Broad bean wing-related proteins, their encoding genes, molecular markers and their applications

Abstract

This invention relates to the fields of plant genetic engineering and molecular biology, specifically to broad bean wing-related proteins, their encoding genes, molecular markers, and their applications. This invention identifies the broad bean wing-related gene VfSWP1, providing a new gene resource and method for breeding broad bean varieties with short wing petals and low outcrossing rates. The functional molecular marker of the VfSWP1 gene developed in this invention can rapidly detect the short wing petal trait in broad beans, offering advantages such as speed, efficiency, time and labor savings, cost reduction, and accurate identification. It helps to efficiently screen short wing petal varieties with low outcrossing rates, shortens the variety purification cycle, greatly improves breeding efficiency, and promotes the rapid fixation of superior target traits, which is of great significance to the molecular breeding and seed industry development of broad beans.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular biology, and in particular to broad bean wing-related proteins, their encoding genes, molecular markers, and their applications. Background Technology

[0002] Broad beans (Vicia faba L.) are an important annual cool-season legume, used as a grain, vegetable, and forage crop. They also possess biological nitrogen-fixing properties, which can improve soil fertility and increase crop yields, giving them significant ecological advantages in promoting sustainable agricultural development. China is the world's largest producer and consumer of broad beans, ranking first globally in both cultivated area and total output. Broad beans are rich in high-quality protein and various nutrients, which can enhance human immunity and play an important role in improving the dietary structure of the population.

[0003] Broad beans are cross-pollinated crops, generally pollinated by bees, with an average cross-pollination rate of around 30%. Their yield depends on the abundance, activity, and efficiency of the pollinators, and is significantly affected by geographical environment and climate change. Furthermore, a high cross-pollination rate leads to reduced varietal uniformity and stability in the widespread application of broad beans, seriously impacting broad bean breeding and industrial development. Therefore, it is necessary to cultivate broad bean varieties with low cross-pollination rates.

[0004] Studies have shown that short-winged broad bean materials exhibit low outcrossing rates. The applicant previously screened a rare short-winged, low-outcrossing-rate broad bean germplasm from local germplasm in Yunnan, China. This germplasm can reduce natural outcrossing rates by decreasing insect pollination, possibly related to the shorter winged petals affecting bee visits to flowers. However, research on the genes controlling the short-winged trait in broad beans and their functions has not been reported. Therefore, functional analysis of the short-winged gene in broad beans and the development of molecular markers can help to rapidly and efficiently screen varieties with low outcrossing rates, promote the rapid fixation of superior target traits, and thus be of great significance for high and stable yields of broad beans and the development of the seed industry. Summary of the Invention

[0005] This invention provides a broad bean wing-related protein, its encoding gene, molecular marker, and its application.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a broad bean wing-related protein, the amino acid sequence of which is shown in SEQ ID NO.2 or SEQ ID NO.4.

[0008] This invention, through constructing a hybrid population using short-winged broad bean germplasm and common-winged broad bean germplasm as parents and conducting phenotypic identification, discovered that the wing length trait in broad bean is controlled by a single-nuclear gene. Based on this, the location, cloning, and sequence identification of wing-related genes were performed, resulting in the gene controlling the wing length trait in broad bean and its encoded protein, which was named VfSWP1 (Short Wing-Petal of Vicia faba). Specifically, the protein shown in SEQ ID NO.2 is a wing-related protein from the short-winged broad bean germplasm, i.e., the short-winged broad bean wing-related protein, and the protein shown in SEQ ID NO.4 is a wing-related protein from the common-winged broad bean germplasm.

[0009] In this invention, a short winglobe refers to a winglobe whose length is equal to or shorter than that of the keel winglobe. A common winglobe refers to a winglobe that is longer than the keel winglobe; in contrast to a short winglobe, a common winglobe is also referred to as a long winglobe.

[0010] The local germplasm VF8137 from Yunnan, China, and the local germplasm H3712 from Anhui, China, exhibit short and normal wing petals, respectively. To identify the short wing petal-related genes in the short-wing petal broad bean VF8137, this invention used QTL mapping and BSA sequencing to determine that the short wing petal gene is located between 990.2 Mb and 1019.3 Mb on chromosome 3 of broad bean. Furthermore, the short wing petal gene VfSWP1 of VF8137 was cloned and sequenced. The results showed that, compared with the common wing lobe germplasm H3712, the short wing lobe germplasm VF8137 had the following changes in its short wing lobe gene VfSWP1: 6 nucleotides TCAACA were inserted at positions 191-196 bp, T was replaced with A at position 199 bp, C was replaced with T at position 249 bp, T was replaced with C at position 484 bp, T was replaced with A at position 685 bp, G was replaced with C at position 689 bp, 6 nucleotides GAAGAA were inserted at positions 694-699 bp, and T was replaced with C at position 912 bp. These changes resulted in the addition of isoleucine and asparagine at positions 64 and 65, the replacement of serine at position 67 (formerly position 65) with threonine, the replacement of serine at position 229 (formerly position 227) with threonine, the replacement of arginine at position 230 (formerly position 228) with proline, and the addition of two glutamic acid residues at positions 232 and 233, thus altering the protein's function.

[0011] Secondly, the present invention provides a nucleic acid molecule encoding the aforementioned broad bean wing-related protein.

[0012] The aforementioned nucleic acid molecules can be DNA or RNA, where DNA can be cDNA or genomic DNA sequences.

[0013] Based on the amino acid sequence and codon rules of the broad bean wing-related protein provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the broad bean wing-related protein. Due to the degeneracy of codons, the nucleotide sequence encoding a single broad bean wing-related protein is not unique, and all nucleic acid molecules capable of encoding the broad bean wing-related protein are within the protection scope of this invention.

[0014] Preferably, the nucleotide sequence of the nucleic acid molecule encoding the broad bean wing-related protein shown in SEQ ID NO.2 is as shown in SEQ ID NO.1. The nucleotide sequence of the nucleic acid molecule encoding the broad bean wing-related protein shown in SEQ ID NO.4 is as shown in SEQ ID NO.3.

[0015] The nucleic acid molecule shown in SEQ ID NO.1 is from short-winged broad bean germplasm, and the nucleic acid molecule shown in SEQ ID NO.3 is from common-winged broad bean germplasm.

[0016] Thirdly, the present invention provides a biological material comprising the above-described nucleic acid molecules or expressing the broad bean wing-related protein, wherein the biological material is an expression cassette, a vector, or a host cell.

[0017] The expression cassette is a recombinant nucleic acid molecule obtained by operatively linking the nucleic acid molecule with transcriptional and / or translational regulatory elements.

[0018] The vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, etc. The plasmid vectors include cloning vectors, expression vectors, etc.

[0019] The host cell is any cell capable of carrying the aforementioned nucleic acid molecules or expressing the broad bean wing-related protein, including microbial cells or plant cells; the microbial cells include, but are not limited to, Escherichia coli, Agrobacterium, yeast, etc.; the plant cells include non-reproductive plant cells or transgenic plant cell lines, etc. "Non-reproductive" means lacking the characteristic of developing into a complete plant.

[0020] Fourthly, the present invention provides the application of the above-described broad bean wing-related protein, the nucleic acid molecule, or the biological material in regulating the length of plant wing petals.

[0021] Preferably, the plant is a legume. Legumes include, but are not limited to, soybeans, broad beans, peanuts, peas, lentils, and common beans.

[0022] More preferably, the plant is a broad bean.

[0023] Fifthly, the present invention provides the application of the above-described broad bean wing-related protein, the nucleic acid molecule, or the biomaterial in the preparation of short-winged plants, breeding of plant wing length traits, improvement of plant wing length germplasm resources, or reduction of plant crossbreeding rates.

[0024] In the above application, inserting TCAACA at position 191-196 bp, replacing T with A at position 199 bp, replacing C with T at position 249 bp, replacing T with C at position 484 bp, replacing T with A at position 685 bp, replacing G with C at position 689 bp, inserting GAAGAA at position 694-699 bp, and replacing T with C at position 912 bp, the corresponding trait changes from ordinary wing to short wing.

[0025] Preferably, the plant is a legume. Legumes include, but are not limited to, soybeans, broad beans, peanuts, peas, lentils, and common beans.

[0026] More preferably, the plant is a broad bean.

[0027] In a sixth aspect, the present invention provides a method for preparing a short-winged plant, the method comprising: causing the plant to express a broad bean wing-related protein with the sequence shown in SEQ ID NO.2.

[0028] The methods for enabling the plant to express the broad bean wing-related protein sequence as shown in SEQ ID NO.2 include, but are not limited to, using transgenic technology to introduce the nucleic acid molecule encoding the broad bean wing-related protein shown in SEQ ID NO.2 into the plant, or introducing the nucleic acid molecule encoding the broad bean wing-related protein shown in SEQ ID NO.2 into the plant through breeding methods such as hybridization, self-pollination and / or backcrossing.

[0029] Preferably, the plant is a legume. Legumes include, but are not limited to, soybeans, broad beans, peanuts, peas, lentils, and common beans.

[0030] More preferably, the plant is broad bean, and the method includes: mutating the broad bean wing-related protein with the sequence as described in SEQ ID NO.4 to the broad bean wing-related protein with the sequence as shown in SEQ ID NO.2.

[0031] In some embodiments of the present invention, the method includes: mutating the broad bean wing-related protein coding gene with the sequence as described in SEQ ID NO.3 to the broad bean wing-related protein coding gene with the sequence as described in SEQ ID NO.1.

[0032] In a seventh aspect, the present invention provides molecular markers related to the length of broad bean wing petals, the molecular markers containing polymorphic sites at positions 191-196bp (- / TCAACA), 199bp (T / A), 685bp (T / A), 689bp (G / C), and 694-699bp (- / GAAGAA) of the sequence shown in SEQ ID NO.1;

[0033] Alternatively, the molecular marker contains polymorphic sites at positions - / TCAACA at 191-196 bp, T / A at 199 bp, C / T at 249 bp, T / C at 484 bp, T / A at 685 bp, G / C at 689 bp, - / GAAGAA at 694-699 bp, and T / C at 912 bp of the sequence shown in SEQ ID NO.1.

[0034] Among them, - / TCAACA and - / GAAGAA represent deletion / insertion mutations, that is, deletion or insertion of TCAACA or GAAGAA. T / A, C / T, T / C, and G / C represent SNP sites.

[0035] Preferably, the molecular marker is a nucleotide sequence containing polymorphic sites at positions - / TCAACA (191-196 bp), T / A (199 bp), T / A (685 bp), G / C (689 bp), and - / GAAGAA (694-699 bp) of the sequence shown in SEQ ID NO.1; or, a nucleotide sequence containing polymorphic sites at positions - / TCAACA (191-196 bp), T / A (199 bp), C / T (249 bp), T / C (484 bp), T / A (685 bp), G / C (689 bp), - / GAAGAA (694-699 bp), and T / C (912 bp) of the sequence shown in SEQ ID NO.1.

[0036] Among the molecular markers mentioned above, the genotype of the sequence shown in SEQ ID NO.1 is TCAACA with 6 nucleotides inserted at positions 191-196bp, A at position 199bp, A at position 685bp, C at position 689bp, and GAAGAA with 6 nucleotides inserted at positions 694-699bp, which corresponds to the short wing lobe trait, and vice versa.

[0037] Among the molecular markers mentioned above, the genotype of the sequence shown in SEQ ID NO.1 is characterized by the insertion of 6 nucleotides TCAACA at positions 191-196 bp, A at position 199 bp, T at position 249 bp, C at position 484 bp, A at position 685 bp, C at position 689 bp, and the insertion of 6 nucleotides GAAGAA at positions 694-699 bp with a position C at position 912 bp, corresponding to the short wing valve trait. The genotype of the sequence shown in SEQ ID NO.1 is characterized by the deletion of 6 nucleotides TCAACA at positions 191-196 bp, T at position 199 bp, C at position 249 bp, T at position 484 bp, T at position 685 bp, G at position 689 bp, the deletion of 6 nucleotides GAAGAA at positions 694-699 bp, and T at position 912 bp, corresponding to the normal wing valve trait.

[0038] The molecular markers mentioned above can be used to detect the short wing lobe gene VfSWP1 in broad beans, thereby determining the wing lobe length trait in broad beans. This method is particularly suitable for identifying broad bean materials or varieties containing the short wing lobe gene VfSWP1 and their breeding derivatives.

[0039] Eighthly, the present invention provides a primer set for detecting the molecular markers described above that are related to the length of broad bean wing petals.

[0040] Based on the known polymorphic sites of molecular markers, those skilled in the art can design primer sets for detecting the SNP molecular markers using conventional techniques, based on the polymorphic sites and their upstream and downstream sequences. Therefore, this invention does not impose any special restrictions on the sequences of primer sets used to detect the molecular markers, and all primers capable of amplifying the molecular markers and performing genotyping are within the scope of protection of this invention.

[0041] In some specific embodiments of the present invention, the primer set includes the primer pairs shown in SEQ ID NO. 5-6 and the primer pairs shown in SEQ ID NO. 7-8.

[0042] SEQ ID NO.5: Upstream primer F: 5'-ACGGCCATGGCTTCTATCAG-3';

[0043] SEQ ID NO.6: Downstream primer R: 5'-GCTGCAACTGTGTGTCATCC-3'; SEQ ID NO.7: Upstream primer F: 5'-TTGCATCAGCATCACTGCAC-3'; SEQ ID NO.8: Downstream primer R: 5'-AGGTCCCACCGAGAACATCT-3'.

[0044] In a ninth aspect, the present invention provides a kit comprising the primer set described above.

[0045] For ease of detection, the kit may also contain other reagents for PCR amplification, including but not limited to PCR buffer, Mg... 2+ DNA polymerase, dNTPs, etc.

[0046] In a tenth aspect, the present invention provides any of the following applications of the molecular markers related to the length of broad bean wing petals described above, or the primer set or the kit described above:

[0047] (1) Application in detecting or assisting in the detection of the wing length trait of broad bean;

[0048] (2) Application in the preparation of reagents for detecting or assisting in the detection of the length of broad bean wing petals;

[0049] (3) Application in the identification or screening of short-winged broad beans;

[0050] (4) Application in the preparation of reagents for the identification or screening of short-winged broad beans;

[0051] (5) Application in the early prediction of the trait of wing length in broad bean;

[0052] (6) Application in the preparation of reagents for early prediction of the wing length trait of broad bean;

[0053] (7) Application in broad bean genetic breeding;

[0054] (8) Application in broad bean variety improvement;

[0055] (9) Application in the creation of new germplasm of broad bean.

[0056] In (3)-(4) above, the short-winged broad bean is a short-winged broad bean with a low cross-crossing rate.

[0057] In (7) above, the genetic breeding is marker-assisted breeding of broad beans.

[0058] In (8) above, the variety improvement is preferably the variety improvement of the wing petal length trait of broad bean.

[0059] In (9) above, the creation of new germplasm is germplasm innovation of the trait of the length of the broad bean wing petals.

[0060] Preferably, in the application, the genotype of the sequence shown in SEQ ID NO.1 is TCAACA with 6 nucleotides inserted at positions 191-196bp, A at position 199bp, A at position 685bp, C at position 689bp, and GAAGAA with 6 nucleotides inserted at positions 694-699bp, corresponding to the short wing lobe trait; otherwise, it corresponds to the ordinary wing lobe trait.

[0061] Alternatively, in the aforementioned application, the genotype of the sequence shown in SEQ ID NO.1 is characterized by the insertion of 6 nucleotides TCAACA at positions 191-196bp, A at position 199bp, T at position 249bp, C at position 484bp, A at position 685bp, C at position 689bp, and the insertion of 6 nucleotides GAAGAA at positions 694-699bp with C at position 912bp, corresponding to the short wing lobe trait; otherwise, it corresponds to the ordinary wing lobe trait.

[0062] By detecting the above polymorphic sites, the genotypes of broad bean wing-related genes can be identified, thereby determining the length of broad bean wing petals. It can also identify short-winged, low-cross-breeding broad bean varieties bred using these materials.

[0063] Specifically, the applications include:

[0064] 1) Extract DNA from the broad bean sample to be tested;

[0065] 2) Using the DNA as a template, PCR amplification reactions were performed using the primer pairs shown in SEQ ID NO.5-6 and SEQ ID NO.7-8, respectively;

[0066] 3) Detect the PCR amplification products and determine the genotype of the molecular marker based on the product bands.

[0067] The 20 μL PCR reaction system consisted of: 2 μL template DNA (20 ng / μL), 9 μL 2×Taq PCR MasterMix (containing 1 U Taq DNA polymerase), 2 μL each of forward and reverse primers (2 μM / μL), and 5 μL double-distilled water (ddH2O).

[0068] The reaction procedure was as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 45 s, for 32-35 cycles; extension at 72℃ for 5 min, and storage at 4℃.

[0069] The beneficial effects of this invention include at least the following: This invention is the first to identify the broad bean wing-related gene VfSWP1 and short wing-related genes, providing new gene resources and methods for breeding broad bean varieties with low outcrossing rates and short wing petals. This invention successfully developed a functional molecular marker for the gene VfSWP1. Using this molecular marker, the short wing petal trait in broad beans can be rapidly detected, offering advantages such as speed, efficiency, time and labor savings, cost reduction, and accurate identification. This helps to efficiently screen short wing petal varieties with low outcrossing rates, shortens the variety purification cycle, greatly improves breeding efficiency, and promotes the rapid fixation of superior target traits, which is of great significance to the molecular breeding and seed industry development of broad beans. Attached Figure Description

[0070] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0071] Figure 1 This is a phenotypic comparison between short wing lobe germplasm VF8137 and ordinary wing lobe germplasm H3712 in Example 1 of the present invention.

[0072] Figure 2 This shows the distribution of wing length in the hybrid F2 population constructed using short wing lobe germplasm VF8137 and ordinary wing lobe germplasm H3712 as parents in Example 1 of this invention.

[0073] Figure 3 The results of genotyping of the VfSWP1 gene functional molecular markers in different short-winged and long-winged germplasms in Example 2 of this invention are shown. Among them, S041, 042, 048, 051, 053, and 055 are F2 generation plants of crosses between VF8137 and H3712 as parents. Detailed Implementation

[0074] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0075] Example 1: Construction and phenotypic identification of a hybrid population using short-winged wing germplasm VF8137 and ordinary wing germplasm H3712 as parents.

[0076] Differences in wing trait between short-winged germplasm VF8137 and common-winged germplasm H3712 are as follows: Figure 1 As shown. The short-winged germplasm VF8137 (maternal parent) and the common-winged germplasm H3712 (paternal parent) were crossed to obtain the F1 generation hybrids, all of which had common-winged petals. The F1 hybrids were self-pollinated to obtain an F2 segregating population of 167 individual plants. In the F2 segregating population, there were 126 common-winged plants and 41 short-winged plants. The distribution of the short-winged trait is shown in the figure. Figure 2 As shown, the segregation ratio conforms to 3:1 (single-gene statistical test χ²). 2 0.05 =0.002<3.84), indicating that the wing lobe length trait is controlled by a single nucleus gene.

[0077] Example 2: Localization, cloning, and sequence identification of the VfSWP1 gene

[0078] Ten leaves each from the short-winged winged germplasm VF8137 (maternal parent) and the common-winged germplasm H3712 (paternal parent) were used to form two parental mixed pools. From the F2 hybrid population, 30 short-winged plants and 30 common-winged plants were used to form two progeny mixed pools. Genomic DNA was extracted from each broad bean, diluted to the same concentration, and mixed in equal volumes. DNA extraction was performed using a modified CTAB method. The extraction method for the test materials was as follows: approximately 1g of dried silica gel leaves were thoroughly ground into powder in liquid nitrogen and placed in a 2.0mL centrifuge tube for freezing and storage. Preheated 2×CTAB extraction solution to 65℃ was mixed with β-mercaptoethanol at a ratio of 1% of the solution volume. 800μL of the preheated CTAB extraction solution was added to each sample, vortexed for 1-2 minutes, and then incubated in a 65℃ water bath for 1 hour (inverting and mixing every 15 minutes). After the water bath heating was completed, the mixture was centrifuged... Add 800 μL of chloroform / isoamyl alcohol (24:1) solution to the centrifuge tube and mix for 15-20 min. Then centrifuge at 10,000 rpm for 15 min. Transfer approximately 600 μL of the supernatant to a new centrifuge tube (amount only a small amount, avoiding contact with the protein layer). Add an equal volume of 95% ethanol, mix well, and incubate at -20°C for 2 h or 4°C overnight to ensure complete precipitation. Centrifuge at 12,000 rpm for 10 min, discard the supernatant, and retain the precipitate. Rinse the white precipitate with 500 μL of 90% ethanol, centrifuge at 10,000 rpm for 5 min, and carefully discard the supernatant. Air dry at room temperature, add 100 μL of ddH2O, and dissolve the precipitate completely. Detect the DNA concentration of the sample using a Nanodrop 2000 / 2000C instrument, and uniformly dilute the DNA concentration of the sample to 50 ng / μL. Store at 4°C for later use.

[0079] Four BSA libraries were constructed using standard procedures and sequenced on the NovaSeqXten sequencing platform. After standard filtering, high-quality data were obtained for BSA analysis. GATK variant detection and BAS analysis based on SNPs and indel-index identified a candidate region (Chr3:990,200,000-1,019,300,000) on chromosome 3 with a Delta-index of 1. Transcriptome sequencing and differential expression analysis of floral tissues from two parents identified 30 differentially expressed genes within the candidate region. Homology annotation of these genes using homology alignment revealed that one gene was a homolog of the Arabidopsis FAF3 gene, which is known to regulate meristem size and floral development pathways in Arabidopsis. Furthermore, compared to H3712, this gene exhibited two non-synonymous SNP mutations and two INDELs in the exon region of VF8137, and its expression level was 0 in the flower. Therefore, this gene was identified as VfSWP1, a candidate gene controlling wing petal length.

[0080] Based on the VfSWP1 gene sequence, sequencing primers 1 and 2 were designed to extract genomic DNA from the parents of the above-mentioned hybrid population and individual plants of the F2 population, and PCR amplification was performed. The 20 μL PCR reaction system consisted of: 2 μL template DNA (20 ng / μL), 9 μL 2×Taq PCRMasterMix (containing 1 U Taq DNA polymerase), 2 μL each of forward and reverse primers (2 μM / μL), and 5 μL double-distilled water (ddH2O). The reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, 32-35 cycles; 72℃ extension for 5 min, and storage at 4℃.

[0081] The amplified products were separated by 2% agarose gel electrophoresis, and the purified PCR products were sequenced to compare the differences in this candidate gene between short-winged and common-winged plants. Sequencing results showed that the VfSWP1 gene sequence from the short-winged germplasm is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. The VfSWP1 gene sequence from the common-winged germplasm is shown in SEQ ID NO.3, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.4.

[0082] like Figure 3As shown, compared with the VfSWP1 gene sequence of the common wing lobe germplasm, the VfSWP1 gene sequence of the short wing lobe germplasm has the following insertions: TCAACA at position 191-196 bp, T replaced with A at position 199 bp, C replaced with T at position 249 bp, T replaced with C at position 484 bp, T replaced with A at position 685 bp, G replaced with C at position 689 bp, GAAGAA at position 694-699 bp, and T replaced with C at position 912 bp.

[0083] Example 3: Functional Molecular Marker and Application of the Broad Bean Short Wing Lobe Gene VfSWP1

[0084] Based on the sequence alignment results of the VfSWP1 gene in short-winged broad bean plants and common-winged broad bean plants, functional molecular markers for the short-winged gene VfSWP1 were further developed. These functional markers include three major SNP sites affecting amino acid variations, located at bases 199, 685, and 689 of the short-winged broad bean VfSWP1 sequence. Plants with these three bases being A, A, and C are short-winged broad bean plants, while plants with these three bases being T, T, and G are common-winged broad bean plants. The functional markers also include two insertion variations affecting amino acid variations, located at positions 191-196 and 694-699 of the short-winged broad bean VfSWP1 sequence. Plants with these two sequences being TCAACA and GAAGAA are short-winged broad bean plants, while plants with these two sequences being deleted are common-winged broad bean plants.

[0085] Based on the sequence of the functional molecular marker, two pairs of primers for detecting the functional molecular marker were designed: Primer 1 includes upstream primer F5'-ACGGCCATGGCTTCTATCAG-3' (SEQ ID NO.5) and downstream primer R5'-GCTGCAACTGTGTGTCATCC-3' (SEQ ID NO.6); Primer 2 includes upstream primer F5'-TTGCATCAGCATCACTGCAC-3' (SEQ ID NO.7) and downstream primer R5'-AGGTCCCACCGAGAACATCT-3' (SEQ ID NO.8).

[0086] Identifying broad bean short-winged germplasm or varieties using the above-mentioned functional molecular markers includes the following steps:

[0087] 1) Extract genomic DNA from the broad beans to be tested, using the same extraction method as described in Example 2;

[0088] 2) Using the extracted DNA as a template, a 620 bp VfSWP1 fragment 1 was amplified by PCR using primer 1 for amplifying the functional molecular marker; a 394 bp VfSWP1 fragment 2 was amplified by PCR using primer 2 for amplifying the functional molecular marker. The PCR reaction system and procedure were the same as described in Example 2.

[0089] 3) Detect the PCR amplification products and use BioEdit software to proofread and assemble the VfSWP1 fragment 1 and fragment 2 sequences of all samples. If the 191-196bp, 199bp, 685bp, 689bp, and 694-699bp sequences in the VfSWP1 gene sequence are TCAACA, A, A, C, and GAAGAA, respectively, then the broad bean to be tested is short-winged petal germplasm.

[0090] Using the aforementioned functional molecular markers, over 160 accessions of the F2 hybrid population (using VF8137 and H3712 as parents) were analyzed. Thirty-six accessions were found to have TCAACA, A, A, C, and GAAGAA sequences at bps 191-196, 199, 685, 689, and 694-699 of the VfSWP1 gene, respectively, all exhibiting the short winged petal phenotype. Therefore, this functional molecular marker can be used to rapidly and accurately identify short-winged broad bean resources or varieties in VF8137 or its derivatives.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A molecular marker associated with the long / short wing trait in Vicia faba, characterized in that, The nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and the polymorphic sites are: / TCAACA at 191-196bp, T / A at 199bp, T / A at 685bp, G / C at 689bp, and / GAAGAA at 694-699bp. Alternatively, the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1, and the polymorphic sites are: / TCAACA at 191-196bp, T / A at 199bp, C / T at 249bp, T / C at 484bp, T / A at 685bp, G / C at 689bp, / GAAGAA at 694-699bp, and T / C at 912bp.

2. A primer set characterized in that, The primer set is used for detecting the molecular marker related to the length of wing petal of Vicia faba as claimed in claim 1.

3. The primer set of claim 2, wherein, The primer set comprises the primer pair shown as SEQ ID NO. 5-6 and the primer pair shown as SEQ ID NO. 7-8.

4. A kit, characterized in that, The kit comprises the primer set as claimed in claim 2 or 3.

5. Use of the molecular marker related to the length of wing petal of Vicia faba as claimed in claim 1 or the primer set as claimed in claim 2 or 3 or the kit as claimed in claim 4 in detecting or assisting in detecting the length of wing petal of Vicia faba.

6. Use of the molecular marker related to the length of wing petal of Vicia faba as claimed in claim 1 or the primer set as claimed in claim 2 or 3 or the kit as claimed in claim 4 in the preparation of reagents for detecting or assisting in detecting the length of wing petal of Vicia faba.

7. Use of the molecular marker related to the length of wing petal of Vicia faba as claimed in claim 1 or the primer set as claimed in claim 2 or 3 or the kit as claimed in claim 4 in the identification or screening of Vicia faba with short wing petal.

8. Use of the molecular marker related to the length of wing petal of Vicia faba as claimed in claim 1 or the primer set as claimed in claim 2 or 3 or the kit as claimed in claim 4 in the preparation of reagents for the identification or screening of Vicia faba with short wing petal.

9. Use of the molecular marker related to the length of wing petal of Vicia faba as claimed in claim 1 or the primer set as claimed in claim 2 or 3 or the kit as claimed in claim 4 in the early prediction of the length of wing petal of Vicia faba.

10. Use of the molecular marker related to the length of wing petal of Vicia faba as claimed in claim 1 or the primer set as claimed in claim 2 or 3 or the kit as claimed in claim 4 in the preparation of reagents for the early prediction of the length of wing petal of Vicia faba.

11. Use of the molecular marker related to the length of wing petal of Vicia faba as claimed in claim 1 or the primer set as claimed in claim 2 or 3 or the kit as claimed in claim 4 in the genetic breeding of Vicia faba.

12. Use of the molecular marker related to the length of wing petal of Vicia faba as claimed in claim 1 or the primer set as claimed in claim 2 or 3 or the kit as claimed in claim 4 in the improvement of Vicia faba varieties.

13. Use of the molecular marker related to the length of wing petal of Vicia faba as claimed in claim 1 or the primer set as claimed in claim 2 or 3 or the kit as claimed in claim 4 in the creation of new germplasm of Vicia faba.

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