CN116904638B - 与藜麦早雌性状连锁的kasp标记及应用 - Google Patents
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Abstract
本发明公开了一种与藜麦早雌性状连锁的分子标记Chr02‑34011758及其应用,该分子标记位于藜麦2号染色体上,为SEQ ID NO.1所示序列的第40位碱基,呈G[C]多态性。利用该分子标记可快速筛选具有早雌特性的藜麦材料,选育藜麦早雌新品种,大大节约了育种成本,提高了育种效率。
Description
技术领域
本发明属于分子遗传学领域,具体涉及一种与藜麦早雌性状相关的KASP标记及应用。
背景技术
藜麦属于苋科藜亚科藜属双子叶植物,雌雄同花,以自交为主,异交率低,但杂种优势明显。目前藜麦杂交育种方法还是基于人工去雄和人工授粉的办法获得杂交后代,并从后代中选出具有优良性状的纯合自交系。
申请号为201811011523.0的专利公开了一种藜麦二元杂交方法,所述杂交方法中采用的母本植株花朵为雌蕊先于雄蕊成熟,且在雌蕊成熟而雄蕊还未成熟时花朵充分张开,使雌蕊全部外露,可以作为雄性不育系使用。因此,藜麦的早雌性状对于杂交育种至关重要,获得具有早雌性状的藜麦能大大提高杂交育种的效率。
KASP技术是一种基于荧光信号的竞争性等位基因特异性PCR,可对样品中的多态性位点进行基因分型检测。与传统PCR方法相比,KASP技术具有通量高、成本低、效率高、准确度高、稳定性好等优点。因此,基于KASP技术开发与早雌性状连锁的分子标记,用于筛选藜麦早雌基因型,对藜麦早雌辅助选择育种和杂种优势利用具有重要价值。
发明内容
本发明提供了一种与藜麦早雌性状连锁的KASP标记及其检测方法,能从现有藜麦种质及其杂交后代新种质中快速鉴定具有早雌特质的藜麦材料。本发明利用具有早雌性状的突变体材料ProA与野生型亲本JQ510杂交,在F2代群体中构建极端表型混池,通过BSA(Bulked segregation analysis)关联分析两个混池间差异序列,在藜麦2号染色体上快速定位到与早雌性状连锁的标记Chr02-34011758。
第一方面,本发明提供了鉴定藜麦早雌性状的分子标记Chr02-34011758及其应用,所述分子标记Chr02-34011758位于藜麦2号染色体上,核苷酸序列如SEQ ID NO.1所示,第40位碱基为G[C]多态性位点。
第二方面,本发明还提供了分子标记Chr02-34011758的检测方法及其应用,所述的分子标记的检测方法采用竞争性等位基因特异性PCR(KASP)方法;所述竞争性等位基因特异性PCR扩增采用的引物对由正向引物Fa、正向引物Fb和反向引物R组成,其中,所述引物Fa的核苷酸序列为
5'-GAAGGTGACCAAGTTCATGCTCTCCTGCTCCAATCAACCCTAGGAG-3';引物Fb的核苷酸序列为
5'-GAAGGTCGGAGTCAACGGATTCTCCTGCTCCAATCAACCCTAGGAC-3';引物R的核苷酸序列为TTCCACTGTACTATATCATCCCAAAGGAT;
第三方面,本发明还提供了一种鉴定或辅助鉴定藜麦早雌性状的方法,包括如下步骤:
(1)利用权利要求3所述检测方法检测待测材料的权利要求1所述的分子标记Chr02-34011758;
(2)如果检测结果为GG或GC,待测材料将表现早雌性状;如果检测结果为CC,待测材料将表现正常性状。
本发明还提供了上述分子标记、检测和鉴定方法在藜麦早雌性状选育中的应用。
本发明的有益效果是:获得了一个在藜麦2号染色体上与早雌性状目标区段连锁分子标记Chr02-34011758。序列分析表明,该标记序列如SEQ ID No.1所示,第40位碱基为G[C]多态性位点。本发明设计开发了Chr02-34011758位点的KASP标记和检测方法,利用该位点的多态性进行基因分型分析,可以快速筛选出具有早雌性状的藜麦材料,可用于藜麦分子标记育种,同时能大大降低藜麦杂交育种的成本,并提高藜麦杂种优势利用的效率。
附图说明
通过阅读参照以下附图所作的对非限制性实施例所作的详细描述,本申请的其它特征、目的和优点将会变得更明显:
图1BSA-seq定位分析图谱(箭头所指为ΔSNP index指示峰)
图2KASP标记的复合区间作图(IciMAPPING软件)
图3标记Chr02-34011758的KASP检测基因分型图谱(深色圆点代表具有早雌表型的A1混池单株,浅色圆点代表A0混池的正常单株)
具体实施方式
下面结合附图对本发明做进一步说明,以下实例仅用于说明本发明但并不限定本发明的范畴。
所述“藜麦”是指藜麦(Chenopodium quinoa willd)属藜科,包括可以与藜麦育种的所有植物品种,包括整株植物、植物细胞、植物器官、植物原生质体、植物可以从中再生的植物细胞组织培养物、植物愈伤组织、植物或植物部分中完整的植物细胞,所述植物部分例如胚、花粉、胚珠、种子、叶、花、枝、果实、茎杆、根、根尖、花药等。
术语“探针”是一段分离的核酸分子,其上面结合有常规的可检测标记或报告分子,例如,放射性同位素、配体、化学发光剂或酶类。
术语“引物”是一段分离的核酸分子,其通过核酸杂交,退火结合到互补的目标DNA链上,在引物和目标DNA链之间形成杂合体,然后在聚合酶(例如DNA聚合酶)的作用下,沿目标DNA链延伸。本发明的引物对涉及其在目标核酸序列扩增中的应用,例如,通过聚合酶链式反应(PCR)或其他常规的核酸扩增方法。
术语“分子标记”指能反映生物个体或种群间基因组中某种差异的特异性DNA片段。“标记”是用作参考点的核苷酸序列或其编码产物(例如蛋白)。对于用于检测重组的标记,它们需要在被监测的种群内检测差异或多态性。对于分子标记,这意味着DNA水平的差异是由于多核苷酸序列差异(例如SSR、RFLP、FLP和SNP)。基因组可变性可为任何一种起源,例如插入、缺失、复制、重复元件、点突变、重组事件或转座因子的存在和序列。分子标记可来源于基因组或表达的核酸(例如EST),并且也可指用作探针或引物对的核酸,所述引物对能够通过使用基于PCR的方法扩增序列片段。
术语“早雌”指的是同株或同花的雌花先于雄花成熟,而且,花朵会完全张开露出雌花,这样目标花粉可以提前给雌蕊授粉达到杂交目的。利用这种特异性花可以突破人工去雄的繁杂及后期选育的麻烦。
实施例
藜麦早雌突变体来源是通过构建突变体库获得,突变体库构建方式如下:
S1.制备1.5mg/ml浓度的EMS;
S2.选取种子颗粒直径在2.5mm以上,色泽为乳白色,适合高原生长的藜麦种子,使其浸泡于1.5mg/ml浓度的EMS溶液中15min。
S3.将处理好的藜麦种子及时在温室中采用育苗盘播种育苗,幼苗长到6片叶后在准备好的大田进行移栽,移栽密度为行距40cm,株距20cm,后期按正常藜麦种植进行管理,成熟后收获其单株种子混合作为突变体库材料。
将EMS诱变获得的早雌藜麦单株ProA与野生型亲本JQ510(黑藜,通过市场购买获得)杂交,在F2代群体中统计早雌性状单株和正常野生型性状单株分别为498和207株(如表1所示),正常:早雌≈1:3,说明早雌性状是显性单基因控制。
表1藜麦F2代群体雌蕊开花时间表型统计
1、通过BSA-seq定位突变位点的候选区间
从F2代群体挑选50株早雌性状单株和50株正常性状单株,采用CTAB法提取藜麦DNA,具体步骤如下:
1)取叶片0.1g,研磨成粉末后加入500-800μL CTAB,65℃温浴0.5-1h;
2)加入700μL氯仿或氯仿:异戊醇(24:1),缓慢摇匀,12000rpm离心15min,取上清400-700μL;
3)加入1mL预冷无水乙醇或异丙醇,充分混匀后,12000rpm离心10min,弃上清;
4)将沉淀用75%酒精洗涤,12000rpm离心5min,弃酒精,倒置吸水干燥;
5)用50μL ddH2O溶解DNA,取3-5μL电泳检测后,用超微量紫外分光光度计测定DNA浓度。
将早雌单株和正常性状单株DNA分别等量混合,构建极端表型混池基因组DNA,记为A1和A0;取原始突变体和正常亲本样品,提取DNA做对照,分别记为ProA、JQ510。
对ProA、JQ510以及混池A1和A0进行二代测序(30×深度,双端二代illumina测序),通过BSA(Bulked segregation analysis)关联分析两个混池间差异序列(包括数据质控、基因组比对、calling SNP和SNP index计算等),分析结果如图1所示,图1箭头所指为ΔSNP index指示峰,暗示该位置存在目标性状的连锁区间。分析结果进一步将突变位点关联到藜麦基因组3条染色体的6个候选区间(区间置信度为99%),如表2所示:
表2 6个候选区间(99%置信度)
上述候选区间内共有12.8万个单核苷酸多态性位点(SNP)位点,经过筛选,其中非同义突变位点有1218个;6000多个插入缺失(INDEL)位点,经过筛选,其中非同义突变有38个。
2、候选区间的初步鉴定
从上述SNP、INDEL位点中挑选出来12个peak值较高且相距较远的位点(每个候选区间2个位点),设计分子标记M1~M12;从早雌性状混池A1中随机挑选3个单株A1-1、A1-2和A1-3,从正常性状混池A0中随机挑选3个单株A0-1、A0-2和A0-3,提取6个单株的DNA作为模板;通过PCR扩增和一代测序的方法,开展候选区间的初步验证。结果表明:分子标记M10和M11可能与目标基因连锁(表3),该区间为Chr02:23140001—4825000。
表3 6个候选区间12个位点的初步鉴定结果
3、目标区段的KASP分子标记开发
基于上述候选区间(Chr02:23140001—4825000)定位的基因组信息及BSA-seq结果,进行了SNP检测和KASP检测标记的开发,在Chr02-22156243、Chr02-28607354、Chr02-34011758和Chr02-44929245这4个SNP位点分别设计了1个KASP标记,每个标记包含1组成套引物(两条正向引物Fa、Fb和一条反向引物R),如表4所示。
表4KASP标记引物组
注:下划线部分为结合荧光基团的通用接头序列,Fa结合FAM荧光基团,Fb结合VIC荧光基团
使用上述引物组,选取F2分离群体中的单株提取DNA,进行KASP标记的多态性检验。
荧光定量PCR反应体系为:引物组0.5μl(10μM),其中Fa、Fb和R比例为1:1:3(体积比);2×KASP Mix 5.0μL;浓度为25~100ng/μL的DNA模板2.0μL;最后添加去离子水至总反应体系10μL。
反应程序为:95℃预变性10分钟;95℃变性15秒,61℃退火延伸60秒,循环10次,每次循环退火延伸温度降低0.6℃;95℃变性15秒,55℃退火延伸60秒,循环35次;25℃保温,采集荧光型号。
将荧光定量PCR的结果用IciMAPPING软件进行单标记分析和复合区间作图,如表5和图2所示。
表5IciMAPPING软件单标记分析结果
通过标记开发和重定位分析,将与目标性状连锁的区域缩小至Chr02:28607354—42503136的区域范围(LOD>2.5)。4个测试标记中,Chr02-34011758标记的LOD和PVE均达到峰值,表明该标记与目标区段连锁,可作为该目标区段的筛选标记。
从标记Chr02-34011758的基因分型图(图3)和基因型/表型数据统计结果(表6)来看,该标记与早雌和正常表型性状相关,能够用于区分这两种表型性状,其中KASP检测基因型为GG或GC的为早雌表型(图3基因分型图上为深色圆点),基因型为CC的为正常表型(图3基因分型图上为浅色圆点)。
表6标记Chr02-34011758的基因型/表型统计分析
注:“-”表示未检测到
以上所述仅是本发明的较佳实施例而已,并非对本发明做任何形式上的限制,虽然本发明已以较佳实施例公开如上,然而并非用以限定本发明,任何熟悉本专业的技术人员,在不脱离本发明技术方案的范围内,当可利用上述揭示的技术内容作出更动或修饰为等同变化的等效实施例,但凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (6)
1.分子标记Chr02-34011758,其特征在于:所述标记位于SEQ ID NO.1所示序列第40位碱基,呈G[C]多态性。
2.权利要求1所述分子标记在藜麦早雌性状选育中的应用。
3.权利要求1所述分子标记Chr02-34011758的检测方法,其特征在于:所述的分子标记的检测方法采用竞争性等位基因特异性PCR(KASP)方法;所述竞争性等位基因特异性PCR扩增采用的引物对由正向引物Fa、正向引物Fb和反向引物R组成,其中,所述引物Fa的核苷酸序列为5'-GAAGGTGACCAAGTTCATGCTCTCCTGCTCCAATCAACCCTAGGAG-3';引物Fb的核苷酸序列为5'-GAAGGTCGGAGTCAACGGATTCTCCTGCTCCAATCAACCCTAGGAC-3';引物R的核苷酸序列为TTCCACTGTACTATATCATCCCAAAGGAT。
4.权利要求3所述的检测方法在藜麦早雌性状选育中的应用。
5.一种鉴定或辅助鉴定藜麦早雌性状的方法,其特征在于,包括如下步骤:
(1)利用权利要求3所述检测方法检测待测材料的权利要求1所述的分子标记Chr02-34011758;
(2)如果检测结果为GG或GC,待测材料将表现早雌性状;如果检测结果为CC,待测材料将表现正常性状。
6.权利要求5所述的鉴定或辅助鉴定藜麦早雌性状的方法在藜麦早雌性状选育中的应用。
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