WO2023236840A1 - 与豇豆花青素含量紧密连锁的snp位点、kasp分子标记引物及其应用 - Google Patents

与豇豆花青素含量紧密连锁的snp位点、kasp分子标记引物及其应用 Download PDF

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WO2023236840A1
WO2023236840A1 PCT/CN2023/097679 CN2023097679W WO2023236840A1 WO 2023236840 A1 WO2023236840 A1 WO 2023236840A1 CN 2023097679 W CN2023097679 W CN 2023097679W WO 2023236840 A1 WO2023236840 A1 WO 2023236840A1
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cowpea
anthocyanin content
genotype
primer
molecular marker
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French (fr)
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张红梅
张威
孟珊
陈华涛
刘晓庆
陈新
崔晓艳
王琼
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江苏省农业科学院
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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

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  • the invention belongs to the field of molecular genetic breeding. Specifically, the present invention relates to SNP sites and KASP markers closely linked to the anthocyanin content of cowpea and their applications.
  • Cowpea is an annual herbaceous plant with high economic value. Its bean sprouts, seedlings and tender pods can all be used as edible vegetables. It is one of the important summer legume vegetables in my country. Cowpeas are rich in nutrients and are loved by consumers. In particular, purple-skinned cowpeas have a relatively high anthocyanin content. They are both an excellent health-care vegetable and a source of anthocyanins (Gao Huajie et al., 2010). Anthocyanins have strong antioxidant capacity and can help the body resist certain cancers, cardiovascular diseases, and some diseases related to aging (Yoshimoto M, et al. 1999; Wang C J, et al. 2000; Kong J M, et al. 2003).
  • the principle is to design two allelic SNP sites A forward primer and a universal reverse primer.
  • Each forward primer has a specific sequence and can be combined with different fluorescent labels.
  • the allelic variation of the DNA of the PCR amplified sample with forward primers and universal reverse primers with different fluorescence binding sequences can be reflected by different fluorescence signals (He C L, et al. 2014).
  • SNP Single nucleotide polymorphism
  • GWAS Genome-wide association
  • KASP markers that are closely linked to cowpea anthocyanin content can be used for early (low generation) breeding selection, which has significant effects on reducing breeding workload and accelerating breeding progress. function, and the economic benefits are obvious at the same time.
  • KASP molecular markers for assisted breeding to achieve early molecular-assisted selection of target traits to improve breeding efficiency.
  • the purpose of the present invention is to identify SNP sites closely linked to cowpea anthocyanin content, and to develop KASP molecular markers and primer pairs based on the SNP site information, so as to provide molecular-assisted selection technology support for early identification and screening of this trait. .
  • the present invention provides a SNP marker closely linked to the anthocyanin content of cowpea.
  • the SNP marker closely linked to the anthocyanin content of cowpea is located in the cowpea genome v1.2 (https://phytozome. jgi.doe.gov), a base A to G substitution occurred at position 2,361,292 bp on chromosome 5.
  • the nucleotide sequence of the wild-type cowpea anthocyanin gene is shown in SEQ ID NO.1. In this SEQ Position 479 of the gene sequence of ID NO.1 was mutated from A to G.
  • the present invention also provides KASP molecular marker primers based on the SNP markers closely linked to cowpea anthocyanin content described in the above technical solution, including two specific primers designed for base differences in key sites and one universal primer.
  • the specific primers are upstream primer F1 and upstream primer F2, and a universal primer is downstream primer R;
  • the nucleotide sequence of the upstream primer F1 is shown in SEQ ID NO.2, and the specific sequence is 5'- gaaggtgaccaagttcatgct ctgaatattttcaaggaatttca-3';
  • nucleotide sequence of the downstream primer R is shown in SEQ ID NO.4, and the specific sequence is 5'- gaaggtcggagtcaacggatt ctgaatattttcaaggaatttcg-3';
  • the nucleotide sequence of the downstream primer R is shown in SEQ ID NO. 4, and the specific sequence is: 5’-tgggttgcgtgagctcattt-3’.
  • the 3' ends of the two specific primers of the present invention are allelic variant bases.
  • the 5' end of the forward primer F1 is added with the fluorescence signal of carboxyfluorescein FAM. Tag (the underlined part of the primer); the 5' end of the forward primer F2 is added with the fluorescent signal tag of hexachlorofluorescein phosphoramidate HEX (the underlined part of the primer), and the fluorescent signal tag of carboxyfluorescein FAM is the British LGC (Laboratory of the The FAM fluorescent linker sequence specific to the KASP reaction reagent of the Government Chemist) company; the fluorescent signal tag of hexachlorofluorescein phosphoramidate HEX is the HEX fluorescent linker sequence specific to the KASP reaction reagent of the British LGC (Laboratory of the Government Chemist) company.
  • the present invention also provides the application of the SNP marker or the KASP molecular marker primer in molecular marker-assisted selection breeding of cowpea anthocyanin traits.
  • the SNP marker or the KASP molecular marker primer provided by the invention can be used to detect whether the genotype of the deoxyribonucleotide at position 2,361,292 bp of cowpea chromosome 5 is AA or GG.
  • Low-generation breeding materials of cowpea with GG genotype were selected to provide technical support for molecular marker-assisted breeding of cowpea anthocyanin content traits.
  • the present invention also provides a kit for detecting the SNP marker, including the KASP molecular marker primer described in the above technical solution.
  • the present invention also provides the use of the SNP marker described in the above technical solution, the KASP molecular marker primer or the kit in screening cowpeas with high anthocyanin content.
  • the invention also provides a method for detecting the anthocyanin content of cowpeas.
  • a method for detecting the SNP markers of the cowpeas to be tested the anthocyanin content of the cowpeas is predicted, which specifically includes the following steps:
  • step (2) Using the cowpea genomic DNA described in step (1) as a template, use the KASP molecular marker primer described in the above technical solution to perform a PCR amplification reaction in a fluorescence quantitative PCR instrument;
  • Genotype typing based on the fluorescence signal When the genotype is AA, it is a cowpea with low anthocyanin content. When the genotype is GG, it is a cowpea with high anthocyanin content.
  • the PCR amplification system described in step (2) preferably includes: cowpea genomic DNA, 2 ⁇ KASP Master mix, KASP Assay Mix, the KASP molecular marker primer described in the above technical solution and water.
  • the KASP Assay Mix is a mixture of upstream primer F1, upstream primer F2 and downstream primer R among the KASP molecular marker primers described in the above technical solution.
  • the reaction conditions of the PCR amplification reaction in step (2) preferably include: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing at 61-55°C for 60 seconds, each cycle is reduced by 0.6°C, 10 cycles ; Denaturation at 94°C for 20sec, annealing at 55°C for 60sec, 26 cycles.
  • the PCR amplification reaction in step (2) is preferably performed in an ABI7500 real-time fluorescence quantitative PCR instrument.
  • the instrument can perform genotyping based on the fluorescence signal.
  • the ABI7500 real-time fluorescence quantitative PCR instrument will directly read the fluorescence data of the PCR reaction product, and the fluorescence scanning results will be automatically converted into graphics; if the typing is insufficient, the amplification will continue and the typing will be checked every 3 cycles. situation, until the classification is complete.
  • the invention also provides a method for screening cowpeas with different anthocyanin contents, which includes the following steps:
  • step (S2) Using the cowpea plant genomic DNA described in step (S2) as a template, use the KASP molecular marker primer described in the above technical solution to perform a PCR amplification reaction and detect the SNP genotype AA or genotype GG;
  • step (S1) is preferably the cetyltrimethylammonium bromide method
  • step (2) of the present invention it is preferred to first add the KASP molecular marker primer to the same PCR reaction system, and set up two blank controls using ultrapure water instead of sample template DNA, and compare them on a fluorescence quantitative PCR instrument. DNA from the cowpea RIL population was PCR amplified.
  • the PCR amplification system of the present invention is preferably 10 ⁇ l, consisting of 25 ng/ ⁇ l cowpea sample DNA template, 2 ⁇ l; 2 ⁇ KASP Master mix 5 ⁇ l; KASP Assay Mix 0.14 ⁇ l and 2.9 ⁇ l water; the KASP Assay Mix It is a mixture of upstream primer F1, upstream primer F2 and downstream primer R among the KASP molecular marker primers described in the above technical solution; the ratio of upstream primer F1: upstream primer F2: downstream primer R in the mixture is 2:2:5.
  • the reaction conditions of the PCR amplification preferably include: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 sec, annealing at 61-55°C for 60 sec, each cycle is reduced by 0.6°C, 10 cycles; denaturation at 94°C for 20 sec, Annealing at 55°C for 60 seconds, 26 cycles.
  • the fluorescence data of the PCR reaction product is directly read through the ABI7500 real-time fluorescence quantitative PCR instrument.
  • the results of the fluorescence scan will be automatically converted into graphics.
  • the molecular marker primer can clearly distinguish the two genotypes. Separately, the dots near the Y-axis are sites carrying A allelic mutations, and the genotype is AA; the dots near the X-axis are sites carrying G allelic mutations, and the genotype is GG.
  • the present invention obtained SNP05_2361292, a tightly linked cowpea anthocyanin, with a phenotypic variation explanation rate of 31.66%-38.59%, located at position 2,361,292 bp on chromosome 5 of cowpea genome v1.2.
  • Select GG or AA genotypes from low-generation breeding materials to provide technical support for molecular marker-assisted breeding of cowpea traits with different anthocyanin contents.
  • the KASP molecular marker developed by the present invention can directly and specifically distinguish and detect the A or G base of the SNP mutation site.
  • the KASP molecular marker has good application value and can realize the advance prediction of the anthocyanin content traits of cowpea. Selection and molecular-assisted breeding.
  • KASP molecular marker primers were used to amplify and genotype 20 cowpea family materials on a real-time fluorescence quantitative PCR instrument. The results showed that the molecular marker primers could clearly separate the two genotypes, which were close to the Y axis.
  • the dots are sites carrying G allelic variation, the genotype is GG, and there are 6 families.
  • the average anthocyanin contents are 144.13 ⁇ g/g FW (June 2020) and 126.84 ⁇ g/g FW (2020). September); the dots near the ⁇ g/g FW (September 2020); the point near the origin of the XY axis is the blank control.
  • Figure 1 is the cowpea anthocyanin QTL result map, specifically the QTL mapping results for cowpea anthocyanin content in June 2020 (ANT2020-06) and September 2020 (ANT2020-06);
  • Figure 2 shows the genotyping of different cowpea families using KASP markers.
  • the black dots near the origin represent the blank control without template DNA; the green dots near the Y-axis and the red dots near the X-axis represent G alleles respectively.
  • S05_2361292 The SNP closely linked to cowpea anthocyanins in the present invention, S05_2361292, is obtained through the following steps:
  • the mixed linear model (MLM) in the GAPIT algorithm package in R language software was used to conduct QTL positioning analysis on cowpea anthocyanin content data in two environments in June 2020 (Spring 4, 2020).
  • the main QTL related to the anthocyanin content of cowpea can be detected both in September 2020 (sowing in August and picking pods in September to detect anthocyanin content) and in September 2020 (in the autumn of 2020 (sowing in August and picking pods in September to detect anthocyanin content)).
  • the closely linked SNP site S05_2361292 has a phenotypic variation explanation rate of 31.66%-38.59%. It is located at position 2,361,292 bp on chromosome 5 of the cowpea genome v1.2. A SNP site closely linked to the anthocyanin content of cowpea was detected. 3, and all are located on chromosome 5 (Table 1, Figure 1).
  • Upstream primer F1 (SEQ ID NO.2) sequence: 5'- GAAGGTGACCAAGTTCATGCT CTGAATATTTTCAAGGAATTTCA-3';
  • Upstream primer F2 sequence (SEQ ID NO.3): 5'- GAAGGTCGGAGTCAACGGATT CTGAATATTTTCAAGGAATTTCG-3';
  • Downstream primer R (SEQ ID NO.4) sequence: 5’-TGGGTTGCGTGAGCTCATTT-3’;
  • Genomic DNA was extracted from 194 families of the RIL population, and PCR amplification was performed using the genomic DNA as a template and the KASP marker-specific primer in Example 2 to obtain a PCR amplification product.
  • PCR amplification is performed in the ABI7500 real-time fluorescence quantitative PCR instrument. After the PCR is completed, the instrument can perform genotyping based on the fluorescence signal.
  • Reaction conditions include pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing at 61-55°C for 60 seconds, and each cycle lowering by 0.6°C, 10 cycles; denaturation at 94°C for 20 seconds, annealing at 55°C for 60 seconds, 26 cycles.
  • the ABI7500 real-time fluorescence quantitative PCR instrument directly reads the fluorescence data of the PCR reaction product.
  • the results are shown in Figure 2.
  • KASP molecular marker primers were used to amplify and genotype 20 families of cowpea RIL populations on a real-time fluorescence quantitative PCR instrument. The results showed that: the molecular marker primers The two genotypes can be clearly separated. The dots near the Y axis are sites carrying G allelic mutations.
  • the genotype is GG, and there are 6 (family numbers are SS-11, SS-13, SS-17, SS-22, SS-155, SS-196), their average anthocyanin contents are 144.13 ⁇ g/g FW (June 2020) and 126.84 ⁇ g/g FW (September 2020) respectively; the circles near the X-axis
  • the points carry A allelic mutation sites, and the genotype is AA, there are 14 (family numbers are SS-44, SS-121, SS-135, SS-138, SS-139, SS-147, SS-148, SS-149, SS-150, SS-151, SS-152, SS-178, SS-198, SS-201), their average anthocyanin content is 10.17 ⁇ g/g FW (June 2020) and 11.03 respectively ⁇ g/g FW (September 2020); the point close to the origin of the XY axis is the blank control (Table 2, Figure 2).

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Abstract

本发明属于分子遗传育种领域，提供了一种与豇豆花青素含量紧密连锁的SNP标记、KASP分子标记引物、试剂盒及其应用。本发明提供的SNP标记位于豇豆基因组v1.2的5号染色体2,361,292 bp位置，发生了碱基A至G的替换，所述豇豆花青素的野生型基因的核苷酸序列如SEQ ID NO.1所示，在该SEQ ID NO.1的基因序列的第479位由A突变为G。

Description

与豇豆花青素含量紧密连锁的SNP位点、KASP分子标记引物及其应用
本申请要求于2022年06月08日提交中国专利局、申请号为CN202210639446.3、发明名称为“与豇豆花青素含量紧密连锁的SNP位点、KASP标记及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于分子遗传育种领域。具体而言，本发明涉及与豇豆花青素含量紧密连锁的SNP位点、KASP标记及其应用。
背景技术
豇豆为一年生草本植物，经济价值较高，其豆芽、幼苗、嫩荚都可作蔬菜食用，是我国重要的夏季豆类蔬菜之一。豇豆含有丰富的营养成分，深受广大消费者喜爱，尤其是紫皮豇豆花青素含量比较高，它既是优良的保健蔬菜又可作为花青素的来源(高华杰等，2010)。花青素具有超强的抗氧化能力，能够帮助人体抵御某些癌症、心血管疾病以及一些与机体老化相关的疾病(Yoshimoto M,et al.1999；Wang C J,et al.2000；Kong J M,et al.2003)。在植物果实发育过程中，花青素含量发生变化，且不同品种之间有差异(Alcalde-Eon C,et al.2014)。对于大多数国家和地区的人民来说，蔬菜在日常饮食中要比水果、保健品占得比重高，所以筛选、培育一些富含花色素的蔬菜，让人们普遍享受到花色素对人体健康的益处。因此，研究快速、有效的豇豆花青素性状分子育种技术，对于分子辅助豇豆花青素性状的遗传改良具有十分重要的意义。
传统高花青素豇豆育种是根据育种后代花青素含量进行的单株选择，这种方式不仅费时耗力而且易受环境干扰准确性不高。利用目标基因存在的碱基差异，开发特异性分子标记进行辅助选择是提高高花青素豇豆选择效率的最佳方法。分子标记在作物育种中具有早期选择、不受环境影响以及准确、快速、高效的优势，已成为一种准确、高效的工具。虽然其中竞争性等位基因特异性PCR(Kompetitive Allele-Specific PCR，KASP)分子标记是建立在等位基因特异性扩增(Amplification Refractory Mutation System，ARMS)和高灵敏度的荧光检测基础之上的一种新型SNP分型方法。其原理是针对等位基因SNP位点设计两 个正向引物和一个通用反向引物，每条正向引物都有特异性序列，可与不同荧光标记结合。带有与不同荧光结合序列的正向引物与通用反向引物PCR扩增样品的DNA，其等位变异就可以通过不同的荧光信号得以反映(He C L,et al.2014)。
研究表明植物花青素含量受多个基因调控并且容易受到环境的影响，为复杂的数量性状(Cavagnaro P F,et al.2014；Choi Y,et al.2020)。单核苷酸多态性(SNP)主要是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。全基因组关联(GWAS)作为一种有效的基因定位工具，可以快速、准确地挖掘与豇豆花青素紧密连锁的SNP。
因此，基于鉴定的与豇豆花青素紧密连锁的SNP，开发与豇豆花青素含量紧密连锁的KASP标记，用于育种早期(低世代)选择，对于减少育种工作量、加速育种进度具有显著的作用，同时经济效益明显。基于挖掘与豇豆花青素紧密连锁的SNP，开发出用于辅助育种的KASP分子标记，实现目标性状的早期分子辅助选择，以提高育种效率尤为重要。
发明内容
本发明的目的是鉴定与豇豆花青素含量紧密连锁的SNP位点，并基于该SNP位点信息开发KASP分子标记及其引物对，为实现该性状的早期鉴定和筛选提供分子辅助选择技术支持。
为解决上述技术问题，本发明提供了一种与豇豆花青素含量紧密连锁的SNP标记，所述与豇豆花青素含量紧密连锁的SNP标记位于豇豆基因组v1.2(https://phytozome.jgi.doe.gov)的5号染色体2,361,292bp位置，发生了碱基A至G的替换，其豇豆花青素的野生型基因的核苷酸序列如SEQ ID NO.1所示，在该SEQ ID NO.1的基因序列的第479位由A突变为G。
本发明还提供了基于上述技术方案所述的与豇豆花青素含量紧密连锁SNP标记的KASP分子标记引物，包含两条针对关键位点碱基差异设计的特异性引物和一条通用引物，两条特异性引物即上游引物F1、上游引物F2，一条通用引物即下游引物R；
所述上游引物F1的核苷酸序列如SEQ ID NO.2所示，具体序列为5’-gaaggtgaccaagttcatgctctgaatattttcaaggaatttca-3’；
所述下游引物R的核苷酸序列如SEQ ID NO.4所示，具体序列为5’-gaaggtcggagtcaacggattctgaatattttcaaggaatttcg-3’；
所述下游引物R的核苷酸序列如SEQ ID NO.4所示，具体序列为：5’-tgggttgcgtgagctcattt-3’。
本发明所述两条特异性引物的3’末端为等位变异碱基，在合成所述的KASP分子标记引物时，所述正向引物F1的5’端加上羧基荧光素FAM的荧光信号标签(引物下划线部分)；正向引物F2的5’端加上六氯荧光素氨基磷酸酯HEX的荧光信号标签(引物下划线部分)，羧基荧光素FAM的荧光信号标签为英国LGC(Laboratory of the Government Chemist)公司KASP反应试剂特定的FAM荧光接头序列；六氯荧光素氨基磷酸酯HEX的荧光信号标签为英国LGC(Laboratory of the Government Chemist)公司KASP反应试剂特定的HEX荧光接头序列。
本发明还提供了所述的SNP标记或所述的KASP分子标记引物在豇豆花青素性状的分子标记辅助选择育种中的应用。
利用本发明提供的SNP标记或所述的KASP分子标记引物可用检测豇豆5号染色体2,361,292bp位置的脱氧核糖核苷酸的基因型是AA还是GG。选择GG基因型豇豆低世代育种材料，为豇豆花青素含量性状的分子标记辅助育种提供技术支持。
本发明还提供了一种用于检测所述的SNP标记的试剂盒，包含上述技术方案所述的KASP分子标记引物。
本发明还提供了上述技术方案所述的SNP标记、所述的KASP分子标记引物或所述的试剂盒在筛选高花青素含量豇豆的用途。
本发明还提供了一种检测豇豆花青素含量高低的方法，通过对待测豇豆进行所述的SNP标记的检测，预测豇豆的花青素含量，具体包括以下步骤：
(1)提取待检测的豇豆基因组DNA；
(2)以步骤(1)所述豇豆基因组DNA为模板，利用上述技术方案所述的KASP分子标记引物在荧光定量PCR仪中进行PCR扩增反应；
(3)根据荧光信号进行基因型分型，当基因型为AA时，其为低花青素含量的豇豆，当基因型为GG时，其为高花青素含量的豇豆。
在本发明中，步骤(2)中所述PCR扩增体系优选包括：豇豆基因组DNA、2×KASP Master mix、KASP Assay Mix、上述技术方案所述的KASP分子标记引物和水。所述KASP Assay Mix为上述技术方案所述KASP分子标记引物中上游引物F1、上游引物F2和下游引物R的混合物。
在本发明中，步骤(2)中所述PCR扩增反应的反应条件优选包括：94℃预变性15min；94℃变性20sec，61～55℃退火60sec，每一个循环降低0.6℃，10个循环；94℃变性20sec，55℃退火60sec，26个循环。
在本发明中，步骤(2)中所述PCR扩增反应优选在ABI7500实时荧光定量PCR仪中进行，PCR结束后该仪器根据荧光信号可进行基因分型。反应完成后，ABI7500实时荧光定量PCR仪会直接对PCR反应产物进行荧光数据读取，荧光扫描的结果会自动转化成图形；若分型不充分，则继续扩增，每3个循环查看分型情况，直至分型全。
本发明还提供了一种筛选不同花青素含量的豇豆的方法，包括以下步骤：
(S1)提取豇豆植株基因组DNA；
(S2)以步骤(S2)所述的豇豆植株基因组DNA为模板，利用上述技术方案所述的KASP分子标记引物进行PCR扩增反应并检测所述的SNP基因型AA或基因型为GG；
(S3)根据基因型在不同家系选择得到不同花青素含量的豇豆单株或株系。
在本发明中，步骤(S1)中所述提取的方法优选为十六烷基三甲基溴化铵法；
在本发明所述步骤(2)中，优选先将所述的KASP分子标记引物加入同一PCR反应体系，并设置2个以超纯水代替样品模版DNA的空白对照，在荧光定量PCR仪上对豇豆RIL群体的DNA进行PCR扩增。本发明所述PCR扩增的体系优选以10μl计，由25ng/μl的豇豆样品DNA模板，2μl；2×KASP Master mix5μl；KASP Assay Mix为，0.14μl和水2.9μl组成；所述KASP Assay Mix为上述技术方案所述KASP分子标记引物中上游引物F1、上游引物F2和下游引物R的混合物；所述混合物中上游引物F1：上游引物F2：下游引物R为2:2:5。
在本发明中，所述PCR扩增的反应条件优选包括：94℃预变性15min；94℃变性20sec，61～55℃退火60sec，每一个循环降低0.6℃，10个循环；94℃变性20sec，55℃退火60sec，26个循环。
本发明所述PCR扩增结束后，通过ABI7500实时荧光定量PCR仪直接对PCR反应产物进行荧光数据读取，荧光扫描的结果会自动转化成图形，该分子标记引物可以清楚的将两种基因型分开，其中靠近Y轴的圆点为携带A等位变异位点，基因型为AA；靠近X轴的圆点为携带G等位变异位点，基因型为GG。
有益效果：与现有技术相比，具备以下优点：
1)本发明首次获得豇豆花青素紧密连锁的SNP05_2361292表型变异解释率达到31.66％-38.59％，位于豇豆基因组v1.2的5号染色体2,361,292bp位置。从低世代育种材料中选择GG或AA基因型，为不同花青素含量的豇豆性状的分子标记辅助育种提供技术支持。
2)本发明开发的KASP分子标记能直接对SNP突变位点的A或G碱基进行特异的区分和检测，该KASP分子标记具有良好的应用价值，可实现对豇豆花青素含量性状的预先选择和分子辅助育种。
3)利用KASP分子标记引物在实时荧光定量PCR仪上对20个豇豆家系材料进行扩增并进行基因分型，结果表明：该分子标记引物可以清楚的将两种基因型分开，其中靠近Y轴的圆点为携带G等位变异位点，基因型为GG，有6个家系，其平均花青素含量分别为144.13μg/g FW(2020年6月)和126.84μg/g FW(2020年9月)；靠近X轴的圆点为携带A等位变异位点，基因型为AA，有14个家系，其平均花青素含量分别为10.17μg/g FW(2020年6月)和11.03μg/g FW(2020年9月)；靠近XY轴原点的点为空白对照。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。
图1为豇豆花青素QTL结果图，具体为2020年6月(ANT2020-06)和2020年9月(ANT2020-06)豇豆花青素含量QTL定位结果；
图2为KASP标记对不同豇豆家系基因分型，其中，靠近原点的黑色圆点代表无模板DNA的空白对照；靠近Y轴的绿色圆点和靠近X轴的红色圆点分别代表携带G等位变异位点的豇豆家系和携有A等位变异位点的豇豆家系。
具体实施方式
实施例1
豇豆花青素含量主效QTL紧密连锁的SNP位点获得
本发明中豇豆花青素紧密连锁的SNP，S05_2361292是通过下列步骤获得的：
(1)以高花青素含量‘苏紫41’为母本(114.99μg/g FW)，‘苏豇1419’为父本(10.78μg/g FW)(江苏省农业种质资源保护与利用平台)，得到杂种F1，F1代单株自交获得F2，在F2使用单粒传法，一直到F7代，获得含有211个家系的重组自交系，构成遗传作图群体。对重组自交系群体花青素表型鉴定，亲 本‘苏紫41’和‘苏豇1419’进行重测序，测序深度为32.6×，重组自交系群体测序深度为5.44×，经过剔除过滤后获得覆盖全基因组的高密度的SNP分子标记图谱。
其中包括DNA提取及高通量测序：采用CTAB法提取RIL豇豆群体2个亲本和211个家系幼嫩叶片的基因组DNA，进行全基因组重测序。
(2)利用紫外分光光度法检测两个环境下(2020年春季和2020年秋季)豇豆荚的花青素含量的测定：
取RIL群体亲本和家系的豇豆荚，将每份样品豇豆荚鲜样(2个亲本和211个家系)充分混匀后，粉碎，籽粒全部通过0.25mm孔径筛，装入样品瓶备用。称取豇豆鲜荚样品0.5g，精确至0.0001g，置于10mL盐酸乙醇(1:1)中，然后依据紫外分光光度法进行花青素的测定。
(3)QTL定位分析：利用R语言软件中的GAPIT算法包中的混合线性模型(MLM)对豇豆花青素含量数据进行QTL定位分析，在两个环境下2020年6月(2020年春季4月份播种6月采荚测花青素含量)和2020年9月(2020年秋季8月份播种9月份采荚检测花青素含量)中均能检测到与豇豆花青素含量相关的主效QTL位点紧密连锁的SNP位点S05_2361292，表型变异解释率达到31.66％-38.59％，位于豇豆基因组v1.2的5号染色体2,361,292bp位置，检测到与豇豆花青素含量紧密连锁的SNP位点3个，且均位于5号染色体上(表1，图1)。
表1豇豆RIL群体花青素含量的QTL定位结果
实施例2
KASP标记特异引物的开发
利用NCBI(https://www.ncbi.nlm.nih.gov/)的Primer-BLAST功能，依据SEQ ID NO.1序列设计三个引物，上游引物F1(SEQ ID NO.2)、上游引物F2(SEQ ID NO.3)和下游引物R(SEQ ID NO.4)，其中F1和F2分别包含FAM和HEX荧光接头序列(下划线)，序列如下：
SEQ ID NO.1：

上游引物F1(SEQ ID NO.2)序列：5’-GAAGGTGACCAAGTTCATGCTCTGAATATTTTCAAGGAATTTCA-3’；
上游引物F2序列(SEQ ID NO.3)：5’-GAAGGTCGGAGTCAACGGATTCTGAATATTTTCAAGGAATTTCG-3’；
下游引物R(SEQ ID NO.4)序列：5’-TGGGTTGCGTGAGCTCATTT-3’；
实施例3
检测不同家系豇豆SNP位点的基因型及其应用
分别提取RIL群体194个家系的基因组DNA，以基因组DNA为模板，采用实施例2的KASP标记专用引物在进行PCR扩增，得到PCR扩增产物。PCR扩增是在ABI7500实时荧光定量PCR仪中进行，PCR结束后该仪器根据荧光信号可进行基因分型。所述的扩增体系均为10μl反应体系：豇豆样品DNA模板，25ng/μl，2μl；2×KASP Master mix 5μl；KASP Assay Mix，F1:F2:R＝2:2:5，0.14μl；水2.9μl。反应条件包括94℃预变性15min；94℃变性20sec，61～55℃退火60sec，每一个循环降低0.6℃，10个循环；94℃变性20sec，55℃退火60sec，26个循环。
反应完成后，ABI7500实时荧光定量PCR仪直接对PCR反应产物进行荧光数据读取，结果如图2。利用KASP分子标记引物在实时荧光定量PCR仪上对豇豆RIL群体20个家系进行扩增并进行基因分型，结果表明：该分子标记引物 可以清楚的将两种基因型分开，其中靠近Y轴的圆点为携带G等位变异位点，基因型为GG，有6个(家系编号为SS-11，SS-13，SS-17，SS-22，SS-155，SS-196)，其平均花青素含量分别为144.13μg/g FW(2020年6月)和126.84μg/g FW(2020年9月)；靠近X轴的圆点为携带A等位变异位点，基因型为AA，有14个(家系编号为SS-44，SS-121，SS-135，SS-138，SS-139，SS-147，SS-148，SS-149，SS-150，SS-151，SS-152，SS-178，SS-198，SS-201)，其平均花青素含量分别为10.17μg/g FW(2020年6月)和11.03μg/g FW(2020年9月)；靠近XY轴原点的点为空白对照(表2，图2)。另外发现，在包含194个豇豆家系的RIL群体中(有3个家系在SNP位点，S05_2361292基因型为缺失)，基因型是GG的94个豇豆家系的平均花青素含量分别为128.75μg/g FW(2020年6月)和127.87μg/g FW(2020年9月)；基因型是AA的97个豇豆家系的平均花青素含量分别为9.81μg/g FW(2020年6月)和10.66μg/g FW(2020年9月)(表2)。从以上结果可以看出，我们通过确定基因型AA或者GG即可确定该豇豆的花青素含量高低，从而筛选不同花青素含量的豇豆。
表2豇豆RIL群体194个家系花青素含量及基因分型表







尽管上述实施例对本申请做出了详尽的描述，但它仅仅是本申请一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本申请保护范围。

Claims (11)

  1. 一种与豇豆花青素含量紧密连锁的SNP标记，其特征在于，所述的SNP标记位于豇豆参考基因组V1.2的5号染色体2,361,292bp位置，发生了碱基A至G的替换。
  2. 基于权利要求1所述的与豇豆花青素含量紧密连锁SNP标记的KASP分子标记引物，其特征在于，所述KASP分子标记引物有上游引物F1、上游引物F2和下游引物R；
    所述上游引物F1的核苷酸序列如SEQ ID NO.2所示；
    所述上游引物F2的核苷酸序列如SEQ ID NO.3所示；
    所述下游引物R的核苷酸序列如SEQ ID NO.4所示。
  3. 一种用于检测权利要求1所述的SNP标记的试剂盒，其特征在于，包含权利要求2所述的KASP分子标记引物。
  4. 权利要求1所述的SNP标记、权利要求2所述的KASP分子标记引物或权利要求3所述的试剂盒在豇豆花青素性状的分子标记辅助选择育种中的应用。
  5. 权利要求1所述的SNP标记、权利要求2所述的KASP分子标记引物或权利要求3所述的试剂盒在筛选高花青素含量豇豆中的应用。
  6. 一种检测豇豆花青素含量高低的方法，其特征在于，所述方法对待测豇豆进行权利要求1所述的SNP标记的检测，预测豇豆的花青素含量高低，包括以下步骤：
    (1)提取待测豇豆的基因组DNA；
    (2)以步骤(1)所述的基因组DNA为模板，利用权利要求2所述的KASP分子标记引物或权利要求3所述试剂盒在荧光定量PCR仪中进行PCR扩增反应；
    (3)根据荧光信号进行基因型分型，当基因型为AA时，所述待测豇豆为低花青素含量的豇豆，当基因型为GG时，所述待测豇豆为高花青素含量的豇豆。
  7. 根据权利要求6所述的方法，其特征在于，步骤(2)中所述PCR扩增反应的体系包括：基因组DNA、2×KASP Master mix、KASP Assay Mix、权利要求2所述的KASP分子标记引物和水。
  8. 根据权利要求6所述的检测豇豆花青素含量的方法，其特征在于，步骤(2)中所述PCR扩增反应的条件包括：94℃预变性15min；94℃变性20sec，61～55℃退火60sec，每一个循环降低0.6℃，10个循环；94℃变性20sec，55℃ 退火60sec，26个循环。
  9. 一种筛选高花青素含量豇豆的方法，其特征在于，包括以下步骤：
    (S1)提取豇豆植株基因组DNA；
    (S2)以步骤(S1)所述的豇豆植株基因组为模板，利用权利要求2所述的KASP分子标记引物或权利要求3所述试剂盒进行PCR扩增反应，得到PCR扩增产物后，检测PCR扩增产物中权利要求1所述SNP标记的基因型；
    (S3)根据基因型在不同家系选择得到高花青素含量豇豆单株或株系，若基因型为GG，则为高花青素含量豇豆单株或株系。
  10. 根据权利要求9所述的筛选不同花青素含量的豇豆的方法，其特征在于，所述步骤(S2)中，检测PCR扩增产物中权利要求1所述SNP标记的基因型方法为：
    利用ABI7500实时荧光定量PCR仪直接对所述PCR扩增产物进行荧光数据读取，荧光扫描的结果自动转化成图形，根据所述图形的结果将基因型判断为AA或GG。
  11. 根据权利要求10所述的筛选不同花青素含量的豇豆的方法，其特征在于，根据所述图形的结果判断基因型时，靠近Y轴的圆点为携带A等位变异位点，基因型为AA；靠近X轴的圆点为携带G等位变异位点，基因型为GG。
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LONARDI STEFANO, MUÑOZ‐AMATRIAÍN MARÍA, LIANG QIHUA, SHU SHENGQIANG, WANAMAKER STEVE I., LO SASSOUM, TANSKANEN JAAKKO, SCHULMAN AL: "The genome of cowpea ( Vigna unguiculata [L.] Walp.)", THE PLANT JOURNAL, BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD., GB, vol. 98, no. 5, 1 June 2019 (2019-06-01), GB , pages 767 - 782, XP093114524, ISSN: 0960-7412, DOI: 10.1111/tpj.14349 *

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Publication number Priority date Publication date Assignee Title
CN117683938A (zh) * 2024-02-02 2024-03-12 山东永盛农业发展有限公司 与番茄果实宽度紧密连锁的kasp分子标记及其应用
CN117683938B (zh) * 2024-02-02 2024-05-07 山东永盛农业发展有限公司 与番茄果实宽度紧密连锁的kasp分子标记及其应用

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