大麦调控侧小穂发育新基因定位

The domestication of cultivated barley led to two-row and six-row differentiation of its spikes. The two-rowed spike is ancestral, being found in the wild progenitor of cultivated barley (Hordeum vulgare ssp. spontaneum), where only the central spikelet is fertile. The cultivated six-row barley contains full fertile triple spikelets and shows significant advantage in seed yield. At least five independent loci are responsible for the row type. The six-rowed spike is controlled by a single allele of SIX-ROWED SPIKE 1 (VRS1), vrs1, that is recessive to the dominant Vrs1 responsible for the two-rowed spike. The phenotypes imposed by VRS1 alleles are modified by INTERMEDIUM-C (VRS5/INT-C) locus. vrs2, vrs3, and vrs4 were derived from artificially induced mutations and generate varying levels of lateral spikelet fertility. Our previous study found that novel loci might exist in barley native to Qinghai-Tibetan Plateau. In this study, we will develop.genetic segregation populations, and the next-generation sequencing based reduced-representation sequencing strategy and bulked segregant analysis (BSA) method will be adopted to high throughput develop molecular marker and map the genes responsible to row type. The tremendous barley genomic resources will also be used for fine mapping by genotyping the recombinants derived from BC3F2 populations. We will further predict the candidates genes by analyses of function annotations, haplotypes distributions and expression difference of genes within the target intervals. This study will provide basis for further cloning of novel genes controlling the row types in barley and insights into their underlying molecular mechanisms. It will also help for better understanding the domestication of cultivated barley on Qinghai-Tibetan Plateau.

大麦的驯化导致了穗型二棱和六棱的分化。二棱穗型来自野生大麦，仅中间小穗能发育成种子。栽培六棱大麦是由二棱隐性突变而来，侧小穗育性正常，具有显着产量优势。至少有5个独立基因位点与棱数有关。VRS1直接决定二棱/六棱性状，INT-C 在不同VRS1等位基因背景下对侧小穗发育起修饰作用。VRS2、VRS3和VRS4来自人工诱变，对侧小穗发育具有增强作用。项目申请人前期研究发现青藏高原大麦中可能存在新的棱数基因。本项目通过构建遗传分离群体，利用简化基因组测序结合混合分离法，对棱数基因进行分子标记定位，构建遗传连锁图；利用大麦基因组序列信息，开发连锁更加紧密的分子标记，在BC3F2群体中筛选重组个体，进一步对棱数基因进行精细定位。通过基因功能注释、基因单倍型分析、表达水平验证等手段，对候选基因进行初步预测，为克隆新的棱数基因，揭示棱型发育的分子机制，深入了解青藏高原大麦的驯化过程奠定基础。

本项目目标是对具有相同VRS1等位基因vrs1.a4的2棱和6棱大麦中控制侧小穗发育的关键基因进行遗传解析。通过本项目实施，主要获得以下主要研究结果：1）遗传分析表明，具有vrs1.a4等位基因的6棱大麦侧小穗发育受一个核基因控制；2）通过BSA分析结合分子标记对大规模遗传群体筛选，将目标基因定位到2H染色体长臂651.7Mb～652.3Mb约0.6Mb的物理区间内，该区间有2个高可信度基因，其中VRS1为最可能的候选基因；3）通过qPCR分析发现，具有vrs1.a4（后改称Vrs1.b4）的2棱大麦中, VRS1基因表达量显著的高于具有vrs1.a4的6棱大麦，其他已知棱数相关基因VRS2、VRS3和VRS4则无显著差异，而Int-C基因与Vrs1.a4表现出一定协同关系；4）通过对多份Vrs1.b4 和vs1.a4 材料以及分离群体中两种棱数表型个体VRS1基因表达水平的分析最终证实，VRS1基因表达显著下调而非基因编码序列的差异是导致携带vrs1.a4大麦6棱性状产生的直接原因；5）VRS1上游调控区的DNA甲基化水平与其表达丰度无显著相关性，而上游1Kb左右的一段‘TA’短串联重复区（STR）存在长度差异，可能与VRS1表达量下调有关。本研究从遗传和分子水平揭示了携带vrs1.a4等位基因的6棱大麦侧小穗正常发育的原因，加深了对VRS1基因调控大麦侧小穗发育分子机制的认识。而导致VRS1基因下调的分子机制值得进一步深入探究。研究结果国内外学术期刊上发表研究论文2篇，培养硕士研究生2名，圆满完成了预期目标。