CN115044697B - Molecular marker related to accumulation of DON toxin in wheat grains and primer and application thereof - Google Patents
Molecular marker related to accumulation of DON toxin in wheat grains and primer and application thereof Download PDFInfo
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Abstract
本发明公开了一种与小麦籽粒DON毒素积累相关的分子标记及其应用,该分子标记是以普通小麦基因组DNA为模板,核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示的引物对进行PCR扩增,在1%的琼脂糖凝胶上电泳后获得结果,扩增产物大小为214bp;该分子标记扩增时间短、效率高,可以应用于小麦群体的基因型分析和抗籽粒毒素积累位点的遗传作图。
The invention discloses a molecular marker related to the accumulation of DON toxin in wheat grains and its application. The molecular marker is based on common wheat genome DNA as a template, and the nucleotide sequences are respectively shown in SEQ ID NO.1 and SEQ ID NO.2. PCR amplification with the primer pair shown, and the results were obtained after electrophoresis on 1% agarose gel. The size of the amplified product was 214bp; the molecular marker amplification time was short and the efficiency was high, and it could be applied to the genotype analysis of wheat populations and genetic mapping of loci for resistance to grain toxin accumulation.
Description
技术领域technical field
本发明属于小麦遗传育种和分子生物学领域,特别是一种与小麦籽粒毒素积累相关的分子标记及其应用。The invention belongs to the field of wheat genetic breeding and molecular biology, in particular to a molecular marker related to wheat grain toxin accumulation and its application.
背景技术Background technique
小麦赤霉病是由禾谷镰刀菌(Fusarium graminearum)引起的重要真菌病害,严重影响小麦的产量和品质。在一般流行年份,赤霉病可引起5-10%的产量损失,大流行年份可导致部分田块绝收,危害小麦粮食安全。禾谷镰刀菌分泌的真菌毒素脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)是一种细胞毒素,被列为3类致癌物,可引起免疫抑制或免疫刺激反应。DON毒素在小麦籽粒中的积累对人类及牲畜的的健康构成了威胁,食用被DON毒素的食物后会出现厌食、呕吐和腹泻等急性中毒症状,严重时可导致循环系统损伤而死亡。根据食品安全国家标准GB2761-2017中对食品中真菌毒素的限量要求,谷物及其制品如玉米(面、渣)、大麦、小麦、麦片和小麦粉中的DON毒素含量不得超过1mg/kg。Wheat scab is an important fungal disease caused by Fusarium graminearum, which seriously affects the yield and quality of wheat. In general epidemic years, head blight can cause 5-10% yield loss, and in pandemic years, some fields may fail to harvest, endangering wheat food security. The mycotoxin deoxynivalenol (DON) secreted by Fusarium graminearum is a cytotoxin, which is listed as a
小麦赤霉病抗性可以分为5种类型:Ⅰ,抗侵染;Ⅱ,抗扩展;Ⅲ,抗籽粒毒素积累;Ⅳ,抗籽粒侵染;Ⅴ,耐病性(Mesterházy 1995;Mesterházy et al.1999)。耐毒素积累抗性特指毒素在小麦籽粒内被降解或产毒被抑制造成的毒素积累减少,具有不同于其他抗性类型的遗传机制。Szabó-Hevér等在小麦“Frontana”3A,4B,7A和7B染色体上检测到抗籽粒毒素积累位点,解释3.1-11.1%的表型变异(Szabó-Hevér et al.,2014)。通过遗传作图,在7A染色体上检测到一个稳定的QTL位点,可以解释16-24%的表型变异(He et al.,2019)。在挪威小麦品种“NK93604”的2AS染色体上检测到一个与抗籽粒毒素积累位点,可以解释26.7%的表型变异,但只在一个环境中可以被检出(Semagn et al.,2007)。此外,在“望水白”的1A,1BL,3BS,5AS,5DL和7A染色体(Yu et al.2008),“Ernie”的4DS和6AL染色体(Liuet al.,2013),“Wuhan-1”的2DS和5AS染色体(Somers et al.,2003),“SHA3/CBRD”的1A,2A,2D,3AS,5BL,5A和6A染色体(Lu et al.,2012)上也检测出微效的QTL位点。利用全基因组关联分析在美国和国际小麦和玉米改良中心(CIMMYT)群体里也检测出与耐毒素积累的位点,这些位点分布在3B,1D,2B,3A,2BL染色体上,解释3-12%的表型变异(Arruda etal.,2016;Wang et al.,2017)。上述位点的标记的开发基于单核苷酸多态性或简单重复序列多态性,难以直接利用,或需要繁琐的分型步骤,不适合大规模的种质筛选或分子标记辅助选择。Wheat head blight resistance can be divided into five types: Ⅰ, resistance to infection; Ⅱ, resistance to expansion; Ⅲ, resistance to grain toxin accumulation; Ⅳ, resistance to kernel infection; Ⅴ, disease tolerance (Mesterházy 1995; Mesterházy et al. 1999). Toxin accumulation resistance specifically refers to the reduction of toxin accumulation caused by the degradation of toxins in wheat grains or the inhibition of toxin production, which has a genetic mechanism different from other types of resistance. Szabó-Hevér et al. detected resistance to grain toxin accumulation loci on chromosomes 3A, 4B, 7A and 7B of wheat "Frontana", explaining 3.1-11.1% of the phenotypic variation (Szabó-Hevér et al., 2014). Through genetic mapping, a stable QTL locus was detected on chromosome 7A, which could explain 16-24% of the phenotypic variation (He et al., 2019). A locus associated with resistance to grain toxin accumulation was detected on the 2AS chromosome of the Norwegian wheat variety "NK93604", which could explain 26.7% of the phenotypic variation, but it was detected in only one environment (Semagn et al., 2007). In addition, in "Wangshuibai" chromosomes 1A, 1BL, 3BS, 5AS, 5DL and 7A (Yu et al.2008), "Ernie" chromosomes 4DS and 6AL (Liu et al., 2013), "Wuhan-1" The 2DS and 5AS chromosomes (Somers et al., 2003), and the 1A, 2A, 2D, 3AS, 5BL, 5A and 6A chromosomes of "SHA3/CBRD" (Lu et al., 2012) have also detected minor QTLs site. Loci associated with toxin-resistant accumulation were also detected in the American and International Wheat and Maize Improvement Center (CIMMYT) populations using genome-wide association analysis. These loci are distributed on chromosomes 3B, 1D, 2B, 3A, and 2BL, explaining that 12% phenotypic variation (Arruda et al., 2016; Wang et al., 2017). The development of markers for the above loci is based on single nucleotide polymorphisms or simple repeat sequence polymorphisms, which are difficult to use directly, or require cumbersome typing steps, and are not suitable for large-scale germplasm screening or molecular marker-assisted selection.
此外,利用小麦籽粒毒素积累相关的分子标记对遗传群体进行分型,在绘制遗传图谱的基础上,精细定位中国小麦品种“苏麦3号”的抗籽粒毒素积累位点是小麦抗赤霉病遗传改良的重要步骤。常用的高通量分型方法基于基因组单核苷酸多态性,分型芯片的设计和制造依赖于进口,分型成本较高,难以形成大规模的研发定位。In addition, the molecular markers related to wheat grain toxin accumulation were used to type the genetic population. On the basis of drawing the genetic map, the locus of grain toxin resistance accumulation in the Chinese wheat variety "Sumai No. An important step in genetic improvement. Commonly used high-throughput typing methods are based on genomic single nucleotide polymorphisms. The design and manufacture of typing chips rely on imports, and the cost of typing is high, making it difficult to form large-scale R&D positioning.
发明内容Contents of the invention
针对上述问题,本发明通过全基因组关联分析和物理位置比对获得了一个新的与小麦籽粒毒素积累相关的优异等位变异位点(位于7B染色体上644438701bp,参考序列为中国春v1.0),并设计了基于聚合酶链式反应的分子标记JAASM3424。此分子标记可以分析不同小麦品种在此位点的基因型与小麦籽粒DON毒素积累的相关性,并用于遗传图谱的绘制。In response to the above problems, the present invention obtained a new excellent allelic variation site related to wheat grain toxin accumulation (located at 644438701bp on chromosome 7B, and the reference sequence is Chinese spring v1.0) through genome-wide association analysis and physical position comparison , and designed a polymerase chain reaction-based molecular marker JAASM3424. This molecular marker can be used to analyze the correlation between the genotypes of different wheat varieties at this locus and the accumulation of DON toxin in wheat grains, and to draw genetic maps.
具体而言,本发明是这样实现的:Specifically, the present invention is realized like this:
首先,本申请提供了一种与小麦籽粒DON毒素积累相关的分子标记JAASM3424,该分子标记是以小麦品种安农8455的DNA为模板,以核苷酸序列分别如SEQ ID NO.1和SEQ IDNO.2所示的序列为引物进行PCR扩增,扩增产物在质量百分数为1%的琼脂糖凝胶上电泳后,获得的大小为214bp的DNA片段。First of all, this application provides a molecular marker JAASM3424 related to the accumulation of DON toxin in wheat grains. The molecular marker is based on the DNA of wheat variety Anong 8455 as a template, and the nucleotide sequences are respectively such as SEQ ID NO.1 and SEQ ID NO. .2 The sequence shown in 2 is a primer for PCR amplification, and the amplified product is electrophoresed on an agarose gel with a mass percentage of 1%, and a DNA fragment with a size of 214bp is obtained.
上述PCR扩增体系:10×buffer 1μl,浓度为25mM的MgCl2溶液0.5μl,浓度为2.5mM的dNTP溶液0.5μl,浓度为10μM的引物I 0.5μl,浓度为10μM的引物II 0.5μl,浓度为5U/μl的Taq聚合酶0.2μl,模板DNA 50ng,ddH2O补足至10μl;所述引物I的核苷酸序列如SEQ IDNO.1所示,所述引物II的核苷酸序列如SEQ ID NO.2所示;The above PCR amplification system: 1 μl of 10×buffer, 0.5 μl of MgCl 2 solution with a concentration of 25 mM, 0.5 μl of dNTP solution with a concentration of 2.5 mM, 0.5 μl of primer I with a concentration of 10 μM, 0.5 μl of primer II with a concentration of 10 μM, Taq polymerase 0.2 μl of 5U/μl, template DNA 50ng, ddH 2 O supplemented to 10 μl; the nucleotide sequence of the primer I is shown in SEQ ID NO.1, and the nucleotide sequence of the primer II is shown in SEQ ID NO.1 As shown in ID NO.2;
PCR扩增程序:94℃3分钟;94℃10秒,60℃30秒,72℃10秒延伸,30个循环;72℃延伸5分钟。PCR amplification program: 94°C for 3 minutes; 94°C for 10 seconds, 60°C for 30 seconds, 72°C for 10 seconds, 30 cycles; 72°C for 5 minutes.
其次,本申请提供了上述分子标记JAASM3424在筛选低DON毒素积累量的小麦种质中的应用。其具体应用步骤如下:以样品小麦DNA为模板,以核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2为引物进行PCR扩增,扩增产物在质量百分数为1%的琼脂糖凝胶上电泳,若电泳产物存在大小为214bp的DNA片段,则判定该样品含有分子标记JAASM3424;反之,则不含分子标记JAASM3424;认为含有分子标记JAASM3424的小麦品种DON毒素积累量高于不含该分子标记JAASM3424的小麦品种。Secondly, the present application provides the application of the above-mentioned molecular marker JAASM3424 in screening wheat germplasm with low accumulation of DON toxin. Its specific application steps are as follows: use the sample wheat DNA as a template, and use nucleotide sequences such as SEQ ID NO.1 and SEQ ID NO.2 as primers to carry out PCR amplification, and the amplified products are prepared in agarose with a mass percentage of 1%. Electrophoresis on the gel, if there is a DNA fragment with a size of 214bp in the electrophoresis product, it is determined that the sample contains the molecular marker JAASM3424; The molecular marker JAASM3424 of the wheat variety.
进一步而言,上述“小麦”包括但不限于望水白、苏麦3号、宁7840、宁麦8号、小偃22、宁98081、济麦20、皖麦44、安农8455、BORLAUG100 F2014、GONDO、IOC 813、ITAPUA 45-DON PANI、KLEIN DON ENRIQUE、LAS ROSAS INTA、LAUREANO ALVAREZ、MARINGA、ONCATIVO-INTA、PERGAMINO GABOTO、PRINIA、PROINTAFEDERAL、PROINTAGRANAR、PROINTAOASIS、QUAIU#1、QUELEA、SABUF、SPINEBILL、THORNBIRD、VALK、VL 616、WAXBILL、济南17、莱州95021、连9791-4、绵2000-1-37、南农9918、宁丰518、宁麦10、宁麦11、宁麦12、宁麦15、宁麦17、宁麦19、宁麦21、宁麦22、宁麦9号、山农9625、生抗1、生抗2、生选3、生选4等国内外小麦品种/品系及其衍生品种/品系;所述衍生品种指通过杂交、回交、自然或人工突变获得的含有上述小麦品种/系遗传物质的小麦品种/品系。Further, the above-mentioned "wheat" includes but not limited to Wangshuibai, Sumai 3, Ning 7840, Ningmai 8, Xiaoyan 22, Ning 98081, Jimai 20, Wanmai 44, Annong 8455, BORLAUG100 F2014 , GONDO, IOC 813, ITAPUA 45-DON PANI, KLEIN DON ENRIQUE, LAS ROSAS INTA, LAUREANO ALVAREZ, MARINGA, ONCATIVO-INTA, PERGAMINO GABOTO, PRINIA, PROINTAFEDERAL, PROINTAGRANAR, PROINTAOASIS, QUAIU#1, QUELEA, SABUF, SPINEBILL THORNBIRD, VALK, VL 616, WAXBILL, Jinan 17, Laizhou 95021, Lian 9791-4, Mian 2000-1-37, Nannong 9918, Ningfeng 518, Ningmai 10, Ningmai 11, Ningmai 12, Ningmai 15 , Ningmai 17, Ningmai 19, Ningmai 21, Ningmai 22, Ningmai 9, Shannong 9625, Shengkang 1, Shengkang 2, Shengxuan 3, Shengxuan 4 and other domestic and foreign wheat varieties/lines and their Derived varieties/lines; said derived varieties refer to wheat varieties/lines containing the genetic material of the above-mentioned wheat varieties/lines obtained by crossing, backcrossing, natural or artificial mutation.
上述PCR扩增体系:10×buffer 1μl,MgCl2溶液(浓度为25mM)0.5μl,dNTP溶液(浓度为2.5mM)0.5μl,引物SEQ ID NO.1(浓度为10μM)0.5μl,引物SEQ ID NO.2(10μM)0.5μl,Taq聚合酶(浓度为5U/μl)0.2μl,模板DNA 50ng,ddH2O补足至10μl;PCR扩增程序:94℃3分钟;94℃10秒,60℃30秒,72℃10秒延伸,30个循环;72℃延伸5分钟。The above PCR amplification system: 10×
第三,本申请提供了一对用于预测小麦籽粒DON毒素积累量的引物对,该引物对的核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示。Thirdly, the present application provides a pair of primers for predicting the accumulation of DON toxin in wheat grains. The nucleotide sequences of the primers are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.
第四,本发明还提供了上述分子标记JAASM3424在绘制小麦籽粒毒素积累位点的遗传图谱中的应用,其具体步骤为:以苏麦3号和安农8455的遗传群体DNA为模板,SEQ IDNO.1和SEQ ID NO.2为引物进行PCR扩增,扩增产物在质量百分数为1%的琼脂糖凝胶上电泳后,若存在大小为214bp的DNA片段,则基因型记为“0”;若不存在上述扩增片段,则基因型记为“2”。收集基因型数据后,采用QTL Icimapping软件绘制遗传图谱。Fourth, the present invention also provides the application of the above-mentioned molecular marker JAASM3424 in drawing the genetic map of wheat grain toxin accumulation sites. The specific steps are: using the genetic population DNA of Sumai 3 and Anong 8455 as templates, SEQ ID NO .1 and SEQ ID NO.2 are primers for PCR amplification. After the amplified product is electrophoresed on an agarose gel with a mass percentage of 1%, if there is a DNA fragment with a size of 214bp, the genotype is recorded as "0". ; If there is no above-mentioned amplified fragment, the genotype is recorded as "2". After genotype data were collected, genetic maps were drawn using QTL Icimapping software.
本申请通过锚定基因组中的单核苷酸多态性变异,设计位点特异性的基于聚合酶链式反应的分子标记,可以降低分型成本,简化分型步骤,提高分子标记辅助选择的效率。与现有技术相比,本发明中的分子标记引物对通过人工比对获得,人为调整引物末端位置锚定变异位点保证了扩增产物的准确性。本发明中的分子标记引物人为引入错配碱基,保证了对靶标序列的特异性扩增。本发明中的分子标记将用于小麦籽粒毒素积累位点的遗传作图,为鉴定相关基因/QTL和连锁标记奠定了基础。本发明中所获得引物对扩增结果特异性好,扩增所需时间短,基因型结果判读准确。This application anchors the single nucleotide polymorphism variation in the genome and designs site-specific molecular markers based on polymerase chain reaction, which can reduce typing costs, simplify typing steps, and improve the efficiency of molecular marker-assisted selection. efficiency. Compared with the prior art, the molecular marker primer pair in the present invention is obtained through manual comparison, and the position of the end of the primer is artificially adjusted to anchor the variation site to ensure the accuracy of the amplification product. The molecular marker primers in the present invention artificially introduce mismatched bases to ensure the specific amplification of the target sequence. The molecular markers in the present invention will be used for genetic mapping of toxin accumulation sites in wheat grains, laying a foundation for identifying related genes/QTLs and linkage markers. The primers obtained in the present invention have good specificity for the amplification result, short time required for amplification, and accurate interpretation of the genotype result.
附图说明Description of drawings
图1为软件设计分子标记P1和P2引物对在不同品种(系)中的扩增结果;Fig. 1 is the amplification result of software design molecular marker P1 and P2 primer pair in different varieties (lines);
其中,M:分子量标准DL2000,1-10泳道分别为小麦品种(系)宁894037、望水白、苏麦3号、宁7840、宁麦8号、小偃22、宁98081、济麦20、皖麦44和安农8455。Among them, M: molecular weight standard DL2000, lanes 1-10 are wheat varieties (lines) Ning 894037, Wangshuibai, Sumai 3, Ning 7840, Ningmai 8, Xiaoyan 22, Ning 98081, Jimai 20, Wanmai 44 and Annong 8455.
图2为分子标记JAASM3424的引物对在不同品种(系)中的扩增结果;Fig. 2 is the amplification result of the primer pair of molecular marker JAASM3424 in different varieties (lines);
其中,M:分子量标准DL2000,1-10泳道分别为小麦品种(系)宁894037、望水白、苏麦3号、宁7840、宁麦8号、小偃22、宁98081、济麦20、皖麦44和安农8455。Among them, M: molecular weight standard DL2000, lanes 1-10 are wheat varieties (lines) Ning 894037, Wangshuibai, Sumai 3, Ning 7840, Ningmai 8, Xiaoyan 22, Ning 98081, Jimai 20, Wanmai 44 and Annong 8455.
图3利用分子标记JAASM3424小麦籽粒毒素积累的优异等位变异进行分型结果示意图;Figure 3 is a schematic diagram of the typing results using molecular marker JAASM3424 wheat grain toxin accumulation excellent allelic variation;
其中,基因型0代表等位变异“C”,基因型2代表等位变异“T”。Among them,
图4分子标记JAASM3424在苏麦3号×安农8455的F2:7重组自交系群中的分型结果示意图;Figure 4 Schematic diagram of the typing results of the molecular marker JAASM3424 in the F 2:7 recombinant inbred line group of Sumai 3×Annong 8455;
其中,M:分子量标准DL2000,1-185泳道为遗传群体重组自交系编号,S为苏麦3号,A为安农8455。Among them, M: molecular weight standard DL2000, lane 1-185 is the number of recombinant inbred line of genetic population, S is Sumai 3, and A is Annong 8455.
图5利用分子标记JAASM3424绘制遗传图谱。Figure 5. Genetic mapping using the molecular marker JAASM3424.
具体实施方式Detailed ways
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
下述实施例涉及的核苷酸序列:Nucleotide sequences involved in the following examples:
SEQ ID NO.1:GAGAATGATGAAGTGCATACATGAGC;SEQ ID NO. 1: GAGAATGATGAAGTGCATACATGAGC;
SEQ ID NO.2:GAGGAAATTTTTGTCTGCAGTCTGC;SEQ ID NO. 2: GAGGAAATTTTTGTCTGCAGTCTGC;
SEQ ID NO.3:ATAGACTAAACCTCTTGGGAAGC;SEQ ID NO. 3: ATAGACTAAACCTCTTGGGAAGC;
SEQ ID NO.4:TTTTGTTCTAAGTAATGGATGGC;SEQ ID NO. 4: TTTTGTTCTAAGTAATGGATGGC;
SEQ ID NO.5:GTATGGCGACTAGGAATACAAAA;SEQ ID NO.5: GTATGGCGACTAGGAATACAAAA;
SEQ ID NO.6:AGAATGCTAAACTTAGGAACGGA。SEQ ID NO. 6: AGAATGCTAAACTTAGGAACGGA.
实施例涉及的材料来源:The source of material that embodiment relates to:
以下实施例涉及的苏麦3号等小麦品种(系):为常规品种(系),由江苏省农业科学院粮食作物研究所小麦遗传育种团队保藏;The wheat varieties (lines) such as Sumai No. 3 involved in the following examples: are conventional varieties (lines), preserved by the wheat genetics and breeding team of the Institute of Grain Crops, Jiangsu Academy of Agricultural Sciences;
赤霉菌:为亚洲镰刀菌Fa0609,由江苏省农业科学院粮食作物研究所小麦遗传育种团队保藏。Gibberella: Fusarium asiatica Fa0609, preserved by the wheat genetics and breeding team of the Institute of Food Crops, Jiangsu Academy of Agricultural Sciences.
实施例1分子标记JAASM3424在不同小麦品种(系)中的验证Example 1 Verification of molecular marker JAASM3424 in different wheat varieties (lines)
本实施例里PCR扩增的DNA模板来自以下小麦品种(系):宁894037、望水白、苏麦3号、宁7840、宁麦8号,其优异等位变异为碱基“T”,且籽粒DON毒素含量较低,分别为:1.76mg/kg、2.29mg/kg、2.38mg/kg、2.63mg/kg和2.85mg/kg;小偃22、宁98081、济麦20、皖麦44、安农8455,其优异等位变异为碱基“C”,且籽粒DON毒素含量较高,分别为:25.80mg/kg、2.83mg/kg、28.7mg/kg、29.3mg/kg和29.8mg/kg。上述小麦品种均种植于江苏农科院粮食作物研究所小麦遗传团队赤霉病鉴定圃(东经118.88°,北纬32.03°),适期播种,行长1米,播种量为40粒。在花期接种赤霉病菌Fa0609(浓度为105个分生孢子/μl)后,收获10个病穗,以测量籽粒DON毒素含量。籽粒DON毒素含量采用酶联免疫反应快速检测试剂盒(北京华安麦科,HEM1896-2)检测。利用CTAB法提取幼嫩叶片基因组DNA(Murray and Thompson,1980),并采用微量分光光度计Nanodrop对基因组DNA进行定量。The DNA templates amplified by PCR in this example come from the following wheat varieties (lines): Ning 894037, Wangshuibai, Sumai No. 3, Ning 7840, Ning Mai No. 8, and their excellent allelic variation is the base "T", And the content of DON toxin in the grain is low, respectively: 1.76mg/kg, 2.29mg/kg, 2.38mg/kg, 2.63mg/kg and 2.85mg/kg;
通过软件Primer Premier5.0对含有变异位点(7B染色体上644438701bp,参考序列为中国春v1.0)的基因组序列进行分子标记引物设计时,参数为:扩增大小200-300bp,退火温度55-65℃,其他参数为默认,不进行人为修改,并选取得分最高的两对引物进行试验。采用软件设计的引物对P1(其核苷酸序列如SEQ ID NO.3和SEQ ID NO.4所示)和P2((其核苷酸序列如SEQ ID NO.5和SEQ ID NO.6所示)以上述小麦品种(系)为模板进行PCR扩增:When using the software Primer Premier5.0 to design molecular marker primers for genomic sequences containing variant sites (644438701bp on chromosome 7B, the reference sequence is Chinese spring v1.0), the parameters are: amplification size 200-300bp, annealing temperature 55- 65°C, other parameters are the default, no artificial modification is performed, and the two pairs of primers with the highest scores are selected for the test. The primer pair P1 (its nucleotide sequence is shown in SEQ ID NO.3 and SEQ ID NO.4) and P2 ((its nucleotide sequence is shown in SEQ ID NO.5 and SEQ ID NO.6) using software design Show) carry out PCR amplification with above-mentioned wheat variety (line) as template:
PCR反应体系为10μl:10×buffer 1μl,MgCl2溶液(浓度为25mM)0.5μl,dNTP溶液(浓度为2.5mM)0.5μl,引物SEQ ID NO.1(浓度为10μM)0.5μl,引物SEQ ID NO.2(10μM)0.5μl,浓度为的Taq聚合酶(浓度为5U/μl)0.2μl,模板DNA 50ng,ddH2O补足至10μl;The PCR reaction system is 10 μl: 10×
PCR扩增程序:94℃3分钟;94℃10秒,60℃30秒,72℃10秒延伸,30个循环;72℃延伸5分钟。PCR amplification program: 94°C for 3 minutes; 94°C for 10 seconds, 60°C for 30 seconds, 72°C for 10 seconds, 30 cycles; 72°C for 5 minutes.
扩增产物用1.0%琼脂糖凝胶电泳检测,电泳结果如图1所示,P1、P2分别为软件设计引物对P1和P2的扩增结果,可见这两个引物对无法对优异等位变异进行有效扩增,所有模板均出现非特异扩增,扩增的特异性和有效性有待提高。The amplified products were detected by 1.0% agarose gel electrophoresis. The electrophoresis results are shown in Figure 1. P1 and P2 are the amplification results of the software-designed primer pairs P1 and P2 respectively. It can be seen that these two primer pairs cannot detect the excellent allelic variation. For effective amplification, non-specific amplification occurs in all templates, and the specificity and effectiveness of amplification need to be improved.
申请人在此基础上,在对变异位点(7B染色体上644438701bp,参考序列为中国春v1.0)进行人工比对的基础上,人为调整引物对末端的锚定位置和调整末端碱基种类,并对锚定位置引物末端第3碱基进行人为突变,由“C”变为“A”,提高扩增特异性,获得分子标记JAASM3424引物对(SEQ ID NO.1和SEQ ID.2)。以上述品种(系)基因组DNA为模板,利用分子标记JAASM3424的引物对SEQ ID NO.1和SEQ ID NO.2进行PCR扩增:On this basis, the applicant artificially adjusted the anchor position of the end of the primer pair and adjusted the base type of the end on the basis of manual comparison of the variation site (644438701bp on chromosome 7B, the reference sequence is Chinese Spring v1.0) , and artificially mutated the third base at the end of the primer at the anchor position, changing from "C" to "A", improving the specificity of amplification, and obtaining the molecular marker JAASM3424 primer pair (SEQ ID NO.1 and SEQ ID.2) . With the above-mentioned variety (line) genomic DNA as a template, PCR amplification of SEQ ID NO.1 and SEQ ID NO.2 is carried out by using the primers of molecular marker JAASM3424:
上述PCR反应体系为10μl体系:10×buffer 1μl,MgCl2溶液(浓度为25mM)0.5μl,dNTP溶液(浓度为2.5mM)0.5μl,引物SEQ ID NO.1(浓度为10μM)0.5μl,引物SEQ ID NO.2(10μM)0.5μl,浓度为的Taq聚合酶(浓度为5U/μl)0.2μl,模板DNA 50ng,ddH2O补足至10μl;The above PCR reaction system is a 10 μl system: 10×
PCR扩增程序:94℃3分钟;94℃10秒,60℃30秒,72℃10秒延伸,30个循环;72℃延伸5分钟。PCR amplification program: 94°C for 3 minutes; 94°C for 10 seconds, 60°C for 30 seconds, 72°C for 10 seconds, 30 cycles; 72°C for 5 minutes.
扩增产物用1.0%琼脂糖凝胶电泳检测,凝胶成像系统记录结果。检测结果如图2所示,分子标记JAASM3424可以对低籽粒毒素积累小麦(泳道1-5:宁894037、望水白、苏麦3号、宁7840和宁麦8号)和高籽粒毒素积累小麦(泳道6-10:小偃22、宁98081、济麦20、皖麦44和安农8455)的等位变异进行有效区分,扩增效率较高,特异性好。The amplified products were detected by 1.0% agarose gel electrophoresis, and the gel imaging system recorded the results. The detection results are shown in Figure 2. The molecular marker JAASM3424 can be used for low grain toxin accumulation wheat (lanes 1-5: Ning 894037, Wangshuibai,
实施例2利用分子标记JAASM3424筛选低籽粒毒素积累的小麦种质Example 2 Screening of Wheat Germplasm with Low Grain Toxin Accumulation Using Molecular Marker JAASM3424
供试小麦群体包含44份小麦品种(系),含22份国外小麦和22份中国小麦(表1)。上述小麦品种均种植于江苏农科院粮食作物研究所小麦遗传团队赤霉病鉴定圃(东经118.88°,北纬32.03°),适期播种,行长1米,播种量为40粒。在花期接种赤霉病菌Fa0609(浓度为105个分生孢子/μl)后,收获10个病穗,测量籽粒DON毒素含量。以实施例1中建立的分子标记JAASM3424的检测方法,对上述小麦品种(系)进行检测,若存在大小为214bp的DNA片段,则基因型记为“0”,判定种质为高DON毒素积累的种质(表1中判读为“高”);若不存在上述扩增片段,则基因型记为“2”,判定种质为低DON毒素积累的种质(表1中判读为“低”)。种质籽粒DON毒素含量检测方法同实施例1。The tested wheat population included 44 wheat varieties (lines), including 22 foreign wheats and 22 Chinese wheats (Table 1). The above-mentioned wheat varieties were all planted in the scab identification nursery of the Wheat Genetics Team of the Institute of Food Crops, Jiangsu Academy of Agricultural Sciences (118.88°E, 32.03°N). After being inoculated with Fusarium rubella Fa0609 (concentration: 10 5 conidia/μl) at the flowering stage, 10 diseased ears were harvested, and the content of DON toxin in the grain was measured. With the detection method of the molecular marker JAASM3424 established in Example 1, the above-mentioned wheat variety (line) is detected, if there is a DNA fragment with a size of 214bp, the genotype is recorded as "0", and the germplasm is judged to be high DON toxin accumulation (interpreted as "high" in Table 1); if there is no above-mentioned amplified fragment, the genotype is recorded as "2", and it is judged that the germplasm is a germplasm with low DON toxin accumulation (interpreted as "low" in Table 1). "). The method for detecting DON toxin content in germplasm grains is the same as that in Example 1.
表1分子标记JAASM3424筛选低籽粒毒素积累的小麦种质Table 1 Molecular marker JAASM3424 screening wheat germplasm with low grain toxin accumulation
检测结果见上表1,根据基因型结果对籽粒DON毒素含量进行预测,分子标记JAASM3424可以分析小麦群体的基因型,并对小麦籽粒毒素积累的优异等位变异进行分型,基因型0和基因型2种质的籽粒DON毒素含量差异显著(p<0.05)(图3)。The test results are shown in Table 1 above. According to the genotype results, the grain DON toxin content can be predicted. The molecular marker JAASM3424 can analyze the genotype of the wheat population and type the excellent allelic variation of wheat grain toxin accumulation,
实施例3利用分子标记JAASM3424绘制遗传图谱Example 3 Drawing Genetic Maps Using Molecular Marker JAASM3424
供试小麦群体为苏麦3号×安农8455的F2:7重组自交系群体共185份、亲本材料苏麦3号和安农8455。以实施例1中建立的分子标记JAASM3424的检测方法,对上述小麦材料进行基因型分析,若存在大小为214bp的DNA片段,则基因型记为“0”;若不存在上述扩增片段,则基因型记为“2”。亲本苏麦3号基因型为“2”,安农8455基因型为“0”,重组自交系群体的分型结果如图4。分型结果准确、易于判读,这表明分子标记JAASM3424引物对扩增效率高、特异性好。The tested wheat populations consisted of 185 F 2:7 recombinant inbred lines of
利用JAASM3424的分型结果对重组自交系群体进行遗传作图,绘制软件为QTLIcimapping,参数为默认,其遗传图谱如图5。优异等位变异区域的遗传图谱全长1.64cM,物理距离全长为6.42Mb。标记序列与小麦基因组序列比对后,标记JAASM3424在遗传图谱中的位置与其在物理图谱中位置对应,这表明使用标记JAASM3424的分型结果进行遗传图谱绘制是可行的,为进一步精细作图、鉴定相关基因/QTL和解析小麦低籽粒毒素积累的机制奠定了基础。The genetic mapping of the recombinant inbred line population was carried out using the typing results of JAASM3424. The drawing software was QTLIcimapping, and the parameters were default. The genetic map is shown in Figure 5. The total length of the genetic map of the excellent allelic variation region is 1.64cM, and the total length of the physical distance is 6.42Mb. After comparing the marker sequence with the wheat genome sequence, the position of the marker JAASM3424 in the genetic map corresponds to its position in the physical map, which indicates that it is feasible to use the typing results of the marker JAASM3424 to draw a genetic map, and it is necessary for further fine mapping and identification The associated genes/QTLs and the mechanism for dissecting the low grain toxin accumulation in wheat laid the foundation.
序列表sequence listing
<110> 江苏省农业科学院<110> Jiangsu Academy of Agricultural Sciences
<120> 与小麦籽粒DON毒素积累相关的分子标记及其引物与应用<120> Molecular markers, primers and applications related to DON toxin accumulation in wheat grain
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