基于混池测序及遗传图谱定位玉米雄穗分枝数基因

Genes mapping of tassel branch number in maize based on mixed-pool sequencing and genetic mapping

【目的】构建玉米雄穗分枝数极端混池和分子遗传图谱,对雄穗分枝数进行QTL定位,为探究玉米雄穗分枝数的发育机制及选育雄穗分枝数少的品种提供理论参考。【方法】将雄穗分枝少的自交系SNM131与雄穗分枝多的自交系HY813组配F_(2)代群体,统计群体各单株的雄穗分枝数,分析其遗传模式。从F_(2)代群体中分别挑选33个雄穗分株少的单株和38个雄穗分枝多的单株构建混池,对2个亲本和2个子代混池分别展开30×、17×和28×覆盖度的全基因组重测序,对测序数据进行关联分析,获得雄穗分枝数性状的定位区间。利用10K芯片对F_(2)代群体276个单株进行单核苷酸多态性(SNP)标记分型,所得数据用于构建遗传连锁图谱,并结合雄穗分枝数表型数据,利用全基因组复合区间作图法(GCIM)进行QTL定位。最后根据定位区间对应的B73参考基因组的物理区段上的基因注释信息,预测调控雄穗分枝数的候选基因。【结果】F_(2)代群体雄穗分枝数平均7.6个,F_(2)代群体在雄穗分枝数性状上偏向母本SNM131,以少雄穗分枝为主。全基因组重测序共获得1812886个SNP标记和24714个插入缺失标记(InDel),并基于混合分组分析法(BSA)进行雄穗分枝数性状分析,最终共获得6个显著关联区间,分布于玉米5、8和10号染色体。其中,10号染色体上一个峰值最高的关联区间(物理位置127.61~129.46 Mb),推测为控制雄穗分枝数性状的主效位点。构建的遗传连锁图谱包含2944个SNP标记,总图距3684.3 cM。基于遗传连锁图谱,利用GCIM法共定位到5个QTL,分别分布于1、4、6和10号染色体,单个QTL可解释1.9%~9.9%的表型变异。主效QTL被定位在10号染色体约0.56 Mb的物理区间(127.81~128.37 Mb),关联区间包含有15个有中、高功能变异位点的候选基因,其中TBN-S基因编码磷脂酰乙醇胺结合蛋白(PEBP),属于TFL1类基因,与花序分生和雄穗分枝数发育密切相关。【结论】通过田间表型鉴定、遗传连锁图谱构建和SNP标记定位,将控制玉米雄穗分枝数的主效QTL定位在10号染色体长臂约0.56 Mb物理区间内,包含15个中、高变异度的基因,预测TBN-S为候选基因。

【Objective】The purpose of the study was to construct the extreme mixing pool and molecular genetic map of maize tassel branch number,and to perform QTL mapping of tassel branch number,so as to provide a theoretical reference for studying the development mechanism of maize tassel branch number and breeding varieties with less tassel branch number.【Method】Firstly,an F_(2)population was developed by using an inbred line SNM131 with fewer tassel branches and an inbred line HY813 with more tassel branches.The number of tassel branches per plant in the population was counted and its genetic model was analyzed.Secondly,33 plants with fewer tassel branches and 38 plants with more tassel branches were selected from the F_(2)population to construct mixed pools.The whole genome re-sequencing of 30×,17×and 28×coverage was carried out on the two parents and two offspring mixed pools,respectively.The sequencing data were subjected to association analysis to determine the mapped region of tassel branch number trait.Additionally,single nucleotide polymorphism(SNP)marker typing was performed on 276 individuals of F_(2)population with 10K chip.The data obtained were utilized to construct genetic linkage map,and QTL mapping was carried out in combination with phenotypic data of tassel branch number and genomic composite interval mapping(GCIM).Finally,the candidate gene determining tassel branch number was predicted based on the gene annotation information on the physical section of the B73 reference genome corresponding to the mapped region.【Result】According to statistics,the average number of tassel branches of F_(2)population was 7.6,indicating that the number of tassel branches of F_(2)population was generally biased towards the female parent SNM131,primarily exhibiting fewer tassel branches.A total of 1812886 single nucleotide polymorphism(SNP)markers and 24714 insertion/deletion(InDel)markers were obtained by genome-wide re-sequencing,and bulked segregant analysis(BSA)of tassel branch number traits was conducted,resulting in the identification of six significant associated regions distributed across maize chromosomes 5,8,and 10.Among them,an associated region with the highest peak value on chromosome 10(physical location 127.61-129.46 Mb)was judged to be the main locus controlling the number of tassel branches.Besides,a genetic map comprising 2944 SNP markers with a total distance of 3684.3 cM was constructed.Based on geneic linkage map,five QTL were mapped on chromosomes 1,4,6 and 10.Each QTL explained 1.9%-9.9%of the phenotypic variation.The major QTL was mapped in a physical interval of 0.56 Mb(127.81-128.37 Mb)in chromosome 10,which contained 15 candidate genes with medium and high functional mutation sites.The TBN-S gene was related to the development of tassel branch number.【Conclusion】Through field phenotypic identification,genetic map construction and SPN marker mapping,the major QTL controlling the tassel branch number in maize is mapped within a range of approximately 0.56 Mb on the short arm of chromosome 10,which contains 15 genes with medium and high mutation.TBN-S gene is predicted as a candidate gene.