裸大麦多棱穗分枝基因的分子鉴定和精细定位

Ybs lines，a mutant of hulless barley with poly-row and branched-spikes, is an important new resource for these studies including ear development in barley.The mutant phenotype of poly-row and branched-spike is controlled by a major recessive gene and minor polygenes in Ybs line, but these genes of Ybs line have still not been identified and mapped. A high throughput sequencing technology based on bulked segregant analysis is employed to re-sequence the genome of mutant parent, branched- spike material and common poly row spike material in F2 generation of Ybs line crossed with recombinant inbred line in the present project, respectively. By using the reference genome sequences of different barley varieties including Morex, mutant parent and common poly row spike material in F2 generation , the genome mutants of branched-spikes material in F2 generation are selected and typed in turn, The genome mutants associated with branched-spike in Ybs line are identified and then be annotated. These genes located in barley chromosome regions, which contain genome mutants associated with branched-spike in Ybs line, are annotated too, and then these mutant genes controlling branched-spike in Ybs line are selected. The technologies including transgenic are used to identified these genes controlling branched-spike.The specific primers of molecular markers are used to amplify genome DNAs of mutant parent, Ybs line and recombinant inbred line, respectively. The amplified DNA fragments are sequenced, their sequences are aligned to the sequences of genome mutant associated with branched-spike in Ybs line , and then markers of the genome mutant associated with branched-spike are selected. A bulked segregate analysis and a F2 population are employed to selected polymorphism SSR markers and the markers of the genome mutant associated with branched-spike. These polymorphism molecular markers are used in finely mapping the genes controlling the inheritance of branched- spike in Ybs line, and in analyzing the association between these genes and traits including number of spikelet. According to results from the analysis on the data, including the identification of mutant genes controlling the inheritance of branched- spike,the genetic mechanism of branched-spike in Ybs line is illustrated.

Ybs株系属稞大麦多棱穗分枝突变体，是大麦穗发育等研究的重要新资源，其穗分枝受1对隐性主基因和微效多基因控制，但未鉴定和定位其基因。本项目采用基于集群分离分析的高通量测序技术，重测序诱变亲本、Ybs株系与重组自交系杂交F2的穗分枝和多棱穗材料基因组。参照Morex、诱变亲本和F2多棱穗等材料的基因组序列，筛选并分型穗分枝F2材料基因组变异。鉴定Ybs株系中穗分枝关联基因组变异，注解关联基因组变异及其染色体区域的基因，筛选穗分枝基因。通过转基因等技术鉴定筛选基因。利用标记特异性引物扩增诱变亲本、Ybs株系和重组自交系的基因组DNA，测序并比对扩增片段序列，筛选穗分枝关联基因组变异标记。基于集群分离分析和F2群体，筛选多态性的SSR标记和关联基因组变异标记，并以之精细定位Ybs株系穗分枝基因，分析穗分枝基因与小穗数等性状的关联性。分析穗分枝基因鉴定等资料，解析Ybs株系穗分枝的遗传机制。

Ybs株系属稞大麦多棱穗分枝突变体，是大麦穗发育等研究的重要基因资源，但其穗分枝基因还未被有效鉴定和定位。本项目通过Ybs突变体、北青7号和重组自交系等材料间穗部特性的比较分析，发现Ybs突变体的穗分枝性状可稳定遗传，具有穗分枝多、分枝较长的特性。通过Ybs突变体与保大麦8号的杂交F1、Ybs与重组自交系的正、反杂交F1、以及F2群体单株的遗传分析证实了Ybs突变体的穗分枝遗传受1对隐性核基因控制，且与小穗数、小穗小花数和百粒重等性状的关联遗传。采用特异性位点扩增片段（SLAF）测序技术，项目组成功开发了Ybs突变与保大麦8号间具有多态性的227,624个多态性SLAF标记。构建了包括14,348个SLAF标记、总长14,348cM的大麦高密度遗传图谱，并将Ybs突变体的穗分枝基因初步定位在Marker310119和Marker2679451之间。基于初步定位区间参考基因组（Morex基因组）序列特异性标记，进一步将Ybs穗分枝基因位于Chr2H染色体基因组的55,177,554bp- 55,404,247bp，该区间携带有3个穗分枝候选基因。开发了3个候选基因的10个特异性分子标记、验证了其多态性。构建了北青7号、光头大麦、重组自交系、Boman和云南高世代的成熟胚和未成熟胚愈伤诱导及其再分化技术体系、以及基于农杆菌的遗传转化技术体系，创制了两个穗分枝候选基因HORVU2Hr1G 020380和HORVU2Hr1G- 020360的46个敲除候选植株。创制了小花数、百粒重、粒长等12个重要性状的2个F5遗传作图群体。