DNA will be cloned by PCR and analyzed for allelic variations of homologues between apomictic and normal lines by multi-sequence alignment. Screening diverse apomictic lines and conventional lines will be carried out using the derived DNA markers. In the future it may be possible to transfer a few of these genes into normal sorghum lines to induce apomixis at a much higher frequency.

无融合生殖具有固定杂种优势的潜力，是植物生殖生物学的重要研究领域。高粱可做为禾谷类作物无融合生殖研究的模式植物，但其分子机制却知之甚少。 以本所选育、国际领先的高频率无融合生殖系2083×常规系E35B杂交F2分离群体及F5代无融合/有性生殖近等基因系为试材, 精细定位控制无融合生殖的位点在染色体区段上。对大孢子母细胞期、减数分裂期等胚珠发育过程的转录组用Solexa Hiseq法作深度测序，比较和筛选出无融合相关的差异表达基因，再根据已公开的全基因组序列，用透射法定位到所获无融合生殖的染色体区段上，得到控制无融合生殖的基因。对基因进行分类并做荧光定量RT-PCR表达分析，克隆和构建转基因植株，分析超量和抑制表达下无融合生殖基因功能。DNA测序和序列列阵比较，分析源于高频无融合生殖系和常规系等位基因DNA多态性，并对多来源无融合生殖系做DNA鉴定,建立分子标记以辅助选育更优系。

作物无融合生殖具有固定杂种优势和降低杂交种种子生产成本的潜力，是植物生殖生物学的重要研究领域。本项目以高粱兼性无融合生殖系SSA-1为核心实验材料，用基因组重测序、转录组学和生物信息学法开展无融合生殖研究，取得如下成果：.1. 对无融合生殖系2083、SSA1等31份高粱种植做了全基因组重测序，初步揭示了高粱丰富的遗传变异；结合文献发表的无融合生殖基因，进行了基因表达分析和SNP\INDEL遗传变异、单体型分析，开发分子标记,进行分子选择育种。开展了无融合生殖性状的全基因组关联分析，已对120份高粱种质构成的自然群体做性状的鉴定，同时进行SLAF-GBS简化基因组测序做基因型分析，利用成熟的技术，定位了无融合生殖基因。.2. 用Illumina HiSEQ4000测序平台。完成了SSA-1胚珠、花药、授粉后10天种子等样品的转录组分析，共获得52.51Gb Clean Data，各样品高质数据均达到7.98Gb，Q30碱基93.17%。分别将各样品的Clean Reads与指定的参考基因组进行序列比对，比对效率从85.41%到93.41%不等。基于比对结果，进行可变剪接预测分析、基因结构优化分析以及新基因的发掘，发掘新基因2,919个，其中2,008个得到功能注释。基于比对结果，进行基因表达量分析。根据基因在不同样品中的表达量识别差异表达基因，并对其进行功能注释和富集分析。SbAGO18a1、SbAGO1c 在正常系与无融合系胚珠间差异表达，SbBBM1在花药和种子表达，胚珠不表达。.3. 在44A × SSA-1 杂交组合后代F2、F5，各种植约120株，采用人工去雄、套袋和结实率统计。对计载数据用QTL mapping和数据转化复合作图，通过联合比较连锁和关联分析，找到2个染色体区段含有影响无融合生殖基因，在区段内找到候选基因SbAGO18a1- Sobic.002G299200.2和SbBBM1 Sobic.002G022600，SbAGO1c- Sobic.004G357700。.4.通过逆转录PCR，将基因扩增出来，构建到超表达和干扰表达GATEWAY转化载体，转化2083、SSA和R111等品系。建立了以R111和2083的幼胚为起始外植体的转化体系。继续观察、解析构建的突变体库以及筛选获得的转基因目标基因抑制、过表达植株，全面揭示无融合生殖的基因机制。