CN116064909A

CN116064909A - Molecular marker related to soybean keel valve opening and application thereof

Info

Publication number: CN116064909A
Application number: CN202211392857.3A
Authority: CN
Inventors: 邱丽娟; 洪慧龙; 邱红梅; 王晓波; 孙如建; 郭兵福; 高华伟; 任洪雷; 袁明; 张勇
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2022-11-08
Filing date: 2022-11-08
Publication date: 2023-05-05

Abstract

The invention relates to the technical field of crop genetic breeding, in particular to a molecular marker related to soybean fossil fragments opening and application thereof. The molecular marker was the presence of an A > C mutation at 33746105 with the gene number Glyma.03g124400. The invention obtains a mutant (OFC) with the flower organ keels being open through mutagenesis, utilizes map-based cloning to determine relevant functional sites, develops identification marks, can conveniently and rapidly detect relevant characters, can screen excellent and abnormal germplasm and provide utilization, and provides material, theory and technical support for soybean heterosis utilization and improvement of outcrossing rate.

Description

Molecular marker related to soybean keel valve opening and application thereof

Technical Field

The invention relates to the technical field of crop genetic breeding, in particular to a molecular marker related to soybean fossil fragments opening and application thereof.

Background

With the growing population, jonathan and Tilman et al predict that current crop yields must double before 2050 to meet the food consumption demands of world population growth. To meet this demand, crop yield needs to increase at a rate of at least 2.4% per year. The cultivated soybean [ Glycine max (L.) Merr ] belongs to the Leguminosae (Leguminosae), papilionate (Papilionate), the genus Glycine (Glycine), annual herbaceous plants of the subgenera Soja are important crops, are main sources of vegetable proteins and oils in people's lives, are also important feed crops, and account for about 56% of the global edible oil yield. Ray et al mention that current annual yield increases of soybeans are about 1.3%, far from reaching the expected requirements. Therefore, searching a high-efficiency breeding strategy to select and breed high-yield and high-quality soybean varieties is the most urgent problem to be solved by current soybean breeders. In the soybean breeding method, the original natural mutation selection breeding, cross breeding, mutation breeding, introduction, heterosis breeding, high light effect breeding, pollen tube channel method breeding, marker assisted selection breeding, transgenic breeding and other methods are also developed, wherein the molecular breeding changes the traditional breeding method based on phenotype selection into the breeding method directly aiming at genotype selection, and the selection efficiency is improved. At present, heterosis is widely applied to various crops, and plays a vital role in increasing the yield of the crops.

The soybean is self-pollinated crop, the outcrossing rate is 0.03% -3.62%, and the mechanical pollination obstacle caused by the special butterfly petal structure causes the difficulty of soybean outcrossing pollination, causes the difficulty of soybean hybrid seed production, and restricts the way of improving the yield of the soybean through heterosis. Therefore, knowing the organ composition of soybean flowers creates petal-opening materials, and improving the soybean outcrossing rate has important value for improving the yield.

Molecular marker breeding is an important auxiliary means of current breeding work, and the key point is to develop efficient molecular markers. The molecular marker which is closely linked with the target character and is stable and convenient and quick to detect directly selects the genotype, can greatly provide the accuracy and the efficiency of selection in breeding and obviously shorten the breeding period. Therefore, the development of the efficient molecular marker has important significance for breeding new varieties of variety improvers.

Disclosure of Invention

In order to solve the problems, the invention provides a molecular marker related to soybean keel valve opening and application thereof. The molecular marker provided by the invention can distinguish the soybeans with open keel petals from the soybeans with closed keel petals, and provides technical support for creating petal opening materials and improving the soybean outcrossing rate.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a molecular marker related to soybean keel valve opening, wherein the molecular marker is that A & gtC mutation exists at 33746105 with the gene number of Glyma.03g124400.

The invention also provides a protein related to soybean fossil fragments opening, and the amino acid sequence of the protein is shown as SEQ ID NO. 1.

The invention also provides a gene for encoding the protein, and the CDS sequence of the gene is shown as SEQ ID NO. 2.

The invention also provides a primer pair for amplifying the molecular marker, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4.

The invention also provides the use of the above molecular markers or the above proteins or the above genes or the above primer pairs in one or more of the following aspects, including:

1) Preparing a product for identifying or screening open-ended soybeans of the dragon bone;

2) Identifying or screening open keel soybean;

3) And (5) auxiliary cultivation of the soybean variety with the open dragon bone flap.

The invention also provides a kit for identifying or screening open keel soybean, which comprises the primer pair.

The invention also provides a method for identifying or screening open keel soybean, comprising the following steps:

performing PCR amplification by taking genomic DNA of soybean to be identified as a template to obtain an amplification product; the primers used in the PCR amplification comprise the primer pair;

judging the properties of the soybean dragon bone flap according to the genotypes of the corresponding sites of the molecular markers in the amplified products, wherein the properties are as follows:

if the genotype of the corresponding site is AA or AC, the soybean to be identified is closed dragon bone flap;

and if the genotype of the corresponding site is CC, the soybean to be identified is an open keel valve.

Preferably, the method for judging the genotype comprises sequencing or electrophoresis;

when the judgment method is electrophoresis, the method comprises the following steps: enzyme digestion is carried out on the amplified product by utilizing aluI enzyme, and electrophoresis analysis is carried out on the enzyme digestion product;

if only one 282bp electrophoresis band exists, the genotype of the corresponding locus of the molecular marker is CC;

if only one 257bp electrophoresis band exists or only two 257bp electrophoresis bands which are respectively 257bp and 25bp electrophoresis bands exist, the genotype of the corresponding locus of the molecular marker is AA;

if two electrophoresis bands of 282bp and 257bp respectively exist or three electrophoresis bands of 282bp, 257bp and 25bp respectively exist, the genotype of the corresponding site of the molecular marker is AC.

Preferably, the reaction process of the PCR amplification comprises: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72℃for 5min.

The invention also provides a main effect QTL with the soybean keel valve open, wherein the main effect QTL is positioned on a soybean chromosome 3, and the physical position is between SSR1043 and SSR1045, and the interval size is 58Kb.

The beneficial effects are that:

the invention provides a molecular marker related to soybean keel valve opening, wherein the molecular marker is that A & gtC mutation exists at 33746105 with the gene number of Glyma.03g124400. The invention obtains a mutant with the flower organ fossil fragments opened through mutagenesis, utilizes map-based cloning to determine relevant functional sites, develops identification marks, can conveniently and rapidly detect relevant characters, can screen excellent and abnormal germplasm and provide utilization, and provides material, theory and technical support for soybean heterosis utilization and improvement of outcrossing rate.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.

FIG. 1 is a comparative graph of wild type WT, ofc flower organ phenotypes provided by the invention;

FIG. 2 is a diagram of candidate gene structure provided by the present invention;

FIG. 3 is a diagram of a preliminary candidate interval for BSA sequencing result analysis provided by the present invention;

FIG. 4 is a fine localization map of the keel valve opening gene provided by the invention;

FIGS. 5-1 and 5-2 show the expression characteristics of genes in a fine positioning candidate interval in tissues such as parent interflower organs, leaves and the like; * P < 0.01, P < 0.05;

FIG. 6 is a diagram showing the alignment difference between the amino acid sequences of the wild type and the mutant of the candidate gene provided by the invention;

FIG. 7 is a verification electrophoresis diagram of 54 parts of known genetic material of domestic and foreign germplasm resources against a molecular marker OFC 17;

fig. 8 is a graph showing the identification results of the dCAPS marker OFC17 of 83 keel valve openings OFC and keel valve closed LY populations provided by the invention.

Detailed Description

The invention utilizes the Keel closed variety 661 to carry out phenotype identification by mutagenesis treatment and combining with full-bloom stage field flower organ observation to obtain Keel open material OFC (namely Keel open mutant, which can also be marked as OFC), utilizes LY (Keel closed mutant) to hybridize with OFC to obtain hybrid seeds, selfs the hybrid seeds in Keel open and Keel closed individuals in F2 generation separation groups to construct Keel closed pools and Keel open pools, carries out BSA-seq sequencing, combines the separation groups to carry out linkage analysis and confirmation interval, screens to obtain a gene Gmofc related to Keel open, and compares Gmofc genes of Keel open material and Keel closed material, screens to obtain a molecular marker related to bean Keel open in a 3 chromosome Gmofc gene (the Gmofc gene is Glyma.03g124400 in a version 2.1 Genbank database) at a second exon physical position 33746105, designs dCAPS marker OF OFC, can identify hybrid and provides a differential soybean support theory and provides a differential cross-strain and a differential soybean support technology by utilizing the characteristics of the Gmofc.

The invention also provides a protein related to soybean fossil fragments opening, the amino acid sequence of the protein is shown as SEQ ID NO.1, and the protein is specifically as follows:

MREILHVQGGQCGNQIGSKFWEVVCDEHGIDPTGKYVGNSDLQLERVNVYYNEASCGRFVPRAVLMDLEPGTMDSVRTGPYGQIFRPDNFVFGQSGAGNNWAKGHYTEGAELIDSVLDVVRKEAENCDCLQGFQVCHSLGGGTGSGMGTLLISKIREEYPDRMMLTFSVFPSPKVSDTVVEPYNATLSVHQLVENADECMVLDNEALYDICFRTLKLTTPSFGDLNHLISATMSGVTCCLRFPGQLNSDLRKLAVNLIPFPRLHFFMVGFAPLTSRGSQQYRALTVPELTQQMWDAKNMMCAADPRHGRYLTASAMFRGKMSTKEVDEQMINVQNKNSSYFVEWIPNNVKSSVCDIAPRGLSMASTFIGNSTSIQEMFRRVSEPFTAMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATAEDDGEYEDEEDDDVEADHM。

the protein is the protein expressed by Gmofc gene in the keel valve opening material ofc, namely the protein expressed by keel valve opening soybean, can regulate and control the opening of the keel valve, and is the amino acid (proline) which is thickened and underlined in the amino acid sequence, wherein the amino acid (proline) at 384 th coded amino acid is changed from glutamine to proline due to the fact that wild soybean plants have A & gtC mutation at the molecular marker.

The invention also provides a gene for encoding the protein, wherein the CDS sequence of the gene is shown as SEQ ID NO.2, and the gene is specifically as follows:

atgagggagatccttcacgttcagggagggcaatgcgggaaccagatcggttcgaagttctgggaagtggtgtgcgacgagcacggcatagatccgacggggaagtacgtcggaaactcagatctacaactcgagcgcgtgaacgtgtactacaacgaagcttcctgcgggcggtttgtcccacgcgcagtgctgatggacctggagcccggaaccatggacagcgtgcggactgggccgtacgggcagatattccggcccgacaacttcgtgttcgggcagtccggcgcgggcaacaactgggccaaggggcactacacggagggcgccgagctcatcgactccgtccttgacgtcgtgcgcaaggaggccgagaactgcgactgcctccaggggttccaggtctgccactcgctcggcggaggaaccggctccgggatggggacgcttttgatttccaagatcagagaggagtaccctgacagaatgatgctcaccttctccgtttttccttcccccaaggtctctgatactgtggttgagccttataacgctactctttctgttcatcagttggtggagaatgctgatgagtgtatggtgcttgataatgaggcgctctacgatatctgcttcaggactctcaagttgaccactcctagctttggtgacttgaaccacttgatctctgcgaccatgagtggtgttacatgctgtcttcgattccctggtcaactcaactctgatctgaggaaattggccgtgaatctcattcccttccctcgtctgcacttcttcatggttggatttgcgcctctcacctctcgcggctctcagcaataccgcgcattgacagtgccagagctgacacagcaaatgtgggatgccaagaacatgatgtgtgctgcagatccaaggcacgggcgttacctcacggcatcagccatgttccgtggcaagatgagcaccaaggaggtggacgagcagatgataaacgtgcagaacaagaactcttcatattttgtcgagtggattcccaacaatgtcaagtcgagcgtgtgtgacattgctcctagagggctctccatggcgtccacattcattggaaactcgacctcgattcaggagatgttcaggagggtgagtgagccgttcacggccatgtttaggaggaaggctttcttgcattggtacaccggggaaggcatggacgagatggagttcacagaggcagagagcaacatgaacgaccttgtttcagagtaccagcagtaccaggatgccactgccgaggatgatggggagtatgaggacgaggaggacgatgatgttgaagcagaccacatgtga. The gene of the invention is a gene for regulating and controlling the opening of the soybean keel valve (namely Gmofc gene of the keel valve opening material), a thickened and underlined base C in SEQ ID No.2 is a mutation site at the molecular marker, and a base of a wild type (namely soybean keel valve closing type) of the gene is A.

The invention also provides a primer pair for amplifying the molecular marker, which comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.3, and the specific steps are as follows: CATGATGTGTGCTGCAGATCCA; the nucleotide sequence of the downstream primer is shown as SEQ ID NO.4, and is specifically as follows: TTCCTCCTAAACATGGCCGTGAGC.

The primer pair provided by the invention can specifically amplify the fragments containing the molecular markers, and the amplified fragments are subjected to enzyme digestion by utilizing aluI, and the genotype of the material to be identified at the molecular marker sites can be judged through electrophoresis, so that the technical support is provided for screening the soybeans with open keel petals.

The invention also provides the use of the above molecular markers or the above proteins or the above genes or the above primer pairs in one or more of the following aspects, including: 1) Preparing a product for identifying or screening open-ended soybeans of the dragon bone; 2) Identifying or screening open keel soybean; 3) And (5) auxiliary cultivation of the soybean variety with the open dragon bone flap.

performing PCR amplification by taking genomic DNA of soybean to be identified as a template to obtain an amplification product; the primers used in the PCR amplification include: the primer pair described above or the primer pair in the kit described above;

judging the characters of the soybean dragon bone flap according to the genotypes of the corresponding sites of the molecular markers in the amplified products, wherein the characters are as follows:

if the genotype of the corresponding locus is AA or AC, the soybean to be identified is closed keel valve;

if the genotype of the corresponding site is CC, the soybean to be identified is open keel valve.

In the present invention, the reaction procedure of the PCR amplification preferably includes: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72℃for 5min.

In the present invention, the reaction system for PCR amplification preferably comprises, in terms of 20. Mu.L, 1 to 2. Mu.L of the template, 0.6. Mu.L of each of the upstream primer and the downstream primer, 0.2. Mu.L of 10 Xeasy Taq Buffer 2. Mu. L, dNTPs 2. Mu. L, easyTaq polymerase and the balance ddH ₂ O。

In the present invention, the method for judging the genotype preferably includes sequencing or electrophoresis, more preferably electrophoresis; the method of electrophoresis preferably comprises non-denaturing polyacrylamide gel electrophoresis;

when the judgment method is electrophoresis, it is preferable to include: enzyme digestion is carried out on the amplified product by utilizing aluI enzyme, and electrophoresis analysis is carried out on the enzyme digestion product;

In the present invention, it is preferable to analyze the electrophoresis band of 282bp and the electrophoresis band of 257bp as a control to determine the size of the electrophoresis band of the cleavage product.

The amplification product is subjected to enzyme digestion by Alu I enzyme, so that the genotype of a sample to be identified can be judged rapidly and accurately according to the enzyme digestion result; because the sizes of the genotype strips are not different, the conventional agarose gel electrophoresis cannot be effectively distinguished, so that the non-denaturing polyacrylamide gel electrophoresis is selected to effectively distinguish the electrophoresis strips after enzyme digestion; in addition, in theory, two electrophoresis bands of 257bp and 25bp respectively are needed after the wild type amplification product is digested, but because of the limitation of the prior electrophoresis technology, in order to distinguish different genotypes, only 257bp and 282bp bands can be simultaneously observed at present, so that in the identification process, whether 257bp and 282bp are observed can distinguish the genotypes of the sample to be identified, and the electrophoresis band of 25bp is not needed to be further observed.

The invention also provides a main effect QTL for soybean keel flap opening, wherein the main effect QTL is positioned on a soybean chromosome 3, and the physical position is between SSR1043 (BARCSOYSSR_03_1043) and SSR1045 (BARCSOYSSR_03_1045), and the interval size is 58Kb. The main effect QTL provided by the invention can realize co-separation of soybeans with soybean dragon bone flap closure property and soybean with soybean dragon bone flap opening property.

For further explanation of the present invention, the following examples are provided to describe in detail a molecular marker related to soybean keel valve opening and its application, but they should not be construed as limiting the scope of the present invention.

In the examples, wild soybean germplasm intermediate 661 (WT) keel valve closing and keel valve opening mutant ofc was provided by institute of crop sciences, academy of agricultural sciences, china, keel valve closing mutant LY and keel valve opening mutant ofc constructed hybrid population F1 was identified by the teacher generation of institute of agriculture and grazing sciences Sun Rujian in allenbel.

Example 1

Construction and fine localization of Gmofc gene group in soybean

1. Genetic population construction: the Beijing Changpin test base of the national academy of agricultural science crop institute in 2018 uses the keel valve opening mutant ofc as a male parent and the keel valve closing mutant LY as a female parent to prepare hybridization combination and obtain hybridization grains. In 2019, the F1 generation plants are propagated and seeds are harvested by the institute of agriculture and animal husbandry in Hulunebel, inner Mongolian, the F2 generation group is planted in the Beijing-Changpin test base of the institute of agricultural science, china, and the F3 generation group is continuously planted in the Hainan-Litsea farm test base of the institute of agricultural science, china, and the phenotype identification is carried out to verify the genotype of the F2 generation group.

2. Population genetic analysis: in 2020, the difference of the opening habits of the keel valve is counted for the F2 group, genetic analysis (table 1) is calculated, and the genetic rule of the opening of the keel valve is verified, so that the result shows that the opening phenotype of the keel valve is controlled by a recessive single gene. Ofc is a keel valve open material and wild type is a closed material as determined by flower organ comparison (FIG. 1)

TABLE 1 Keel valve patency Gene Gmofc genetic analysis

3. Primary localization of BSA with soybean keel valve open: and selecting 30 keel valve closed individuals and 30 keel valve open individuals from the F2 group of the combined LY X ofc, constructing a keel valve closed pool and a keel valve open pool, and parent materials thereof, and performing deep resequencing on the DNA mixed pools of 4 materials on a Hiseq2500 platform. Taking the Williams 82 genome sequence as a reference, exploring single nucleotide polymorphism sites between two pool genome sequences, and obtaining 1 candidate region related to the characters by utilizing an SNP-index association algorithm, wherein the total length is 18.33Mb; ED association algorithm, which obtains 1 candidate region related to the character, the total length is 18.90Mb, and the intersection of the two methods obtains 1 candidate region related to the character, and the total length is 18.33Mb. The associated region contains 938 genes altogether, 10 of which are nonsensical mutant genes (FIG. 3).

Fine positioning of soybean keel flap opening: extracting single plant genome DNA of the F2 population of LY× ofc, preliminarily determining candidate gene positions by utilizing initial positioning interval candidate gene annotation information and combining phenotype (keel valve opening or closing character), developing polymorphism markers through parent difference InDel to identify the genotype of the F2338 strain of the population, combining F3 phenotype, and narrowing the candidate interval between SSR1043 (BARCOYSSR_03_1043) and SSR1045 (BARCOYSSR_03_1045) with interval size of 58Kb (figure 4).

The 58Kb interval was analyzed for annotation genes in the reference genome using the soybean genome annotation website (https:// soybase. Org /), and the results are shown in Table 2, which indicate that the localization interval contains 3 annotation genes, 1 known encoding protein Gene and 2 genes with unknown functions, and no reported soybean keel flap opening related genes are found in the interval, indicating that the Gmofc (i.e., gene1 in Table 2) keel flap opening Gene located by the invention is a novel Gene.

TABLE 2 Gene annotation information within a localization interval

By using a fluorescence quantitative PCR technology, comparing Gene expression differences (fig. 5-1 and 5-2) between a candidate interval Gene in a wild keel valve closure intermediate product 661, a group parent keel valve closure LY and a keel valve opening mutant ofc sepal tissue, a petal tissue, a stalk tissue, a bud tissue and a flower bud tissue, the result shows that the Gene expression quantity of each tissue between two parents is highest and the difference is extremely obvious, and besides the Gene1 expression quantity of each tissue between the two parents is lower and the difference is not obvious. Combining gene annotation and sequencing results to obtain Gnofc as a candidate gene.

Example 2

Separation and structural analysis of Gmofc gene in soybean

Isolation of Gmofc Gene

Extracting total RNA from a wild soybean variety ZP661, carrying out reverse transcription on the total RNA to obtain cDNA as a template, and respectively carrying out PCR amplification by using a forward primer (shown as SEQ ID NO. 5: ATGAGGGAGATCCTTCACGTTCAG) and a reverse primer (shown as SEQ ID NO. 6: TGTTGAAGCAGACCACATGTGA), wherein the PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 120s,35 cycles; extending at 72 ℃ for 8min; the PCR amplification reaction system comprises: 5. Mu.L of template (100 ng/. Mu.L), 1. Mu.L of forward primer and 10 Xeasy Taq Buffer (Mg-free) each ²⁺ ) 25 mu L, dNTPs (2.5 mM) 10. Mu.L, easy Taq polymerase (full gold, AP 111) 0.6. Mu.L and ddH ₂ O7.4. Mu.L. A cDNA fragment of the Gmofc gene with the length of 1350bp is obtained, as shown in SEQ ID NO. 7:

atgagggagatccttcacgttcagggagggcaatgcgggaaccagatcggttcgaagttctgggaagtggtgtgcgacgagcacggcatagatccgacggggaagtacgtcggaaactcagatctacaactcgagcgcgtgaacgtgtactacaacgaagcttcctgcgggcggtttgtcccacgcgcagtgctgatggacctggagcccggaaccatggacagcgtgcggactgggccgtacgggcagatattccggcccgacaacttcgtgttcgggcagtccggcgcgggcaacaactgggccaaggggcactacacggagggcgccgagctcatcgactccgtccttgacgtcgtgcgcaaggaggccgagaactgcgactgcctccaggggttccaggtctgccactcgctcggcggaggaaccggctccgggatggggacgcttttgatttccaagatcagagaggagtaccctgacagaatgatgctcaccttctccgtttttccttcccccaaggtctctgatactgtggttgagccttataacgctactctttctgttcatcagttggtggagaatgctgatgagtgtatggtgcttgataatgaggcgctctacgatatctgcttcaggactctcaagttgaccactcctagctttggtgacttgaaccacttgatctctgcgaccatgagtggtgttacatgctgtcttcgattccctggtcaactcaactctgatctgaggaaattggccgtgaatctcattcccttccctcgtctgcacttcttcatggttggatttgcgcctctcacctctcgcggctctcagcaataccgcgcattgacagtgccagagctgacacagcaaatgtgggatgccaagaacatgatgtgtgctgcagatccaaggcacgggcgttacctcacggcatcagccatgttccgtggcaagatgagcaccaaggaggtggacgagcagatgataaacgtgcagaacaagaactcttcatattttgtcgagtggattcccaacaatgtcaagtcgagcgtgtgtgacattgctcctagagggctctccatggcgtccacattcattggaaactcgacctcgattcaggagatgttcaggagggtgagtgagcagttcacggccatgtttaggaggaaggctttcttgcattggtacaccggggaaggcatggacgagatggagttcacagaggcagagagcaacatgaacgaccttgtttcagagtaccagcagtaccaggatgccactgccgaggatgatggggagtatgaggacgaggaggacgatgatgttgaagcagaccacatgtga。

the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.8, and has 449 amino acids (WT in FIG. 6) in total, and is specifically as follows:

MREILHVQGGQCGNQIGSKFWEVVCDEHGIDPTGKYVGNSDLQLERVNVYYNEASCGRFVPRAVLMDLEPGTMDSVRTGPYGQIFRPDNFVFGQSGAGNNWAKGHYTEGAELIDSVLDVVRKEAENCDCLQGFQVCHSLGGGTGSGMGTLLISKIREEYPDRMMLTFSVFPSPKVSDTVVEPYNATLSVHQLVENADECMVLDNEALYDICFRTLKLTTPSFGDLNHLISATMSGVTCCLRFPGQLNSDLRKLAVNLIPFPRLHFFMVGFAPLTSRGSQQYRALTVPELTQQMWDAKNMMCAADPRHGRYLTASAMFRGKMSTKEVDEQMINVQNKNSSYFVEWIPNNVKSSVCDIAPRGLSMASTFIGNSTSIQEMFRRVSEQFTAMFRRKAFLHWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATAEDDGEYEDEEDDDVEADHM。

gmofc Gene structural analysis

Extracting DNA in young leaves of a wild soybean variety zp661, taking the genomic DNA as a template, and amplifying by two sections of overlapping primers (products obtained by amplifying the two pairs of primers are respectively sequenced and then the sequencing results are combined) to obtain a Gmofc genome fragment, wherein the primers are respectively a primer pair 1OFC1244-1F, a primer pair GACTCAATCGAATTGCACCCCA (SEQ ID NO. 9) and a primer pair 2OFC 1244-1R, a primer pair 2OFC1244-2F, wherein the primers are respectively a primer pair 1OFC1244-1F, a primer pair 2OFC primer pair 1F, a primer pair 2OFC primer pair 2F, and a primer pair 1F, wherein the primer pair 1 is prepared by the primer pair is prepared by the steps of sequencing and sequencing: CCAAGGGCATATCCGTCACA (SEQ ID NO. 11) and OFC1244-2R: GGTGGCTCCTTTACCAGTTCA (SEQ ID NO. 12), the reaction procedure and the reaction progress of the primer pair 1 and the primer pair 2 are the same as in step 1.

The Gmofc genome fragment has a total length of 2939bp and comprises 2 exons and 2 introns (figure 2), and is specifically shown as SEQ ID NO. 13:

actctcactataaaaactccctcactcactcttcttccaaaaactatctctctctctgtttatctccgcactccgtcttcatcaatcagcatcaccaccgctccaacacaggtacttactctttcgcttcttctcaaattctctctctctagatctagaattagaggttaattagtaagttaattatggtagtaattagcaaggttttgaatatctgtcacggttgcgttgctattgttgatatcgaggatagttaaaaaatcttgatgttgcggctgaaatcaccgttccttggtaattagtaagtaattaagtaacgagtgagtggtttttgaaatgcaggggaaaatgagggagatccttcacgttcagggagggcaatgcgggaaccagatcggttcgaagttctgggaagtggtgtgcgacgagcacggcatagatccgacggggaagtacgtcggaaactcagatctacaactcgagcgcgtgaacgtgtactacaacgaagcttcctgcgggcggtttgtcccacgcgcagtgctgatggacctggagcccggaaccatggacagcgtgcggactgggccgtacgggcagatattccggcccgacaacttcgtgttcgggcagtccggcgcgggcaacaactgggccaaggggcactacacggagggcgccgagctcatcgactccgtccttgacgtcgtgcgcaaggaggccgagaactgcgactgcctccaggggttccaggtctgccactcgctcggcggaggaaccggctccgggatggggacgcttttgatttccaagatcagagaggagtaccctgacagaatgatgctcaccttctccgtttttccttcccccaaggtctctgatactgtggttgagccttataacgctactctttctgttcatcagttggtggagaatgctgatgagtgtatggtgcttgataatgaggcgctctacgatatctgcttcaggactctcaagttgaccactcctagctgtaagttcatcagaaaccctaatttctgatccgaatgttagagtgtgtgtgtgtttagttataataatttatttttgaagtaactagtgtttagtttcgtcatgcgaaaccgtagtttctgatctgattgttattgagtctcgatgtggtcgaggacctgcggtttctaaaattggtttagaataaaaatgattttagttcaaattaattttaaagtaactagtgtttagtttcatcatgtgaaagtgttgagtttgtgcgtggttgaggtttctaaaattgatatattttgaataaaaatgattttagttcaaattaatcttgaagtaggtgtgttaagtttacatcatgaattgattttgactgtagtcaaaacttgattttatatgcttggttattgcttgctaaattgtttatctcaaaattttggtataaatataatcactttacttcaaatcaatttaaccataattaatttttggaaatcaattttgtaatcattcatccatacacaatacatgcaatttatgtttttagatgtttttattctataaacgagttattagtgtaaattattttattcattgcaaacattacttatcttataattattttaattcaaaatcaattcaaaatcaaatccttaatgtaaaattagtcatgttaacatgtacttaaaattgtgttatagtaggtagtgcttcaaagtgattctgatgccaaacatggcgagatgatgtgtgaattgtgattgtttgtgttgcagttggtgacttgaaccacttgatctctgcgaccatgagtggtgttacatgctgtcttcgattccctggtcaactcaactctgatctgaggaaattggccgtgaatctcattcccttccctcgtctgcacttcttcatggttggatttgcgcctctcacctctcgcggctctcagcaataccgcgcattgacagtgccagagctgacacagcaaatgtgggatgccaagaacatgatgtgtgctgcagatccaaggcacgggcgttacctcacggcatcagccatgttccgtggcaagatgagcaccaaggaggtggacgagcagatgataaacgtgcagaacaagaactcttcatattttgtcgagtggattcccaacaatgtcaagtcgagcgtgtgtgacattgctcctagagggctctccatggcgtccacattcattggaaactcgacctcgattcaggagatgttcaggagggtgagtgagcagttcacggccatgtttaggaggaaggctttcttgcattggtacaccggggaaggcatggacgagatggagttcacagaggcagagagcaacatgaacgaccttgtttcagagtaccagcagtaccaggatgccactgccgaggatgatggggagtatgaggacgaggaggacgatgatgttgaagcagaccacatgtgaagagactgaatgtgaaagatgcctgtgtcttacttatgctatgtgatgcgtaagtgtgtttgctctgctctccaacgtgaagaagattgtaattactaatagtgtatgtacggtttgcgggataaaactttgatgagcttggtgtggagaatggaaaaaatagggggttagtgggagtggagtttgggaacaatgttatgcggcacctatctgtttgttcttatattggcttgcttttatgttgttatattgagtcatgtttccgtaatatttgttgttcttatgcatgacttttgtctacgttggattcttggaatgtctttggagaaagccttgcctcatgtttttgtctatactgtgttgaggtcaaggtgtgatttaggatactgatcgaatctagttatcctttgagaaagtggaagattaaccccttgctctacatatatgcataacagagcaacaaagattccaatca。

example 3

Development and utilization of functional markers of Gmofc gene in soybean

By utilizing the sequence difference of the wild soybean zp661 and the mutant ofc and designing a primer according to the conserved sequences at the two ends of the SNP locus, 1 dCAPS mark is successfully developed and used for identifying the population, and the information of the dCAPS mark is shown in Table 3.

TABLE 3dCAPS Mark information

The 1238F 2 recessive genotypes of the keel valve opening mutant ofc and the keel valve closing mutant LY of example 1 were identified as follows:

performing PCR amplification by taking genomic DNA of soybean to be identified as a template to obtain an amplification product;

enzyme digestion is carried out on the amplified product by utilizing aluI enzyme, and electrophoresis analysis is carried out on the enzyme digestion product;

if only one 282bp electrophoresis band exists, the genotype of the corresponding locus of the molecular marker is CC, which is shown as the opening of the keel valve;

if only one 257bp electrophoresis band exists, the genotype of the corresponding site of the molecular marker is AA, which is shown as closure of the keel valve;

if two electrophoresis bands of 282bp and 257bp are respectively arranged, the genotype of the corresponding site of the molecular marker is AC, which is shown as closure of the keel valve.

The PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72℃for 5min.

The PCR amplification reaction system comprises: template (100 ng/. Mu.L) 2. Mu.L, upstream primer and downstream primer (shown in Table 3) each 0.6. Mu.L, 10 XEasyTaq Buffer (Mg-free) ²⁺ ) 2 mu L, dNTPs (2.5 mM) 2 mu L, easy Taq polymerase (full gold, AP 111) 0.2 mu L and ddH ₂ O 12.6μL。

The method for non-denaturing polyacrylamide gel electrophoresis is as follows:

to the digested product, 4. Mu.L of 10×loading Buffer was added, and after mixing, 1.5. Mu.L of the sample was taken and detected by Non-denaturing polyacrylamide gel electrophoresis (Non-denaturing Polyacrylamide Gel Electrophoresis, non-9 PAGE). And (3) plate manufacturing: cleaning plate and concave glass, standing on a horizontal table top, scrubbing with 70% ethanol to ensure no residual adhesive on the plates, buckling the two plates, and concaveThe plate is arranged under the flat plate, the middle cavity is used for filling gel, and the two sides of the plate are clamped by the clamps, so that no liquid leakage is ensured. And (3) glue filling: 30mL of the pre-formulated gel was prepared, 1% by volume of Ammonium Persulfate (APS) and 1% by volume of TMED were added, mixed horizontally and poured gently between the two plates. Inserting a cleaned comb, wherein the comb teeth extend into the gel by about 1.0 cm; setting at normal temperature for 30 min-1 hr, and observing at any time to prevent gel shrinkage crack deformation. And (3) an upper plate: removing the solidified gel, washing the gel clean (removing residual glue), slightly pulling out the comb, and ensuring that the sample application holes are complete and no residual glue exists; the two plates are relatively and vertically arranged and fixed on the electrophoresis tank by the clip, and the buffer solution is ensured not to leak; 0.5 XTBE buffer was added to each of the upper and lower electrophoresis tanks, so that the lower side was allowed to pass through the card tank and the upper side was allowed to pass through the spotting well. Spotting: cleaning the sample application hole by using an injector, blowing out impurities such as bubbles, broken glue and the like from the sample application hole, injecting 1.0-1.5 mu L of non-denatured PCR product added with a small amount of bromophenol blue indicator into the sample application hole by using a micropipette, and injecting D2000 DNAmaror into the sample application holes at the two ends of the sample application hole as a strip mark. Electrophoresis: and running for 30-40 min at the voltage of 250V, observing a bromophenol blue indication band at any time, and running the gel for a period of time which depends on the size of the band. Silver staining: removing the electrophoresis tank clamp after electrophoresis, removing the gel plate, prying open the plate and the concave plate, taking out the gel, and slowly adding 1% AgNO ₃ Silver staining is carried out in the aqueous solution, the gel is ensured to be completely contacted with the silver staining solution by gently shaking, the gel is taken out after 3 to 5min, and the gel is washed by deionized water for 30 seconds. Developing: the gel was placed in a developer (1L deionized water, 1.5g NaOH and 5-10 mL formaldehyde were added and mixed well) for development, after the gel was observed at any time until the bands on the gel were clear, the gel was removed into deionized water for washing to terminate the development and wash the developer. Sealing: cleaning a table top, spreading a layer of preservative film, spraying a little clear water, spreading the developed gel on the preservative film, spraying a little clear water, sealing with the preservative film, filtering out excessive water and air in the preservative film by using a plastic plate, and placing the gel in a fume hood for a period of time in parallel to remove formaldehyde.

The 1238 keel flap open plants were all identical in genotype (figure 8 is a photograph of 83 individuals for dCAPS marker development and identification).

The genotypes of the domestic and foreign planting resources are identified by using the developed dCAPS markers (the identification method is the same as that above), so that 39 keel valve closed genotypes are the same as the genotypes of the wild zp661, the dCAPS marker development efficiency of the research is verified, and the success of dCAPS marker development is obtained (FIG. 7 is a 45-variety dCAPS marker development identification photograph).

Table 4 45 soybean varieties with known flower organ, fossil fragments and valve opening characteristics at home and abroad

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Note that, in table 4, the numbers 3 to 47 are provided by the institute of crop sciences of the national academy of agricultural sciences, and the phenotype was identified as a keel valve closing material.

In conclusion, the molecular marker provided by the invention can distinguish the soybean with the open keel valve from the soybean with the closed keel valve, and provides technical support for creating a petal-open material and improving the soybean outcrossing rate.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A molecular marker associated with soybean keel valve opening, wherein said molecular marker is the presence of a > C mutation at 33746105 under the gene number glyma.03g124400.

2. A protein related to soybean fossil fragments opening, which is characterized in that the amino acid sequence of the protein is shown in SEQ ID NO. 1.

3. A gene encoding the protein of claim 2, wherein the CDS sequence of the gene is shown in SEQ ID No. 2.

4. A primer pair for amplifying the molecular marker of claim 1, wherein the primer pair comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID No.3, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 4.

5. Use of a molecular marker according to claim 1 or a protein according to claim 2 or a gene according to claim 3 or a primer pair according to claim 4 in one or more of the following aspects, comprising:

2) Identifying or screening open keel soybean;

6. A kit for identifying or screening open keel soybean, comprising the primer pair of claim 4.

7. A method of identifying or screening open keel soybeans comprising the steps of:

performing PCR amplification by taking genomic DNA of soybean to be identified as a template to obtain an amplification product; the primers used in the PCR amplification include: the primer pair of claim 4;

8. The method of claim 7, wherein the genotyping method comprises sequencing or electrophoresis;

9. The method of claim 7, wherein the reaction procedure for PCR amplification comprises: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s, annealing at 58℃for 30s, elongation at 72℃for 30s,35 cycles; extending at 72℃for 5min.

10. The main effect QTL is positioned on a soybean chromosome 3, and the physical position is between SSR1043 and SSR1045, and the interval size is 58Kb.