WO2024055406A1 - 一种大豆皂苷相关的kasp标记及其应用 - Google Patents

一种大豆皂苷相关的kasp标记及其应用 Download PDF

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WO2024055406A1
WO2024055406A1 PCT/CN2022/131935 CN2022131935W WO2024055406A1 WO 2024055406 A1 WO2024055406 A1 WO 2024055406A1 CN 2022131935 W CN2022131935 W CN 2022131935W WO 2024055406 A1 WO2024055406 A1 WO 2024055406A1
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soybean
saponin
soybeans
kasp
content
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French (fr)
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陈华涛
唐威
张红梅
张威
刘晓庆
王琼
许文静
崔晓艳
陈新
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江苏省农业科学院
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Publication of WO2024055406A1 publication Critical patent/WO2024055406A1/zh

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    • 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
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

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  • the invention belongs to the field of molecular breeding technology, and in particular relates to a soybean saponin-related KASP marker and its application.
  • Soybean saponin also known as soybean saponin, is extracted from the seeds of soybeans and other leguminous plants. Its molecules are a type of compound formed by the condensation of oligosaccharides and oleanene triterpenes. At present, chemical methods are used to study soybean saponins, and it is confirmed that there are 5 types of soybean saponins, namely soybean saponin A, soybean saponin B, soybean saponin C, soybean saponin D, and soybean saponin E. There are 3 naturally occurring soy saponins. The species are soybean saponin A, soybean saponin B, and soybean saponin E.
  • soybean saponins have various physiological functions such as anti-lipid oxidation, anti-free radicals, enhanced immune regulation, anti-tumor, anti-virus, etc., and can be used in food, medicine, cosmetics and other fields, and have broad market prospects.
  • soybean saponins play an important role in human health, the current utilization of soybean resources is low and there are few specialized soybean varieties. Breeding specialized soybeans has good development prospects. Soybean saponins are controlled by multiple genes and are easily affected by external environmental factors such as soil environment and climate change. In conventional screening, the saponins of each soybean germplasm are measured, which has the problems of long identification cycle, high cost, time-consuming and labor-intensive, and cannot meet the needs of stable and efficient screening.
  • the purpose of the present invention is to provide a soybean saponin-related KASP marker and its application.
  • the invention provides a soybean saponin-related KASP marker.
  • the KASP marker includes one or both of S07_43139773 G/T and S07_43139033 A/G.
  • the S07_43139773 G/T is the 43139773bp position of chromosome No. 7 of the soybean genome.
  • the bases on are G/T, and the S07_43139033 A/G is A/G.
  • the bases at the 43139033bp position on chromosome 7 of the soybean genome are A/G.
  • the soybeans whose base is G at S07_43139773 G/T are soybeans with low saponin Aa content, and the soybeans whose base is T are soybeans with high saponin Aa content.
  • the S07_43139033 A/G base is a soybean with a high soybean saponin Ab content, and the base is a soybean with a low soybean saponin Ab content.
  • the present invention also provides a primer pair for detecting the above-mentioned KASP mark.
  • the primer pair of the KASP mark includes one or both of the S07_43139773 G/T primer pair and the S07_43139033 A/G primer pair;
  • the sequence of the upstream primer F1 of the S07_43139773 G/T is shown in SEQ ID No.1
  • the sequence of the upstream primer F2 is shown in SEQ ID No.2
  • the sequence of the downstream primer R is shown in SEQ ID No.3;
  • the sequence of the upstream primer F1 of the S07_43139033 A/G is shown in SEQ ID No. 4, the sequence of the upstream primer F2 is shown in SEQ ID No. 5, and the sequence of the downstream primer R is shown in SEQ ID No. 6.
  • the present invention also provides a kit for detecting the above-mentioned KASP marker, which kit contains the above-mentioned primer pair.
  • the present invention also provides an application of the above-mentioned KASP marker, primer pair or kit in improving soybean saponin germplasm breeding.
  • the invention also provides a method for screening soybeans with high saponin content, which includes the following steps:
  • PCR amplification reactions were performed using the above primer pairs, and genotyping was performed through the fluorescence signal of the PCR amplification product.
  • the genotype at position 43139773 bp on chromosome 7 in the soybean genome is selected to be TT and the genotype at position 43139033 on chromosome 7 in the soybean genome is selected to be one or both of AA, then the soybean to be tested is high in saponins. Content soybeans.
  • the present invention has the following beneficial effects:
  • the present invention provides a soybean saponin-related KASP marker and its application.
  • the present invention conducts genome-wide correlation analysis on the soybean saponin Aa, Ab, and Bb content of 264 soybean germplasm natural populations, and utilizes the significant correlation between the two saponin components.
  • KASP markers were developed for relevant SNP sites, and two KASP markers were designed.
  • the accuracy of S07_43139773 G/T of the present invention for detecting soybean saponin content is 93.2%
  • the accuracy of S07_43139033 A/G for detecting soybean saponin content is 93.2%.
  • the KASP marker of the present invention can accurately genotype the soybean saponin content trait.
  • the KASP marker of the present invention can be used for the breeding of soybean germplasm with high soybean saponin content.
  • the KASP marker of the present invention not only has high accuracy and low cost, but is also simple and fast to operate. It only needs to extract the genomic DNA of plant leaves. You can perform PCR amplification.
  • Figure 1 shows the resequencing results of the natural population, where A is the source distribution of each variety, B is the evolutionary tree, C is the principal component analysis, and D is the linkage disequilibrium analysis;
  • Figure 2 shows the Manhattan plot and QQ plot of the genome-wide association analysis results of three soybean saponins.
  • a and B are Hainan soybean saponins Aa and Ab in 2020 respectively;
  • C and D are Nanjing soybean saponins Aa and Ab in 2021 respectively;
  • FIG 3 shows the genotyping of different soybean germplasm by KASP markers, where A and B are genotyping S07_43139773 and S07_43139033 respectively.
  • the invention provides a soybean saponin-related KASP marker.
  • the KASP marker includes one or both of S07_43139773 G/T and S07_43139033 A/G.
  • the S07_43139773 G/T is the 43139773bp position of chromosome No. 7 of the soybean genome.
  • the bases on are G/T, and the S07_43139033 A/G is A/G.
  • the bases at the 43139033bp position on chromosome 7 of the soybean genome are A/G.
  • the KASP marker when the KASP marker includes one or both of S07_43139773 G/T and S07_43139033 A/G, both are related to the soybean saponin content.
  • the KASP marker of the present invention is preferably one of S07_43139773 G/T and S07_43139033 A/G.
  • the KASP markers of the present invention are preferably S07_43139773 G/T and S07_43139033 A/G.
  • the soybeans whose base is G at the S07_43139773 G/T position are preferably soybeans with low saponin Aa content, and the soybeans whose base is T are those with high saponin Aa content.
  • the S07_43139033 A/G base is a soybean with a high soybean saponin Ab content, and the base is a soybean with a low soybean saponin Ab content.
  • the present invention also provides a primer pair for detecting the above-mentioned KASP mark.
  • the primer pair of the KASP mark includes one or both of the S07_43139773 G/T primer pair and the S07_43139033 A/G primer pair;
  • the sequence of the upstream primer F1 of the S07_43139773 G/T is shown in SEQ ID No.1
  • the sequence of the upstream primer F2 is shown in SEQ ID No.2
  • the sequence of the downstream primer R is shown in SEQ ID No.3;
  • the sequence of the upstream primer F1 of the S07_43139033 A/G is shown in SEQ ID No. 4, the sequence of the upstream primer F2 is shown in SEQ ID No. 5, and the sequence of the downstream primer R is shown in SEQ ID No. 6.
  • the KASP labeled primer pair includes one or both of the S07_43139773 G/T primer pair and the S07_43139033 A/G primer pair.
  • the KASP labeled primer pair is preferably one of the S07_43139773 G/T primer pair and the S07_43139033 A/G primer pair.
  • the KASP labeled primer pair includes S07_43139773 G/T primer pair and S07_43139033 A/G primer pair.
  • the present invention also provides a kit for detecting the above-mentioned KASP marker, which kit contains the above-mentioned primer pair.
  • the kit preferably includes a PCR amplification reaction solution, and further preferably, the PCR amplification reaction solution includes one or both of the S07_43139773 G/T primer pair and the S07_43139033 A/G primer pair.
  • the PCR amplification reaction solution also includes soybean sample DNA template and 2 ⁇ KASP Master mix.
  • the present invention also provides an application of the above-mentioned KASP marker, primer pair or kit in improving soybean saponin germplasm breeding.
  • the invention also provides a method for screening soybeans with high saponin content, which includes the following steps:
  • PCR amplification reactions were performed using the above primer pairs, and genotyping was performed through the fluorescence signal of the PCR amplification product.
  • one or both of the genotypes at the 43139773bp position on chromosome 7 in the soybean genome are selected as TT and the genotype at the 43139033 bp position on chromosome 7 in the soybean genome is selected as AA, then the soybean to be tested is High saponin content in soybeans.
  • the soybeans to be tested are soybeans with high soybean saponin Aa content; if the genotype is GG, then the soybeans to be tested are soybeans with low soybean saponin Aa content. , the soybean saponin Aa content of soybean germplasm with genotype TT is higher than that of GG.
  • Select the genotype at the 43139033bp position of chromosome 7 in the soybean genome as AA then the soybeans to be tested are soybeans with high soybean saponin Ab content, and the genotype is GG, then the soybeans to be tested are soybeans with low soybean saponin Ab content, and the genotype is AA.
  • the soybean germplasm has a higher soybean saponin Ab content than GG.
  • the genotype at the 43139773bp position on chromosome 7 in the soybean genome is TT and the genotype at the 43139033bp position on chromosome 7 in the soybean genome is AA, then the soybean saponin content of the soybean to be tested is significantly increased. .
  • the soybean genomic DNA to be tested is used as a template, and the above-mentioned primer pairs are used to perform PCR amplification reactions respectively.
  • KASP-PCR amplification reaction conditions are: the first stage is 94°C for 15min; the second stage is 94°C for 20s, 61 ⁇ 55°C for 1min, and the annealing temperature is reduced by 0.6°C in each cycle, a total of 10 cycles; the third stage is 94°C for 20s, 55°C for 1 min, a total of 26 cycles; the fourth stage is 37°C for 1 min.
  • Linkage disequilibrium (LD) analysis showed (D in Figure 1) that the LD value of all accessions was approximately 106 kb, the LD value of wild soybean accessions was approximately 33 kb, and the LD value of non-wild soybean accessions was approximately 120 kb.
  • the high-performance liquid chromatography method was used to detect the soybean saponin content of natural populations of soybeans harvested in Hainan base in 2020 and Nanjing Liuhe base in 2021, and genome-wide association analysis was performed on the two years of soybean saponin content phenotypic data.
  • the visualization results of GWAS analysis are shown in Figure 2.
  • soybean saponin standard solution must be prepared and used immediately.
  • HPLC chromatography conditions are as follows, chromatographic column: Thermo BDS Hypersil C18 (250mm ⁇ 4.6mm, 5 ⁇ m); mobile phase A: 0.2% (V/V) acetic acid aqueous solution, mobile phase B: 100% (V/V) acetonitrile solution ; Flow rate: 1.0mL/min; Column temperature: 30°C; Injection volume: 10 ⁇ L; HPLC elution procedure is shown in Table 1. Each sample was injected three times, and the content of the soybean saponin component in the sample was calculated based on the standard curve drawn with the standard. The average value of the three injections was used as the final detection result of the soybean saponin component.
  • soybean saponin Aa there are 1,678 SNPs associated with soybean saponin Aa in Nanjing in 2021, and there are 1,345 significantly associated SNPs, 182 more SNPs than in 2020, which are present on chromosomes 7, 9 and 14; while soybean saponin Ab is associated with The situation of soybean saponin Aa is different. Compared with 2020, the number of SNPs significantly related to soybean saponin Ab is 589 fewer, reaching 229, which are distributed on chromosomes 7 and 9 respectively; it is worth noting that in 2021, soybean saponin Bb There are 4 more significantly associated SNP sites than in 2020, and there are 2 significant sites on chromosomes 5 and 14 respectively (see Table 2).
  • a kind of soybean saponin-related KASP marker is S07_43139773 G/T and S07_43139033 A/G.
  • Each pair of primers includes two specific forward primers F1 and F2 and a universal reverse primer R, where F1 and F2 respectively contain 6-carboxyfluorescein (FAM) and hexachloro-6-methylfluorescein (HEX) fluorescent linker sequence (underlined), the primer sequence was synthesized by Qingke Biotech (Nanjing), and the sequence is shown in Table 5.
  • F1 and F2 respectively contain 6-carboxyfluorescein (FAM) and hexachloro-6-methylfluorescein (HEX) fluorescent linker sequence (underlined)
  • FAM 6-carboxyfluorescein
  • HEX hexachloro-6-methylfluorescein
  • a soybean saponin-related KASP marker is S07_43139773 G/T.
  • the KASP labeling primer pair includes two specific forward primers F1 and F2 and a universal reverse primer R, wherein F1 and F2 contain 6-carboxyfluorescein (FAM) and hexachloro-6-methylfluorescein (HEX) fluorescent linker sequences (underlined), respectively.
  • F1 and F2 contain 6-carboxyfluorescein (FAM) and hexachloro-6-methylfluorescein (HEX) fluorescent linker sequences (underlined), respectively.
  • the primer sequences are synthesized by Qingke Biotechnology (Nanjing), and the upstream primer F1, upstream primer F2 and downstream primer R are the sequences shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively.
  • a soybean saponin-related KASP marker is S07_43139033 A/G.
  • the KASP labeled primer pair includes two specific forward primers F1 and F2 and a universal reverse primer R, where F1 and F2 respectively contain 6-carboxyfluorescein (FAM) and hexachloro-6-methylfluorescein (HEX) fluorescent linker sequence (underlined), the primer sequence was synthesized by Qingke Biotechnology (Nanjing), the upstream primer F1, upstream primer F2 and downstream primer R are SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 respectively. sequence shown.
  • a kind of soybean saponin-related KASP marker is S07_43139773 G/T and S07_43139033 A/G.
  • the KASP labeled primer pair includes two specific forward primers F1 and F2 and a universal reverse primer R, where F1 and F2 respectively contain 6-carboxyfluorescein (FAM) and hexachloro-6-methylfluorescein (HEX) fluorescent linker sequence (underlined), the primer sequence was synthesized by Qingke Biotechnology (Nanjing), the upstream primer F1, the upstream primer F2 and the downstream primer R are the sequences shown in SEQ ID No.1 ⁇ SEQ ID No.3 and SEQ Sequences shown in ID No.4 ⁇ SEQ ID No.6.
  • F1 and F2 respectively contain 6-carboxyfluorescein (FAM) and hexachloro-6-methylfluorescein (HEX) fluorescent linker sequence (underlined)
  • FAM 6-carboxyfluorescein
  • HEX hexachloro-6-methylfluorescein
  • kits for S07_43139773 G/T KASP labeling includes S07_43139773 G/T upstream primer F1, upstream primer F2 and downstream primer R, respectively SEQ ID No.1, SEQ ID No.2, SEQ The sequence shown in ID No.3.
  • kits for S07_43139033 A/G KASP labeling includes S07_43139033 A/G upstream primer F1, upstream primer F2 and downstream primer R, respectively SEQ ID No. 4, SEQ ID No. 5, SEQ The sequence shown in ID No.6.
  • kits for S07_43139773 G/T and S07_43139033 A/G KASP labeling includes S07_43139773 G/T and S07_43139033 A/G upstream primer F1, upstream primer F2 and downstream primer R, respectively SEQ ID No. .1 ⁇ SEQ ID No.3 and the sequence shown by SEQ ID No.4 ⁇ SEQ ID No.6.
  • a method for screening soybeans with high soybean saponin content the steps are as follows:
  • the CTAB method was used to extract DNA from soybean leaves.
  • the configuration system of 100mL 2 ⁇ CTAB buffer is shown in Table 6.
  • nucleic acid extraction solution 24:1 chloroform: isoamyl alcohol
  • nucleic acid extraction solution 24:1 chloroform: isoamyl alcohol
  • step (12) Repeat step (12) once and leave it at room temperature overnight. Add 50 ⁇ L of water the next day to dissolve it and store it in a -20°C refrigerator for long-term storage.
  • nucleic acid protein detector uses a nucleic acid protein detector to detect the concentration of extracted DNA, and then use ultrapure water to dilute the spare DNA to 50ng/ ⁇ L.
  • the PCR amplification system was: the total volume was 10 ⁇ L, where Including soybean sample DNA template (50ng/ ⁇ L) 5 ⁇ L, 2 ⁇ KASP Master mix 5 ⁇ L, primer mixture 0.14 ⁇ L; PCR reaction conditions are: first stage 94°C 15min; second stage 94°C 20sec, 61 ⁇ 55°C 1min, each The annealing temperature was reduced by 0.6°C for each cycle, for a total of 10 cycles; the third stage was 94°C for 20sec, 55°C for 1min, for a total of 26 cycles; the fourth stage was 37°C for 1min.
  • the blue dots near the Y-axis are soybean varieties with low saponin content carrying G allelic variation sites, and the red dots near the X-axis The dots are soybean varieties with high saponin content carrying the A allelic mutation site, and the black dots are the blank control.
  • the soybean saponin Ab content of soybean varieties with genotype AA was higher than that of GG in both years, and the soybean saponin Ab content increased by 177.84% and 230.38%.
  • the present invention conducts whole-genome correlation analysis on the soybean saponin Aa, Ab, and Bb contents of 264 soybean germplasm natural populations, which makes the soybean saponin SNP sites obtained by the correlation analysis more reliable, and enables the discovery of soybean saponin candidate genes and the development of genetic markers. More precise. And the SNP sites significantly related to the two saponin components are used to develop KASP markers.
  • the KASP markers of the present invention not only have high accuracy and low cost, but are also simple and fast to operate, providing a basis for in-depth research on soybean saponin-related genes in the future. And lay the foundation for the screening of high saponin content in soybeans and molecular marker-assisted breeding of soybeans.

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Abstract

本发明提供了一种大豆皂昔相关的KASP标记及其应用,属于分子育种技术领域，所述KASP标记包括S07_43139773G/T和SO7_43139033A/G中的一种或两种，所述S07_43139773G/T为大豆基因组第7号染色体43139773bp位置上的碱基为G/T,所述S07_43139033A/G为大豆基因组第7号染色#43139033bp位置上的碱基为A/G。本发明的KASP标记能够用于大豆高大豆皂昔含量种质的选育，只需要提取植物叶片基因组DNA,就可以进行PCR扩增，有助于大豆皂昔有关基因的深入研究和大豆高皂昔含量的筛选以及大豆分子标记辅助育种。

Description

一种大豆皂苷相关的KASP标记及其应用
本申请要求于2022年09月16日提交中国专利局、申请号为202211126454.4、发明名称为“一种大豆皂苷相关的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
本发明属于分子育种技术领域，尤其涉及一种大豆皂苷相关的KASP标记及其应用。
背景技术
大豆皂苷，又称大豆皂甙，是由大豆及其它豆类植物种子中提取出来的，其分子是由低聚糖及齐墩果烯三萜缩合形成的一类化合物。目前使用化学法研究大豆皂苷，确认大豆皂苷元有5个种类，即大豆皂苷元A、大豆皂苷元B、大豆皂苷元C、大豆皂苷元D、大豆皂苷元E，天然存在的大豆皂苷有3种，即大豆皂苷元A、大豆皂苷元B、大豆皂苷元E。研究表明，大豆皂苷具有抗脂质氧化、抗自由基、增强免疫调节、抗肿瘤、抗病毒等多种生理功能，以应用于食品、药品、化妆品等领域，具有广阔的市场前景。
虽然大豆皂苷对人类健康发挥着重要作用，但目前对大豆资源的利用程度较低，专用型大豆品种少，培育专用型大豆具有很好的发展前景。大豆皂苷受多基因控制，容易受到土壤环境，气候变化等外界环境因素的影响。在常规的筛选中，每个大豆种质的皂苷都进行测定，存在鉴定周期长、成本高、费时费力的问题，不能满足稳定高效的筛选需求。
发明内容
有鉴于此，本发明的目的在于提供一种大豆皂苷相关的KASP标记及其应用。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种大豆皂苷相关的KASP标记，所述KASP标记包括S07_43139773 G/T和S07_43139033 A/G中的一种或两种，所述 S07_43139773 G/T为大豆基因组第7号染色体43139773bp位置上的碱基为G/T，所述S07_43139033 A/G为大豆基因组第7号染色体43139033bp位置上的碱基为A/G。
优选的，所述S07_43139773 G/T处碱基是G的大豆为皂苷Aa含量低的大豆，碱基是T的大豆为皂苷Aa含量高的大豆。
优选的，所述S07_43139033 A/G碱基是A的大豆皂苷Ab含量高的大豆，碱基是G的大豆皂苷Ab含量低的大豆。
本发明还提供了一种用于检测上述KASP标记的引物对，所述KASP标记的引物对包括S07_43139773 G/T引物对和S07_43139033 A/G引物对中的一种或两种；
所述S07_43139773 G/T的上游引物F1的序列如SEQ ID No.1所示，上游引物F2的序列如SEQ ID No.2所示，下游引物R的序列如SEQ ID No.3所示；
所述S07_43139033 A/G的上游引物F1的序列如SEQ ID No.4所示，上游引物F2的序列如SEQ ID No.5所示，下游引物R的序列如SEQ ID No.6所示。
本发明还提供了一种用于检测上述KASP标记的试剂盒，所述试剂盒包含上述的引物对。
本发明还提供了一种上述KASP标记、引物对或试剂盒在提高大豆皂苷种质育种中的应用。
本发明还提供了一种筛选高皂苷含量大豆的方法，包括如下步骤：
以待测大豆基因组DNA为模板，利用上述的引物对分别进行PCR扩增反应，通过PCR扩增产物的荧光信号，进行基因分型。
优选的，选择大豆基因组中第7号染色体43139773bp位置上的基因型为TT和大豆基因组中第7号染色体43139033位置上的基因型为AA中的一种或两种，则待测大豆为高皂苷含量大豆。
相对于现有技术，本发明具有如下有益效果：
本发明提供了一种大豆皂苷相关的KASP标记及其应用，本发明对264份大豆种质自然群体大豆皂苷Aa、Ab、Bb含量进行了全基因组关联分析，并利用与两种皂苷组分显著相关的SNP位点进行KASP标记的开 发，设计得到2个KASP标记，本发明的S07_43139773 G/T检测大豆皂苷含量的准确率为93.2％，S07_43139033 A/G检测大豆皂苷含量的准确率为93.2％，故本发明的KASP标记可准确对大豆皂苷含量性状进行基因分型，通过基因分型发现S07_43139773 G/T基因型为TT大豆的大豆皂苷Aa含量高于GG的大豆皂苷Aa含量，S07_43139033 A/G基因型为AA大豆的大豆皂苷Ab含量高于GG的大豆皂苷Ab含量。因此，本发明的KASP标记能够用于大豆高大豆皂苷含量种质的选育，且本发明的KASP标记不仅准确度高、成本较低，而且操作简单、快捷，只需要提取植物叶片基因组DNA，就可以进行PCR扩增。
说明书附图
图1为自然群体重测序结果，其中A为各个品种来源分布，B为进化树，C为主成分分析，D为连锁不平衡分析；
图2为3种大豆皂苷全基因组关联分析结果的曼哈顿图和QQ图，A、B分别是2020年海南大豆皂苷Aa、Ab；C、D分别是2021年南京大豆皂苷Aa、Ab；
图3为KASP标记对不同大豆种质基因分型，其中A、B分别是对S07_43139773、S07_43139033进行基因分型。
具体实施方式
下面结合实施例和附图对本发明进一步说明。
本发明提供了一种大豆皂苷相关的KASP标记，所述KASP标记包括S07_43139773 G/T和S07_43139033 A/G中的一种或两种，所述S07_43139773 G/T为大豆基因组第7号染色体43139773bp位置上的碱基为G/T，所述S07_43139033 A/G为大豆基因组第7号染色体43139033bp位置上的碱基为A/G。
在本发明中，当KASP标记包括S07_43139773 G/T和S07_43139033 A/G中的一种或两种时，均与大豆皂苷含量相关。作为一优选的实施方式，本发明KASP标记优选的为S07_43139773 G/T和S07_43139033 A/G中的 一种。作为另一优选的实施方式，本发明KASP标记优选为S07_43139773 G/T和S07_43139033 A/G。
在本发明中，所述S07_43139773 G/T处碱基是G的大豆优选为皂苷Aa含量低的大豆，碱基是T的大豆为皂苷Aa含量高的大豆。所述S07_43139033 A/G碱基是A的大豆皂苷Ab含量高的大豆，碱基是G的大豆皂苷Ab含量低的大豆。
本发明还提供了一种用于检测上述KASP标记的引物对，所述KASP标记的引物对包括S07_43139773 G/T引物对和S07_43139033 A/G引物对中的一种或两种；
所述S07_43139773 G/T的上游引物F1的序列如SEQ ID No.1所示，上游引物F2的序列如SEQ ID No.2所示，下游引物R的序列如SEQ ID No.3所示；
所述S07_43139033 A/G的上游引物F1的序列如SEQ ID No.4所示，上游引物F2的序列如SEQ ID No.5所示，下游引物R的序列如SEQ ID No.6所示。
在本发明中，所述KASP标记的引物对包括S07_43139773 G/T引物对和S07_43139033 A/G引物对中的一种或两种。作为一优选的实施方式，所述KASP标记的引物对优选为S07_43139773 G/T引物对和S07_43139033 A/G引物对中的一种。作为另一优选的实施方式，所述KASP标记的引物对包括S07_43139773 G/T引物对和S07_43139033 A/G引物对。
本发明还提供了一种用于检测上述KASP标记的试剂盒，所述试剂盒包含上述的引物对。
在本发明中，所述试剂盒优选的包括PCR扩增反应液，进一步优选的，所述PCR扩增反应液包括S07_43139773 G/T引物对和S07_43139033 A/G引物对中的一种或两种，所述PCR扩增反应液还包括大豆样品DNA模板和2×KASP Master mix。
本发明还提供了一种上述KASP标记、引物对或试剂盒在提高大豆皂苷种质育种中的应用。
本发明还提供了一种筛选高皂苷含量大豆的方法，包括如下步骤：
以待测大豆基因组DNA为模板，利用上述的引物对分别进行PCR扩增反应，通过PCR扩增产物的荧光信号，进行基因分型。
在本发明中，选择大豆基因组中第7号染色体43139773bp位置上的基因型为TT和大豆基因组中第7号染色体43139033位置上的基因型为AA中的一种或两种，则待测大豆为高皂苷含量大豆。选择大豆基因组中第7号染色体43139773bp位置上的基因型为GG和大豆基因组中第7号染色体43139033位置上的基因型为GG中的一种或两种，则待测大豆为低皂苷含量大豆。
在本发明中，选择大豆基因组中第7号染色体43139773bp位置上的基因型为TT，则待测大豆为高大豆皂苷Aa含量大豆，基因型为GG，则待测大豆为低大豆皂苷Aa含量大豆，基因型为TT的大豆种质的大豆皂苷Aa含量高于GG的大豆皂苷Aa含量。选择大豆基因组中第7号染色体43139033bp位置上的基因型为AA，则待测大豆为高大豆皂苷Ab含量大豆，基因型为GG，则待测大豆为低大豆皂苷Ab含量大豆，基因型为AA的大豆种质的大豆皂苷Ab含量高于GG的大豆皂苷Ab含量。在本发明中，本发明通过大豆基因组中第7号染色体43139773bp位置上的基因型为TT和大豆基因组中第7号染色体43139033bp位置上的基因型为AA，则待测大豆的大豆皂苷含量显著提高。
在本发明中，以待测大豆基因组DNA为模板，利用上述的引物对分别进行PCR扩增反应。所述PCR扩增体系优选的包括45～55ng/μL大豆样品DNA模板5μL，2×KASP Master mix 5μL，KASP Assay Mix(F1:F2:R＝1:1:1)0.14μL。KASP-PCR扩增反应条件为：第一阶段94℃15min；第二阶段94℃20s，61～55℃1min，每个循环退火温度降低0.6℃，共10个循环；第三阶段94℃20s，55℃1min，共26个循环；第四阶段37℃1min。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
(1)关联群体：选出来自全国各地的具有代表性的264份大豆，包含52份地方种和212份栽培种，另外收集野生豆19份，组成了微核心种质资源，共283份，大豆材料主要来自黄淮海地区和中国南方生态区，少 部分来自中国北方生态区(如图1中的A所示)。
(2)重测序：对283份材料进行重测序，平均测序深度为12.4×，获得SNP标记10210329个，基于3319306个高质量SNP构建了大豆系统发育树，可以有效地将野生大豆、地方品种和改良品种区分开来，其中地方种和改良品种从野生大豆进化、人工选择而来(图1中的B)；主成分分析(图1中的C)表明野生大豆能够有效地同地方种和改良种区分开来，地方种和改良品种更为接近，野生大豆和非野生大豆之间存在较大差异。连锁不平衡(LD)分析表明(图1中的D)，所有材料的LD值约为106kb，野生大豆的LD值约为33kb，而非野生大豆材料的LD值约为120kb。
(3)大豆皂苷GWAS分析：用于全基因组关联分析自然群体的SNP标记来自重测序工作，该高密度物理图谱共包含2597425个SNP。全基因组关联分析采用基于R软件的GAPIT算法包进行计算，采用混合线性模型(MLM)进行全基因组关联分析以控制假阳性关联位点。以P≤1/2597425＝3.85×10 -7，-LogP≥6.4作为显著阈值，当SNP的-LogP≥6.4时，则被认为是显著关联位点，而当SNP的阈值处于5≤-LogP＜6.4时，则被认为是潜在关联位点。
采用高效液相色谱方法分别检测了2020年收获于海南基地和2021年收获与南京六合基地的大豆自然群体的大豆皂苷含量，对两年的大豆皂苷含量表型数据进行全基因组关联分析。GWAS分析可视化结果如图2所示。
采用Agilent 1260高效液相色谱仪(HPLC)检测大豆皂苷的含量，检测器使用蒸发光散射检测器(ELSD)，大豆皂苷含量的检测步骤如下：
(1)大豆皂苷标准样品的制备
称取四种大豆皂苷标品各0.01g(精确至0.0001g)，用甲醇(色谱纯)配置成浓度为1mg/mL的母液，再梯度稀释浓度为0.6、0.5、0.4、0.3、0.2、0.1、0.08、0.08、0.04、0.02mg/mL，过0.22m滤膜备用，其中配置的大豆皂苷标品溶液必须现配现用。
(2)大豆皂苷的提取
将收取的大豆种子放入烘箱以80℃烘干至恒重，之后用植物组织研 磨仪器充分磨碎成粉末；准确称取1g(精确至0.01g)大豆粉放入15ml离心管中，加入70％(V/V)乙醇水溶液10ml；40℃超声水浴提取40min(40kHz)，期间不时的摇晃离心管以便充分提取皂苷；离心机3000rpm离心5min；用1ml针管吸取上清液经0.22m滤膜过滤至进样瓶中，放入-20℃冰箱待用。
(3)高效液相检测
HPLC色谱条件如下所示，色谱柱：Thermo BDS Hypersil C18(250mm×4.6mm，5μm)；流动相A：0.2％(V/V)乙酸水溶液，流动相B：100％(V/V)乙腈溶液；流速：1.0mL/min；柱温：30℃；进样量：10μL；HPLC洗脱程序见表1。每个样品重复进样三次，根据标品绘制的标准曲线计算样品中大豆皂苷组分的含量，取三次进样测得的平均值来作为大豆皂苷组分最终的检测结果。
表1 HPLC洗脱程序
Figure PCTCN2022131935-appb-000001
表2自然群体中与大豆皂苷关联的SNP位点数量
Figure PCTCN2022131935-appb-000002
由表2和图2结果表明，2020年海南与大豆皂苷Aa关联(-log 10(P)≥5)的SNPs共有1450个，显著关联(-log 10(P)≥6.4)的SNPs的SNPs共有1163个，分别分布在2号、7号和17号染色体上；与大豆皂苷Ab关联的SNPs一共1331个，显著关联的SNPs共有818个，分布在7号染色体；与Bb关联的SNPs只有10个，并没有显著关联的位点。
2021年南京与大豆皂苷Aa关联的SNPs一共存在1678个，显著性关联的SNPs共有1345个，比2020年多182个SNPs，存在于7号、9号和14号染色体上；而大豆皂苷Ab与大豆皂苷Aa情况不同，与2020年相比，大豆皂苷Ab显著性相关的SNPs反而少589个，为229个，分别分布在7号和9号染色体上；值得注意的是，2021年大豆皂苷Bb比2020年多出了4个显著性关联的SNP位点，在5号和14号染色体上分别存在2个显著位点(见表2)。
表3各个皂苷显著相关的SNPs
Figure PCTCN2022131935-appb-000003
由表3可知，2020年三亚大豆皂苷Aa和Ab极显著SNP位置分别位于7号染色体43139773bp和43139612bp处，-log 10(P)值极大值分别为10.63和13.44；2021年南京大豆皂苷Aa与Ab极显著SNP位置也都在7号染色体上，分别在S07染色体43918953bp和43217800bp处，-log 10(P)值极大值分别为13.26和8.72，大豆皂苷Bb在S05染色体33311337处，-log 10(P)值极大值为7.66。
表4两年自然群体中与大豆皂苷关联的SNP共位点数量
Figure PCTCN2022131935-appb-000004
表4结果表明，2020年与2021年自然群体与大豆皂苷关联的SNP位点存在许多共位，两年中大豆皂苷Aa与Ab的SNP共位点都在7号染色体上，分别为1306和301个，显著性关联(-log10(P)≥6.4)的SNP共位点分别为928和227个，但是大豆皂苷Bb不存在共位现象。
实施例2
一种大豆皂苷相关的KASP标记，所述KASP标记为S07_43139773 G/T和S07_43139033 A/G。
每一对引物包括两个特异性的正向引物F1和F2和一个通用的反向引物R，其中F1和F2分别包含6-羧基荧光素(FAM)和六氯-6-甲基荧光素(HEX)荧光接头序列(下划线)，引物序列由擎科生物(南京)合成，序列如表5所示。
表5 KASP标记的特异性引物
Figure PCTCN2022131935-appb-000005
实施例3
一种大豆皂苷相关的KASP标记，所述KASP标记为S07_43139773 G/T。
KASP标记引物对包括两个特异性的正向引物F1和F2和一个通用的反向引物R，其中F1和F2分别包含6-羧基荧光素(FAM)和六氯-6-甲基荧光素(HEX)荧光接头序列(下划线)，引物序列由擎科生物(南京)合成，上游引物F1、上游引物F2和下游引物R分别为SEQ ID No.1、SEQ ID No.2、SEQ ID No.3所示的序列。
实施例4
一种大豆皂苷相关的KASP标记，所述KASP标记为S07_43139033 A/G。
KASP标记引物对包括两个特异性的正向引物F1和F2和一个通用的反向引物R，其中F1和F2分别包含6-羧基荧光素(FAM)和六氯-6-甲基荧光素(HEX)荧光接头序列(下划线)，引物序列由擎科生物(南京)合成，上游引物F1、上游引物F2和下游引物R分别为SEQ ID No.4、SEQ ID No.5、SEQ ID No.6所示的序列。
实施例5
一种大豆皂苷相关的KASP标记，所述KASP标记为S07_43139773 G/T和S07_43139033 A/G。
KASP标记引物对包括两个特异性的正向引物F1和F2和一个通用的反向引物R，其中F1和F2分别包含6-羧基荧光素(FAM)和六氯-6-甲基荧光素(HEX)荧光接头序列(下划线)，引物序列由擎科生物(南京)合成，上游引物F1、上游引物F2和下游引物R分别为SEQ ID No.1～SEQ ID No.3所示的序列和SEQ ID No.4～SEQ ID No.6所示的序列。
实施例6
一种用于S07_43139773 G/T KASP标记的试剂盒，所述试剂盒包括S07_43139773 G/T上游引物F1、上游引物F2和下游引物R，分别为SEQ ID No.1、SEQ ID No.2、SEQ ID No.3所示的序列。
实施例7
一种用于S07_43139033 A/G KASP标记的试剂盒，所述试剂盒包括 S07_43139033 A/G上游引物F1、上游引物F2和下游引物R，分别为SEQ ID No.4、SEQ ID No.5、SEQ ID No.6所示的序列。
实施例8
一种用于S07_43139773 G/T和S07_43139033 A/G KASP标记的试剂盒，所述试剂盒包括S07_43139773 G/T和S07_43139033 A/G上游引物F1、上游引物F2和下游引物R，分别为SEQ ID No.1～SEQ ID No.3所示的序列和SEQ ID No.4～SEQ ID No.6所示的序列。
实施例9
一种筛选高大豆皂苷含量大豆的方法，步骤如下：
1)提取待测大豆的DNA：
摘取基地播种的R6时期新鲜大豆叶片放入2mL的离心管中，离心管底部放有小钢珠，再放入液氮或者带有冰盒的泡沫盒中冷冻，保存用于提取基因组DNA。
利用CTAB法提取大豆叶片的DNA，100mL 2×CTAB buffer的配置体系如表6所示。
表6 DNA提取液体系
Figure PCTCN2022131935-appb-000006
配置好DNA提取液之后，DNA提取的具体操作步骤如下所示：
(1)将2×CTAB buffer带到通风橱加入2％的β-巯基乙醇，每10mL CTAB加入200μL的β-巯基乙醇。
(2)将之前收取样品的2mL离心管与磨样盒一起放入液氮进行冷冻，液氮刚好覆盖磨样盒。
(3)将离心管对称放入磨样盒，放入植物组织研磨仪中进行磨样， 植物组织以40kHz运行30s，运行完毕之后再次将样品放入液氮。
(4)将样品带到通风橱，加入600μL的2×CTAB buffer，盖上盖子迅速混匀。
(5)65℃水浴半小时，每5min摇晃一次离心管。
(6)水浴结束后带到通风橱进行第一次抽提，加入等体积核酸提取液24:1(氯仿：异戊醇)，上下颠倒混匀，摇晃3～5min。
(7)离心机10000r/min离心5min，吸取上清600μL到新的1.5mL离心管中。
(8)将上一步样品带到通风橱进行第二次抽提，加入等体积核酸提取液24:1(氯仿：异戊醇)，上下颠倒混匀。
(9)离心机10000r/min离心5min，吸取上清400μL到新的1.5mL离心管中。
(10)在离心管中加入20μL 5M的NaCl，再加入400μL放入-20℃冰箱预冷后的异丙醇，轻轻混匀，放入-20℃冰箱30min以上。
(11)离心机12000r/min离心5min，倒去废液。
(12)加入600μL 70％的乙醇，用手指将沉淀弹起，之后再用离心机12000r/min离心5min后倒去废液。
(13)重复步骤(12)一次后常温放置过夜，第二天加入50μL的水进行溶解，放入-20℃冰箱长期保存。
用核酸蛋白检测仪检测提取的DNA浓度，再用超纯水将备用的DNA稀释至50ng/μL。
2)针对与大豆皂苷Aa、Ab、Bb显著关联的SNP位点S07_43139773(G/T)和S07_43139033(A/G)开发KASP标记，标记序列如表5所示，以步骤1)提取得到大豆基因组的DNA为模板，用相应的引物F1、F2、R分别进行PCR扩增，反应在QuantStudio5实时荧光定量PCR仪中进行，得到PCR扩增产物，其中PCR扩增体系为：总体积为10μL，其中包括大豆样品DNA模板(50ng/μL)5μL，2×KASP Master mix 5μL，引物混合物0.14μL；PCR反应条件为：第一阶段94℃15min；第二阶段94℃20sec，61～55℃1min，每个循环退火温度降低0.6℃，共10个循环；第三阶段94℃20sec，55℃1min，共26个循环；第四阶段37℃1min。
3)反应结束后，在QuantStudio5实时荧光定量PCR仪读取反应产物的荧光数据，利用KASP分子标记引物在实时荧光定量PCR仪上对24份大豆进行扩增并进行基因分型。
表7不同基因型大豆种质的皂苷含量
Figure PCTCN2022131935-appb-000007
图3结果表明：两种分子标记引物对都可以清楚的将两种基因型分开。
图3和表7结果表明，对于针对大豆皂苷Aa显著关联的SNP标记设计的KASP标记—S07_43139773 G/T，其中靠近Y轴的蓝色圆点为携带T等位变异位点的高皂苷含量的大豆品种，靠近X轴的红色圆点为携带G等位变异位点的皂苷含量低的大豆品种，黑色圆点为空白对照。基因型为TT的大豆品种的大豆皂苷Aa含量在两年中都要高于GG的大豆皂苷Aa含量，且大豆皂苷Aa含量提高159.02％和94.47％。
对于针对大豆皂苷Ab显著关联的SNP标记设计的KASP标记—S07_43139033 A/G，其中靠近Y轴的蓝色圆点为携带G等位变异位点的皂苷含量低的大豆品种，靠近X轴的红色圆点为携带A等位变异位点的高皂苷含量的大豆品种，黑色圆点为空白对照。基因型为AA的大豆品种的大豆皂苷Ab含量在两年中都要高于GG的大豆皂苷Ab含量，且大豆皂苷Ab含量提高177.84％和230.38％。
本发明对264份大豆种质自然群体大豆皂苷Aa、Ab、Bb含量进行了全基因组关联分析，这使关联分析得到的大豆皂苷SNP位点更加可靠，大豆皂苷候选基因的发掘和遗传标记的开发更加精确。并利用与两种皂苷组分显著相关的SNP位点进行KASP标记的开发，本发明的KASP标记不仅准确度高、成本较低，而且操作简单、快捷，为今后对于大豆皂苷有 关基因的深入研究和大豆高皂苷含量的筛选以及大豆分子标记辅助育种奠定基础。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。对这些实施例的多种修改对本领域的专业技术人员来说是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (8)

  1. 一种大豆皂苷相关的KASP标记，其特征在于，所述KASP标记包括S07_43139773 G/T和S07_43139033 A/G中的一种或两种，所述S07_43139773 G/T为大豆基因组第7号染色体43139773bp位置上的碱基为G/T，所述S07_43139033 A/G为大豆基因组第7号染色体43139033bp位置上的碱基为A/G。
  2. 根据权利要求1所述的KASP标记，其特征在于，所述S07_43139773 G/T处碱基是G的大豆为皂苷Aa含量低的大豆，碱基是T的大豆为皂苷Aa含量高的大豆。
  3. 根据权利要求1所述的KASP标记，其特征在于，所述S07_43139033 A/G碱基是A的大豆皂苷Ab含量高的大豆，碱基是G的大豆皂苷Ab含量低的大豆。
  4. 一种用于检测权利要求1所述KASP标记的引物对，其特征在于，所述KASP标记的引物对包括S07_43139773 G/T引物对和S07_43139033 A/G引物对中的一种或两种；
    所述S07_43139773 G/T的上游引物F1的序列如SEQ ID No.1所示，上游引物F2的序列如SEQ ID No.2所示，下游引物R的序列如SEQ ID No.3所示；
    所述S07_43139033 A/G的上游引物F1的序列如SEQ ID No.4所示，上游引物F2的序列如SEQ ID No.5所示，下游引物R的序列如SEQ ID No.6所示。
  5. 一种用于检测权利要求1所述KASP标记的试剂盒，其特征在于，所述试剂盒包含权利要求4所述的引物对。
  6. 权利要求1～3任意一项所述KASP标记、权利要求4所述引物对或权利要求5所述试剂盒在提高大豆皂苷种质育种中的应用。
  7. 一种筛选高皂苷含量大豆的方法，其特征在于，包括如下步骤：
    以待测大豆基因组DNA为模板，利用权利要求4所述的引物对分别进行PCR扩增反应，通过PCR扩增产物的荧光信号，进行基因分型。
  8. 根据权利要求7所述的方法，其特征在于，选择大豆基因组中第 7号染色体43139773bp位置上的基因型为TT和大豆基因组中第7号染色体43139033位置上的基因型为AA中的一种或两种，则待测大豆为高皂苷含量大豆。
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