CN113462804B - 一种snp分子标记及其应用 - Google Patents
一种snp分子标记及其应用 Download PDFInfo
- Publication number
- CN113462804B CN113462804B CN202110895567.XA CN202110895567A CN113462804B CN 113462804 B CN113462804 B CN 113462804B CN 202110895567 A CN202110895567 A CN 202110895567A CN 113462804 B CN113462804 B CN 113462804B
- Authority
- CN
- China
- Prior art keywords
- snp
- barley
- molecular marker
- salt
- snp molecular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000003147 molecular marker Substances 0.000 title claims abstract description 24
- 241000209219 Hordeum Species 0.000 claims abstract description 47
- 235000007340 Hordeum vulgare Nutrition 0.000 claims abstract description 45
- 230000015784 hyperosmotic salinity response Effects 0.000 claims abstract description 22
- 239000012634 fragment Substances 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000009395 breeding Methods 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- 239000011734 sodium Substances 0.000 abstract description 13
- 150000003839 salts Chemical class 0.000 abstract description 12
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 abstract description 10
- 229910052708 sodium Inorganic materials 0.000 abstract description 10
- 210000000349 chromosome Anatomy 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 3
- 230000002596 correlated effect Effects 0.000 abstract description 2
- 238000010219 correlation analysis Methods 0.000 abstract description 2
- 239000000463 material Substances 0.000 description 16
- 230000035882 stress Effects 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 7
- 238000000034 method Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 102000054766 genetic haplotypes Human genes 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 229910001415 sodium ion Inorganic materials 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000012214 genetic breeding Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 235000009566 rice Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 241000219194 Arabidopsis Species 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 230000009418 agronomic effect Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000012098 association analyses Methods 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000012272 crop production Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000002354 inductively-coupled plasma atomic emission spectroscopy Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000008723 osmotic stress Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000000258 photobiological effect Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/172—Haplotypes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Botany (AREA)
- Mycology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种SNP分子标记及其应用,该SNP分子标记的位点位于大麦一号染色体第527797394个碱基处;SNP碱基差异为C或T。本发明通过对大麦盐胁迫下根部相对干物质重和钠含量全基因组关联分析鉴定对耐盐性有提高作用的SNP位点,公开了一个与大麦盐胁迫下根部相对干物重及钠离子含量显著相关联的SNP分子标记,该分子标记检测准确高效、扩增方便稳定,可用于分子标记辅助选择,提高不同耐盐性大麦品种的鉴定效率。
Description
技术领域
本发明涉及分子生物学及遗传育种技术领域,尤其涉及一种SNP分子标记及其应用。
背景技术
土壤盐化是限制全球作物生产的主要逆境胁迫之一,研究作物的耐盐机理并培育作物耐盐新品种对世界粮食安全具有重要的意义。开发与利用耐盐遗传资源并培育耐盐作物新品种,必须明确作物的耐盐机制。盐胁迫对作物造成的伤害主要是渗透胁迫和离子毒害,以及由此产生的次级氧化胁迫,进而造成光合生理损伤、矿质营养代谢失调等,最终导致作物产量降低,品质恶化。
大麦作为古老也是耐盐性最强的农作物之一,具有适应性广、抗逆性强、生育期短、早熟、丰产等特点,又因其营养价值高,兼有食用、饲用和酿造等多种用途,是工业发达国家、干旱半干旱地区发展最迅速的谷物之一。大麦的种植面积和总产量仅次于小麦、水稻和玉米,位居全球第四。
培育耐盐作物是提高盐渍化地区作物产量最有效的途径,探究作物抗盐过程中的限制因素和相关基因是培育耐盐作物的有效途径。植物耐盐性是由微效多基因控制的复杂性状。随着SNP序列和新一代测序技术的快速发展,全基因组关联分析(Genome wideassociation study,GWAS)正成为一种新的、有效的基因检测方法。与传统的QTL定位相比,GWAS可以利用全基因组定位的SNPs作为分子标记来分析复杂性状。此外,GWAS可以处理多达数百万个SNP标记和数千个自然遗传资源作为绘图群体。目前已成功应用于水稻、拟南芥、玉米、小麦、大麦和其他作物重要农艺性状的候选基因挖掘。
GWAS在植物中被广泛使用,因为它能够有效地将基因型与表型联系起来,并利用自然群体同时检测许多自然等位基因变异。因此,通过GWAS开发与大麦耐盐性相关的分子标记并应用于辅助育种和作物改良具有重大的理论意义和实践作用。
发明内容
本发明的目的是提供了一种与大麦耐盐性状连锁的SNP分子标记,同时提供了该SNP分子标记在筛选大麦耐盐性方面的应用,该分子标记S1H_527797394,应用于筛选不同耐盐性的大麦种质材料,同时在大麦遗传育种领域可用于培育具有较强耐盐性的大麦新品种。
具体技术方案如下:
本发明提供了一种与大麦耐盐性状连锁的SNP分子标记,所述SNP分子标记的位点位于大麦一号染色体第527797394个碱基处;SNP碱基差异为C或T。
进一步地,所述SNP分子标记的序列如SEQ ID NO.3所示;
即:ACAGCCCACGTGGCTTTTCGGCCCGCGTCCACTCGCCCGCCGCCAGGTGGGCGGCACCAGC(/T)ACCCGACACCCCTCGCACCTGACCCACCGCACCCACGGCCACTGACACATGG。
进一步地,所述耐盐性状的表征指标为大麦盐胁迫下根部相对干物质重和钠含量。
进一步地,本发明根据上述SNP位点设计了扩增引物,用于扩增所述SNP分子标记的引物对序列,如下所示:
上游引物F为:5’-ACAGCCCACGTGGCTTTTC-3’(SEQ ID NO.1);
下游引物R为:5’-ACGGCCACTGACACATGG-3’(SEQ ID NO.2)。
扩增产物为113bp,序列如SEQ ID NO.3所示,SNP位点位于所述扩增片段的第61bp处。
进一步地,本发明还提供了一种用于扩增所述SNP分子标记的引物对,序列如下所示:
上游引物F为:5’-ACAGCCCACGTGGCTTTTC-3’;
下游引物R为:5’-ACGGCCACTGACACATGG-3’。
利用上述引物对对不用大麦基因型进行PCR扩增并测序分析,可有效筛选不同耐盐性的大麦种质资源,具体为根部高相对干物重,高钠离子含量的大麦材料在该SNP位点上为T类型,而低相对干物重及钠离子含量的大麦材料为C类型。
本发明还提供了所述SNP分子标记以及所述引物对在大麦分子标记辅助育种中的应用。
本发明还提供了所述SNP分子标记以及所述引物对在筛选不同耐盐性的大麦种质资源中的应用。
具体的,所述应用包括以下步骤:
(1)提取大麦植株的基因组DNA;
(2)以步骤(1)所述基因组DNA为模板,利用所述引物对进行PCR扩增反应(使用Vazyme Rapid Taq Master Mix);
(3)对扩增产物进行测序,从而进行基因分型和单倍型分析;
若SNP位点上的碱基为T类型,则该待测大麦植株为高耐盐性材料;若SNP位点上的碱基为C类型,则该待测大麦植株为低耐盐性材料。
与现有技术相比,本发明具有以下有益效果:
本发明通过对大麦盐胁迫下根部相对干物质重和钠含量全基因组关联分析鉴定对耐盐性有提高作用的SNP位点,公开了一个与大麦盐胁迫下根部相对干物重及钠离子含量显著相关联的SNP分子标记S1H_527797394,该分子标记检测准确高效、扩增方便稳定,可用于分子标记辅助选择,提高不同耐盐性大麦品种的鉴定效率。
附图说明
图1为实施例1中100份大麦材料根部相对干物重(a)及钠离子含量(b)GWAS单倍型分析的箱型图。
具体实施方式
下面结合实施例对本发明提供的分子标记和应用进行详细说明,但不能把它们理解为对本发明保护范围的限定。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所做的修改或替换,均属于本发明的范围。
若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
本发明所使用的大麦材料可从浙江大学作物科学研究所得到。本发明所使用生化试剂均为市售。
实施例1
(1)供试材料
利用100份全球大麦微核心种质资源为材料,其中包含34份二棱大麦和66份六棱大麦。
(2)性状测定
用2%H2O2溶液对大麦种子进行消毒,并置于生长室的湿滤纸上发芽。种子发芽7天后,将幼苗移植到含有五分之一的Hoagland溶液(pH 6.0)的15L黑色塑料容器中,水培营养液每3天更换一次。于2019年秋季在浙江大学紫金港校区网室和温室(250μmol·m-2s-1日光灯,23℃14h/18℃10h)进行水培育苗及盐处理试验。
在幼苗成长14天时向水培溶液中加入100mM氯化钠,第二天再添加氯化钠至200mM并持续处理两周。用去离子水冲洗植株后取根部,用吸水纸轻轻晾干,在80℃烘箱中干燥3天后测定根部样品干重。在2ml 69%HNO3中消化干燥的根样品后采用ICP-OES/MS法测定消解液中Na+含量。相对干重=处理干重/对照干重×100%。
(3)GWAS分析及SNP分子标记确定
结合上述测定的大麦根部相对干物质重、Na+含量和该群体的12564个SNP标记,采用TASSEL软件进行GWAS分析,结果显示位于一号染色体上的一个SNP标记与大麦盐胁迫下根部相对干物质重和钠含量显著关联,该SNP位于一号染色体的第527797394个碱基处,可在两个环境中重复检测,-LOG10(P)值均大于4,所述SNP位点为碱基C/T的差异。
(4)单倍型分析
结合SNP标记与100份供试材料的根部相对干物质重、Na+含量表型数据进行单倍型分析,结果如图1所示。其中,SNP分型共分为两个组,深色为C型,浅色为T型,T型基因型大麦根部相对干物质重、Na+含量显著高于C型大麦基因型。
实施例2
(1)供试材料
利用14份全球大麦微核心种质资源为材料,分别进行盐胁迫下根部相对干物质重和钠含量的测定及S1H_527797394目标区域的分析。具体如表1所示,包括7份高相对干物质重和钠含量材料及7份低相对干物质重和钠含量材料。
表1 14份不同耐盐性的大麦种质材料
大麦种质 | SNP检测结果 | Na+含量 | 相对干重 | 指标类型 |
W09 | T | 0.37 | 52.47 | 高 |
W10 | T | 0.67 | 52.29 | 高 |
W30 | T | 0.34 | 52.00 | 高 |
W32 | T | 0.46 | 58.33 | 高 |
W33 | T | 0.47 | 50.77 | 高 |
W41 | T | 0.52 | 49.22 | 高 |
W58 | T | 1.22 | 46.29 | 高 |
W81 | C | 0.18 | 38.90 | 低 |
W15 | C | 0.18 | 43.05 | 低 |
W50 | C | 0.19 | 36.11 | 低 |
W97 | C | 0.22 | 42.00 | 低 |
W55 | C | 0.25 | 37.76 | 低 |
W51 | C | 0.25 | 39.05 | 低 |
W66 | C | 0.29 | 32.70 | 低 |
(2)SNP标记的获得
根据上述SNP位点信息,结合大麦全基因组序列信息,开发SNP标记引物,上游引物F为:5’-ACAGCCCACGTGGCTTTTC-3’;下游引物R为:5’-ACGGCCACTGACACATGG-3’,扩增大小为113bp,所述SNP位于扩增片段的第61bp处,利用上述引物,对大麦一号染色体527797394处碱基的变异进行检测。
(3)DNA提取
以苗期新鲜叶片为材料,采用CTAB法提取DNA,详细步骤如下:
a)在2ml可立管内放入一大两小用75%乙醇清洗的钢珠并加入400μl CTAB提取缓冲液;
b)取大麦嫩叶5g左右放入可立管中,放入全自动磨样机中55HZ磨样1min后置于65℃水浴1h,每隔15min摇匀,水浴后置于室温冷却;
c)加等体积的氯仿:异戊醇(24:1)溶液,并充分摇匀;经10000rpm离心10min,将上清液转入一个新的1.5ml离心管中;
d)加入上清液体积2/3预冷的异丙醇,缓慢上下摇匀30s使异丙醇与水层充分混合,在-20℃下静置20min以沉淀DNA;
e)12000rpm离心4min,弃去上清夜;加入400μl无水乙醇静置20min洗涤DNA;
f)10000rpm离心ddH2O溶解DNA,保存于-20℃待用。
(4)PCR
PCR扩增反应体系为:2×Rapid Taq Master Mix(Vazyme)25μl,10μmol/L PrimerF 1μl,10μmol/L Primer R 1μl,100ng/μl模板DNA 1μl,无菌水22μl,反应总量50μl。
在PCR仪上进行PCR反应,程序如下:95℃预变性3min;95℃变性15s,58℃退火15s,72℃延伸3s,35个循环;72℃延伸5min,12℃保存。
(5)反应结束后将反应产物测序进行基因分型鉴定,结果显示SNP位点变异如表1所示。
以上结果说明我们制备的分子标记可以应用于大麦盐胁迫下根部相对干物质重和钠含量的分子标记辅助选择,以提高选择的准确度。
序列表
<110> 浙江大学
<120> 一种SNP分子标记及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
acagcccacg tggcttttc 19
<210> 2
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
acggccactg acacatgg 18
<210> 3
<211> 113
<212> DNA
<213> 大麦(Hordeum vulgare L.)
<400> 3
acagcccacg tggcttttcg gcccgcgtcc actcgcccgc cgccaggtgg gcggcaccag 60
yacccgacac ccctcgcacc tgacccaccg cacccacggc cactgacaca tgg 113
Claims (2)
1.与大麦耐盐性状连锁的SNP分子标记在大麦耐盐性相关的分子标记辅助育种中的应用,其特征在于,所述SNP分子标记的序列如SEQ ID NO.3所示;SNP碱基差异为C或T,SNP位点位于所述扩增片段的第61bp处。
2.SNP分子标记在筛选不同耐盐性的大麦种质资源中的应用,其特征在于,所述SNP分子标记的序列如SEQ ID NO.3所示;SNP碱基差异为C或T,SNP位点位于所述扩增片段的第61bp处。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110895567.XA CN113462804B (zh) | 2021-08-05 | 2021-08-05 | 一种snp分子标记及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110895567.XA CN113462804B (zh) | 2021-08-05 | 2021-08-05 | 一种snp分子标记及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113462804A CN113462804A (zh) | 2021-10-01 |
CN113462804B true CN113462804B (zh) | 2023-11-28 |
Family
ID=77884067
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110895567.XA Active CN113462804B (zh) | 2021-08-05 | 2021-08-05 | 一种snp分子标记及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113462804B (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111690764A (zh) * | 2020-07-03 | 2020-09-22 | 浙江大学 | 一种与大麦啤酒混浊性状相关的InDel分子标记及其应用 |
CN111979345A (zh) * | 2020-08-07 | 2020-11-24 | 中国科学院遗传与发育生物学研究所 | 盐胁迫条件下小麦生物量和产量相关kasp标记的开发及应用 |
-
2021
- 2021-08-05 CN CN202110895567.XA patent/CN113462804B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111690764A (zh) * | 2020-07-03 | 2020-09-22 | 浙江大学 | 一种与大麦啤酒混浊性状相关的InDel分子标记及其应用 |
CN111979345A (zh) * | 2020-08-07 | 2020-11-24 | 中国科学院遗传与发育生物学研究所 | 盐胁迫条件下小麦生物量和产量相关kasp标记的开发及应用 |
Non-Patent Citations (1)
Title |
---|
vcZ232Y78;Ensembl;Ensembl plants;1 * |
Also Published As
Publication number | Publication date |
---|---|
CN113462804A (zh) | 2021-10-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN103952402B (zh) | 一种与植物根系性状相关的snp位点及其应用 | |
CN106755561B (zh) | 一种与大豆根干重相关的qtl、snp分子标记及应用 | |
Shiri | Identification of informative simple sequence repeat (SSR) markers for drought tolerance in maize | |
Kaewmungkun et al. | Development of new aromatic rice lines with high eating and cooking qualities | |
AL-Fatlawi et al. | Evaluation of seven different wheat cultivars for their resistance to drought in terms of growth indicators and yield | |
CN111479460A (zh) | 具有抗霜霉病花球或叶球的甘蓝植物 | |
CN106755413B (zh) | 水稻氮素吸收利用位点qNUE6及其分子标记方法 | |
CN113462804B (zh) | 一种snp分子标记及其应用 | |
WO2012127558A1 (ja) | 新品種、植物品種の鑑別方法、及びイネ個体を早生化する方法 | |
CN113637791B (zh) | 一种同时鉴定辣椒雄性不育三系杂交种恢复性和真实性的分子标记及其鉴定方法 | |
CN113862387B (zh) | 水稻耐旱性调控基因OsNAC6的分子标记及其应用 | |
CN113462807B (zh) | Snp分子标记及其在提高大麦耐盐性中的应用 | |
CN113462806B (zh) | 与大麦耐盐能力相关的snp分子标记及其应用 | |
Patel et al. | Genetic diversity analysis for salinity tolerance in Indian mustard [Brassica juncea (L.)] using SSR markers | |
CN112662802B (zh) | 小麦根长分子标记及其应用 | |
CN113462805B (zh) | 一种与大麦耐盐性相关的snp分子标记及其应用 | |
Zhang et al. | Breeding of the new Saccharina variety “Sanhai” with high-yield | |
CN111004857B (zh) | 大豆分枝数主效qtl位点的分子标记引物及其应用 | |
CN103667484B (zh) | 油菜品系6f313中含油量性状主效基因位点及应用 | |
CN113528700A (zh) | 检测小麦穗发芽抗性的kasp分子标记试剂盒及检测方法和应用 | |
CN101955988B (zh) | 一种抗白粉病近等基因系Brock/京4117的遗传检测方法 | |
CN111485032A (zh) | 一种鉴定黄瓜雌性系的方法及其使用的snp引物组合 | |
Zhumabaeva et al. | Morphogenetic and Biochemical Study on University Common Bean Collection | |
CN111304359B (zh) | 一种与水稻种子萌发耐盐紧密连锁的分子标记及应用 | |
WANG et al. | Construction of near isogenic lines for pericarp color and evaluation on their near isogenicity in rice |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |