CN112708693B - 与玉米大斑病情指数相关的ZmCaMBP1基因SNP分子标记及应用 - Google Patents
与玉米大斑病情指数相关的ZmCaMBP1基因SNP分子标记及应用 Download PDFInfo
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Abstract
与玉米大斑病情指数相关的ZMCaMBP1基因SNP分子标记及应用属于作物遗传育种分子标记应用技术领域，本发明具体涉及玉米第5号染色体上1个与大斑病情指数相关的SNP分子标记及应用。该SNP分子标记位于ZMCaMBP1(Zm00001d016856)基因的第8内含子区，是从玉米自交系群体的全基因组关联分析中得到，其核苷酸序列如SEQIDN01所示。本发明的ZMCaMBP1基因SNP分子标记可用于玉米育种过程中大斑病抗性性状的辅助选择，能提高选择的准确性、加快抗大斑病新品种的培育进程。
Description
技术领域
本发明属于作物遗传育种分子标记应用技术领域，具体涉及与玉米大斑病情指数相关的SNP分子标记及应用。
背景技术
玉米大斑病(Northern corn leaf blight)是由大斑突脐蠕孢菌(Exserohilumturcicum)引起的一种产生大型病斑症状为主的严重叶枯性病害。病斑可以沿叶脉迅速扩展并不受叶脉限制，形成条形或长梭形病斑，严重时病斑融合，造成叶片大面积枯死，导致产量大幅度降低。一般流行年份大斑病导致玉米减产20％，严重发生年份的易感品种减产50％以上。玉米大斑病抗性品种的选育与推广不仅可降低发病率，减少产量损失，也可以减少化学药剂防治对生态环境的破坏，还可以减少生产成本投入，提高玉米生产的经济效益。过往对玉米大斑病质量抗性基因Ht1、Ht2、Ht3、HtN的QTL定位研究取得了一定的进展，将它们分别定位于玉米染色体的特定区域即2.07、8.05、7.04、8.06，但Ht基因至今没有被克隆的报道(王贺等，玉米科学，2011)。
常规育种中将分散于各个种质中的优良基因聚合于同一个体的选择针对性不强，培育优良新品种的过程缓慢、难度较大。分子标记由于能对基因型进行直接的选择，具有快速、准确的优点。将分子标记技术与常规育种相结合，即分子标记辅助选择培育作物新品种正引起广泛的重视。随着玉米基因组测序的完成，玉米遗传图谱精度的提高，分子标记辅助基因聚合正日益体现出巨大的优势和应用前景。但目前与玉米大斑病抗性相关的分子标记在数量与质量上都无法满足生产实践的需求。单核苷多态性(SNP)在玉米基因组中广泛存在，SNP遗传稳定，检测方便，基于SNP的全基因组关联分析(Genome-wide AssociationStudy，GWAS)广泛应用于作物重要农艺性状遗传位点的检测，可以满足GWAS对于大样本、高密度标记的要求。与传统的QTL相比，GWAS具有更高的分辩率，可以更准确的识别和定位与目标性状相关联的新的基因，精确的筛选出相关农艺性状的分子标记从而应用于作物遗传育种领域。
钙调素(calmodulin，CaM)也称为钙离子结合蛋白，是一种普遍存在于真核生物细胞内的、参与基因调节转录及酶活性的多功能蛋白质。CaM作为最重要的一类Ca2+传感蛋白可以通过与其下游钙调素结合蛋白(CaMBP)作用而调节细胞的多种生理功能。在植物细胞的细胞质、细胞膜、细胞核及至细胞器中都检测到CaMBP的存在，钙调素自身并没有酶的活性，序列高度保守，它通过与钙调素结合蛋白相互作用，调节钙调素结合蛋白的空间结构传递信号的(Lei Zhang et al.Trends in Plant Science，2003)。钙调素结合蛋白是动物(包括人类)细胞骨架调控、肌球蛋白运动机理、肿瘤诱发以及免疫反应等领域的研究热点。在植物领域的研究表明，钙调素结合蛋白在开花调控、花粉发育及花粉萌发中发挥重要的作用，还参与细胞的代谢调节、逆镜胁迫反应并具有真核细胞的细胞骨架功能。
细胞内钙离子浓度的瞬时升高是触发植物防御反应的早期信号，Min Chul Kim等(Journal of Biological Chemistry，2002)研究表明，在活体细胞实验中缺失CaMBP的MLO对植物抗病原性的负调节能力降低一半，表明MLO的功能部分依赖于CaMBP与CaM的结合。病原菌侵染位置引发过敏反应(hypersensitive response，HR)，HR反应也会激活病程相关基因(pathogenesis-related gene，PR)的表达，PR的产生是植物系统抗病性的产生标志(Carlos M Hernandez-Garcia et al.，BMC Plant Biology，2013)。Dae Sung Kim等(Planta，2014)研究表明，HR反应标记基因的表达可以正向调节病原体诱导的细胞死亡并增强植物对病原菌的抗性，钙调蛋白CaCaM1的瞬时表达增加了ROS爆发和过敏性细胞死亡，从而改善了辣椒的防御反应。但至今为至，人们对玉米ZmCaMBP1基因的作用与功能了解是非常有限的，ZmCaMBP1基因参与玉米大斑病的病程进展相关的文献更是鲜见报道。通过GWAS研究自然群体中突变位点与性状的关联，进而探究未知基因的功能是一个非常有效的技术手段。
发明内容
本发明的目的在于克服现有技术的缺陷，提供一种与玉米大斑病情指数极显著相关的SNP分子标记，作为玉米育种过程中大斑病抗性相关的辅助选择标记，以提高选择的准确性，加快育种进程。
本发明的与玉米大斑病情指数相关的SNP分子标记，其位于玉米第5号染色体ZmCaMBP1基因的内含子区，其核苷酸序列如SEQ ID NO1所示，序列198位的碱基R为A或G。
本发明的与玉米大斑病情指数相关的SNP分子标记，能在玉米大斑病抗性品种培育辅助选择中应用，其基因型G/G为抗大斑病侵染的极显著分子标记；基因型A/G和A/A为易感大斑病的极显著分子标记。
本发明提供与玉米大斑病情指数显著相关的SNP分子标记的制备方法，包括如下步骤：
1、提取玉米基因组DNA；
2、简化基因组测序，检出高质量的候选标记；
3、使用软件GAPIT(3.1.0)中的CMLM模型(compressed mixed linear model)对玉米大斑病情指数进行关联分析；
4、检出显著位点不同基因型组间比较。
本发明与玉米大斑病情指数相关的ZmCaMBP1基因SNP分子标记，运用全基因组关联分析的方法，根据ZmCaMBP1基因基因的序列多态性与玉米大斑病情指数存在极显著的关联，即该基因中靠近3’端的第8内含子区域，位于玉米第5号染色体178419997位置，存在与玉米大斑病抗性相关的SNP分子标记，该位点基因型G/G与A/A+A/G之间大斑病情指数差异极显著(P＜0.0001)，组间均值差为1.231级。本发明的与玉米大斑病情指数相关的SNP分子标记，能在玉米大斑病抗性品种培育辅助选择中应用，其基因型G/G为抗大斑病侵染的极显著分子标记，基因型A/A和A/G为易感大斑病的极显著分子标记。以此作为玉米大斑病抗性选择的辅助分子标记，提高选择的准确性，加快育种进程。
附图说明
图1为玉米大斑病情指数次数分布的方柱形图
图2为玉米基因组DNA检测的部分电泳图
图3为玉米大斑病情指数全基因组关联分析的曼哈顿图
其中：横坐标代表每个SNP所在的基因组位置；纵坐标代表CMLM模型下每个SNP位点P值的以10为底的负对数。
图4为玉米大斑病情指数的全基因组关联分析的QQ图
其中：横坐标代表假设P值服从均匀[0，1]分布，所预期观察的P值以10为底的负对数；纵坐标代表观察到P值以10为底的负对数。
图5为ZmCaMBP1基因Chr5：178419997位点不同基因型大斑病情指数的箱形图
具体实施方式
实施例1
1.1GWAS群体的构建
玉米全基因组关联分析群体包括431份不同亲缘关系的自交系。所有的自交系都由吉林大学植物科学学院提供，种植于吉林大学植物科学学院教学与科研实验基地(吉林省长春市绿园区)。小区排列遵循随机区组设计，区组3次重复，单行小区，行长3m，行距65厘米，株距20厘米，田间管理措施与大田相同。
1.2样品的采集和表型数据的调查
小区玉米幼苗出苗后的5-6叶期，采集3株幼苗，清洗干净后置于-20℃冰箱冻存。
玉米病叶病情分级的鉴定依据地方标准：DB52T 1501.9-2020
0：叶片上无病斑
1：叶片上有零星病斑，病斑占叶面积少于或等于5％
3：叶片上有少量病斑，占叶面积6％-10％
5：叶片上有较多病斑，占叶面积11％-30％
7：叶片上有大量病斑，病斑相连，占叶面积31％-70％
9：叶片病斑占叶面积70％以上，叶片枯死。
在玉米的乳熟期每个小区调查3株植株，每株调查果穗上下各2片叶的发病情况，病情指数的计算方法如下：
以区组三次重复的平均值作为GWAS分析的表型数据，调查数据采用Excel 2013软件进行整理。
1.3玉米基因组DNA的提取与检测
(1)取50-100mg玉米幼苗用液氮研磨成粉末，转移至1.5ml离心管中，加入400ulBuffer PCL，8ul β-巯基乙醇，震荡混匀。65℃水浴45min至样品完全裂解。
(2)加入200ul Buffer PP，充分颠倒混匀，-20℃冰箱放置5min，室温10000rpm离心5min，将上清液(500-550ul)转移到新的1.5ml离心管中。若上清液混浊，可再加入等体积氯仿混匀，12000rpm离心取上清。
(3)加入等体积的异丙醇，颠倒5-8次使之充分混匀，室温放置2-3min。室温10000rpm离心5min，弃上清。
(4)加入1ml 75％乙醇，颠倒漂洗1-3min，10000rpm离心2min，弃上清。重复上述操作一次，开盖室温倒置5-10min至残留的乙醇完全挥发。
(5)得到的DNA用50-100ul TE Buffer溶解。提取的DNA可立即进行下一步实验或-20℃保存。
(6)1％琼脂糖凝胶(200V电泳30min)检测DNA完整性，Qubit2.0定量检测DNA样本浓度。
1.4研究结果
供试群体玉米大斑病情指数样本均值4.32级，中位数为4.45级，平均偏差1.79，极差8.75级，标准差2.17级，变异系数0.5007，95％置信区间为4.11-4.54级，病情指数次数分布见图1。
玉米幼苗基因组DNA采用Rapid Plant Genomic DNA Isolation Kit提取后，在裂解液作用下裂解消化RNA，经有机相洗脱蛋白等杂质，得到纯净DNA，DNA样本经电泳检测合格后(＞10ng/ul)，可用于基因组文库构建，电泳检测结果见图2。
实施例2
2.1测序文库的构建
(1)200ng基因组DNA用限制性内切酶EcoRI酶切，酶切消化完全后磁珠纯化回收。
(2)酶切后纯化的DNA用T4 DNA Ligase连接Barcode adapters PI，连接产物磁珠纯化回收，记录合格的P1引物标签。
(3)所有样本按要求等比例混合为一个总DNA mix，使用covaris220将DNA片段化，打断后的DNA的碎片长度约为200-500bp。
(4)End Repair&dA-tailing后，连接Adaptor P2，连接产物磁珠纯化回收。
(5)利用KAPA 2G Robust PCR Kit扩增并富集接头连接产物，利用磁珠对PCR反应产物进行纯化分选，通过2％琼脂糖凝胶电泳检测构建完成的文库PCR纯化产物质量，合格的PCR产物进行二代测序。
2.2简化基因组测序(restriction site-associated DNA sequencing，RAD)
(1)采用Illumina NovaSeq 6000测序平台进行双末端测序(PE150)，读长(Reads)为2×150bp。
(2)产出数据的质控与过虑：对碱基检出的错误发生几率进行预测，如果质量分值(Q-score)为低质量(Q≤5(E))的碱基数占整条read的一半以上，则去掉该read，同时还去除用于样品识别的标签序列。
(3)利用STACKS-1.08(http：//creskolab.uoregon.edu/stacks/)分别对所有样品进行stack聚类分析，检测获得各样本Tag序列，以及SNP信息。
(4)数据比对以及SNP-Callling：采用BWA软件(版本0.7.17-r1188)进行基因组比对，将质控合格的数据比对B73参考基因组序列。
2.3基因型检测与GWAS
(1)变异检测和筛选：使用PiCard软件标记重复，SamTools(1.9)对bam进行排序，BcfTools(1.9)进行call SNP。
(2)SNP质控：使用质控软件Vcf Tools(0.1.16)对SNP进行质控，质控标准为MAF＜0.05，缺失率＞0.8，HW(哈迪-温伯格指数)＞0.0001。
(3)SNP注释：软件Snp Eff(版本4.3t)利用基因组结构注释数据(GTF文件)对VCF文件中的SNP/InDel信息进行注释，即是否能够对基因编码蛋白造成影响，包括SNP的突变类型和突变位置，氨基酸的变异类型和效应等。
(4)GWAS：使用软件GAPIT(3.1.0)中的CMLM模型(compressed mixed linearmodel)对性状进行关联分析。
2.4研究结果
SNP质控后，获得549211个合格的SNP用于全基因组关联分析，关联分析时基于贝叶斯信息准则(Bayesian information criterion，BIC)的模型选择，找到GWAS模型中最优的PCs数量，用以拟合最优的性状因子。玉米果穗行粒数性状的GWAS分析结果见图3和图4：图3是关联分析结果的曼哈顿图，X轴是每个SNP所在的基因组上的位置，Y轴为CMLM模型下每个SNP位点P值的以10为底的负对数。图4是关联分析的QQ图，Y轴是观察到P值以10为底的负对数，X轴是假设P值服从均匀[0，1]分布，所预期观察的P值以10为底的负对数；5.2全基因组关联分析。
当检出的SNP的P值小于10-4时，我们认为这个SNP达到了基因组水平的显著，ZmCaMBP1基因(Zm00001d016856)的靠近3’端的第8内含子区域检出1个SNP位点符合上述条件，该位点位于玉米第5号染色体178419997位置，生物信息学功能注释表明该基因编码产物是钙调素结合蛋白(calmodulin binding protein 1)。钙调素结合蛋白具有多种重要的功能，在开花调控、花粉发育及花粉萌发中发挥重要的作用，还参与细胞的代谢调节、逆镜胁迫反应、病原物侵染的防御反应并具有真核细胞的细胞骨架功能，细胞内钙离子浓度的瞬时升高是触发植物防御反应的早期信号。结果详见表1。
表1玉米行粒数性状显著位点的关联分析
实施例3
检出显著位点不同基因型的组间比较
3.1ZmCaMBP1基因Chr5：178419997位点组间比较
该位点包括三种基因型，即A/A、G/G、A/G，不同基因型样本的病情指数分布的箱形图见图5。A/G组中位数为4.88级、下四分位数Q1为3.83级、上四分位数Q3为5.83级；A/A组中位数为5.30级、下四分位数Q1为3.86级、上四分位数Q3为6.63级；G/G组中位数为3.96级、下四分位数Q1为2.25级、上四分位数Q3为5.35级。
不同基因型组间表型均值差异的t测验结果见表2，由表2可知基因型A/A与G/G组间差异极显著(P＜0.0001)，组间均值差为1.311级；G/G与A/G之间差异不显著(P＝0.103)，A/A与A/G之间差异也不显著(P＝0.387)；G/G与A/A+A/G之间差异极显著(P＜0.0001)，组间均值差为1.231级。因此，基因型G/G是抗大斑病极显著的分子标记，基因型A/A和A/G是易感大斑病极显著的分子标记。
表2 ZmCaMBP1基因Chr5：178419997位点病情指数的组间均值比较
序列：
Zea mays cultivar B73 Chr5：178419800-178420100，B73 RefGen_v4，
上述序列中198位的碱基R为A或G，导致供试玉米自交系群体基因多态性及玉米大斑病情指数极显著差异。
序列表
申 请 人：吉林大学
发明名称：与玉米大斑病情指数相关的ZMCaMBP1基因SNP分子标记及应用
SEQ ID NO.1的序列
（i）序列特征：（A）长度：301bp,Chr5:178419800-178420100；（B）类型：核苷酸；（C）链性：单链。
（ii）分子类型：核苷酸
（iii）序列描述：SEQ ID NO.1
1 GTATATCCCT TGTGAGCTTA TCCATTACCA AGGCAAAGGC TCAAAGCTGA TCCTTGATGT
61 AGTTGCTTTA TGACATTTTA AATACTTATT AGTCAACAGT TGTTGGTTAA GTATAGAAAA
121 TGATACTGTT TATGATTATG TGATATGGAT GTGCAGTTCA CACGAGTCAT GAATTTAATT
181 AATTTACCAC ACGTATARAA AATGATAGTG CTTATGATTA TGGGATATGG ATGTGAGTTT
241 GCATGAGTCG TGTATTTGAT TAGTTTATTA CCATGCGTGT GCTGTACTGA CATAACTGAA
301 T
Claims (1)
1.与玉米大斑病情指数相关的ZMCaMBP1基因SNP分子标记在玉米大斑病抗性品种培育辅助选择中的应用，所述与玉米大斑病情指数相关的ZMCaMBP1基因SNP分子标记，位于玉米第5号染色体ZMCaMBP1基因的内含子区，其核苷酸序列如SEQ ID N01所示，序列198位的碱基R为A或G，其中基因型G/G为抗大斑病侵染的极显著分子标记；基因型A/G和A/A为易感大斑病的极显著分子标记。
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