CN112575004A - 通过基因编辑得到的与不结球白菜自交亲和性状相关的等位基因及其应用 - Google Patents

通过基因编辑得到的与不结球白菜自交亲和性状相关的等位基因及其应用 Download PDF

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CN112575004A
CN112575004A CN202011607747.5A CN202011607747A CN112575004A CN 112575004 A CN112575004 A CN 112575004A CN 202011607747 A CN202011607747 A CN 202011607747A CN 112575004 A CN112575004 A CN 112575004A
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高长斌
汪爱华
张雪丽
宋莉萍
林处发
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Wuhan Academy of Agricultural Sciences
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Abstract

本发明涉及基因工程技术领域，尤其涉及通过基因编辑得到的与不结球白菜自交亲和性状相关的等位基因及其应用。本发明所提供的等位基因，其核苷酸序列如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3或SEQ ID No.4所示。本发明还进一步提供了所述等位基因的分子标记，以及所述等位基因、分子标记的应用。通过基因编辑、远缘杂交、连续回交和分子标记辅助选择等技术手段成功获得了自交亲和的不结球白菜材料。本发明打破了不结球白菜的自交不亲和性，采用本发明方法创建的自交亲和材料将能极大提高育种效率，降低制种成本。

Description

通过基因编辑得到的与不结球白菜自交亲和性状相关的等位 基因及其应用
技术领域
本发明涉及基因工程技术领域，尤其涉及通过基因编辑得到的与不结球白菜自交亲和性状相关的等位基因及其应用。
背景技术
植物的自交不亲和性(self-incompatibility,SI)是指雌蕊柱头能够识别自身的花粉，使自身的花粉不能萌发或伸长，导致不能正常授粉或授粉不能正常结籽的现象。自交不亲和性广泛分布于十字花科蔬菜作物(白菜、甘蓝、萝卜、西兰花等)中，在遗传上受S位点复等位基因控制。S位点主要包括两个关键的识别基因：SRK，控制着柱头的识别特异性；SP11/SCR，控制着花粉的识别特异性。当花粉落到自身的柱头上时，花粉SP11/SCR蛋白与柱头SRK蛋白进行识别，引起SRK蛋白的自体磷酸化以及之后的一系列信号传导途径，最终表现为柱头对自花花粉的抑制作用以及异化花粉的识别作用。
CRISPR/Cas系统是近年来快速发展的一种基因定点编辑技术，有实验方法较简单、价格低廉、实验周期短等优势，成为了研究植物基因功能的有效方法。在sgRNA的引导下，Cas9蛋白利用两种内切酶活性，在特异性位点进行切割产生了DNA双链断裂(DSBs)。DNA双链断裂则会引起DNA链发生非同源末端链接(NHEJ)的修复机制，而这种修复机制的错配率很高，造成了碱基的缺失或者添加，从而实现对目的基因的编辑。基于CRISPR/Cas9系统的原理，可以通过转基因技术手段实现目标基因的定点编辑，创建新的等位基因，在植物分子育种中具有广阔的应用前景。前人通过对油菜品系‘Westar’进行研究，发现其A基因组上的柱头识别基因BnSRK1具有完整的功能，能识别来自白菜花粉中的BrSP11-47基因，导致自交不亲和反应发生。由于自交不亲和性的影响，不结球白菜的自交结实率很低，严重影响育种材料的繁殖和种子生产。因此，打破不结球白菜的自交不亲和性，创建自交亲和的材料将能极大提高育种效率，降低制种成本。
发明内容
本发明的目的在于提供一种与不结球白菜自交亲和性状相关的基因，该基因能使不结球白菜表现为自交亲和的性状。
本发明的技术方案是通过基因编辑得到的与不结球白菜自交亲和性状相关的等位基因，其核苷酸序列如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3或SEQ ID No.4所示。
本发明还提供了核苷酸序列如SEQ ID No.1所示的等位基因的分子标记，其引物的核苷酸序列如SEQ ID No.19和SEQ ID No.20所示。
本发明还提供了所述的通过基因编辑得到的与不结球白菜自交亲和性状相关的等位基因在不结球白菜育种中的应用。
本发明还提供了所述的核苷酸序列如SEQ ID No.1所示的等位基因的分子标记在不结球白菜育种中的应用。
本发明还提供了所述与不结球白菜自交亲和性状相关的等位基因的获得方法，包括如下步骤：根据BnSRK1基因的结构信息，在其外显子1区域设计2个靶位点，对靶位点片段进行PCR扩增，将扩增片段与基因编辑表达载体进行重组，转化大肠杆菌，获得阳性克隆；通过农杆菌介导的方法转化甘蓝型油菜品系‘Westar’下胚轴，获得转基因再生植株，扩增靶位点，扩增产物进行测序分析，靶位点序列发生突变的植株中的BnSRK1即为基因编辑得到的等位基因。
由于白菜的转基因技术体系不成熟，因此选择油菜为载体进行转化获得等位基因，再通过远缘杂交、连续回交的方式将等位基因导入到白菜基因组中。选用的甘蓝型油菜品系‘Westar’的自交不亲和基因是有功能的，而且研究很清楚，因此，选择该品系的自交不亲和基因BnSRK1作为研究基础最合适。
具体的，2个靶位点的核苷酸序列如SEQ ID No.5和SEQ ID No.6所示。
本发明还提供了获得自交亲和不结球白菜的方法，包括如下步骤：
(1)通过CRISPR/Cas9基因编辑技术，对来源于油菜品系‘Westar’中的柱头识别基因BnSRK1进行定点编辑，得到突变的等位基因BnSRK1m，其核苷酸序列如SEQ ID No.1、SEQID No.2、SEQ ID No.3或SEQ ID No.4所示。
(2)将含有突变等位基因BnSRK1m的油菜转基因株系作为母本，与不结球白菜自交系‘QR44’进行人工杂交授粉得到远缘杂种F1。
(3)以‘QR44’为轮回亲本，进行连续回交，在每个回交世代用分子标记进行辅助选择，跟踪突变等位基因BnSRK1m，得到BC4的单株。
(4)挑选BC4中包含有BnSRK1m基因的单株进行自交，得到BC4F2的群体。
(5)分析BC4F2群体中单株的基因型，调查自交亲和性，筛选得到BnSRK1m基因型为纯合，表现为自交亲和的材料，即为自交亲和不结球白菜。
本发明的有益效果：本发明通过设计特定的靶位点，采用基因编辑技术对不结球白菜自交亲和性状相关的基因BnSRK1进行了编辑，获得了其等位基因。并将其进一步应用，通过远缘杂交、连续回交和分子标记辅助选择等技术手段成功获得了自交亲和的不结球白菜材料。本发明打破了不结球白菜的自交不亲和性，采用本发明方法创建的自交亲和材料将能极大提高育种效率，降低制种成本。
附图说明
图1.基于BnSRK1基因靶位点的sgRNA表达框示意图；Pu6-26：U6-26基因的启动子，Target1：靶位点1ab-T1，gRNA-Sc：gRNA Scaffold，Tu6-29：u6-29基因的终止子，Pu6-29：U6-29基因的启动子，Target2：靶位点1a-T2，Tu6-26：U6-26基因的终止子。
图2.转基因株系中BnSRK1基因靶位点序列分析；序列的前3个碱基代表NGG位点，Wt代表编辑前的序列，m1,m2,m3和m4分别代表靶位点不同的碱基变异。
图3.BnSRK1m基因特异性分子标记在毛细管电泳仪中检测结果。横坐标代表DNA片段的迁移时间，纵坐标代表DNA片段的浓度，蓝色的峰代表目标片段，粉红色的峰代表marker。
图4.BC1分离群体中目标基因的分子标记检测结果；横坐标代表DNA片段的迁移时间，纵坐标代表DNA片段的浓度，中间的峰代表目标片段，两边的峰代表marker，右侧纵坐标的1～13代表检测的13个BC1植株序号。
图5.545PKSE-Cas9-F/R在BC1分离群体中的PCR扩增结果；M：marker；泳道1-9：K15-73-2×QR44；泳道10-13：K15-73-1×QR44；B：空白对照，N：阴性对照，P：阳性对照。
图6.BnSRK1m纯合基因型植株亲和性表型分析；a(左图)，花粉萌发观察；b(右图)，角果伸长观察。
具体实施方式
下面结合实施例对本发明提供的一种与不结球白菜自交亲和性状相关的基因及其应用进行详细的说明，但是不能将其理解为对本发明保护范围的限定。
实施例1自交不亲和基因BnSRK1的定点编辑
根据已公开的BnSRK1基因的序列(NCBI数据库序列号AB270771)，进行靶位点设计。BnSRK1基因具有7个外显子，6个内含子，序列总长3851bp；根据BnSRK1基因的结构信息，利用CRISPR-P(http://crispr.hzau.edu.cn/CRISPR2/)网站，在其外显子1区域设计2个靶位点，见表1。对靶位点片段进行PCR扩增，所用引物见表2，将扩增片段与基因编辑表达载体PKSE401(购自武汉天问生物科技有限公司)重组后电转化大肠杆菌DH5α，挑选克隆子摇菌，并挑选阳性克隆子进行送样检测，检测用相关引物见表3。具体的载体构建操作步骤参见：刘聪，硕士论文，基于CRISPR/Cas9系统定点编辑甘蓝型油菜自交不亲和基因，2018。最终构建的重组载体命名为17KN22。载体中拼接的sgRNA表达框序列如SEQ ID No.23所示，表达框结构如图1所示。Pu6-26启动靶位点和gRNA-Sc的转录表达，靶位点的表达产物引导gRNA-Sc表达的产物(酶)对宿主基因组中靶位点的dna进行剪切，从而实现基因的定点编辑。
表1靶位点序列
基因 靶位点名称 序列(5’—3’) 序列号
BnSRK1 17KN22-1ab-T1 TTGAGGGTTCCAATGGAAT SEQ ID No.5
BnSRK1 17KN22-1a-T2 CGAGAAGCTCTGCCATCAC SEQ ID No.6
表2靶位点PCR扩增引物
Figure BDA0002873987620000031
Figure BDA0002873987620000041
表3菌液PCR引物与测序引物
引物 序列(5’—3’) 用途 序列号
U626-IDF TGTCCCAGGATTAGAATGATTAGGC 菌液PCR检测 SEQ ID No.12
U626-IDR GTCAGGCTGCAGTAGTTTCCATTAA 菌液PCR检测 SEQ ID No.13
U629-seqR AGCCCTCTTCTTTCGATCCATCAAC 测序 SEQ ID No.14
将载体17KN22通过农杆菌介导的方法转化甘蓝型油菜品系‘Westar’下胚轴(转化方法参考高长斌，博士论文，甘蓝型油菜自交不亲和分子恢复机理研究及应用，2013)，获得转基因再生植株105棵。经PCR鉴定阳性植株有48株，阳性率为47.5％。在目标基因BnSRK1外显子1的靶位点区域设计引物(SRK1-CO1：AGCGATAACCCTCTCCCCAAT，SEQ ID No.15；SRK1-CO2：CCATTAGCGAGAAGCTCTGCC，SEQ ID No.16)扩增阳性转基因植株的DNA，并进行T-A克隆，测序。结果表明48个转基因单株中有10个单株发生了不同程度的基因编辑(图2)，但是这些编辑均发生在靶位点1ab-T1，而在靶位点1a-T2处未发现基因编辑现象。进一步统计分析发现，在靶位点1ab-T1处NGG上游3个碱基处存在4种不同类型的碱基变异，分别为缺失4个碱基(CCAT)，缺失2个碱基(CC)，缺失1个碱基(C)和一个碱基替换(T)(图2)。4中不同变异等位基因的核苷酸序列分别如SEQ ID No.1～4所示。挑取缺失4个碱基(CCAT)的等位基因命名为BnSRK1m，挑选对应的株系K15-73-1和K15-73-2进行后续的转育研究。
实施例2突变等位基因BnSRK1m的转育
1、亲本、杂种F1基因型鉴定及分子标记的开发
将含有突变等位基因BnSRK1m的甘蓝型油菜T1代转基因株系K15-73-1和K15-73-2分别与轮回亲本小白菜自交不亲和系‘QR44’进行远缘杂交得到F1代种子。使用自交不亲和基因的通用引物IISP11-1L/IISP11-1R(引物系列IISP11-1L：TCATAAGTCATGAGATATGCTACTT，SEQ ID No.17；IISP11-1R：TTATGATTTAACTTTGCAACAGTAGC，SEQ ID No.18)对父本自交不亲和系‘QR44’进行扩增，PCR产物进行克隆、测序、比对分析，结果表明其包含一个Ⅱ类的自交不亲和基因，基因型为Ⅱ类SP11-44，与转基因油菜中的自交不亲和基因完全不同。
同时根据突变等位基因BnSRK1m的序列开发了特异性的分子标记mSRK1-L/SRK1-CO-R(引物序列mSRK1-L：GATAACCCTCTCCCCAATTGGA，SEQ ID No.19；SRK1-CO-R：ATCACGAAGTTTCCATTAGCGA，SEQ ID No.20)，产物大小为141bp(图3)。该标记能有效检测出远缘杂交的子代植株中是否含有BnSRK1m基因，可用于跟踪目标基因。
2、回交后代的分子标记辅助选择
在温室种植远缘杂种F1代的植株，于开花期去掉花蕾的雄蕊，同时授以轮回亲本‘QR44’的花粉，构建转基因油菜株系K15-73-1和K15-73-2与‘QR44’的回交分离群体BC1；继续种植BC1分离群体的植株，于苗期提取叶片组织的DNA，采用毛细管电泳仪对BnSRK1m基因特异性分子标记mSRK1-L/SRK1-CO-R进行检测。结果表明，在检测的13个BC1植株中，有9个单株中能有效扩增出分子标记mSRK1-L/SRK1-CO-R的特异性条带，在其余的4个单株中未出现目标条带(图4)。挑取含有目标基因的单株用于后续的分析。
3、Cas9转基因成分检测分析
为了在回交转育过程中去除外源转基因片段，使用基因编辑系统中Cas9基因的特异性分子标记545PKSE-Cas9-F/R(545PKSE-Cas9-F：CCCTTCTGCGTGGTCTGATT，SEQ IDNo.21；545PKSE-Cas9-R:GGGACCTACCACGATCTCCT，SEQ ID No.22)来检测BC1代全部植株中的Cas9转基因成分。检测结果如图5所示，545PKSE-Cas9-F/R能在BC1代的13个植株中检测到5个，其中与K15-73-2株系相关的共有9个单株，其中1个单株含有转基因成分；与K15-73-1株系相关的4份材料都含有Cas9转基因成分，该结果表明杂种后代植株中的Cas9转基因成分可以在回交转育的过程中去除。挑取不含有转基因成分的植株继续与轮回亲本‘QR44’进行回交，构建BC2的分离群体。
4、亲和性表型调查分析
以‘QR44’为轮回亲本，采用相同的回交方式与检测方法最终得到BC4分离群体，挑选含有目标基因的单株进行剥蕾自交，得到BC4F2分离群体。在分离群体中，挑选BnSRK1m纯合基因型的单株共6株进行自交亲和性的表型分析。于开花期，授粉24h之后将处理过的雌蕊置于荧光显微镜下，观察花粉管的萌发状态(花粉管观察方法参考高长斌，博士论文，2013)。通过荧光显微镜，可以观察到全部6个单株的花粉均能正常萌发，长出花粉管并且能顺利穿过柱头的乳突细胞，进入花柱中，到达胚珠完成受精(图6a)。所有植株在套袋自交后角果能够正常生长，植株表现为自交亲和(图6b)；以上结果表明通过基因编辑、远缘杂交、连续回交和分子标记辅助选择等技术手段成功获得了自交亲和的不结球白菜材料。
序列
SEQ ID No.23sgRNA表达框序列
GATAATCTTCAAAAGGCCCCTGGGAATCTGAAAGAAGAGAAGCAGGCCCATTTATATGGGAAAGAACAATAGTATTTCTTATATAGGCCCATTTAAGTTGAAAACAATCTTCAAAAGTCCCACATCGCTTAGATAAGAAAACGAAGCTGAGTTTATATACAGCTAGAGTCGAAGTAGTGATTGTTGAGGGTTCCAATGGAATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTGCAAAATTTTCCAGATCGATTTCTTCTTCCTCTGTTCTTCGGCGTTCAATTTCTGGGGTTTTCTCTTCGTTTTCTGTAACTGAAACCTAAAATTTGACCTAAAAAAAATCTCAAATAATATGATTCAGTGGTTTTGTACTTTTCAGTTAGTTGAGTTTTGCAGTTCCGATGAGATAAACCAATATTAATCCAAACTACTGCAGCCTGACAGACAAATGAGGATGCAAACAATTTTAAAGTTTATCTAACGCTAGCTGTTTTGTTTCTTCTCTCTGGTGCACCAACGACGGCGTTTTCTCAATCATAAAGAGGCTTGTTTTACTTAAGGCCAATAATGTTGATGGATCGAAAGAAGAGGGCTTTTAATAAACGAGCCCGTTTAAGCTGTAAACGATGTCAAAAACATCCCACATCGTTCAGTTGAAAATAGAAGCTCTGTTTATATATTGGTAGAGTCGACTAAGAGATTGCGAGAAGCTCTGCCATCACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTGCAAAATTTTCCAGATCGATTTCTTCTTCCTCTGTTCTTCGGCGTTCAATTTCTGGGGTTTTCTCTTCGTTTTCTGTAACTGAAACCTAAAATTTGACCTAAAAAAAATCTCAAATAATATGATTCAGTGGTTTTGTACTTTTCAGTTAGTTGAGTTTTGCAGTTCCGATGAGATAAACCAATATTAATCCAAACTACTGCAGCCTGACAGACAAATGAGGATGCAAACAATTTTAAAGTTTATCTAAC
SEQ ID No.1m1碱基变异的等位基因序列
ATGAAAGGTGTACGAAACATCTATGACCACCATTCTTACACCTTCTTGCTCGTCTTCGTTGTCATGATTCTATTTCATCCTGCCCTTTCGATCTATATCAACACTTTGTCGTCTACAGAATCTCTTACAATTTCAAACAATAGAACACTTGTATCTCCCGGTGATGTTTTCGAGCTCGGTTTCTTCAAAACCACCTCAAGTTCTCGTTGGTATCTCGGGATATGGTACAAGCAATTGCCCGAGAAAACCTATGTATGGGTTGCCAACAGGGATAACCCTCTCCCCAATTTGGAACCCTCAAAATATCCAACATGAACCTTGTCCTCCTTGATCACTCTAATAAATCTGTTTGGTCCACGAATCTTACTAGACGTAATGAGAGAACTCCGGTGATGGCAGAGCTTCTCGCTAATGGAAACTTCGTGATGAGAGACTCCAATAACAACGATGCAAGTGAATTCTTGTGGCAAAGTTTCGATTACCCTACAGATACTTTGCTTCCAGAGATGAAACTGGGTTACAACCTCAAAAAAGGGCTAAACAGGTTCCTTATATCATGGAGAAGTTCAGATGATCCGTCAAGCGGGGATTACTCGTACAAGCTCGAACCCCGAAGGCTTCCTGAGTTTTATCTACTGCAAGGAGACGTTCGAGAGCATCGGAGTGGTCCATGGAACGGAATCCGATTTAGTGGGATACTAGAGGACCAAAAGCTGAGTTACATGGAGTACAATTTCACAGAGACTAGTGAGGAGGTCGCTTATACATTCCGAATGACCAACAACAGCTTCTACTCGAGATTGACACTAAGCTCCACAGGGTATTTTGAGCGACTGACGTGGGCTCCGTCATCAGTGATATGGAACGTCTTCTGGTCTTCTCCAGCAAACCCCCAGTGCGATATGTACAGGATGTGTGGGCCTTACTCTTACTGTGACGTGAACACATCACCATCGTGTAACTGTATACAAGGGTTCGATCCCAGGAATTTGCAGCAGTGGGCTCTGAGAATCTCATTAAGGGGGTGTAAAAGGAGGACGCTGCTGAGCTGCAATGGAGATGGTTTTACCAGGATGAAGAATATGAAGTTGCCAGAAACTACGATGGCCATTGTCGACCGCAGTATAGGTGAGAAAGAATGTAAGAAGAGGTGCCTTACCGATTGTAATTGTACCGCGTTTGCAAATGCGGATATCCGGAATGGTGGGACGGGTTGTGTGATTTGGACTGGAAATCTCGCTGATATGCGGAATTACGTTGCTGACGGTCAAGACCTTTATGTCAGATTGGCTGCGGCTGATCTCGGTTAGCTTCGTCTCCCTTGAATATTGCAACTATACAAATATCCAAGATTTAGTTCTATACAAATACGTACACACACATATATTCAGATCCCCTTCTGTTTCCATTAAAAATTAAAACGTAAATTAAAAGATGATTATTTTATGAATTATTAAACTATCATTTATTTGAAATATAAAGTATCACTTTAAAATTTTCTTTTGTAACTATAAGTTTATAACAACAAATAAACTTTATTAAGTCTAATAAATCATTGAATTTTTCTAATGACGTAAATAGAACTATTTATATAATTAAATAAATATTAAATTTTGTAATACTAATCTTTAAATTTTAAAATAAAAGTTGATTTATTTGATCAATAATGTTTATGAAGGTTTTTTTTAATTTGAAAAAGTTTTCAACTGAAGTGAGGAAAACAGTTTTATTTGTGGTTTTGGTCAATAATTTTTTTTTCAAATAATAATCACGATAATTTTTTTCAAATAATAATAGTTTTATATTTTTTTCATTAAAATATTAATTCTCAAAAGCCGGAACTAATCGACCTTGAATTCTTAATATTTTCCTCACAGTACTCTTTACTATTATGATTTTTGTGTATTTTCTATGACATCCTTTACTTTTATGGGGTTTCCCAAAAATAATCTTTTTAAATATTAAACAGTGCCGAATTCAGTATTATTATTTATTAAGATAATAAAATATTAGTCAACACACATATGAGACTAATGTGGGTATATTTTTGAATGAACCTCTATATATTAGTGCTCAAAAAGTACATTCAAGGGAAGTATAAAAATGTTGTAAATAAGGGAAGTATAAAAATCTATGTAAAGATTGTAGTAGGTTTATAAAAGTCATATTCGCAAATATTTTTGAGGACTTTATCACCTTTTCCCCACAGTTAAGAAGAGTAACGCGAATGGGAAAATCATAAGTTTGATTGTTGGAGTTAGTGTTCTGCTTCTTCTGATCATGTTTTGCCTCTGGAAAAGGAAACAAAATCGAGAAAAATCAAGTGCAGCATCTATTGGTAACTATAACAACTTTCATTTGTTATGATATCTATATGTAAATTTTGATTTTATATTTTGTTTGTTTCGGTGTCTGAGTGGAAACAGCAAATCGACAGAGAAACCAAAATTTGCCTATGAACGGGATAGTACTATCAAGCAAGAGACAGTTGTCTGGAGAGAACAAAATTGAGGAGCTGGAACTTCCATTGATAGAGTTGGAAGCTATTGTCAAAGCCACCGAAAATTTCTCCAATTCTAACAAAATTGGACAAGGTGGTTTTGGTATTGTTTACAAGGTAGAAAGGATATTACACTGAAAATATACACACAATCTACCATTATGCTATACCTTAAGTATGTGCTATCAGGGGATATTACTTGACGGGCAAGAAATCGCGGTAAAAAGGCTATCAAAGACGTCAGTTCAAGGGGTTGATGAGTTTATGAATGAGGTGACATTAATCGCGAGGCTTCAACATGTAAATCTTGTCCAAATTCTTGGCTGTTGCATTGACGCAGATGAGAAGATGCTGATATATGAGTATTTGGAAAATTTAAGCCTCGATTCTTATCTCTTCGGTTAGAGCCTCATTCTTTTAAAGCTCTATACAATAGTTGAATGTGGGTAGAAATAAGCTAATCTGATTTGGATGTGTCGATTTGTAGGAAAAACTCGAAGGTCTAAGCTAAATTGGAAGGAGAGATTCGACATTACCAATGGTGTTGCTCGAGGGCTTTTATATCTTCATCAAGACTCCCGCTTTAGGATAATCCACAGAGATTTGAAAGTAAGTAACATTTTGCTTGATAGAAATATGGTCCCAAAGATCTCGGATTTTGGAATGGCCAGGATATTTGAAAGAGACGAGACGGAAGCTAACACAATGAAGGTGGTCGGAACTTAGTAAGCAATCAAAATATCACAAACATCTGTATCTTTAAAAATACAAAGAGTTTAACTTTTCTTTACAGCGGCTACATGTCCCCAGAGTACGCAATGGGTGGGATATTCTCGGAAAAATCAGATGTTTTCAGTTTTGGAGTCATGGTTCTTGAAATTATTACTGGGAAGAGAAACAGAGGATTCGACGAAGACAATCTTCTAAGCTGTGTAAGTATAAGAACCAACAATTCAATCTGCTTTCTGAGATTGCTCAAACATTGAATGTTTTTATCTTAATAAACAGGCATGGAGAAATTGGAAGGAAGGAAGAGCGCTAGAAATAGTAGATCCAGTCATCGTAAATTCATTTTCACCACTGTCATCACCATTTCAACTACAAGAAGTCCTAAAATGCATACAAATTGGTCTCTTGTGTGTTCAAGAACTTGCAGAGAACAGACCAACCATGTCGTCTGTGGTTTGGATGCTTGGCAATGAAGCAACAGAGATTCCTCAGCCTAAATCGCCAGGTTGCGTCAGAAGAAGTCCTTACGAACTTGATCCTTCATCAAGTAGGCAGCGCGACGATGATGAATCCTGGACGGTGAACCAGTACACCTGCTCAGTAATCGATGCCCGGTAA
SEQ ID No.2m2碱基变异的等位基因序列
ATGAAAGGTGTACGAAACATCTATGACCACCATTCTTACACCTTCTTGCTCGTCTTCGTTGTCATGATTCTATTTCATCCTGCCCTTTCGATCTATATCAACACTTTGTCGTCTACAGAATCTCTTACAATTTCAAACAATAGAACACTTGTATCTCCCGGTGATGTTTTCGAGCTCGGTTTCTTCAAAACCACCTCAAGTTCTCGTTGGTATCTCGGGATATGGTACAAGCAATTGCCCGAGAAAACCTATGTATGGGTTGCCAACAGGGATAACCCTCTCCCCAATTATTGGAACCCTCAAAATATCCAACATGAACCTTGTCCTCCTTGATCACTCTAATAAATCTGTTTGGTCCACGAATCTTACTAGACGTAATGAGAGAACTCCGGTGATGGCAGAGCTTCTCGCTAATGGAAACTTCGTGATGAGAGACTCCAATAACAACGATGCAAGTGAATTCTTGTGGCAAAGTTTCGATTACCCTACAGATACTTTGCTTCCAGAGATGAAACTGGGTTACAACCTCAAAAAAGGGCTAAACAGGTTCCTTATATCATGGAGAAGTTCAGATGATCCGTCAAGCGGGGATTACTCGTACAAGCTCGAACCCCGAAGGCTTCCTGAGTTTTATCTACTGCAAGGAGACGTTCGAGAGCATCGGAGTGGTCCATGGAACGGAATCCGATTTAGTGGGATACTAGAGGACCAAAAGCTGAGTTACATGGAGTACAATTTCACAGAGACTAGTGAGGAGGTCGCTTATACATTCCGAATGACCAACAACAGCTTCTACTCGAGATTGACACTAAGCTCCACAGGGTATTTTGAGCGACTGACGTGGGCTCCGTCATCAGTGATATGGAACGTCTTCTGGTCTTCTCCAGCAAACCCCCAGTGCGATATGTACAGGATGTGTGGGCCTTACTCTTACTGTGACGTGAACACATCACCATCGTGTAACTGTATACAAGGGTTCGATCCCAGGAATTTGCAGCAGTGGGCTCTGAGAATCTCATTAAGGGGGTGTAAAAGGAGGACGCTGCTGAGCTGCAATGGAGATGGTTTTACCAGGATGAAGAATATGAAGTTGCCAGAAACTACGATGGCCATTGTCGACCGCAGTATAGGTGAGAAAGAATGTAAGAAGAGGTGCCTTACCGATTGTAATTGTACCGCGTTTGCAAATGCGGATATCCGGAATGGTGGGACGGGTTGTGTGATTTGGACTGGAAATCTCGCTGATATGCGGAATTACGTTGCTGACGGTCAAGACCTTTATGTCAGATTGGCTGCGGCTGATCTCGGTTAGCTTCGTCTCCCTTGAATATTGCAACTATACAAATATCCAAGATTTAGTTCTATACAAATACGTACACACACATATATTCAGATCCCCTTCTGTTTCCATTAAAAATTAAAACGTAAATTAAAAGATGATTATTTTATGAATTATTAAACTATCATTTATTTGAAATATAAAGTATCACTTTAAAATTTTCTTTTGTAACTATAAGTTTATAACAACAAATAAACTTTATTAAGTCTAATAAATCATTGAATTTTTCTAATGACGTAAATAGAACTATTTATATAATTAAATAAATATTAAATTTTGTAATACTAATCTTTAAATTTTAAAATAAAAGTTGATTTATTTGATCAATAATGTTTATGAAGGTTTTTTTTAATTTGAAAAAGTTTTCAACTGAAGTGAGGAAAACAGTTTTATTTGTGGTTTTGGTCAATAATTTTTTTTTCAAATAATAATCACGATAATTTTTTTCAAATAATAATAGTTTTATATTTTTTTCATTAAAATATTAATTCTCAAAAGCCGGAACTAATCGACCTTGAATTCTTAATATTTTCCTCACAGTACTCTTTACTATTATGATTTTTGTGTATTTTCTATGACATCCTTTACTTTTATGGGGTTTCCCAAAAATAATCTTTTTAAATATTAAACAGTGCCGAATTCAGTATTATTATTTATTAAGATAATAAAATATTAGTCAACACACATATGAGACTAATGTGGGTATATTTTTGAATGAACCTCTATATATTAGTGCTCAAAAAGTACATTCAAGGGAAGTATAAAAATGTTGTAAATAAGGGAAGTATAAAAATCTATGTAAAGATTGTAGTAGGTTTATAAAAGTCATATTCGCAAATATTTTTGAGGACTTTATCACCTTTTCCCCACAGTTAAGAAGAGTAACGCGAATGGGAAAATCATAAGTTTGATTGTTGGAGTTAGTGTTCTGCTTCTTCTGATCATGTTTTGCCTCTGGAAAAGGAAACAAAATCGAGAAAAATCAAGTGCAGCATCTATTGGTAACTATAACAACTTTCATTTGTTATGATATCTATATGTAAATTTTGATTTTATATTTTGTTTGTTTCGGTGTCTGAGTGGAAACAGCAAATCGACAGAGAAACCAAAATTTGCCTATGAACGGGATAGTACTATCAAGCAAGAGACAGTTGTCTGGAGAGAACAAAATTGAGGAGCTGGAACTTCCATTGATAGAGTTGGAAGCTATTGTCAAAGCCACCGAAAATTTCTCCAATTCTAACAAAATTGGACAAGGTGGTTTTGGTATTGTTTACAAGGTAGAAAGGATATTACACTGAAAATATACACACAATCTACCATTATGCTATACCTTAAGTATGTGCTATCAGGGGATATTACTTGACGGGCAAGAAATCGCGGTAAAAAGGCTATCAAAGACGTCAGTTCAAGGGGTTGATGAGTTTATGAATGAGGTGACATTAATCGCGAGGCTTCAACATGTAAATCTTGTCCAAATTCTTGGCTGTTGCATTGACGCAGATGAGAAGATGCTGATATATGAGTATTTGGAAAATTTAAGCCTCGATTCTTATCTCTTCGGTTAGAGCCTCATTCTTTTAAAGCTCTATACAATAGTTGAATGTGGGTAGAAATAAGCTAATCTGATTTGGATGTGTCGATTTGTAGGAAAAACTCGAAGGTCTAAGCTAAATTGGAAGGAGAGATTCGACATTACCAATGGTGTTGCTCGAGGGCTTTTATATCTTCATCAAGACTCCCGCTTTAGGATAATCCACAGAGATTTGAAAGTAAGTAACATTTTGCTTGATAGAAATATGGTCCCAAAGATCTCGGATTTTGGAATGGCCAGGATATTTGAAAGAGACGAGACGGAAGCTAACACAATGAAGGTGGTCGGAACTTAGTAAGCAATCAAAATATCACAAACATCTGTATCTTTAAAAATACAAAGAGTTTAACTTTTCTTTACAGCGGCTACATGTCCCCAGAGTACGCAATGGGTGGGATATTCTCGGAAAAATCAGATGTTTTCAGTTTTGGAGTCATGGTTCTTGAAATTATTACTGGGAAGAGAAACAGAGGATTCGACGAAGACAATCTTCTAAGCTGTGTAAGTATAAGAACCAACAATTCAATCTGCTTTCTGAGATTGCTCAAACATTGAATGTTTTTATCTTAATAAACAGGCATGGAGAAATTGGAAGGAAGGAAGAGCGCTAGAAATAGTAGATCCAGTCATCGTAAATTCATTTTCACCACTGTCATCACCATTTCAACTACAAGAAGTCCTAAAATGCATACAAATTGGTCTCTTGTGTGTTCAAGAACTTGCAGAGAACAGACCAACCATGTCGTCTGTGGTTTGGATGCTTGGCAATGAAGCAACAGAGATTCCTCAGCCTAAATCGCCAGGTTGCGTCAGAAGAAGTCCTTACGAACTTGATCCTTCATCAAGTAGGCAGCGCGACGATGATGAATCCTGGACGGTGAACCAGTACACCTGCTCAGTAATCGATGCCCGGTAA
SEQ ID No.3m3碱基变异的等位基因序列：
ATGAAAGGTGTACGAAACATCTATGACCACCATTCTTACACCTTCTTGCTCGTCTTCGTTGTCATGATTCTATTTCATCCTGCCCTTTCGATCTATATCAACACTTTGTCGTCTACAGAATCTCTTACAATTTCAAACAATAGAACACTTGTATCTCCCGGTGATGTTTTCGAGCTCGGTTTCTTCAAAACCACCTCAAGTTCTCGTTGGTATCTCGGGATATGGTACAAGCAATTGCCCGAGAAAACCTATGTATGGGTTGCCAACAGGGATAACCCTCTCCCCAATTCATTGGAACCCTCAAAATATCCAACATGAACCTTGTCCTCCTTGATCACTCTAATAAATCTGTTTGGTCCACGAATCTTACTAGACGTAATGAGAGAACTCCGGTGATGGCAGAGCTTCTCGCTAATGGAAACTTCGTGATGAGAGACTCCAATAACAACGATGCAAGTGAATTCTTGTGGCAAAGTTTCGATTACCCTACAGATACTTTGCTTCCAGAGATGAAACTGGGTTACAACCTCAAAAAAGGGCTAAACAGGTTCCTTATATCATGGAGAAGTTCAGATGATCCGTCAAGCGGGGATTACTCGTACAAGCTCGAACCCCGAAGGCTTCCTGAGTTTTATCTACTGCAAGGAGACGTTCGAGAGCATCGGAGTGGTCCATGGAACGGAATCCGATTTAGTGGGATACTAGAGGACCAAAAGCTGAGTTACATGGAGTACAATTTCACAGAGACTAGTGAGGAGGTCGCTTATACATTCCGAATGACCAACAACAGCTTCTACTCGAGATTGACACTAAGCTCCACAGGGTATTTTGAGCGACTGACGTGGGCTCCGTCATCAGTGATATGGAACGTCTTCTGGTCTTCTCCAGCAAACCCCCAGTGCGATATGTACAGGATGTGTGGGCCTTACTCTTACTGTGACGTGAACACATCACCATCGTGTAACTGTATACAAGGGTTCGATCCCAGGAATTTGCAGCAGTGGGCTCTGAGAATCTCATTAAGGGGGTGTAAAAGGAGGACGCTGCTGAGCTGCAATGGAGATGGTTTTACCAGGATGAAGAATATGAAGTTGCCAGAAACTACGATGGCCATTGTCGACCGCAGTATAGGTGAGAAAGAATGTAAGAAGAGGTGCCTTACCGATTGTAATTGTACCGCGTTTGCAAATGCGGATATCCGGAATGGTGGGACGGGTTGTGTGATTTGGACTGGAAATCTCGCTGATATGCGGAATTACGTTGCTGACGGTCAAGACCTTTATGTCAGATTGGCTGCGGCTGATCTCGGTTAGCTTCGTCTCCCTTGAATATTGCAACTATACAAATATCCAAGATTTAGTTCTATACAAATACGTACACACACATATATTCAGATCCCCTTCTGTTTCCATTAAAAATTAAAACGTAAATTAAAAGATGATTATTTTATGAATTATTAAACTATCATTTATTTGAAATATAAAGTATCACTTTAAAATTTTCTTTTGTAACTATAAGTTTATAACAACAAATAAACTTTATTAAGTCTAATAAATCATTGAATTTTTCTAATGACGTAAATAGAACTATTTATATAATTAAATAAATATTAAATTTTGTAATACTAATCTTTAAATTTTAAAATAAAAGTTGATTTATTTGATCAATAATGTTTATGAAGGTTTTTTTTAATTTGAAAAAGTTTTCAACTGAAGTGAGGAAAACAGTTTTATTTGTGGTTTTGGTCAATAATTTTTTTTTCAAATAATAATCACGATAATTTTTTTCAAATAATAATAGTTTTATATTTTTTTCATTAAAATATTAATTCTCAAAAGCCGGAACTAATCGACCTTGAATTCTTAATATTTTCCTCACAGTACTCTTTACTATTATGATTTTTGTGTATTTTCTATGACATCCTTTACTTTTATGGGGTTTCCCAAAAATAATCTTTTTAAATATTAAACAGTGCCGAATTCAGTATTATTATTTATTAAGATAATAAAATATTAGTCAACACACATATGAGACTAATGTGGGTATATTTTTGAATGAACCTCTATATATTAGTGCTCAAAAAGTACATTCAAGGGAAGTATAAAAATGTTGTAAATAAGGGAAGTATAAAAATCTATGTAAAGATTGTAGTAGGTTTATAAAAGTCATATTCGCAAATATTTTTGAGGACTTTATCACCTTTTCCCCACAGTTAAGAAGAGTAACGCGAATGGGAAAATCATAAGTTTGATTGTTGGAGTTAGTGTTCTGCTTCTTCTGATCATGTTTTGCCTCTGGAAAAGGAAACAAAATCGAGAAAAATCAAGTGCAGCATCTATTGGTAACTATAACAACTTTCATTTGTTATGATATCTATATGTAAATTTTGATTTTATATTTTGTTTGTTTCGGTGTCTGAGTGGAAACAGCAAATCGACAGAGAAACCAAAATTTGCCTATGAACGGGATAGTACTATCAAGCAAGAGACAGTTGTCTGGAGAGAACAAAATTGAGGAGCTGGAACTTCCATTGATAGAGTTGGAAGCTATTGTCAAAGCCACCGAAAATTTCTCCAATTCTAACAAAATTGGACAAGGTGGTTTTGGTATTGTTTACAAGGTAGAAAGGATATTACACTGAAAATATACACACAATCTACCATTATGCTATACCTTAAGTATGTGCTATCAGGGGATATTACTTGACGGGCAAGAAATCGCGGTAAAAAGGCTATCAAAGACGTCAGTTCAAGGGGTTGATGAGTTTATGAATGAGGTGACATTAATCGCGAGGCTTCAACATGTAAATCTTGTCCAAATTCTTGGCTGTTGCATTGACGCAGATGAGAAGATGCTGATATATGAGTATTTGGAAAATTTAAGCCTCGATTCTTATCTCTTCGGTTAGAGCCTCATTCTTTTAAAGCTCTATACAATAGTTGAATGTGGGTAGAAATAAGCTAATCTGATTTGGATGTGTCGATTTGTAGGAAAAACTCGAAGGTCTAAGCTAAATTGGAAGGAGAGATTCGACATTACCAATGGTGTTGCTCGAGGGCTTTTATATCTTCATCAAGACTCCCGCTTTAGGATAATCCACAGAGATTTGAAAGTAAGTAACATTTTGCTTGATAGAAATATGGTCCCAAAGATCTCGGATTTTGGAATGGCCAGGATATTTGAAAGAGACGAGACGGAAGCTAACACAATGAAGGTGGTCGGAACTTAGTAAGCAATCAAAATATCACAAACATCTGTATCTTTAAAAATACAAAGAGTTTAACTTTTCTTTACAGCGGCTACATGTCCCCAGAGTACGCAATGGGTGGGATATTCTCGGAAAAATCAGATGTTTTCAGTTTTGGAGTCATGGTTCTTGAAATTATTACTGGGAAGAGAAACAGAGGATTCGACGAAGACAATCTTCTAAGCTGTGTAAGTATAAGAACCAACAATTCAATCTGCTTTCTGAGATTGCTCAAACATTGAATGTTTTTATCTTAATAAACAGGCATGGAGAAATTGGAAGGAAGGAAGAGCGCTAGAAATAGTAGATCCAGTCATCGTAAATTCATTTTCACCACTGTCATCACCATTTCAACTACAAGAAGTCCTAAAATGCATACAAATTGGTCTCTTGTGTGTTCAAGAACTTGCAGAGAACAGACCAACCATGTCGTCTGTGGTTTGGATGCTTGGCAATGAAGCAACAGAGATTCCTCAGCCTAAATCGCCAGGTTGCGTCAGAAGAAGTCCTTACGAACTTGATCCTTCATCAAGTAGGCAGCGCGACGATGATGAATCCTGGACGGTGAACCAGTACACCTGCTCAGTAATCGATGCCCGGTAA
SEQ ID No.4m4碱基变异的等位基因序列：
ATGAAAGGTGTACGAAACATCTATGACCACCATTCTTACACCTTCTTGCTCGTCTTCGTTGTCATGATTCTATTTCATCCTGCCCTTTCGATCTATATCAACACTTTGTCGTCTACAGAATCTCTTACAATTTCAAACAATAGAACACTTGTATCTCCCGGTGATGTTTTCGAGCTCGGTTTCTTCAAAACCACCTCAAGTTCTCGTTGGTATCTCGGGATATGGTACAAGCAATTGCCCGAGAAAACCTATGTATGGGTTGCCAACAGGGATAACCCTCTCCCCAATTACATTGGAACCCTCAAAATATCCAACATGAACCTTGTCCTCCTTGATCACTCTAATAAATCTGTTTGGTCCACGAATCTTACTAGACGTAATGAGAGAACTCCGGTGATGGCAGAGCTTCTCGCTAATGGAAACTTCGTGATGAGAGACTCCAATAACAACGATGCAAGTGAATTCTTGTGGCAAAGTTTCGATTACCCTACAGATACTTTGCTTCCAGAGATGAAACTGGGTTACAACCTCAAAAAAGGGCTAAACAGGTTCCTTATATCATGGAGAAGTTCAGATGATCCGTCAAGCGGGGATTACTCGTACAAGCTCGAACCCCGAAGGCTTCCTGAGTTTTATCTACTGCAAGGAGACGTTCGAGAGCATCGGAGTGGTCCATGGAACGGAATCCGATTTAGTGGGATACTAGAGGACCAAAAGCTGAGTTACATGGAGTACAATTTCACAGAGACTAGTGAGGAGGTCGCTTATACATTCCGAATGACCAACAACAGCTTCTACTCGAGATTGACACTAAGCTCCACAGGGTATTTTGAGCGACTGACGTGGGCTCCGTCATCAGTGATATGGAACGTCTTCTGGTCTTCTCCAGCAAACCCCCAGTGCGATATGTACAGGATGTGTGGGCCTTACTCTTACTGTGACGTGAACACATCACCATCGTGTAACTGTATACAAGGGTTCGATCCCAGGAATTTGCAGCAGTGGGCTCTGAGAATCTCATTAAGGGGGTGTAAAAGGAGGACGCTGCTGAGCTGCAATGGAGATGGTTTTACCAGGATGAAGAATATGAAGTTGCCAGAAACTACGATGGCCATTGTCGACCGCAGTATAGGTGAGAAAGAATGTAAGAAGAGGTGCCTTACCGATTGTAATTGTACCGCGTTTGCAAATGCGGATATCCGGAATGGTGGGACGGGTTGTGTGATTTGGACTGGAAATCTCGCTGATATGCGGAATTACGTTGCTGACGGTCAAGACCTTTATGTCAGATTGGCTGCGGCTGATCTCGGTTAGCTTCGTCTCCCTTGAATATTGCAACTATACAAATATCCAAGATTTAGTTCTATACAAATACGTACACACACATATATTCAGATCCCCTTCTGTTTCCATTAAAAATTAAAACGTAAATTAAAAGATGATTATTTTATGAATTATTAAACTATCATTTATTTGAAATATAAAGTATCACTTTAAAATTTTCTTTTGTAACTATAAGTTTATAACAACAAATAAACTTTATTAAGTCTAATAAATCATTGAATTTTTCTAATGACGTAAATAGAACTATTTATATAATTAAATAAATATTAAATTTTGTAATACTAATCTTTAAATTTTAAAATAAAAGTTGATTTATTTGATCAATAATGTTTATGAAGGTTTTTTTTAATTTGAAAAAGTTTTCAACTGAAGTGAGGAAAACAGTTTTATTTGTGGTTTTGGTCAATAATTTTTTTTTCAAATAATAATCACGATAATTTTTTTCAAATAATAATAGTTTTATATTTTTTTCATTAAAATATTAATTCTCAAAAGCCGGAACTAATCGACCTTGAATTCTTAATATTTTCCTCACAGTACTCTTTACTATTATGATTTTTGTGTATTTTCTATGACATCCTTTACTTTTATGGGGTTTCCCAAAAATAATCTTTTTAAATATTAAACAGTGCCGAATTCAGTATTATTATTTATTAAGATAATAAAATATTAGTCAACACACATATGAGACTAATGTGGGTATATTTTTGAATGAACCTCTATATATTAGTGCTCAAAAAGTACATTCAAGGGAAGTATAAAAATGTTGTAAATAAGGGAAGTATAAAAATCTATGTAAAGATTGTAGTAGGTTTATAAAAGTCATATTCGCAAATATTTTTGAGGACTTTATCACCTTTTCCCCACAGTTAAGAAGAGTAACGCGAATGGGAAAATCATAAGTTTGATTGTTGGAGTTAGTGTTCTGCTTCTTCTGATCATGTTTTGCCTCTGGAAAAGGAAACAAAATCGAGAAAAATCAAGTGCAGCATCTATTGGTAACTATAACAACTTTCATTTGTTATGATATCTATATGTAAATTTTGATTTTATATTTTGTTTGTTTCGGTGTCTGAGTGGAAACAGCAAATCGACAGAGAAACCAAAATTTGCCTATGAACGGGATAGTACTATCAAGCAAGAGACAGTTGTCTGGAGAGAACAAAATTGAGGAGCTGGAACTTCCATTGATAGAGTTGGAAGCTATTGTCAAAGCCACCGAAAATTTCTCCAATTCTAACAAAATTGGACAAGGTGGTTTTGGTATTGTTTACAAGGTAGAAAGGATATTACACTGAAAATATACACACAATCTACCATTATGCTATACCTTAAGTATGTGCTATCAGGGGATATTACTTGACGGGCAAGAAATCGCGGTAAAAAGGCTATCAAAGACGTCAGTTCAAGGGGTTGATGAGTTTATGAATGAGGTGACATTAATCGCGAGGCTTCAACATGTAAATCTTGTCCAAATTCTTGGCTGTTGCATTGACGCAGATGAGAAGATGCTGATATATGAGTATTTGGAAAATTTAAGCCTCGATTCTTATCTCTTCGGTTAGAGCCTCATTCTTTTAAAGCTCTATACAATAGTTGAATGTGGGTAGAAATAAGCTAATCTGATTTGGATGTGTCGATTTGTAGGAAAAACTCGAAGGTCTAAGCTAAATTGGAAGGAGAGATTCGACATTACCAATGGTGTTGCTCGAGGGCTTTTATATCTTCATCAAGACTCCCGCTTTAGGATAATCCACAGAGATTTGAAAGTAAGTAACATTTTGCTTGATAGAAATATGGTCCCAAAGATCTCGGATTTTGGAATGGCCAGGATATTTGAAAGAGACGAGACGGAAGCTAACACAATGAAGGTGGTCGGAACTTAGTAAGCAATCAAAATATCACAAACATCTGTATCTTTAAAAATACAAAGAGTTTAACTTTTCTTTACAGCGGCTACATGTCCCCAGAGTACGCAATGGGTGGGATATTCTCGGAAAAATCAGATGTTTTCAGTTTTGGAGTCATGGTTCTTGAAATTATTACTGGGAAGAGAAACAGAGGATTCGACGAAGACAATCTTCTAAGCTGTGTAAGTATAAGAACCAACAATTCAATCTGCTTTCTGAGATTGCTCAAACATTGAATGTTTTTATCTTAATAAACAGGCATGGAGAAATTGGAAGGAAGGAAGAGCGCTAGAAATAGTAGATCCAGTCATCGTAAATTCATTTTCACCACTGTCATCACCATTTCAACTACAAGAAGTCCTAAAATGCATACAAATTGGTCTCTTGTGTGTTCAAGAACTTGCAGAGAACAGACCAACCATGTCGTCTGTGGTTTGGATGCTTGGCAATGAAGCAACAGAGATTCCTCAGCCTAAATCGCCAGGTTGCGTCAGAAGAAGTCCTTACGAACTTGATCCTTCATCAAGTAGGCAGCGCGACGATGATGAATCCTGGACGGTGAACCAGTACACCTGCTCAGTAATCGATGCCCGGTAA。

Claims (7)

1.通过基因编辑得到的与不结球白菜自交亲和性状相关的等位基因，其核苷酸序列如SEQ ID No.1、SEQ ID No.2、SEQ ID No.3或SEQ ID No.4所示。
2.核苷酸序列如SEQ ID No.1所示的等位基因的分子标记，其特征在于，其引物的核苷酸序列如SEQ ID No.19和SEQ ID No.20所示。
3.权利要求1所述的通过基因编辑得到的与不结球白菜自交亲和性状相关的等位基因在不结球白菜育种中的应用。
4.权利要求2所述的核苷酸序列如SEQ ID No.1所示的等位基因的分子标记在不结球白菜育种中的应用。
5.与不结球白菜自交亲和性状相关的等位基因的获得方法，其特征在于，包括如下步骤：根据BnSRK1基因的结构信息，在其外显子1区域设计2个靶位点，对靶位点片段进行PCR扩增，将扩增片段与基因编辑表达载体进行重组，转化大肠杆菌，获得阳性克隆；通过农杆菌介导的方法转化甘蓝型油菜品系‘Westar’下胚轴，获得转基因再生植株，扩增靶位点，扩增产物进行测序分析，靶位点序列发生突变的植株中的BnSRK1即为基因编辑得到的等位基因。
6.如权利要求5所述的方法，其特征在于，2个靶位点的核苷酸序列如SEQ ID No.5和SEQ ID No.6所示。
7.获得自交亲和不结球白菜的方法，包括如下步骤：
(1)通过CRISPR/Cas9基因编辑技术，对来源于油菜品系‘Westar’中的柱头识别基因BnSRK1进行定点编辑，得到突变的等位基因BnSRK1m，其核苷酸序列如SEQ ID No.1、SEQ IDNo.2、SEQ ID No.3或SEQ ID No.4所示；
(2)将含有突变等位基因BnSRK1m的油菜转基因株系作为母本，与不结球白菜自交系‘QR44’进行人工杂交授粉得到远缘杂种F1；
(3)以‘QR44’为轮回亲本，进行连续回交，在每个回交世代用分子标记进行辅助选择，跟踪突变等位基因BnSRK1m，得到BC4的单株；
(4)挑选BC4中包含有BnSRK1m基因的单株进行自交，得到BC4F2的群体；
(5)分析BC4F2群体中单株的基因型，调查自交亲和性，筛选得到BnSRK1m基因型为纯合，表现为自交亲和的材料，即为自交亲和不结球白菜。
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