Molecular marker related to yield traits of arrow leek pea, identification method and application

Abstract

The application discloses a molecular marker related to yield traits of arrow pea, an identification method and application, and belongs to the technical field of biology. The molecular marker is located on chromosome 5 of an arrow pea reference genome (download address: http: / / dx.doi.org / 10.5524 / 100954). The nucleotide sequence of the insertion / deletion fragment is shown as SEQ ID NO. 3. The nucleotide sequences of a primer pair for amplifying the molecular marker are shown as SEQ ID NO. 1-2. The breeding efficiency can be greatly improved by using the molecular marker, and high-yield arrow pea materials can be quickly screened.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to molecular markers, identification methods, and applications related to yield traits of arrowhead pea. Background Technology

[0002] Currently, crop breeding technology systems mainly consist of traditional breeding and molecular breeding. In breeding practice, the targeted selection of target traits highly depends on the association analysis between genotype and phenotype. Molecular marker-assisted breeding, as a key technical pathway in molecular breeding, utilizes the close linkage between molecular markers and target trait genes. By detecting the presence of specific molecular markers, the carrier status of the target gene can be indirectly determined, thereby achieving efficient screening of the target trait. This technology overcomes many limitations of traditional breeding, possessing significant advantages such as high detection efficiency, high accuracy, and immunity to environmental interference. It can significantly shorten the breeding cycle, improve breeding efficiency, and effectively solve technical problems such as complex phenotypic identification and long selection cycles in conventional breeding. Molecular marker technology, with genome-wide association analysis (GWAS) at its core, has become one of the key research hotspots and core competitive directions in the current breeding field.

[0003] Arrowhead pea ( Vicia sativa L . *Vicia sativa* is an important self-pollinating annual legume with multiple values, including feed, green manure, and ecological restoration. Rich in minerals, vitamins, and protein with high digestibility, it is not only a high-quality silage feed for ruminants but also provides starch and protein for human diets. Furthermore, it can fix nitrogen through symbiotic relationships with rhizobia, reducing the need for chemical fertilizers. It is a low-cost, highly adaptable, and eco-friendly crop, currently widely cultivated in Turkey, Australia, New Zealand, and China. Despite its promising application prospects, improving its yield traits still faces significant challenges. Traditional breeding methods rely on natural variation and phenotypic observation, which are time-consuming and inefficient. Determining high-yield traits requires substantial time, financial investment, and manpower, making it difficult to meet the urgent demand for high-quality, high-yield feed in the rapidly developing livestock industry. Existing varieties also generally suffer from low yields and poor stability, hindering the efficient operation of the feed supply chain. To accelerate the breeding process and improve yield and economic benefits, this study combined *Vicia sativa* GWAS with third-generation molecular marker technology to develop the InDel molecular marker, which is closely related to yield traits. This technology can accurately and efficiently locate key gene loci that regulate yield, breaking through the bottleneck of traditional breeding and providing core scientific basis and technical support for the breeding of new high-yielding arrowhead pea varieties. Summary of the Invention

[0004] One of the objectives of this invention is to provide a molecular marker associated with yield traits in arrowhead peas.

[0005] The second objective of this invention is to provide the application of the aforementioned molecular markers related to the yield traits of arrowhead pea.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention discloses a pair of molecular markers related to yield traits in pea shoots, located on chromosome 5 of pea shoots, and the molecular markers are named Vs_Chr5_243849158.

[0007] The molecular marker Vs_Chr5_243849158 is inserted / deleted on chromosome 5 of the arrowhead pea reference genome in the fragment TCAACAACTGAAACATTTAGCGGTCCTCGTCCAAATCTTGCAACAAGACTTCAAAGAAGAGCAAGTATTCGTGAAAAATAAGTTCATCGTCAACTTCAAGGAGATCTAGTC (shown in SEQ ID NO.3). Primer pairs for amplifying molecular markers associated with yield traits in arrowhead peas were used. The primer pair sequence corresponding to the molecular marker Vs_Chr5_243849158 is as follows: Vs_Chr5_243849158-F: TGGAAGACGAACGACATACATA (shown in SEQ ID NO.1); Vs_Chr5_243849158-R: AAGAACTCACATTGGGTCCTAC (shown as SEQ ID NO.2).

[0008] This invention also discloses the application of the above-mentioned molecular marker primer pairs in marker-assisted breeding of yield traits in *Vitis salvia*. Specifically, the molecular markers of this invention can be used in future marker-assisted breeding. DNA is extracted from leaves during the seedling stage, and the presence of the molecular markers of this invention is detected to identify yield traits in *Vitis salvia*. The detection can be performed using PCR, specifically using the above-mentioned molecular marker primer pairs, or it can be performed using sequencing methods.

[0009] This invention also discloses the application of the above-mentioned molecular markers in identifying yield traits in *Vaccaria buergeriana*, particularly in screening and identifying high-yielding *Vaccaria buergeriana*. The specific steps for identifying whether *Vaccaria buergeriana* possesses high-yielding traits are as follows: (1) Using the DNA of the tested germplasm as the template for PCR amplification, and the primer pair corresponding to the label Vs_Chr5_243849158 as the primers, the PCR amplification reaction system is shown in Table 1: Table 1. Reaction system for PCR amplification

[0010] PCR amplification program: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 55℃ annealing for 5 s, 72℃ extension for 5 s, 35 cycles; 72℃ extension for 1 min; store at 4℃.

[0011] (2) Detection of PCR products by agarose gel electrophoresis, and judgment of arrowhead pea yield based on the results: If the PCR amplification product has both a characteristic band of 449 bp as shown in SEQ ID NO.4 and a characteristic band of 338 bp as shown in SEQ ID NO.5, then *Vaccaria buergeriana* is a high-yielding type; if the PCR amplification product has only one characteristic band of 449 bp as shown in SEQ ID NO.4, then *Vaccaria buergeriana* is a low-yielding type.

[0012] Alternatively, reagent kits containing the aforementioned molecular marker primer pairs can be prepared to identify yield traits in *Viburnum fasciatum* materials. Furthermore, these kits can be used to identify or assist in identifying the genotype of *Viburnum fasciatum*. All of these applications can be performed using conventional methods.

[0013] Specific identification methods may include: (1) Extract genomic DNA from the target *Vitis hyacinthus* pea; (2) Using the genomic DNA extracted in step (1) as a template, PCR amplification was performed using the primer pair of the molecular marker Vs_Chr1_112142528; (3) Perform electrophoresis and / or sequencing on the PCR amplification products. If the PCR amplification products have both a characteristic band of 449 bp as shown in SEQ ID NO.4 and a characteristic band of 338 bp as shown in SEQ ID NO.5, then the arrow-shaped pea is a high-yielding genotype; if the PCR amplification products have only a characteristic band of 449 bp as shown in SEQ ID NO.4, then the arrow-shaped pea is a low-yielding genotype.

[0014] The present invention has the following advantages: (1) The molecular markers in this invention are closely related to the yield trait of arrow-shaped peas and can be applied to molecular marker-assisted breeding of arrow-shaped pea yield traits, to the screening of germplasm resources of high-yielding arrow-shaped peas, and to the genetic improvement of arrow-shaped peas.

[0015] (2) The molecular markers of the present invention have the characteristics of convenient detection, stable amplification products and high specificity, and can be applied to arrow pea breeding practice quickly and in high throughput. Attached Figure Description

[0016] Figure 1The results of the genome-wide association analysis of the dry weight trait of arrowhead pea are based on the Manhattan diagram obtained by EMMAX software. The red sites shown are the InDel positions associated in this invention.

[0017] Figure 2 This is a box plot of dry weight (DW) values ​​for different genotypes at locus Vs_Chr5_243849158 in a genome-wide association study (GWAS). 0 / 0 indicates a low-yielding genotype at locus Vs_Chr5_243849158, and 0 / 1 indicates a high-yielding genotype. Dots represent extreme values, and the values ​​above them indicate differential values. P value.

[0018] Figure 3 The partial sequence differences between the 0 / 0 genotype and the 0 / 1 genotype within the molecular marker Vs_Chr5_243849158 region are shown.

[0019] Figure 4 Electrophoresis image of molecular markers amplified from some arrowhead pea germplasm resources. The concentration of the agarose gel is 1.5%. Detailed Implementation

[0020] The present invention will now be described in detail through specific embodiments. These embodiments are provided so that this invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0021] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials used can be purchased commercially.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be used in this invention. The preferred embodiments and materials described herein are for illustrative purposes only. Example 1: Development of molecular markers associated with arrowhead pea yield

[0023] This invention uses the dry weight (DW, in g) index to evaluate the yield trait of *Viola yezoensis*. A higher DW value indicates higher yield, and a lower value indicates lower yield. After measuring the DW in the *Viola yezoensis* population, GWAS analysis located an InDel site in the *Viola yezoensis* population. Figure 1The blue locus, named Vs_Chr5_243849158, is located at locus 243849158 on chromosome 5 of the *Vaccinium bracteatum* reference genome (downloadable at: http: / / dx.doi.org / 10.5524 / 100954). The first allele with InDel insertion has a genotype of 0 / 0; the second allele with InDel deletion has a genotype of 0 / 1. The DW box plot shows the distribution of different genotypes at locus Vs_Chr5_243849158 in the *Vaccinium bracteatum* population. Figure 2 This indicates that the dry weight of *Vicia sativa* genotype 0 / 1 was significantly higher than that of genotype 0 / 0. The insertion / deletion fragment at locus 243849158 on chromosome 5 of *Vicia sativa* is TCAACAACTGAAACATTTAGCGGTCCTCGTCCAAATCTTGCAACAAGACTTCAAAGAAGAGCAAGTATTCGTGAAAAATAAGTTCATCGTCAACTTCAAGGAGATCTAGTC (shown in SEQ ID NO. 3). Figure 3 The 0 / 0 type has the insert fragment of SEQ ID NO.3, while the 0 / 1 type lacks the insert fragment of SEQ ID NO.3.

[0024] Based on the InDel variant and its upstream and downstream sequences, the following primers were designed using Primer 5.0 software: Vs_Chr5_243849158-F: TGGAAGACGAACGACATACATA (shown in SEQ ID NO.1); Vs_Chr5_243849158-R: AAGAACTCACATTGGGTCCTAC (shown as SEQ ID NO.2).

[0025] Then, the primers were used to perform PCR amplification on the test samples. The results showed that the PCR product of the high-yielding arrowhead pea sample had two characteristic bands of 146 bp and 36 bp, while the PCR product of the low-yielding arrowhead pea sample had a characteristic band of 146 bp.

[0026] Example 2: Accuracy verification of the molecular markers described in this invention A total of 222 germplasm accessions were identified, and the specific germplasm materials used are shown in Table 2: Table 2. Dry weight indices and genotypes corresponding to the Vs_Chr5_243849158 locus in 222 *Vaccinium bracteatum* germplasm materials.

[0027]

[0028]

[0029] Note: In the table, genotype. / . represents genotype deletion.

[0030] 1) Using the genomic DNA of the arrowhead pea to be identified as a template, PCR amplification was performed using the primer pair to obtain the PCR product; The PCR amplification reaction system is as follows: template DNA 10–100 ng, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 5 μL of 2 × Taq PCR Master Mix, and deionized water to a final volume of 10 μL. The preferred PCR amplification reaction program is: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 55℃ annealing for 5 s, 72℃ extension for 5 s, 35 cycles; 72℃ extension for 5 min; and storage at 4℃. Separation is performed by electrophoresis on a 1.5% agarose gel. After loading, electrophoresis is performed at 140 V DC for 1 h, and the PCR banding patterns of each sample are then read.

[0031] 2) Determine the genotype of *Vaccaria buergeriana* based on the size of the PCR product: if the PCR product of the *Vaccaria buergeriana* to be identified contains the fragment shown in SEQ ID NO.3, then the *Vaccaria buergeriana* to be identified is type 0 / 0; if the PCR product of the *Vaccaria buergeriana* to be identified lacks the fragment shown in SEQ ID NO.3, then the *Vaccaria buergeriana* to be identified is type 0 / 1. Specifically, when the PCR product of the *Vaccaria buergeriana* to be identified contains the fragment shown in SEQ ID NO.3, and the band length of the PCR product is 449 bp (SEQ ID NO.4), then the *Vaccaria buergeriana* to be identified is type O / O.

[0032] The sequence of SEQ ID NO.4 is as follows: TGGAAGACGAACGACATACATATGGAGGTAATTTTGATTACTCTTATGATAATATGGATAACAACAACTCAACAACTGAAACATTTAGCGGTCCTCGTCCAAATCTTGCAACAAGACTTCAAAGAAGAGCAAGTATTCGTGAAAAATAAGTTCATCGTCAACTTCAAGGAGATCTAGTCGAGTATATTTGGGAACGTTTTGGACATGAAGATGATAAAATTTAAG TTTGATTAATTATGTGATGTTATTTATTTTTATTGTTTTTAATTATATGTCATGTATTTGAGATTAATTTTAATTATCATTTTAAATTATAAGTTTAATTTGAATTTTATTTTATATCAATTTTAATTTAAATAATTAAAATTATTATTTTAAATGCTATAAAATTGAATATGAATAAAATATTAATACAAAGTAAAATTGTAGGACCCAATGTGAGTTCTT.

[0033] When the PCR product of the *Vaccinium bracteatum* to be identified simultaneously amplifies the bands corresponding to SEQ ID NO.4 (449 bp) and SEQ ID NO.5 (338 bp), then the *Vaccinium bracteatum* to be identified is of type 0 / 1.

[0034] The sequence of SEQ ID NO.5 is as follows: TGGAAGACGAACGACATACATATGGAGGTAATTTTGATTACTCTTATGATAATATGGATAACAACAACGAGTATATTTGGGAACGTTTTGGACATGAAGATGATAAAATTTAAGTTTGATTAATTATGTGATGTTATTTATTTTTATTGTTTTTAATTATATGTCATGT ATTTGAGATTAATTTTAATTATCATTTAAATTATAAGTTTAATTTGAATTTTATTTTATATCAATTTTAATTTAAATAATTAAAATTATTTTAAATGCTATAAAATTGAATATGAATAAAATATTAATACAAAGTAAAATTGTAGGACCCAATGTGAGTTCTT.

[0035] Analysis of the 222 *Viola yezoensis* accessions in Table 2 revealed that, excluding 17 accessions with missing genotypes, 189 accessions had a genotype of 0 / 0 at the Vs_Chr5_243849158 locus, with a mean yield (DW) of 17.05015873 g, classifying them as low-yielding *Viola yezoensis*. Conversely, 16 accessions had a genotype of 0 / 1 at the Vs_Chr5_243849158 locus, with a mean DW of 22.871875 g, classifying them as high-yielding *Viola yezoensis*. Analysis of variance showed a significant difference in DW between high-yielding and low-yielding *Viola yezoensis*. P <0.01).

[0036] Twenty-four accessions from 222 *Viburnum fasciatus* accessions (germplasm numbers 94, 64, 98, 57, 29, 61, 470, 17, 191, 38, 22, 81, 172, 291, 18, 169, 235, 402, 177, 120, 510, 330, 382, ​​409) were selected for validation. Figure 4 It can be seen that 12 *Vaccinium bracteatum* samples amplified two characteristic bands of 449 bp and 338 bp, respectively, belonging to the 0 / 1 type. Statistical analysis showed that the average depth-to-weight (DW) of these 12 samples was 25.62416667 g. Another 12 samples amplified a single characteristic band of 449 bp, belonging to the 0 / 0 type. Statistical analysis showed that the average DW of these 12 0 / 0 samples was 5.978333333 g, which was lower than the average DW of the 12 0 / 1 samples. Analysis of variance showed that the DW difference between the 0 / 0 and 0 / 1 types was significant. P <0.01). The 0 / 1 type arrowhead pea material obtained by PCR testing was expected to be a high-yielding material, and the actual result was consistent with the expectation. Therefore, the InDel molecular marker of this invention can effectively identify the yield trait of arrowhead pea and can be used for the prediction and screening of high-yielding arrowhead pea varieties.

[0037] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. The molecular marker Vs_Chr5_243849158 associated with the yield trait of arrowhead pea, characterized in that, The nucleotide sequence of the molecular marker Vs_Chr5_243849158 is shown in SEQ ID NO.4 and SEQ ID NO.

5. This molecular marker Vs_Chr5_243849158 is an insertion / deletion fragment TCAACAACTGAAACATTTAGCGGTCCTCGTCCAAATCTTGCAACAAGACTTCAAAGAAGAGCAAGTATTCGTGAAAAATAAGTTCATCGTCAACTTCAAGGAGATCTAGTC on chromosome 5 of the *Vaccinium bracteatum* reference genome. The primer pair sequences for amplifying the molecular marker Vs_Chr5_243849158 are as follows: Vs_Chr5_243849158-F: TGGAAGACGAACGACATACATA; Vs_Chr5_243849158-R:AAGAACTCACATTGGGTCCTAC.

2. To test the application of the primer pair of the molecular marker Vs_Chr5_243849158 described in claim 1 in molecular marker-assisted breeding of arrowhead pea.

3. The application according to claim 2, characterized in that, The molecular marker Vs_Chr5_243849158 is used to identify or assist in identifying the yield trait of arrowhead pea.

4. A method for identifying the yield trait of arrowhead pea, characterized in that, The method includes the following steps: (1) Extract genomic DNA from the target *Vitis hyacinthus* pea; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer pair of the molecular marker Vs_Chr5_243849158 described in claim 1, and perform electrophoresis detection and / or sequencing on the PCR amplification products; (3) The determination is based on the electrophoresis bands and / or sequencing results of step (2), and the specific criteria are as follows: If the PCR amplification product has both a characteristic band of 449 bp as shown in SEQ ID NO.4 and a characteristic band of 338 bp as shown in SEQ ID NO.5, then *Vaccaria buergeriana* is a high-yielding type; if the PCR amplification product has only one characteristic band of 449 bp as shown in SEQ ID NO.4, then *Vaccaria buergeriana* is a low-yielding type.

5. A reagent kit for identifying the yield of arrowhead peas, characterized in that, The kit contains a primer pair of the molecular marker Vs_Chr5_243849158 as described in claim 1.

6. The use of the kit according to claim 5 in identifying the yield genotype of arrowhead pea.

7. The application according to claim 6, characterized in that, The method for identifying high-yield and low-yield genotypes in arrowhead pea using the aforementioned kit is as follows: (1) Extract genomic DNA from the target *Vitis hyacinthus* pea; (2) Using the genomic DNA extracted in step (1) as a template, perform PCR amplification using the primer pair of the molecular marker described in claim 1; (3) Perform electrophoresis and / or sequencing on the PCR amplification products. If the PCR amplification products have both a characteristic band of 449 bp as shown in SEQ ID NO.4 and a characteristic band of 338 bp as shown in SEQ ID NO.5, then the arrow-shaped pea is a high-yielding genotype; if the PCR amplification products have only a characteristic band of 449 bp as shown in SEQ ID NO.4, then the arrow-shaped pea is a low-yielding genotype.

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