CN111534629A - 利用二代测序快速开发甘蓝型油菜抗草甘磷连锁分子标记 - Google Patents
利用二代测序快速开发甘蓝型油菜抗草甘磷连锁分子标记 Download PDFInfo
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Abstract
本发明公开了一种利用二代测序快速开发的甘蓝型油菜抗草甘磷连锁SSR和InDel分子标记,所述分子标记包括5个SSR引物对和4个InDel引物对。分子连锁标记的开发,可使抗草甘磷基因在甘蓝型油菜不同种质资源中的转育更为有效快速和准确,本发明开发的抗草甘磷基因连锁分子标记结合田间分离世代转育抗草甘磷基因对为转育甘蓝型油菜抗草甘磷新品系是提供快捷有效的办法。
Description
技术领域
本发明属于油菜育种技术领域。具体涉及一种利用二代测序组学技术快速鉴定甘蓝型油菜抗草甘磷基因候选区间并开发连锁SSR和InDel标记的方法。
背景技术
油菜是重要的油料作物,菜籽油约占中国食用植物油消费量的23%,是我国食用植物油主要来源之一。油菜地杂草危害导致油菜生产效率低、成本高,成为制约油菜生产发展重要因素。我国油菜,特别是稻田油菜因杂草危害一般田块产量损失在10%以上,严重田块则达50%以上。采用人工除草,劳动强度大,效果差;采用化学除草,只能施用选择性除草剂,除草不彻底,同时选择性类除草剂因分解慢,残留多,有时危害下季作物。生产实践中发现,草甘膦以外的除草剂都极易使杂草产生抗药性。草甘膦(N-膦酸甲基甘氨酸)是一种内吸传导型广谱灭生性除草剂,通过抑制5-磷酸烯醇式丙酮酸莽草酸-3磷酸合酶(EPSPS)的活性阻断莽草酸路径,使植物芳香族氨酸合成受阻(Gruys et al.,1993),从而导致植物死亡。利用从农杆菌(AgobacteriumsP.CP4)中分离出的EPSP合成酶一CP4酶,该酶对草甘麟不敏感,对PEP具有很高的亲和力,它可以取代植物内源EPSP合成酶体系,使莽草酸途径正常进行,从而达到抗草甘嶙的效果。目前草甘磷抗性作物绝大部分含有编码非敏感EPSP合成酶一CP4酶的转基因(Zelaya and Owen,2005)(Zelayawen and Owen,2005)。1996年,Monsanto(孟山都)公司克隆了CP4-EPSPS基因,并转化了大豆,获得了最早的商业化的大豆抗草甘膦品种(Pollegioni et al.,2011)。到目前为止,许多变异版本的EPSPS基因相继被克隆。孟山都公司在1974年推出了草甘膦异丙胺盐将其投放市场,商品名为农达(Roundup)(Duke and Powles,2008;Funke et al.,2006)。美国农民1996~2004年间种植抗草甘膦大豆增收64亿美元,种植抗草甘膦玉米增收5.64亿美元;而在加拿大种植抗草甘膦油菜每公顷可增加净收入27美元。
在抗草甘膦转基因油菜的研究中,钟蓉等(1997)在由农杆菌介导的油菜下胚轴、子叶的遗传转化中,对抗草甘膦基因进行转化,成功获得转化苗。王景雪等(2005)将抗草甘膦和抗虫基因的双抗载体转入湘油15号,获得了双抗的油菜植株。王开芳(2015)构建了含有EPSPs和BnGRF2双价基因的植物表达载体pCEGRF,将该载体导入油菜中,可获得抗草甘膦除草剂和高含油率高的油菜品种。万丽丽等(2016)将PCAMBIA-GOX-CP4EPSP植物双元表达载体转化到甘蓝型油菜PolCMS恢复系“7-5”获得的转基因植株。经试验验证该转基因植株是具有草甘膦抗性。李杰华(2018)利用农杆菌介导的油菜下胚轴遗传转化方法,将抗草甘膦和抗草铵膦的基因转入甘蓝型油菜甲9707品系中,获得了同时具有抗草甘膦和草铵膦的转基因油菜。王朋宝等(2019)通过农杆菌介导法将构建好的抗草甘膦和磷高效吸收基因的表达载体导入甘蓝型油菜中,获得的转基因阳性植株同时具有草甘膦的抗性和对磷素具有高效吸收的特性。目前为止,全球共有498个转基因转化事件,其中335个为抗除草剂转化事件,而棉花、大豆、油菜和玉米的抗除草剂转化事件306个,占抗除草剂转化事件总数的91.34%。我国在转基因油菜育种已开展广泛研究,但由于政策对转基因作物的限制,目前除草剂油菜品种不能推广种植,随着国家产业政策调整和对转基因事件的深入认识,油菜转基因品种的推广和种植指日可待。因此,获得稳定高产优质、抗灭生性除草剂草甘膦基因的油菜品系,能很好地解决油菜生产的草害问题,为我国油菜生产储备新材料和新技术。
抗草甘磷基因转育不同遗传背景甘蓝型油菜进入F2或BC1分离世代群体选择时有两个局限性。第一、利用抗草苷磷基因元件信息检测分离世代群体单株工作量极大,涉及到基因元件专利,田间育种选育工作一线工作人员难以有效利用。第二、对分离群体苗期直接喷施草甘磷进行选择,这种方式最为直观有效,直接可在苗期选择鉴别出抗草甘磷单株。但在实践中我们发现,喷施后的抗草甘磷单株进入现蕾开花阶段时,雄蕊退化严重,表现出明显的雄性不育现象,自交繁殖难以收获到抗草甘磷单株自交种,导致无法进行下一步的选育。迄今为止,还未有利用分子标记辅助选育甘蓝型油菜抗草甘磷材料的文献报道,也未查阅到利用二代组学技术快速开发甘蓝型油菜抗草甘磷连锁分子标记的报道。因此,利用组学方法快速开发抗草甘磷基因连锁分子标记结合田间分离世代转育抗草甘磷基因对辅助转育甘蓝型油菜抗草甘磷新品系应该是快捷有效的办法。
发明内容
有鉴于此,本发明目的之一在于提供一种甘蓝型油菜抗草甘磷连锁SSR和InDel分子标记。
所述分子标记包括以下5个SSR引物对和4个InDel引物对中至少一对,
SSR-10F:CTTCCGCACGCATCAGAAGAAT,SSR-10R:TGCTTGCGAGTTTTCCGGTTAA,SSR-25F:CGCCGTCGTTAATGTTGTGTTT,SSR-25R:GCGCCAGTAGTCGATTGATTGA,SSR-26F:CCTCAGGGGCATCTACGCTTAT,SSR-26R:GGAGCAGAAGGTGGCGGTTTTG,SSR-37F:CGGAGACCAGGGGCACATACTT,SSR-37R:AGCAGCACTTTGGTAGGATGTC,SSR-38F:GTTTGAAGAGCACGGCCAAGAA,SSR-38R:CATGGCGCAGGAGAACTACTA,InDel-1F:CATGCAAAGGTGGAGGGAGT,InDel-1R:ACCTACCATCGTCAAGTTTTTGT,InDel-2F:TGGGCCTATAGTGAATTGGGC,InDel-2R:TGCTAGATTGCTCTCTTGTGTGT,InDel-3F:TGGACACAAAGCCAAGCGAC,InDel-3R:GTGGTGATGGCGTTGTCAGT,InDel-4F:ACCACATTCACGAATCACGA,InDel-4R:TCGTCGTATTTTAGCCCTGACA。
进一步,所述SSR引物和InDel引物大小为:SSR-10F:248bp,SSR-10R:250bp,SSR-25F:210bp,SSR-25R:190bp,SSR-26F:223bp,SSR-26R:198bp,SSR-37F:220bp,SSR-37R:196bp,SSR-38F:195bp,SSR-38R:220bp,InDel-1F:180bp,InDel-1R:180bp,InDel-2F:245bp,InDel-2R:245bp,InDel-3F:145bp,InDel-3R:145bp,InDel-4F:200bp,InDel-4R:250bp。
本发明目的之二在于提供一种根前述的分子标记的开发方法。
所述方法包括以下步骤:(1)通过Mutmap+将抗草甘磷基因cp4-epsps基因定位在甘蓝型油菜A01染色体19.5-20.5Mb物理区间之内;(2)使用MISA软件识别参考组Darmar-bzh A01染色体抗草甘磷基因候选区间19.5-20.5Mb微卫星和复合微卫星序列,使用设计软件Primer3设计所述候选区间内SSR引物;(3)使用BWA和GATK对亲本和抗感子代重测序数据预处理,通过IGV软件可视化亲本和抗感子代池A01染色体19.5-20.5Mb内InDel变异,IGV软件锁定此区间内InDel的起始和终止位置并设计InDel引物;(4)利用所述SSR引物和InDel引物进行PCR扩增得扩增产物。
进一步,步骤(4)所述PCR扩增的反应体系为:10×PCR buffer(含Mg2+)1.9μL,2.5mmol·L-1dNTPs 0.25μL,Taq酶(2.5U·μL-1)0.31μL,各待检测单株的DNA 2.2μL,前/后引物各0.36μL,ddH2O补充至终体积为11μL。
进一步,步骤(4)所述PCR扩增的程序为:94℃5min预变性,94℃30s变性,52℃—60℃30s退火,72℃30s,延伸,72℃5min延伸,共35个循环。
进一步,所述PCR扩征的程序结束后4℃保存。
进一步,将所述扩增产物进行聚丙烯酰胺凝胶电泳。
进一步,所述聚丙烯酰胺凝胶为200mL 5×TBE,78g丙烯酰胺,2g甲叉丙烯酰胺,加入超纯水定容至1L。
进一步,所述电泳具体为:8%PAGE点样,以电压300V,1×TBE缓冲液,电泳0.5-1h。
本发明目的之三在于提供一种扩增与甘蓝型油菜抗草甘磷基因连锁的甘蓝型油菜基因组DNA多态性片段的应用。
本发明目的之四在于提供一种开发甘蓝型油菜抗草甘磷产品中的应用。
本发明的有益效果在于:
本发明开发的抗草甘磷基因连锁分子标记结合田间分离世代转育抗草甘磷基因对为转育甘蓝型油菜抗草甘磷新品系是提供快捷有效的办法。
本发明提供的分子标记可使抗草甘磷基因在甘蓝型油菜不同种质资源中的转育更为有效快速和准确。
附图说明
图1—图19 19条染色体SNP-index和delta(SNP-index)滑窗分析图。
图20—图23 A01染色体20Mb区间附近的插入缺失。
图24—图27 InDel标记在抗草甘磷材料和感草甘磷材料杂交F2代单株中的扩增效果图(1-12单株抗草甘磷,13-24单株感草甘磷)。
图28—图32 SSR标记在抗草甘磷材料和感草甘磷材料杂交F2代单株中的扩增效果图(1-12单株抗草甘磷,13-24单株感草甘磷)。
具体实施方式
所举实施例是为了更好地对本发明进行说明,但并不是本发明的内容仅局限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整,仍属于本发明的保护范围。
本发明实施例通过来自加拿大抗草甘磷材料Roundup与本单位纯系甘蓝型黄籽油菜L804杂交配制F2分离世代,苗期F2群体先取叶片保存,然后喷施草甘磷鉴定抗性构建抗草甘磷和感草甘磷子代池。
实施例1利用二代测序鉴定甘蓝型油菜抗草甘磷基因候选区间及开发连锁分子标记
(1)结合BSA法,对亲本Roundup、L804以及抗草甘磷和感草甘磷子代池进行二代30X重测序,用组学分析流程Mutmap+,将抗草甘磷基因cp4-epsps基因定位在甘蓝型油菜A01染色体19.5-20.5Mb物理区间之内,滑窗分析图如图1-图19所示,在A01染色体20Mb左右有明显最大的峰值。
(2)使用MISA软件识别甘蓝型油菜参考组Darmar-bzh A01染色体抗草甘磷基因候选区间19.5-20.5Mb微卫星和复合微卫星序列(A01染色体20Mb区间附近的串联重复序列如表1所示),结合命令运行引物设计软件Primer3,快速高效批量设计候选区间内SSR引物(引物序列如表2所示),根据分离群体中单株筛选开发连锁SSR标记。
(3)使用BWA和GATK对亲本Roundup、L804和抗草甘磷和感草甘磷子代池重测序数据预处理,通过IGV软件可视化亲本和抗感子代池A01染色体19.5-20.5Mb内InDel变异,A01染色体20Mb区间附近的插入缺失如图20-图23所示,IGV软件锁定此区间内InDel的起始和终止位置并设计InDel引物(引物序列如表3所示),根据分离群体中单株筛选开发连锁InDel标记。
表1 A01染色体20Mb区间附近的串联重复序列
编号 | 串联重复序列 |
1 | (AT)6 |
2 | (T)11 |
3 | (TTA)6 |
4 | (T)10 |
5 | (CT)12 |
表2 SSR引物序列
表3InDel引物序列
实施例2 PCR扩增
根据实施例1得到单株的DNA和引物进行PCR扩增。
PCR扩增条件:
(1)PCR反应体系:10×PCR buffer(含Mg2+)1.9μL,2.5mmol·L-1 dNTPs 0.25μL,Taq酶(2.5U·μL-1)0.31μL,各待检测单株的DNA 2.2μL,前/后引物各0.36μL,ddH2O补充至终体积为11μL。
(2)PCR循环程序:94℃5min预变性;94℃30s变性,52℃—60℃30s退火,72℃30s,延伸,72℃5min延伸,共35个循环;4℃保存。
根据上述的PCR扩增步骤和条件对下表4所示的引物进行扩增,PCR循环程序中的52℃—60℃对应表中各种引物序列的具体的扩增温度,如SSR-10的PCR循环程序为“94℃5min预变性;94℃30s变性,58℃30s退火,72℃30s,延伸,72℃5min延伸,共35个循环;4℃保存”,SSR-25的PCR循环程序为“94℃5min预变性;94℃30s变性,56℃30s退火,72℃30s,延伸,72℃5min延伸,共35个循环;4℃保存”,对表4所示所有的标记进行扩增得扩增产物。
表4开发的与cp4-epsps基因连锁的分子标记特征
实施例3 PCR产物检测
(1)在聚丙烯酰胺凝胶(200mL 5×TBE,78g丙烯酰胺,2g甲叉丙烯酰胺,加入超纯水定容至1L)(8%PAGE)中点样,以电压300V,1×TBE缓冲液电泳0.5-1h,电泳完毕后,普通蒸馏水漂洗1min;
(2)1.0L 0.1%g/mlAgNO3银染液银染5min左右;
(3)普通蒸馏水快速漂洗3次;
(4)1L显影液(12g NAOH,1500μL HCHO溶于1L的纯水中)中显色,不断摇动约10-15min,直到DNA条带清晰可见;
(5)自来水中漂洗3次,晾干拍照,记录多态性结果,结果如图24-图32所示,图24-图27为InDel标记在抗草甘磷材料和感草甘磷材料杂交F2代单株中的扩增效果图,图28-图32为SSR标记在抗草甘磷材料和感草甘磷材料杂交F2代单株中的扩增效果图,图24-图32中,1-12单株抗草甘磷,13-24单株感草甘磷。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (10)
1.甘蓝型油菜抗草甘磷连锁SSR和InDel分子标记,其特征在于,所述分子标记包括以下5个SSR引物对和4个InDel引物对中至少一对,
SSR-10F:CTTCCGCACGCATCAGAAGAAT,SSR-10R:TGCTTGCGAGTTTTCCGGTTAA,
SSR-25F:CGCCGTCGTTAATGTTGTGTTT,SSR-25R:GCGCCAGTAGTCGATTGATTGA,
SSR-26F:CCTCAGGGGCATCTACGCTTAT,SSR-26R:GGAGCAGAAGGTGGCGGTTTTG,
SSR-37F:CGGAGACCAGGGGCACATACTT,SSR-37R:AGCAGCACTTTGGTAGGATGTC,
SSR-38F:GTTTGAAGAGCACGGCCAAGAA,SSR-38R:CATGGCGCAGGAGAACTACTA,
InDel-1F:CATGCAAAGGTGGAGGGAGT,InDel-1R:ACCTACCATCGTCAAGTTTTTGT,
InDel-2F:TGGGCCTATAGTGAATTGGGC,InDel-2R:TGCTAGATTGCTCTCTTGTGTGT,
InDel-3F:TGGACACAAAGCCAAGCGAC,InDel-3R:GTGGTGATGGCGTTGTCAGT,
InDel-4F:ACCACATTCACGAATCACGA,InDel-4R:TCGTCGTATTTTAGCCCTGACA。
2.根据权利要求1所述的分子标记,其特征在于,所述SSR引物和InDel引物大小为:SSR-10F:248bp,SSR-10R:250bp,SSR-25F:210bp,SSR-25R:190bp,SSR-26F:223bp,SSR-26R:198bp,SSR-37F:220bp,SSR-37R:196bp,SSR-38F:195bp,SSR-38R:220bp,InDel-1F:180bp,InDel-1R:180bp,InDel-2F:245bp,InDel-2R:245bp,InDel-3F:145bp,InDel-3R:145bp,InDel-4F:200bp,InDel-4R:250bp。
3.根据权利要求1所述的分子标记的开发方法,其特征在于,所述方法包括以下步骤:(1)通过Mutmap+将抗草甘磷基因cp4-epsps基因定位在甘蓝型油菜A01染色体19.5-20.5Mb物理区间之内;(2)使用MISA软件识别参考组Darmar-bzh A01染色体抗草甘磷基因候选区间19.5-20.5Mb微卫星和复合微卫星序列,使用设计软件Primer3设计所述候选区间内SSR引物;(3)使用BWA和GATK对亲本和抗感子代重测序数据预处理,通过IGV软件可视化亲本和抗感子代池A01染色体19.5-20.5Mb内InDel变异,IGV软件锁定此区间内InDel的起始和终止位置并设计InDel引物;(4)利用所述SSR引物和InDel引物进行PCR扩增得扩增产物。
4.根据权利要求3所述的开发方法,其特征在于,所述PCR扩增的反应体系为:10×PCRbuffer(含Mg2+)1.9μL,2.5mmol·L-1dNTPs 0.25μL,Taq酶(2.5U·μL-1)0.31μL,各待检测单株的DNA 2.2μL,前/后引物各0.36μL,ddH2O补充至终体积为11μL。
5.根据权利要求4所述的开发方法,其特征在于,所述PCR扩增的程序为:94℃5min预变性,94℃30s变性,52℃—60℃30s退火,72℃30s,延伸,72℃5min延伸,共35个循环。
6.根据权利要求3所述的方法,其特征在于,将所述扩增产物进行聚丙烯酰胺凝胶电泳。
7.根据权利要求6所述的方法,其特征在于,所述聚丙烯酰胺凝胶为200mL 5×TBE,78g丙烯酰胺,2g甲叉丙烯酰胺,加入超纯水定容至1L。
8.根据权利要求5所述的方法,其特征在于,所述电泳具体为:8%PAGE点样,以电压300V,1×TBE缓冲液,电泳0.5-1h。
9.根据权利要求1所述的分子标记用于扩增与甘蓝型油菜抗草甘磷基因连锁的甘蓝型油菜基因组DNA多态性片段的应用。
10.根据权利要求1所述的分子标记在开发甘蓝型油菜抗草甘磷产品中的应用。
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