CN113215301B - 簇毛麦5vs染色体臂特异分子标记引物及应用 - Google Patents
簇毛麦5vs染色体臂特异分子标记引物及应用 Download PDFInfo
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Abstract
本发明公开了与簇毛麦5VS共分离的分子标记,属于农业生物技术领域。以中国春‑簇毛麦T5DL.5VS易位系NAU415和中国春‑簇毛麦T5AL.5VS易位系NAU421易位系为材料,通过染色体分选和二代测序获得了4个与簇毛麦5VS染色体臂共分离的分子标记,利用群体进行验证,以上4个标记在BC6F2群体中与白粉病抗性共分离。该标记作为簇毛麦5VS染色体臂新的特异分子标记,为T5AL.5VS和T5DL.5VS两个补偿性易位染色体在小麦育种的高效利用提供分子标记和理论依据,简化选择方法和提高育种效率,进而加快优质抗病品种的育种进程。
Description
技术领域
本发明设计簇毛麦5VS染色体臂特异分子标记的开发及其引物序列,可应用于分子标记辅助育种,属于农业生物技术领域。
背景技术
普通小麦(Triticum aestivum L.,2n=6x=42,AABBDD)是异源六倍体,经历了两次染色体加倍过程,其遗传多样性受其原始二倍体供体基因组多样性尤其是D基因组多样性的制约。此外,现代小麦品种还由于长期人工定向选择,新的育成品种普遍呈现出遗传基础日益狭窄的趋势,突出表现为产量水平进一步提高的瓶颈制约效应。因此,提高小麦基因组遗传多样性,是培育适应气候变化及高产品种的重要举措。
普通小麦包括三个基因源,其中小麦三级基因源包括簇毛麦属(Dasypyrum,V)、黑麦属(Scale,R)和除含D和S基因组以外的山羊草属(Aegilops,CUM)等23个属,携带丰富的有利于小麦改良的抗病、耐逆基因(Colmer等,2006,Use of wild relatives to improvesalt tolerance in wheat,J Exp Bot 57,1059~1078;Friebe等,1996,Characterization of wheat-alien translocations conferring resistance todiseases and pests:Current status,Euphytica 91,59~87;Chen等,1995,Developmentand molecular cyto-genetic analysis of wheat-Haynaldia villosa 6VS/6ALtranslocation lines specifying resistance to powdery mildew,Theor Appl Genet91,1125~1128;Triebe等,1991,Identification of alien chromatin specifyingresistance to wheat streak mosaic and greenbug in wheat germ plasm by C-banding and insitu hybridization,Theor Appl Genet 81,381~389)。小麦育种家为了利用这些优异基因,已经通过染色体工程技术创制了大量的附加系、双二倍体、易位系、渐渗系等。由于这些野生种质资源遗传变异巨大,与小麦染色体的同源性较差,利用这些遗传变异仍然具有很大的挑战。同时,外源染色体导入到小麦背景之后仍然难以高通量的追踪与鉴定。细胞学方法如染色体核型鉴定、基因组原位杂交(Genomic in SituHybridization,GISH)和荧光原位杂交(Fluorescence in Situ Hybridization,FISH)已经被广泛的用于小麦外源片段的检测和分析,但是通量较低(Lukaszewski等,2005,Limitations of in situ hybridization with total genomic DNA in routinescreening for alien introgressions in wheat,Cytogenet Genome Res 109,373~377)。分子标记检测则比较高效、方便,但是三级基因源的基因组序列信息很少,分子标记依然缺乏,迫切需要高通量开发分子标记的方法用于外源染色体片段的追踪与鉴定。
近年来,随着测序技术的迅猛发展,中国春(Appels等,2018,Shifting thelimits in wheat research and breeding using a fully annotated referencegenome,Science 6403,eaar7191)、野生二粒小麦(Avni等,2017,Wild emmer genomearchitecture and diversity elucidate wheat evolution and domestication,Science 6403,eaar7191)、乌拉尔图小麦(Ling等,2018,Genome sequence of theprogenitor of wheat A subgenome Triticum Urartu,Nature557,424~428)、节节麦(Luo等,2017,Genome sequence of the progenitor of the wheat D genome Aegilopstauschii,Nature 551,498~502;Jia等,2013,Aegilops tauschii draft genomesequence reveals a gene repertoire for wheat adaptation,Nature 496,91~95)、西藏半野生小麦(Guo等,2020,Origin and adaptation to high altitude of Tibetansemi-wild wheat,Nature Communication 11,5085)等基因组测序工作相继完成。尽管如此,针对小麦特定染色体,基因组作图和测序工作仍是艰巨的任务,而且性价比低。流式细胞技术能够从复杂基因组中分离单个染色体从而减少样本的复杂性,减少大量繁杂、无目的的工作,是目前小麦染色体分离和基因克隆的重要技术。截至目前,该技术不但在普通小麦和硬粒小麦中成功应用(Dolezel等,2012,Chromosomes in the flow to simplifygenome analysis,Funct Integr Genomic 12,397~416),而且在小麦野生资源中也已成功应用(Tiwari等,2014,SNP Discovery for mapping alien introgressions in wheat,BMC Genomics 15,273;Molnar等,2011,Chromosome isolation by flow sorting inAegilops umbellulata and Ae.comosa and their allotetraploid hybridsAe.biuncialis and Ae.Geniculata,Plos One 6(11),e27708;Molnar等,2015,Flowsorting of C-genome chromosomes from wild relatives of wheat Aegilopsmarkgrafii,Ae.triuncialis and Ae.cylindrica,and their molecular organization,Ann Bot 116,189~200;Molnar等,2014,Flow cytometric chromosome sorting fromdiploid progenitors of bread wheat,T.urartu,Ae.speltoides and Ae.Tauschii,Theor Appl Genet 127,10919~104;Molnar等,2013,Syntenic relationships betweenthe U and M genomes of Aegilops,wheat and the model species Brachypodium andrice as revealed by COS markers,Plos One 8(8),e70844;Gradzielewska等,2006,Thegenus Dasypyrum-Part 2.Dasypyrum villosum-a wild species used in wheatimprovement,Euphytica 152,441~54;Wang等,2017,Development of intron targeting(IT)markers specific for chromosome arm 4VS of Haynaldia villosa bychromosome sorting and next-generation sequencing,BMC Genomics 18,167;Xing等,2021,Long-range assembly of sequences helps to unravel the genome structureand small variation of the wheat–Haynaldia villosa translocated chromosome6VS.6AL,Plant Biotechnology Journal,2021,https://doi.org/10.1111/pbi.13570),包括卵穗山羊草(Ae.geniculata)、伞穗山羊草(Ae.umbellulata)、顶芒山羊草(Ae.comosa)、尾状山羊草(Ae.markgrafii)、三芒山羊草(Ae.triuncialis)、圆柱山羊草(Ae.cylindrica)、乌拉尔图(T.urartu)、斯卑尔托山羊草(Ae.speltoides)、节节麦(Ae.tauschii)、两芒山羊草(Ae.biuncialis)和簇毛麦(D.villosum)。流式细胞染色体分离技术与二代DNA测序技术组合的方法已经用于小麦野生资源特异染色体分子标记的开发,如簇毛麦4VS、6VS特异分子标记的开发(Wang等,2017,Development of introntargeting(IT)markers specific for chromosome arm 4VS of Haynaldia villosa bychromosome sorting and next-generation sequencing,BMC Genomics 18,167;Xing等,2021,Long-range assembly of sequences helps to unravel the genome structureand small variation of the wheat–Haynaldia villosa translocated chromosome6VS.6AL,Plant Biotechnology Journal,2021,https://doi.org/10.1111/pbi.13570)。
随着测序技术的发展和序列信息的丰富,分子标记的类型逐渐多样化。初期的限制性片段长度多态性(restriction fragment length polymorphism,RFLP)、扩增片段长度多态性(amplified fragment length polymorphism,AFLP)等标记已经不能够满足高通量试验的需求。根据序列特征和信息开发的标记越来越多、种类越来越丰富,简单重复序列(simple sequence repeats,SSR)、序列标签位点(sequence tagged sites,STS)、酶切扩增多态性序列(cleaved amplified polymorphic sequence,CAPS)、衍生的酶切扩增多态性序列(derived cleaved amplified polymorphic sequence,dCAPS)、单核苷酸多态性(single nucleotide-acid polymorphism,SNP)、内含子标签(intron targeting,IT)和保守直系同源基因序列(Coserved Ortholog Sequence,COS)等类型的标记应运而生。野生资源在没有序列信息之前,只有IT类型标记开发相对成功。然而,随着测序技术的发展,野生资源的基因组序列信息越来越丰富,上述类型的标记将会在野生资源的性状定位、基因克隆和分子标记选择与追踪等方面发挥越来越显著的作用,也为小麦野生资源优异基因的快速鉴定和利用奠定基础。
簇毛麦(Dasypyrum villosum L.)作为小麦的野生近缘物种,携带许多优异的抗性基因,是小麦遗传改良重要的三级基因源库。研究者最早于1908年开展了小麦与簇毛麦的远缘杂交,尽管一百多年来不同学者利用二倍体、四倍体和六倍体小麦与簇毛麦进行了大量的远缘杂交工作,仅携带抗白粉病基因Pm21的6AL.6VS易位系被广泛用于抗病育种亲本(De Pace等,2011,Dasypyrum.In:Kole C(ed)Wild crop relatives:genomic andbreeding resources,Cereals 4(1),185~292)。目前,不同麦区中抗性最强、抗谱最广的品种大多携带簇毛麦6VS染色体臂及其抗白粉病基因Pm21,携带该基因的品种目前已超过40个,而且区域试验中携带Pm21基因的品系更多。由于育种工作者大多重视免疫或高抗类型品种的选择,可以预见Pm21基因在生产上的利用还会不断增加。反思国内外大量使用1BL/1RS易位系进行抗病育种的历史经验,大面积推广种植相对单一的抗病性品种,极易对病原菌群体的毒性基因或小种产生定向选择压力,产生新的毒性小种。基因克隆发现Pm21基因亦编码CC-NBS-LRR抗病结构域蛋白(Xing等,2018,Pm21 from Haynaldia villosaencodes a CC-NBS-LRR protein conferring powdery mildew resistance in wheat,Mol Plant 11,874~878),大面积使用该基因产生毒性新小种的可能性很大。因此,需要发掘便于育种利用的新抗性资源。
发明内容
本发明是针对上述技术问题,提供簇毛麦5VS染色体臂特异分子标记引物,以中国春-簇毛麦T5DL.5VS易位系NAU415、T5AL.5VS易位系NAU421为材料,筛选与寻找簇毛麦5VS染色体臂特异的分子标记,为T5AL.5VS和T5DL.5VS两个补偿性易位染色体在小麦育种的高效利用提供分子标记和理论依据。
簇毛麦5VS染色体臂特异的分子标记引物Xmp1479,该引物是与簇毛麦5VS特异的COS标记Xmp1479的引物,簇毛麦5VS与小麦5AS、5BS及5DS存在异质性,5VS在小麦背景中与5AS、5BS及5DS不发生重组,标记Xmp1479与5VS染色体臂共分离,引物序列如下:
左引物:5’-GATGGTCAGCAACTCGCT-3’(SEQ ID NO.1);
右引物:5’-TCACCACCTGCCCGTAC-3’(SEQ ID NO.2)。
所述分子标记引物的应用,其特征在于,用所述与簇毛麦5VS共分离的COS标记Xmp1479的引物PCR扩增小麦植株DNA,如果出现Xmp1479标记的135bp的带型,则表示该植株中携带簇毛麦5VS染色体臂。所述的应用是以易位系NAU415、易位系NAU421或其衍生品种为扩增小麦植株DNA对象的。
PCR反应体系10μl:内含模板10~20ng、左右引物各2pmol、MgCl2 15nmol、0.1UTaq DNA聚合酶与1×PCR缓冲液;
反应循环程序如下:94℃预变性3min,然后按“94℃,30s;58.7℃,40s;72℃,40s”进行35个循环扩增,最后72℃延伸10min;
簇毛麦5VS染色体臂特异的分子标记引物Xmp1520,该引物是与簇毛麦5VS特异的COS标记Xmp1520的引物,簇毛麦5VS与小麦5AS、5BS及5DS存在异质性,5VS在小麦背景中与5AS、5BS及5DS不发生重组,标记Xmp1520与5VS染色体臂共分离,引物序列如下:
左引物:5’-TTGTTCGCCTCCATGAGA-3’(SEQ ID NO.3);
右引物:5’-GATCTTCGCCTGCTCCCT-3’(SEQ ID NO.4)。
所述分子标记引物的应用,其特征在于,用所述与簇毛麦5VS共分离的COS标记Xmp1520的引物PCR扩增小麦植株DNA,如果出现Xmp1520标记的134bp的带型,则表示该植株中携带簇毛麦5VS染色体臂。所述的应用是以易位系NAU415、易位系NAU421或其衍生品种为扩增小麦植株DNA对象的。
PCR反应体系10μl:内含模板10~20ng、左右引物各2pmol、MgCl2 15nmol、0.1UTaq DNA聚合酶与1×PCR缓冲液;
反应循环程序如下:94℃预变性3min,然后按“94℃,30s;59.5℃,40s;72℃,40s”进行35个循环扩增,最后72℃延伸10min;
簇毛麦5VS染色体臂特异的分子标记引物Xmp1526,该引物是与簇毛麦5VS特异的COS标记Xmp1526的引物,簇毛麦5VS与小麦5AS、5BS及5DS存在异质性,5VS在小麦背景中与5AS、5BS及5DS不发生重组,标记Xmp1526与5VS染色体臂共分离,引物序列如下:
左引物:5’-TTCGTTACACGCTACTGC-3’(SEQ ID NO.5);
右引物:5’-AGATTCAGGTTGACACCG-3’(SEQ ID NO.6)。
所述分子标记引物的应用,其特征在于,用所述与簇毛麦5VS共分离的COS标记Xmp1526的引物PCR扩增小麦植株DNA,如果出现Xmp1526标记的223bp的带型,则表示该植株中携带簇毛麦5VS染色体臂。所述的应用是以易位系NAU415、易位系NAU421或其衍生品种为扩增小麦植株DNA对象的。
PCR反应体系10μl:内含模板10~20ng、左右引物各2pmol、MgCl2 15nmol、0.1UTaq DNA聚合酶与1×PCR缓冲液;
反应循环程序如下:94℃预变性3min,然后按“94℃,30s;54℃,40s;72℃,40s”进行35个循环扩增,最后72℃延伸10min;
簇毛麦5VS染色体臂特异的分子标记引物Xmp1550,该引物是与簇毛麦5VS特异的COS标记Xmp1550的引物,簇毛麦5VS与小麦5AS、5BS及5DS存在异质性,5VS在小麦背景中与5AS、5BS及5DS不发生重组,标记Xmp1550与5VS染色体臂共分离,引物序列如下:
左引物:5’-GTGGCAGCGTCAGAGTAA-3’(SEQ ID NO.7);
右引物:5’-CCAATGTTGGTTTTCATGTG-3’(SEQ ID NO.8)。
所述分子标记引物的应用,其特征在于,用所述与簇毛麦5VS共分离的COS标记Xmp1550的引物PCR扩增小麦植株DNA,如果出现Xmp1550标记的155bp的带型,则表示该植株中携带簇毛麦5VS染色体臂。所述的应用是以易位系NAU415、易位系NAU421或其衍生品种为扩增小麦植株DNA对象的。
PCR反应体系10μl:内含模板10~20ng、左右引物各2pmol、MgCl2 15nmol、0.1UTaq DNA聚合酶与1×PCR缓冲液;
反应循环程序如下:94℃预变性3min,然后按“94℃,30s;56℃,40s;72℃,40s”进行35个循环扩增,最后72℃延伸10min;
本发明还提供一种选育抗白粉病小麦的方法,其特征在于,上述任一分子标记引物PCR扩增小麦植株DNA,根据检测结果选育小麦品种。
进一步的,筛选符合下列条件的品种进行选育:
Xmp1479标记在135bp处出现特异性条带;
或Xmp1520标记在134bp处出现特异性条带;
或Xmp1526标记的223bp处出现特异性条带;
或Xmp1550标记的155bp处出现特异性条带。
有益效果
簇毛麦5VS染色体臂携带抗白粉病基因Pm55、抗条锈病基因Yr5V和籽粒硬度基因Dina/Dinb,本发明利用分子标记方法开发出新的簇毛麦5VS共分离的COS标记,在小麦育种实践、抗病种质和优异弱筋种质创制上都有很重要的价值。本发明通过对5DL.5VS易位染色体分拣测序开发5VS染色体特异分子标记,并将T5AL.5VS和T5DL.5VS易位染色体导入南农0686遗传背景以分析5VS的遗传效应,为T5AL.5VS和T5DL.5VS两个补偿性易位染色体在小麦育种的高效利用提供分子标记和理论依据。其优点是:本发明中与簇毛麦5VS共分离的COS标记,是在对创制的中国春-簇毛麦T5DL.5VS易位系NAU415、T5AL.5VS易位系NAU421及其杂交后代单株中获得的新标记,可以用于簇毛麦5VS的快速鉴定与追踪奠定基础,为抗白粉病、抗条锈病和优异弱筋小麦品种选育提供理论依据。
附图说明
通过下面的详细描述并结合附图,将更清楚的理解本发明的目的、特征和其他优点,其中:
图1为5VS上Xmp1479、Xmp1520、Xmp1526和Xmp1550标记在中国春、中国春-簇毛麦T5DL.5VS易位系NAU415、T5DL.5VS易位系NAU415、T5AL.5VS易位系NAU421、T5AL.5VS易位系NAU421,线段处所示为该标记扩增的5AS、5DS和5VS的带型。
图2为易位系的GISH/FISH鉴定结果和有/无易位染色体植株白粉病抗性表现。
具体实施方式
下面实施例中所用方法如无特别说明均为常规方法。
实施例1、簇毛麦5VS特异分子标记的获得
1、植物材料
中国春-簇毛麦T5DL.5VS易位系NAU415、T5AL.5VS易位系NAU421、高产品种南农0686由南京农业大学细胞遗传研究所选育。对照材料中国春由江苏省农业科学院种质资源与生物技术研究所种质资源评价与创新研究室保存提供。T5DL.5VS易位系NAU415和T5AL.5VS易位系NAU421分别与南农0686杂交,每个世代经细胞学鉴定后,选择携带易位染色体单株再与南农0686杂交,回交6次后获得携带单条易位染色体的BC6F1单株,并收获BC6F1单株自交种子。2018年秋播种植BC6F2种子于江苏省农科院南京试验田,BC6F2群体经白粉病抗性鉴定、分子细胞学鉴定后,获得纯合易位染色体单株和无外源单株。2019年秋播种植纯合易位染色体单株和无外源单株于江苏省农科院南京试验田,每个单株种植5行,行长1.2m,每行种植15株。为减少病害对农艺性状分析的影响,2020年春季在小麦抽穗期对实验材料喷施包含杀菌剂和杀虫剂的混合液。田间管理与当地大田生产一致。
2、细胞学鉴定
根尖染色体制片方法如下:(1)将干种子在室温下完成浸种,随后将萌动后的种子置于垫有湿润滤纸的培养皿中并放入25℃恒温箱生长,待根长长至1.5-2.5cm时,倒入0.1g/L的amiprophos-methyl(APM)水溶液,2h后清洗,剪取根尖;(2)把剪取的根尖放入湿润的带孔离心管中,N2O(0.8-1.2MPa)处理1.5h,向离心管中加入90%的乙酸并放置4℃冰箱中固定5-10min,然后转移至70%的乙醇中保存备用;(3)取出根尖用45%的乙酸解离5min后制片,制好的片子放-70℃超低温冰箱冷冻,至少6h后可揭片,后在100%酒精脱水大于10min,吹干后用于原位杂交实验。混合利用簇毛麦基因组DNA标记的绿色荧光探针(绿色)和小麦D基因组专化的oligo-pAs1寡聚核苷酸探针(红色)进行原位杂交。利用OlympusBX60型荧光显微镜观察原位杂交分裂相,使用SPOT CCD(SPOT Cooled Color Digital,DP72,Olympus,Japan)摄取图像。
3、T5DL.5VS易位染色体分选及二代测序
利用流式细胞仪和荧光染色手段对中国春-簇毛麦T5DL.5VS易位系NAU415中的易位染色体进行分拣,并在捷克Dolezel Jaroslav实验室通过Illumina HiSeq 2500对未扩增的染色体DNA进行测序组装。首先制备供试材料根尖有丝分裂染色体的悬浮液,通过荧光标记的GAA-FITC寡核苷酸探针对悬浮液进行荧光原位杂交(FISHIS),并使用双变量流式细胞术从DAPI染色的染色体悬浮液中提取了250万条染色体,并利用pSc119.2、Afa和45SrDNA进行分拣染色体的组成检测及纯度测定。在获得的clean-reads中利用生物信息学的方法筛选出5VS的scaffolds序列,序列拼接利用软件SOAPdenovo(version2.0)。
利用流式细胞仪对中国春-簇毛麦5DL.5VS易位系NAU415中的易位染色体进行分拣测序,得到了5DL.5VS易位染色体的96.3Gb的有效序列,测序深度为50×,分拣纯度为89%。经组装后获得57,258个scaffolds,拼接长度最短1000bp,最长的序列185,441bp,N50为18.3Kb。将5DL.5VS易位染色体分拣测序的57,258个scaffolds按照长度区间进行数目分类统计,1-2kb的scaffolds占的数目最多,共计15,051个,大于100kb的scaffolds总共31个。将分拣测序所得的5DL.5VS易位染色体序列中的5VS和5DL序列分别筛选出来,获得约162.958Mb的5VS序列,共计18,544个scaffolds。
4、簇毛麦5VS特异分子标记的开发及检测
标记开发分为四种方式。第一种是在小麦5AS、5BS、5DS均存在且为单拷贝,利用这些基因序列与簇毛麦5V染色体序列进行BLASTn比对,鉴定在簇毛麦中存在且为低拷贝的序列开发标记;第二种、第三种和第四种分别是只在5AS、5BS、5DS一个亚基因组上存在的基因,利用这些基因与簇毛麦5V染色体序列进行BLASTn比对,鉴定在簇毛麦中存在且为低拷贝的序列开发标记。以上基因序列和簇毛麦5VS基因序列的比对利用DNAMAN软件分析,在四个基因组中均存在差异的序列用于标记开发。引物设计利用DNAMAN软件进行。
基于Blastn所得到的5VS上的18,544个scaffolds,选择比对到中国春上的位置位于初定位对应的中国春区间内的scaffolds进行标记开发。结果表明,674个基因在5VS、5AS、5BS、5DS中均存在,5AS、5BS、5DS上特异的基因分别有2350、2261、1939个,能够比对到5VS基因组序列的分别有322、316、269个基因。利用上述的674个、322个、316个和269个基因,平均每5Mb选择一个与中国春基因序列有大小差异的基因开发标记,共开发了29对、15对、11对和14对标记,其中分别有24对、14对、9对和12对标记存在多态,多态率依次为82.76%、93.33%、81.82%和85.71%。根据标记检测四个亚基因组(5AS、5BS、5DS和5VS)的类型,将上述59对多态标记分为了4类。这些标记中,5VS与5AS共显性标记有12对,5VS与5DS共显性标记有21对。其中,Xmp1479、Xmp1520是5VS与5AS共显性标记,Xmp1526和Xmp1550是5VS与5DS共显性标记。
实施例2、簇毛麦5VS特异分子标记在以中国春-簇毛麦T5DL.5VS易位系NAU415和中国春-簇毛麦T5AL.5VS易位系NAU421易位系为亲本的BC6F2及BC6F2:3群体中的应用
1、DNA提取
从嫩叶中提取总DNA,–4℃贮存备用。PCR反应在SENSO仪器上进行,反应体系为10μL,混合液中包含0.5U Taq酶(2×Taq Mix,擎科生物科技有限公司),5pM的引物以及20ng基因组DNA。扩增程序为94℃5min;94℃30s,退火温度47-65℃(根据不同引物改变,表1)45s,72℃45s,35个循环;72℃10min,10℃保存。PCR产物在8%非变性的聚丙烯酰胺凝胶电泳分离,硝酸银染色。
2、白粉病抗性鉴定
对2019年种植于江苏省农科院南京试验田的BC6F2群体进行混合菌株接种鉴定,以南农0686作对照,材料种植的两边过道各种一行白粉病诱发材料南农0686。利用南农0686在春化室繁殖白粉菌混合生理小种,并用抖落法向田间诱发行抖落新鲜孢子,待鉴定材料充分发病后,在抽穗期、灌浆期调查白粉病抗性,根据植株叶片上是否有白粉菌孢子分为抗病(R)和感病(S)两种类型。
3、分子标记的验证
利用5VS与5AS共显性分子标记Xmp1479和Xmp1520对T5AL.5VS易位系的BC6F2群体的282个单株进行鉴定,仅扩增出5AS特异条带的单株有72株,同时扩增出5AS和5VS特异条带单株有142株,仅扩增出5VS特异条带的单株有68株,经卡方测验符合1:2:1分离(χ2=0.13,df=3,P=0.94)。利用5VS与5DS共显性分子标记Xmp1526和Xmp1550对T5DL.5VS易位系的BC6F2群体的435个单株进行鉴定(图1),仅扩增出5DS特异条带的单株有110株,同时扩增出5AS和5VS特异条带单株有230株,仅扩增出5VS特异条带的单株有95株,经卡方测验符合1:2:1分离(χ2=2.47,df=3,P=0.29)。白粉病鉴定结果表明,所有扩增出5VS特异条带的单株成株期叶片均未出现白粉菌孢子,为抗病(R)类型。而未扩增出5VS的单株成株期叶片出现大量白粉菌孢子,为感病(S)类型(图2c,d)。群体验证的结果表明,本研究所开发的共显性分子标记可以对5VS染色体臂进行分子标记辅助选择。
在分子标记鉴定的基础上,从T5AL.5VS易位系的BC6F2群体中选择5株仅扩增出5AS特异条带单株和5株仅扩增出5VS特异条带单株,同样从T5DL.5VS易位系的BC6F2群体中选择5株仅扩增出5DS特异条带单株和5株仅扩增出5VS特异条带单株。经GISH/FISH鉴定,未扩增出5VS特异条带的单株均为无易位染色体单株,而仅扩增出5VS特异条带单株分别为纯合的T5AL.5VS易位系和T5DL.5VS易位系(图2a,b)。这20个单株衍生的F2:3家系与其背景亲本南农0686的农艺性状分析结果表明,纯合T5AL.5VS和T5DL.5VS易位系株系的株高较无外源单株和南农0686相比,显著降低大约10.0cm;千粒重也显著降低约1-3g;籽粒硬度降低幅度更大,约50%-80%。所有纯合易位系株系均为软质类型,而无易位染色体株系与其亲本南农0686相同为硬质类型。相比之下,T5AL.5VS易位系较T5DL.5VS易位系降低的幅度更大,二者差异达显著水平。其它农艺性状包括抽穗期、开花期、单株有效穗数和主茎穗长均没有显著差异,但主茎每穗粒数却增加了5-10粒左右(表1)。
序列表
<110> 江苏省农业科学院
<120> 簇毛麦5VS染色体臂特异分子标记引物及应用
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gatggtcagc aactcgct 18
<210> 2
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tcaccacctg cccgtac 17
<210> 3
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ttgttcgcct ccatgaga 18
<210> 4
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gatcttcgcc tgctccct 18
<210> 5
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttcgttacac gctactgc 18
<210> 6
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agattcaggt tgacaccg 18
<210> 7
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gtggcagcgt cagagtaa 18
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ccaatgttgg ttttcatgtg 20
Claims (3)
1.与簇毛麦共分离的COS标记Xmp1520、Xmp1526或Xmp1550的引物在选育抗白粉病小麦品种中的应用,其特征在于,采用所述引物PCR扩增小麦植株DNA,当出现以下情况时,表示该植株中存在簇毛麦5VS染色体臂为抗病植株:
Xmp1520标记在134bp处出现特异性条带;
或Xmp1526标记在223bp处出现特异性条带;
或Xmp1550标记在155bp处出现特异性条带;
与簇毛麦共分离的COS标记Xmp1520的引物序列如下:
左引物:5’-TTGTTCGCCTCCATGAGA-3’;
右引物:5’-GATCTTCGCCTGCTCCCT-3’;
与簇毛麦共分离的COS标记Xmp1526的引物序列如下:
左引物:5’-TTCGTTACACGCTACTGC-3’;
右引物:5’-AGATTCAGGTTGACACCG-3’;
与簇毛麦共分离的COS标记Xmp1550的引物序列如下:
左引物:5’-GTGGCAGCGTCAGAGTAA-3’;
右引物:5’-CCAATGTTGGTTTTCATGTG-3’;
所述小麦为易位系NAU415、易位系NAU421或其杂交后代。
2.根据权利要求1所述的应用,其特征在于,
PCR反应条件为:
PCR反应体系10µl:
内含模板10~20ng、左右引物各2pmol、MgCl2 15nmol、0.1U Taq DNA聚合酶与1×PCR 缓冲液;
反应循环程序如下:94℃预变性3min,然后按“94℃,30s;59.5或54或56℃,40s;72℃,40s”进行35个循环扩增,最后72℃延伸10min;
PCR产物用质量比8%的非变性聚丙烯酰胺胶进行电泳分离,并用银染法显色读取分离条带。
3.一种选育抗白粉病小麦的方法,其特征在于,采用与簇毛麦共分离的COS标记Xmp1520、Xmp1526或Xmp1550的引物PCR扩增小麦植株DNA,筛选符合下列条件的品种进行选育:
Xmp1520标记在134bp处出现特异性条带;
或Xmp1526标记在223bp处出现特异性条带;
或Xmp1550标记在155bp处出现特异性条带;
与簇毛麦共分离的COS标记Xmp1520的引物序列如下:
左引物:5’-TTGTTCGCCTCCATGAGA-3’;
右引物:5’-GATCTTCGCCTGCTCCCT-3’;
与簇毛麦共分离的COS标记Xmp1526的引物序列如下:
左引物:5’-TTCGTTACACGCTACTGC-3’;
右引物:5’-AGATTCAGGTTGACACCG-3’;
与簇毛麦共分离的COS标记Xmp1550的引物序列如下:
左引物:5’-GTGGCAGCGTCAGAGTAA-3’;
右引物:5’-CCAATGTTGGTTTTCATGTG-3’;
所述小麦为易位系NAU415、易位系NAU421或其杂交后代。
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