CN113584212B - 用于鉴定木薯薯肉氢氰酸含量的snap引物组及应用 - Google Patents
用于鉴定木薯薯肉氢氰酸含量的snap引物组及应用 Download PDFInfo
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Abstract
本发明涉及生物检测技术领域,公开了用于鉴定木薯薯肉氢氰酸含量的SNAP引物组,包括引物对一和引物对二,所述引物对一包括正向引物一和反向引物一,所述引物对二包括正向引物二和反向引物二;还公开了应用。本发明的应用方法简单,鉴定和筛选过程高效快速,条带准确易读,应用成本低,可大大提高分子标记辅助选择在木薯育种中的实用性及高效性。
Description
技术领域
本发明涉及生物检测技术领域,具体涉及用于鉴定木薯薯肉氢氰酸含量的SNAP引物组及应用。
背景技术
木薯是大戟科木薯属植物,耐旱抗贫瘠,广泛种植于非洲、美洲和亚洲等100余个国家或地区,是世界三大薯类作物之一,热区第三大粮食作物,全球第六大粮食作物,被誉为“淀粉之王”。进入二十一世纪,随着人们生活水平的提高,对物质生活有了更加多样化和功能化的需求,木薯作为一种特色薯类杂粮在美食界崭露头角。然而,由于木薯块根中含有一种叫生氰糖苷(以下简称氰苷)的物质,其会分解释放出有毒物质氢氰酸(HCN),在食用木薯块根时需要十分小心,如有不慎就会中毒,食用前需要用清水浸泡。木薯块根中HCN的含量因品种不同而有较大的差异,新鲜木薯中HCN含量低于50mg/kg时,不用泡水即可食用。因此,选育低HCN食用木薯品种具有重要的意义。
目前,传统木薯育种方法主要集中在资源引进与系统选育方面,包括杂交或实生种选育,周期相对较长,效率低下,而具有领先优势的分子标记辅助育种方法为木薯品种选育或资源筛选提供了一条高效的途径。单核苷酸扩增多态性(single-nucleotideamplified polymorphism:SNAP)标记技术是通过将一条PCR引物的3’末端安排在SNP(single nucleotide polymorphism)位点上,并引入错配的碱基,另一条PCR引物按照传统方法设计,可通过标准的琼脂糖凝胶电泳可以明确判定PCR扩增条带的“有”和“无”,来判断相关性状。该方法操作简单,简易高效,因此,迫切需要开发出适用于木薯薯肉氢氰酸含量的SNAP分子标记,提高低HCN木薯选育种的效率,降低木薯品种选育或资源筛选过程的繁琐度及成本。
发明内容
基于以上问题,本发明提供用于鉴定木薯薯肉氢氰酸含量的SNAP引物组及应用,本发明简单、高效快速、应用成本低,可简易快速实现利用分子标记检测木薯薯肉HCN含量的目的,大大提高分子标记辅助选择在木薯育种中的实用性及高效性。
为解决以上技术问题,本发明提供了用于鉴定木薯薯肉氢氰酸含量的SNAP引物组,包括引物对一和引物对二,所述引物对一包括正向引物一和反向引物一,所述引物对二包括正向引物二和反向引物二,所述正向引物一和正向引物二的核苷酸序列见SEQ IDNO.1,所述反向引物一和反向引物二的核苷酸序列分别见SEQ ID NO.2和SEQ I DNO.3。
为解决以上技术问题,本发明还提供了引物组在制备用于鉴定或筛选木薯种质资源的试剂盒中的应用。
进一步的,所述试剂盒可用于PCR扩增检测,PCR扩增反应体系为50μL体系,具体如下:浓度为50ng/μL的模板DNA 2μL,2×Taq Master Mix 25μL,浓度为10μmol/L的正向引物1μL,浓度为10μmol/L的反向引物1μL,ddH2O 21μL。
进一步的,PCR扩增程序如下:在95℃条件下预变性3min,95℃变性30s,60.5℃退火30s,72℃延伸30s,进行32个循环,72℃延伸5min,4℃保存。
与现有技术相比,本发明的有益效果是:本发明的应用方法简单,鉴定和筛选过程高效快速,条带准确易读,应用成本低,可大大提高分子标记辅助选择在木薯育种中的实用性及高效性。
附图说明
图1为本发明的实施例的鉴定电泳结果图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,下面结合实施例和附图,对本发明作进一步的详细说明,本发明的示意性实施方式及其说明仅用于解释本发明,并不作为对本发明的限定。
实施例:
本实施例在广西农业科学院建基地,选取主栽木薯品种“新选048(Xinxuan048)”及其自交所得的45份木薯,以及6个木薯品种的叶片,合计共52份材料,采用CTAB法从木薯叶片中提取基因组DNA。
发明人开发出两对等位基因特异性单核苷酸扩增多态性(SNAP)引物,并通过分子标记方法来区分低HCN木薯与高HCN木薯,准确性可达92.3%。将上述引物组应用于食用木薯种质鉴定和筛选,可为拓宽木薯选育途径提供重要信息,还可以丰富木薯种质资源,选育食用木薯优良品种。
本实施例开发的两对引物包括引物对一和引物对二,所述引物对一包括正向引物一和反向引物一,所述引物对二包括正向引物二和反向引物二,所述正向引物一和正向引物二的核苷酸序列见SEQ ID NO.1,所述反向引物一和反向引物二的核苷酸序列分别见SEQID NO.2和SEQ ID NO.3,具体如下:
正向引物一:GCATGACGATTTCCGGATGG,
反向引物一:CGGAGACTGCTTTGGAAACTGT;
正向引物二:GCATGACGATTTCCGGATGG,
反向引物二:CGGAGACTGCTTTGGAAACTGC。
可利用本实施例的引物对一和引物对二分别对木薯基因组DNA进行PCR扩增,从而得到PCR扩增产物,并进行电泳检测。PCR扩增反应体系为50μL体系,具体如下:浓度为50ng/μL的模板DNA 2μL,2×Taq Master Mix 25μL,浓度为10μmol/L的正向引物1μL,浓度为10μmol/L的反向引物1μL,ddH2O 21μL。PCR扩增程序如下:在95℃条件下预变性3min,95℃变性30s,60.5℃退火30s,72℃延伸30s,进行32个循环,72℃延伸5min,4℃保存,得到PCR扩增产物。
使用浓度为1.5%的琼脂糖凝胶电泳对上述PCR产物进行电泳分离,电泳方法为:取6μL PCR产物与1μL 6×Loading buffer混匀后,用含有GelRed核酸染色剂的1.5%琼脂糖凝胶进行电泳分离,缓冲液为1×TAE,在110V恒电压下电泳约25min,电泳结束后,利用紫外凝胶成像系统成像后拍照,根据电泳结果进行木薯块根肉质HCN含量的判定,判定标准如下:
若引物对1能扩增出355bp的条带,则该木薯材料的薯肉为纯合的低HCN;
若引物对2能扩增出355bp的条带,则该木薯材料的薯肉为纯合的高HCN;
若引物对1和引物对2均能扩增出355bp的条带,则该木薯材料的薯肉为杂合型高HCN。
本实施例对上述材料的鉴定电泳结果见附图1,其中M为DL2000,1-28为新选048自交系后代。本实施例的52份材料的相关情况如下表:
结果显示:低HCN含量的有24个材料,分别为白3、白9、白14、白16、黄5、黄8、黄18、浅黄2、浅黄9、浅黄20、浅黄21、浅黄24、浅黄25、浅黄37、浅黄51、黄1、白1、黄9、浅黄8、浅黄42、P2、沙田、GR891和文昌红心,且24份材料检测的HCN含量均在50mg/kg以下,与基因型一致,表明本实施例的引物组的检测结果准确。
高HCN含量的材料有28个,其中,基因型纯合的有11份,分别为白10、黄23、浅黄17、浅黄56、白20、黄10、黄12、黄13、浅黄18、浅黄35和浅黄50,其中,除浅黄17和黄13的HCN含量在20~50mg/kg外,其他材料的HCN含量均在50mg/kg以上,与基因型一致;基因型杂合的有17份,分别为Xinxuan048、白5、白19、黄20、黄21、黄22、浅黄40、浅黄61、浅黄63、黄32、黄37、浅黄36、浅黄38、浅黄46、浅黄48、M5和M64,HCN含量除黄20和黄21在30~50mg/kg外,其他材料的HCN含量均大于50mg/kg,与基因型一致。
综上,52份材料中,基因型与检测的HCN含量一致的材料有48份,分子标记检测的准确性高达92.3%,表明本实施例的引物组的检测结果准确。
本实施例可仅通过简单的PCR扩增及琼脂糖凝胶电泳检测即实现在木薯苗期通过提取木薯叶片DNA进行分子标记检测,从而快速的鉴别木薯薯肉HCN含量,准确的选育出适合于育种目标的木薯材料;同时凝胶电泳的条带易读,整套操作价格低廉,大大提高了分子标记辅助选择在木薯育种中的实用性及高效性,本实施例的引物组可用于制备用于鉴定或筛选木薯种质资源的试剂盒中。
如上即为本发明的实施例。上述实施例以及实施例中的具体参数仅是为了清楚表述发明验证过程,并非用以限制本发明的专利保护范围,本发明的专利保护范围仍然以其权利要求书为准,凡是运用本发明的说明书及附图内容所作的等同结构变化,同理均应包含在本发明的保护范围内。
序列表
<110> 广西壮族自治区农业科学院
<120> 用于鉴定木薯薯肉氢氰酸含量的SNAP引物组及应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gcatgacgat ttccggatgg 20
<210> 2
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cggagactgc tttggaaact gt 22
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cggagactgc tttggaaact gc 22
Claims (4)
1.用于鉴定木薯薯肉氢氰酸含量的SNAP引物组,其特征在于,包括引物对一和引物对二,所述引物对一包括正向引物一和反向引物一,所述引物对二包括正向引物二和反向引物二,所述正向引物一和正向引物二的核苷酸序列见SEQ ID NO.1,所述反向引物一和反向引物二的核苷酸序列分别见SEQ ID NO.2和SEQ ID NO.3。
2.权利要求1所述的引物组在制备用于鉴定或筛选木薯种质资源的试剂盒中的应用。
3.根据权利要求2所述的应用,其特征在于,所述试剂盒可用于PCR扩增检测,PCR扩增反应体系为50μL体系,具体如下:浓度为50ng/μL的模板DNA 2μL,2×Taq Master Mix 25μL,浓度为10μmol/L的正向引物1μL,浓度为10μmol/L的反向引物1μL,ddH2O 21μL。
4.根据权利要求3所述的应用,其特征在于,PCR扩增程序如下:在95℃条件下预变性3min,95℃变性30s,60.5℃退火30s,72℃延伸30s,进行32个循环,72℃延伸5min,4℃保存。
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