A molecular marker related to the anti-cracking trait of arrow leek pea on chromosome 1 and application thereof

Abstract

The application discloses a molecular marker related to a crack resistance trait of arrow pea located at chromosome 1 and application, and belongs to the technical field of biotechnology. By using a molecular marker development and whole genome association analysis technology, the crack resistance trait of the arrow pea is positioned and analyzed, and an InDel molecular marker Vs_Chr1_123211351 which is related to the crack resistance trait, stable and reliable and has polymorphism is obtained. The nucleotide sequence of the insertion or deletion fragment is shown as in SEQ ID NO. 3. The nucleotide sequences of primer pairs for amplifying the InDel molecular marker are shown as in SEQ ID NO. 1-2. The application has a very important role in predicting, identifying or assisting in identifying the crack resistance phenotype of the arrow pea or breeding the crack resistance arrow pea. The method in the application is simple and fast, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to molecular markers related to the crack resistance trait of arrowhead pea. Technical Background

[0002] *Vicia sativa*, also known as arrowhead vetch, is an annual or biennial cleistopodium plant belonging to the genus *Vicia* in the legume family (Fabaceae). Native to the Mediterranean coast of Europe and western Asia, it is cultivated worldwide. *Vicia sativa* is characterized by its short growth cycle, strong adaptability, and high nutritional value. As a legume, it possesses nitrogen-fixing and soil-improving abilities. Cultivated varieties were introduced to Northwest China in the 1940s and expanded to other regions after the 1960s. It is now widely planted in high-altitude areas of North China, Northwest China, and the middle and lower reaches of the Yangtze River. *Vicia sativa* is an important forage and green manure crop in high-altitude areas of China, and its application in the Qinghai-Tibet Plateau has successfully alleviated the problem of winter forage scarcity in these regions.

[0003] Pod splitting is a common problem in legumes, causing significant losses in seed production. This is especially true for cultivated crops, where pod splitting results in substantial economic losses, making pod resistance a priority for breeders. The arrowhead vetch (Vicia sativa) has straight pods, and pod splitting is also prevalent, with most varieties exhibiting high splitting rates, which is detrimental to seed production. Breeding of arrowhead vetch started relatively late, and research on its pod splitting trait is not yet in-depth. Therefore, developing molecular markers associated with pod resistance through genome-wide association analysis is an effective approach. Furthermore, the development of pod resistance markers can accelerate the breeding of pod-resistant arrowhead vetch, and is of great significance for molecular-assisted breeding and new variety selection in arrowhead vetch. Summary of the Invention

[0004] One of the objectives of this invention is to provide a molecular marker associated with the crack resistance trait of arrowhead pea.

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

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention discloses a pair of molecular markers related to the crack resistance trait of arrowhead pea, located on chromosome 1 of arrowhead pea, and the molecular markers are named Vs_Chr1_123211351.

[0008] Primer pairs were used to amplify molecular markers associated with crack resistance in *Vallis fulva*. The primer pair sequence corresponding to the molecular marker Vs_Chr1_123211351 is as follows:

[0009] Vs_Chr1_123211351-F: CATATGCACTGACATCTAAGAT (as shown in SEQ ID NO.1);

[0010] Vs_Chr1_123211351-R: ACAGACACCCTGAATACGA (as shown in SEQ ID NO.2).

[0011] This invention also discloses the application of the above-mentioned molecular marker primer pairs in marker-assisted breeding of pod-cracking resistance in *Vitis salvia*. In other words, the molecular markers of this invention can be used in future marker-assisted breeding. By extracting DNA from leaves during the seedling stage and detecting the presence of the molecular markers of this invention, the pod-cracking trait of *Vitis salvia* can be identified. The detection can be performed using PCR, specifically using the above-mentioned molecular marker primer pairs, or it can be performed using sequencing methods.

[0012] This invention also discloses the application of the above-mentioned molecular markers in identifying the crack-resistant trait of *Vitis pulcherrima*, especially in screening and identifying crack-resistant *Vitis pulcherrima*. Specifically, the specific steps for identifying whether *Vitis pulcherrima* has the crack-resistant trait are as follows:

[0013] (1) Using the DNA of the tested germplasm as the template for PCR amplification, and the primer pair corresponding to the label Vs_Chr1_123211351 as the primers, the PCR amplification reaction system is shown in Table 1:

[0014] Table 1. Reaction system for PCR amplification

[0015]

[0016] PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 20 s, 35 cycles; 72℃ extension for 5 min; store at 4℃.

[0017] (2) Agarose gel electrophoresis detection of PCR products, and judgment of the crack resistance of arrowhead peas based on the results:

[0018] If the PCR amplification product is a characteristic band of 100 bp as shown in SEQ ID NO.4, then *Vaccaria buergeriana* is a cleavable type; if the PCR amplification product is a characteristic band of 86 bp as shown in SEQ ID NO.5, then *Vaccaria buergeriana* is a cleavable type.

[0019] Alternatively, reagent kits containing the aforementioned molecular marker primer pairs can be prepared to identify the crack resistance trait of *Vicia sativa* materials. Furthermore, these kits can be used to identify or assist in the identification of *Vicia sativa* genotypes. All of these applications can be performed using conventional methods.

[0020] The present invention has the following advantages:

[0021] (1) The molecular markers in this invention are closely related to the crack resistance trait of arrow-shaped peas and can be applied to molecular marker-assisted breeding of crack resistance trait of arrow-shaped peas, to the screening of germplasm resources of crack-resistant arrow-shaped peas, and to the genetic improvement of arrow-shaped peas.

[0022] (2) The molecular markers of the present invention have the characteristics of convenient detection, stable amplification products and high specificity, and can be applied rapidly and in high throughput to the breeding practice of arrowhead pea. Attached Figure Description

[0023] Figure 1 The results of the genome-wide association analysis of the crack resistance trait in arrowhead pea are presented in the Manhattan diagram obtained based on the MLM model analysis. The red sites shown are the InDel positions associated in this invention.

[0024] Figure 2 Box plots of PSRI (Potamogeton Species Resilience Index) corresponding to different genotypes at locus Vs_Chr1_123211351 in a genome-wide association analysis. 0 / 0 indicates a genotype at locus Vs_Chr1_123211351 that is prone to cracking, while 1 / 1 indicates a genotype at locus Vs_Chr1_123211351 that is resistant to cracking. Dots represent extreme values. One-way ANOVA was used to assess the differences.

[0025] Figure 3 The sequence differences between the 0 / 0 genotype and the 1 / 1 genotype within the molecular marker Vs_Chr1_123211351 region are shown.

[0026] Figure 4 Electrophoresis image of molecular markers amplified from some arrowhead pea germplasm resources. The concentration of agarose gel is 2%.

[0027] In the diagram, M represents a DNA marker. Detailed Implementation

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Example 1: Development of molecular markers related to crack resistance in arrowhead pea.

[0032] This invention uses the percentage of intact pods after simulated natural pod drop in *Viola yezoensis* as the PSRI (Periplaneta spp.) crack resistance index to evaluate the cracking trait of *Viola yezoensis*. A higher PSRI indicates greater crack resistance, while a lower value indicates greater susceptibility to cracking. After measuring the PSRI in a *Viola yezoensis* population, GWAS analysis located an InDel site in the *Viola yezoensis* population. Figure 1 The red locus, named Vs_Chr1_123211351, is located at locus 123211351 on chromosome 1 of the *Vaccinium bracteatum* reference genome. The first allele is genotype 0 / 0, and the second allele is genotype 1 / 1. Box plot of PSRI distribution for different genotypes at locus Vs_Chr1_123211351 in the *Vaccinium bracteatum* population (…). Figure 2 This indicates that the genotype 1 / 1 *Vitex acutus* exhibits significantly higher crack resistance than the genotype 0 / 0. The insertion / deletion fragment at locus 123211351 on chromosome 1 of *Vitex acutus* is TCAGTGTTGCTGTG (shown in SEQ ID NO. 3). Figure 3 The 0 / 0 type has an inserted fragment of SEQ ID NO.3, while the 1 / 1 type has a missing fragment of SEQ ID NO.3.

[0033] Based on the InDel variant and its upstream and downstream sequences, the following primers were designed using Primer 5.0 software:

[0034] Vs_Chr1_123211351-F: CATATGCACTGACATCTAAGAT (as shown in SEQ ID NO.1);

[0035] Vs_Chr1_123211351-R: ACAGACACCCTGAATACGA (as shown in SEQ ID NO.2).

[0036] Then, the primers were used to perform PCR amplification on the test samples. The results showed that the PCR product of the resistant *Vaccinium bracteatum* sample had only an 86bp characteristic band, while the PCR product of the easily cracked *Vaccinium bracteatum* sample had only a 100bp characteristic band.

[0037] Example 2: Accuracy verification of the molecular markers described in this invention

[0038] Eighteen germplasm accessions were identified, and the specific germplasm materials used are shown in Table 2:

[0039] Table 2. Pod splitting status and genotype of 18 germplasm materials

[0040] Germplasm name genotype Crack Resistance Index (PSRI) S33 0 / 0 0.0871 S62 0 / 0 0.9624 S64 0 / 0 0.0871 S65 0 / 0 0.9315 S67 0 / 0 0.9445 S79 0 / 0 0.0835 S109 0 / 0 0.9472 S143 0 / 0 0.1216 S164 0 / 0 0.1004 S172 0 / 0 0.0454 S208 0 / 0 0.1037 S17 1 / 1 0.9844 S113 1 / 1 0.9568 S200 1 / 1 0.9775 S303 1 / 1 0.9649 S458 1 / 1 0.9079 S467 1 / 1 0.1804 S509 1 / 1 0.9681

[0041] 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;

[0042] 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, 15 μL of 2×Taq PCR Master Mix, and deionized water to a final volume of 30 μL. The preferred PCR amplification reaction program is: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 52℃ annealing for 30 s, 72℃ extension for 20 s, 35 cycles; 72℃ extension for 10 min; storage at 4℃. Separation is performed by electrophoresis on a 2% agarose gel. After loading, electrophoresis is performed at 5 V / cm DC for 2 h, and the PCR banding patterns of each sample are then read.

[0043] 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 1 / 1.

[0044] Specifically, when the PCR product of the *Vaccaria serrata* to be identified contains the fragment shown in SEQ ID NO.3, and the band length of the PCR product is 100bp (SEQ ID NO.4), then the *Vaccaria serrata* to be identified is of type O / O.

[0045] The sequence of SEQ ID NO.4 is as follows:

[0046]

[0047] When the PCR product of the *Vaccaria buergeriana* to be identified is missing the fragment shown in SEQ ID NO.3, and the band length of the PCR product is 86 bp (SEQ ID NO.5), then the *Vaccaria buergeriana* to be identified is type 1 / 1.

[0048] The sequence of SEQ ID NO.5 is as follows:

[0049]

[0050] from Figure 4 As shown in Table 2, 11 *Viburnum cuspidatum* materials amplified a 100bp characteristic band, indicating the 0 / 0 type. Statistical analysis revealed that the average PSRI of these 11 materials was 0.4013. Seven *Viburnum cuspidatum* materials amplified an 86bp characteristic band, indicating the 1 / 1 type. Statistical analysis showed that the average PSRI of these seven materials was 0.8486, which was higher than the average PSRI of the 11 0 / 0 type materials. Analysis of variance showed a significant difference in PSRI between the 0 / 0 and 1 / 1 types (P < 0.05). The 1 / 1 type *Viburnum cuspidatum* materials, genotyped using PCR detection, were expected to be crack-resistant, and this was indeed the case. Therefore, the InDel molecular marker of this invention can effectively identify the crack-resistant trait in *Viburnum cuspidatum* and can be used for the prediction and screening of crack-resistant *Viburnum cuspidatum* varieties.

[0051] 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. A molecular marker located on chromosome 1 associated with crack resistance in arrowhead pea, characterized in that, The nucleotide sequences of the molecular marker are shown in SEQ ID NO.4 and SEQ ID NO.

5. This molecular marker is an insertion / deletion of the fragment shown in SEQ ID NO.3 on chromosome 1 of the alfalfa reference genome. The primer pair sequences for amplifying the molecular marker are as follows: Vs_Chr1_123211351-F: CATATGCACTGACATCTAAGAT; Vs_Chr1_123211351-R:ACAGACACCCTGAATACGA.

2. The application of the primer pair of the molecular marker described in claim 1 in the auxiliary identification of the crack resistance trait of arrowhead pea.

3. A method for auxiliary identification of crack resistance in arrowhead peas, 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 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: PCR amplification was performed using primers Vs_Chr1_123211351-F and Vs_Chr1_123211351-R. If the PCR amplification product is a characteristic band of 100 bp as shown in SEQ ID NO.4, then *Vaccaria salsa* is a cleavable type; if the PCR amplification product is a characteristic band of 86 bp as shown in SEQ ID NO.5, then *Vaccaria salsa* is a cleavable type.

4. The application of a reagent kit in assisting in the identification of crack-resistant genotypes in arrowhead peas, characterized in that, The kit contains the primer pair described in claim 1, and the method for auxiliary identification of the crack-resistant genotype of *Vallis fulva* using the 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 product is a characteristic band of 100 bp as shown in SEQ ID NO.4, then the arrowhead pea is a cleavage-prone genotype; if the PCR amplification product is a characteristic band of 86 bp as shown in SEQ ID NO.5, then the arrowhead pea is a cleavage-resistant genotype.

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