CN114164289A - 一种大豆细胞核雄性不育InDel标记及其应用 - Google Patents
一种大豆细胞核雄性不育InDel标记及其应用 Download PDFInfo
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Abstract
本发明涉及遗传育种技术领域,尤其涉及一种与大豆细胞核雄性不育显著相关的InDel标记及其应用和利用该InDel标记鉴定大豆植株育性的方法。该InDel标记位于大豆第13号染色体第22776268bp‑22815034bp之间,利用该InDel标记可以快速鉴定大豆植株细胞核雄性不育株,同时降低了筛选时间,提高了筛选效率和筛选准确率。
Description
本申请主张2021年4月20日申请的申请号为202110426981.6的“一种大豆细胞核雄性不育InDel标记及创制雄性不育系的方法”的优先权,原受理机构为中国。
技术领域
本发明涉及遗传育种技术领域,尤其涉及一种与大豆细胞核雄性不育显著相关的InDel标记及其应用和利用该InDel标记鉴定大豆植株育性的方法。
技术背景
大豆是世界上最主要经济作物之一,营养丰富,蛋白质含量40%左右,明显超出其他主要作物的水平,油份含量能达20%左右。大豆中还含有大豆异黄酮、膳食纤维等多种人体必需的营养物质。但是目前来说,中国大豆单产低仍然是目前大豆产业急需突破的问题。我国大豆年需求量超过1亿吨,而本国产量仅能满足10%左右。因此,提高产量是目前大豆研究的首要任务。
杂种优势对作物产量的提高有明显的促进作用,多数作物的杂交种比常规种增产能达到20%左右。大豆雄性不育正是其杂交育种中利用的最重要的农艺性状,也是目前生产上杂交育种利用最多性状。与此同时,大豆雄性不育系的利用前景广泛,有效的解决大豆杂交困难,增加了天然异交率。因此对大豆雄性不育性状的研究、不育基因的克隆和不育分子机制的解析以及不育系的创制是大豆杂交应用、新一代杂交技术的利用以及提高大豆产量的工作基础。
常规育种年限长、效率低且成本高,而分子标记反映生物个体基因组DNA片段的差异,直接反应种质资源本质,具有高效、准确以及经济等优点,大幅缩短育种周期,是传统育种技术的有力补充。SNP分子标记具有数量多、遗传稳定等优点,基于酶切扩增多态性序列标记技术(CAPS)的SNP分子标记检测方法,主要是将PCR扩增产物中含SNP位点的DNA片段进行限制性酶切分析,是SNP分子标记的检测方法之一。但CAPS标记仍具有耗时长、成本较高及操作繁琐的缺点。大豆植株的育性对大豆育种发展具有重要的作用,而现有技术中并未见报道可对大豆植株是否可育进行有效判断的分子生物学方法。
发明内容
为了解决现有技术中存在的问题,本发明的目的之一在于提供一种与大豆植株细胞核雄性不育显著相关的InDel标记,所述InDel标记的核苷酸序列如SEQ ID NO.1所示,该InDel标记位于大豆第13号染色体第22776268bp-22815034bp之间。
本发明还提供该InDel标记在鉴定大豆植株细胞核雄性不育中的应用,以及所述InDel标记在制备检测大豆植株细胞核雄性不育试剂或试剂盒中的应用。
优选的,上述应用均通过核酸扩增技术检测大豆植株中所述InDel标记的表达水平,用于核酸扩增的引物对序列为:
正向序列SEQ ID NO.2:TGCCACTAACACCATCGACA
反向序列SEQ ID NO.3:ACTCATGCGGTTTGTGGGAG
本发明的目的之二在于提供一种利用双引物对鉴定大豆植株育性的方法,包括以下步骤:
S1.设计鉴定可育大豆植株的引物对,所述引物对序列为:
正向引物SEQ ID NO.4:TAAACCTCGTCGTCGTTCATT
反向引物SEQ ID NO.5:CTGCTAGTCTGCCCATACGC
S2.提取待测大豆植株基因组DNA;
S3.以用于InDel标记核酸扩增的引物对序列SEQ ID NO.2、SEQ ID NO.3和所述SEQ ID NO.4、SEQ ID NO.5为引物,对S2提取的DNA进行PCR扩增,获得扩增产物;其中,
正向序列SEQ ID NO.2:TGCCACTAACACCATCGACA
反向序列SEQ ID NO.3:ACTCATGCGGTTTGTGGGAG
S4.对S4扩增产物进行琼脂糖凝胶电泳,根据电泳所得条带结果判断待测大豆植株的育性;若所得条带为高带1333bp,则判断待测大豆植株为可育植株,若所得条带为低带661bp,则判断待测大豆植株为细胞核雄性不育植株;
所述高带的核苷酸序列如SEQ ID NO.6所示,所述低带的核苷酸序列如SEQ IDNO.7所示。
优选的,所述S3中,PCR扩增程序为:取100ng/μL的模板DNA和10μM的引物各1.0μL,加入1.0U KODone酶12.5μL和ddH2O 8.5μL;反应条件为:94℃预变性2min;98℃变性10s,起始退火温度63℃,每一个循环降落0.2℃,共退火15s,68℃延伸30s,设38次循环;最后68℃延伸2min,4℃保存。
本发明的有益效果在于:
1.InDel标记是由插入/缺失位点两侧的基因序列所设计的特异性引物,根据PCR产物片段大小可直接快速的进行检测,具有准确性高、稳定性好以及成本低等优势。InDel标记相比较现有的CAPS标记来说,具有耗时短、成本低及操作简便的优点。本发明利用InDel标记可以快速鉴定大豆植株细胞核雄性不育株。
2.本发明采用双引物对对待测大豆基因组DNA进行PCR扩增,实现被测大豆植株细胞核雄性不育的鉴定,降低了筛选时间,提高了筛选效率和筛选准确率。
附图说明
图1为实施例1中大豆天然雄性不育株与可育株的对比,其中图1A为不育和可育植株株型及结荚情况对比,不育植株显示豆荚败育,可育植株正常结荚,图1A左侧为不育株结荚部位放大图;图1B为不育和可育植株花粉散粉特性对比,不育植株显示花粉败育,无法散粉,而可育植株花粉正常散粉;图1C是体式镜下可育和不育花药形态对比,不育植株光滑,无花粉产生,而可育植株有花粉产生。
图2为对群体中不育和可育大豆的带有柱头的花药进行RNA提取后的转录组测序结果,Gm200和Gm300在可育样品中高表达,在不育样品中表达量为0。
图3为RT-PCR验证Gm200和Gm300的转录组的测序结果,编号1-3个为不育个体,4-6为可育个体,tublin为内参基因。
图4为实施例中扩增产物琼脂糖电泳图,2K Marker,编号1,4,12为可育的材料,其余编号为不可育材料,高带为1333bp,用来鉴定可育植株标记,低带为661bp,用来鉴定细胞核雄性不育标记。
具体实施方式
为了便于理解,下面结合实施例对本发明的技术方案做出更为具体的说明:
实施例1
如图1所示,利用前期在田间发现的1株大豆天然雄性不育株,于2013年利用其与其他大豆品种进行杂交,进而构建不育群体。2014年种植收获杂交种。随后将收获的F1代的种子种植于试验基地获得F2群体。随后2016-2019年将该群体连续繁殖,种植于安徽农业大学高新技术产业园。
本实施例中,对上述F2群体进行遗传学分析,在F2不育群体内892株大豆植株进行形状调查和统计,发现不育株共694株,可育株198,用SPSS12.0软件对得到的数据进行卡方检测分离比符合3:1的比例,如表1所示,χ2 3:1<χ2 0.05=3.841,表明该群体的不育性状是由一个单隐性核基因控制的;田间性状调查结果显示,回交后代中性状分离比为1:1。综合上述调查的田间群体分离情况的数据来看,说明该雄性不育突变体育性受1对隐性核基因控制。
表1F2群体遗传学分析
接下来对三个不育样品(编号ms1-1,ms1-2,ms1-3)以及三个可育样品(F1-1,F-2,F1-3)的花器官提取RNA进行转录组测序,各样品高质量Clean reads比率均大于94%、数据过滤前后碱基组成平衡。如表2、表3所示,所有样品共检测到55953个基因的表达数据,其中47339(84.60%)个基因为已知基因,剩余基因为检测到的新基因。各组中,可育组共检测到46878个基因,不育组共检测到46779个基因,说明不育和可育组转录组测序基因覆盖率均满足要求,可以进行下一步分析。表中比率=已知基因数目/参考基因组的基因总数。
表2所有样品检测基因数目统计
表3各组检测基因数目统计
选取群体中不育和可育大豆的带有柱头的花药进行RNA提取,反转录,如图2所示,发现两个在不育组中表达量显著下调的基因Gm200和Gm300。对Gm200和Gm300的转录组测序结果进行RT-PCR验证,从phytozome上下载Gm200和Gm300的CDS序列别分别设计引物,如图3所示,Gm200和Gm300在可育个体中表达,在不育个体中表达量几乎为零,与可育个体差异明显,进一步验证了转录组测序结果。
不育大豆个体重测序结果显示,在13号染色体存在一个结构变异,该变异导致13号染色体38.7kb(22776268-22815034)的染色体片段缺失,其中包括Gm200、Gm300这两个基因(22782042-22814720)。该13号染色体第22776268-bp-22815034bp之间的染色体片段缺失为大豆植株细胞核雄性不育的InDel标记,核苷酸序列如说明书序列表SEQ ID NO.1所示。
实施例2
利用上述InDel标记鉴定大豆植株细胞核雄性育性,首先在13号染色体缺失的染色体片段两端设计引物对序列如下:
1F:TGCCACTAACACCATCGACA SEQ ID NO.2
1R:ACTCATGCGGTTTGTGGGAG SEQ ID NO.3
再依据大豆可育植株未缺失的片段Gm200基因设计一对引物对,用于对其进行鉴定,该引物对序列如下:
2F:TAAACCTCGTCGTCGTTCATT SEQ ID NO.4
2R:CTGCTAGTCTGCCCATACGC SEQ ID NO.5
为了实现快速检测与鉴定大豆植株的育性,本实施例中,采用上述两对引物对共同对提取的多组待测大豆基因组DNA进行PCR扩增;
PCR扩增体系为:2.0μL 100ng/μL的模板DNA、10μM的引物各1.0μL,1.0U KODone酶12.5μL,ddH2O 8.5μL;反应条件为:94℃变性2min,98℃变性10s,起始退火温度63℃,每一个循环降落0.2℃,共退火15s,68℃延伸30s,共循环38次;最后68℃延伸2min,4℃保存。获得扩增产物,对扩增产物进行琼脂糖电泳。扩增产物为高带,用于验证可育植株,扩增产物为低带,用于验证不育植株,其结果如图3所示。
从图3可以看出,1,4,12号扩增产物为高带,其序列大小为1333bp,表明1,4,12均为可育的材料;其余组扩增产物为低带,序列大小为661bp,表明均为不育材料。
以上实施方式仅用以说明本发明的技术方案,而并非对本发明的限制;尽管参照前述实施方式对本发明进行了详细的说明,本领域的普通技术人员应当理解:凡在本发明创造的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明创造的保护范围之内。
序列表
<110> 安徽农业大学
<120> 一种大豆细胞核雄性不育InDel标记及其应用
<141> 2021-07-30
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 661
<212> DNA
<213> Glycine max
<400> 1
tgccactaac accatcgaca ttactatcac catcaccgtc attgtcataa ccaccactac 60
tcccatattc cctacacctt ctgtaacgat gttcaacatg caattgtgca ccatatattg 120
aactttgaat ggctttccat gtggttttgt tcttgtaacg atcatactcc ttgtgttggc 180
ttggcacttt aaagttcagt gtcaatcgtt ttgcttttaa gaaaaagttg ctgggcaatc 240
acacaactta tcttttcttt acaaaactaa gaagaaaagt cttacaccaa atccaaacct 300
ttaatacagc caggttgtaa ttgacatagg tatgaataac tcctaaaaga attcttattc 360
aagattcata tttatagtat aagtatgaat tttatatata aaaaaaaact ttacattatt 420
ttaactagtg atctataatt tatattacaa ttttagtttt ttaaaattat atgaacgtct 480
cgttttacat gtattgttcg tatataaaaa aggaaataaa aatcataaat ttatttccgt 540
tatcggatta gacatttatg aactcttgag cattaactcg tagagaaccg caatttacct 600
acaattctaa aagtgaagct cccacaaaac taaaagtgaa gctcccacaa accgcatgag 660
t 661
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 2
tgccactaac accatcgaca 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 3
actcatgcgg tttgtgggag 20
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 4
taaacctcgt cgtcgttcat t 21
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 5
ctgctagtct gcccatacgc 20
Claims (5)
1.一种大豆植株细胞核雄性不育InDel标记,其特征在于,所述InDel标记的核苷酸序列如SEQ ID NO.1所示,该InDel标记位于大豆第13号染色体第22776268bp-22815034bp之间。
2.如权利要求1所述的InDel标记的应用,其特征在于,所述InDel标记在鉴定大豆植株细胞核雄性不育中的应用,或所述InDel标记在制备检测大豆植株细胞核雄性不育试剂或试剂盒中的应用。
3.如权利要求2所述的应用,其特征在于,通过核酸扩增技术检测大豆植株中所述InDel标记的表达水平,用于核酸扩增的引物对序列为:
正向序列SEQ ID NO.2:TGCCACTAACACCATCGACA
反向序列SEQ ID NO.3:ACTCATGCGGTTTGTGGGAG
4.一种利用如权利要求1所述的InDel标记鉴定大豆植株育性的方法,其特征在于,包括以下步骤:
S1.设计鉴定可育大豆植株的引物对,所述引物对序列为:
正向引物SEQ ID NO.4:TAAACCTCGTCGTCGTTCATT
反向引物SEQ ID NO.5:CTGCTAGTCTGCCCATACGC
S2.提取待测大豆植株基因组DNA;
S3.以用于InDe1标记核酸扩增的引物对序列SEQ ID NO.2、SEQ ID NO.3和所述SEQ IDNO.4、SEQ ID NO.5为引物,对S2提取的DNA进行PCR扩增,获得扩增产物;其中,
正向序列SEQ ID NO.2:TGCCACTAACACCATCGACA
反向序列SEQ ID NO.3:ACTCATGCGGTTTGTGGGAG
S4.对S4扩增产物进行琼脂糖凝胶电泳,根据电泳所得条带结果判断待测大豆植株的育性;若所得条带为高带1333bp,则判断待测大豆植株为可育植株,若所得条带为低带661bp,则判断待测大豆植株为细胞核雄性不育植株;
所述高带的核苷酸序列如SEQ ID NO.6所示,所述低带的核苷酸序列如SEQ ID NO.7所示。
5.如权利要求4所述的方法,其特征在于,所述S3中,PCR扩增程序为:100ng/μL的模板DNA和10μM的引物各1.0μL,1.0U KODone酶12.5μL,ddH2O 8.5μL;反应条件为:94℃预变性2min;98℃变性10s,起始退火温度63℃,每一个循环降落0.2℃,共退火15s,68℃延伸30s,设38次循环;最后68℃延伸2min,4℃保存。
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