A library of ssr markers developed based on mung bean genome and application thereof

Abstract

The present application relates to the technical field of molecular markers, and in particular to a kind of SSR marker based on dolichos lablab genome and application, the present application discloses 30 pairs of SSR markers of dolichos lablab developed using dolichos lablab genome data, the forward and reverse primers corresponding to the 30 pairs of markers are as shown in SEQ ID NO:1-60.The 30 pairs of SSR markers disclosed in the present application show good stability and polymorphism in dolichos lablab genome, show good universality, and provide great help for molecular breeding of dolichos lablab.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to an SSR marker developed based on the lentil genome and its application. Background Technology

[0002] Hyacinth bean (Lysimachia foenum-graecum) is an annual or perennial herbaceous vine belonging to the genus *Lysimachia* in the legume family (Fabaceae). It is mainly distributed in India and tropical countries. In my country, it is more widely cultivated in the south. Hyacinth beans have a wide range of uses; they can be used for biological control of weeds, are a source of high-quality silage, and are also a vegetable rich in protein and carotene. They are easy to cultivate. Furthermore, hyacinth beans are also an ideal traditional Chinese medicine with certain medicinal value. Therefore, hyacinth beans have high economic value and broad application prospects. However, current research and breeding efforts on hyacinth beans, both domestically and internationally, lag significantly behind those on other legumes.

[0003] Classifying hyacinth bean germplasm resources is of great significance for hyacinth bean breeding. However, selecting new hyacinth bean varieties with superior traits using traditional breeding methods is time-consuming and costly. Molecular markers have wide applications in germplasm resource research and marker-assisted breeding. Among them, SSR markers are widely used due to their abundance, high reproducibility, and widespread presence in biological genomes. Currently, SSR markers are widely used in legumes such as soybean and alfalfa, but research on the application of SSR markers developed using the hyacinth bean genome is relatively limited. Summary of the Invention

[0004] This invention provides an SSR marker developed based on the hyacinth bean genome and its application. This invention develops 30 SSR markers for hyacinth bean based on the hyacinth bean genome and describes the application of these SSR markers in the diversity analysis of hyacinth bean germplasm resources.

[0005] To achieve the above objectives, this invention provides an SSR marker for hyacinth genome development, comprising forward and reverse primers corresponding to the following 30 loci:

[0006]

[0007]

[0008] Furthermore, the above-mentioned SSR markers of hyacinth bean were applied in the genetic diversity analysis of hyacinth bean germplasm resources.

[0009] Furthermore, the primers for the above-mentioned lentil SSR markers were obtained by the following method:

[0010] 1) Based on the hyacinth bean genome data with the National Gene Bank sequence number CNP0000096, the SSR sequences in the genome sequence were detected using MISA, and primers were designed for the detected SSR sequences using Primer3.

[0011] 2) Using the SSR primer pairs obtained in step 1), DNA was extracted from 8 lentil materials from different sources out of 115 lentil materials for PCR amplification, and SSR primers were screened based on the amplification results.

[0012] 3) Extract DNA from the 115 lentil materials described in step 2) as a template, add fluorescent labels to the SSR markers screened in step 2) and perform PCR amplification, and use the amplification products for fluorescent capillary electrophoresis to verify the SSR primers screened in step 2).

[0013] Furthermore, the 8 portions of hyacinth bean material mentioned in step 2) are hyacinth bean seedlings.

[0014] Further, the PCR reaction system described in steps 2) and 3) has a total volume of 25 μL, and its components include DNA template (20-50 ng / μL), SSR forward primer (10 μM), SSR reverse primer (10 μM), dNTP mix (5 μM), Taq Buffer (containing MgCl2) 2.5 μL, and Taq enzyme 5 U / μL.

[0015] Further, the PCR reaction conditions described in steps 2) and 3) are as follows: 95℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds (annealing temperature decreased by 0.5℃ per cycle), 72℃ extension for 30 seconds, for 10 cycles; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, for 30 cycles; 72℃ extension for 10 minutes.

[0016] The SSR markers developed in this invention have excellent technical effects: 1) The developed SSR markers are based on hyacinth bean genome data, possessing reliability and rich polymorphism. The 30 pairs of developed SSR markers were validated by capillary electrophoresis using 115 hyacinth bean germplasm resources. The results showed that the 30 primer pairs amplified an average of 4.13 bands in the population, with an average of 3.37 alleles detected per locus; 2) The developed SSR markers have strong universality. SSR detection analysis of the collected 115 hyacinth bean germplasm resources revealed that all 30 pairs of SSR primers amplified polymorphic bands in all 115 resources, demonstrating good polymorphism. These 115 hyacinth bean germplasm resources came from 5 different regions: 34 from Shanghai, 14 from Henan, 9 from Northeast China, 47 from the United States, and 11 from India; 3) The developed SSR markers exhibit rich polymorphism and strong stability, providing significant assistance for the identification of hyacinth bean germplasm resources and molecular marker-assisted breeding.

[0017] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0018] Figure 1 These are the polymorphism results of primers for the SSR marker Sat10_96 in eight lentil materials;

[0019] Figure 2 These are the results of capillary electrophoresis of some fluorescent primers for some materials;

[0020] Figure 3 It is a UPGMA phylogenetic tree based on SSR tags. Detailed Implementation

[0021] The following description, with reference to the accompanying drawings, illustrates several preferred embodiments of the present invention to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0022] Example 1: Characterization of SSR Markers in the Hyacinth Bean Genome

[0023] The hyacinth bean genome data were obtained from the National Gene Bank (accession number CNP0000096). SSR sequences in the genome were detected using MISA with the following parameters: single-10, double-6, triple-5, quadruple-5, five-5, and six-5. The minimum spacing between SSRs was 200 bp. Primers were designed using Primer3 for the detected SSR sequences.

[0024] Distribution and structural characteristics of SSRs in hyacinth beans:

[0025] The assembled genome of the hyacinth bean has a total sequence length of 395,472,305 bp, with 5,524 SSR loci detected, averaging one SSR locus every 71.6 kb. Analysis of SSR locus types revealed that SSR repeats range from single nucleotide to hexanucleotide repeats, with dinucleotide repeats being the predominant type, accounting for 50.58% of the total SSR loci; followed by tetranucleotide repeats, accounting for 24.02%; single nucleotide, trinucleotide, pentanucleotide, and hexanucleotide repeats were the least common, accounting for 1.79%, 9.70%, 8.58%, and 5.32% of the total SSR loci, respectively. Among all SSR loci, those with 11-20 repeats were the most numerous, accounting for 52.70% of the total; followed by five repeats, accounting for 25.11%; other repeat counts each accounted for less than 10% (Table 1). The length of SSR loci in lentils ranged from 10 to 2805 bp, with a total length of 188,080 bp and an average length of 34.05 bp. Among them, the number of SSR loci with a length of 40-50 bp was the largest, accounting for 34.59% of the total; followed by SSR loci with a length of 30-40 bp and 20-30 bp, accounting for 30.03% and 18.81% of the total, respectively; the number of loci with a length of less than 20 bp and a length of more than 50 bp was relatively small, accounting for 1.14% and 15.42% of the total, respectively.

[0026] Table 1. Types and distribution ratio of SSRs in broad beans

[0027]

[0028] The 5524 SSR loci in the hyacinth genome contain 6 classes of motifs, totaling 269 different repeating motifs. These include 1, 3, 16, 59, 83, and 107 types of single-nucleotide to hexanucleotide repeating motifs, respectively. Among the single-nucleotide, dinucleotide, and trinucleotide types, the motif types are relatively concentrated. The single-nucleotide type contains only one type, A / T; the dinucleotide type includes three repeating motifs, with the AT / TA type accounting for the highest proportion (45.55% of all SSR loci); and the trinucleotide type accounts for 90.67% of the repeating motifs, namely ATA / TAT, AAT / ATT, TAA / TTA, and GAA / TTC. Among the tetranucleotide, pentanucleotide, and hexanucleotide types, the motif types are more dispersed, with no particularly concentrated motif types found (Table 2).

[0029] Table 2. Characteristics of SSR motifs from hyacinth bean.

[0030]

[0031]

[0032] Example 2: Molecular Marker Screening and Application

[0033] Primer screening and material evaluation were conducted on 115 collected hyacinth bean germplasm resources using developed SSR markers. Primer parameters were as follows: primer sequence length 20 bp, GC content 40%–60%, annealing temperature 59–62℃, Tm value difference between upstream and downstream primers ≤2℃, expected amplification product length 250–300 bp, and no secondary structure or dimers.

[0034] Primer screening

[0035] DNA was extracted from 8 lentil samples from different origins out of 115 lentil samples for PCR amplification, and SSR primers were screened based on the amplification results. Figure 1 Polymorphism results of primers for the SSR marker Sat10_96 in 8 lentil materials.

[0036] The PCR reaction system has a total volume of 25 μL, which includes DNA template (20-50 ng / μL), SSR forward primer (10 μM), SSR reverse primer (10 μM), dNTP mix (5 μM), Taq Buffer (containing MgCl2) 2.5 μL, and Taq enzyme 5 U / μL.

[0037] The PCR reaction conditions were: 95℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds (annealing temperature decreased by 0.5℃ per cycle), 72℃ extension for 30 seconds, for 10 cycles; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 30 seconds, for 30 cycles; 72℃ extension for 10 minutes.

[0038] Filtering results

[0039] 16,572 primer pairs were designed for 5,524 SSR loci, and 100 primer pairs were randomly selected for validation. SSR primers were screened using 8 materials with significant differences in origin. The results showed that 87 of the 100 primer pairs amplified clear bands. These 87 primer pairs were then validated using fluorescently labeled capillary electrophoresis on 115 lentil germplasm resources. The results showed that 30 primer pairs exhibited polymorphism in their amplification results. Figure 2 These are capillary electrophoresis results for some materials using partial fluorescent primers, showing clear and reliable allele signals. A total of 30 primer pairs were screened, and their amplification products exhibited polymorphism.

[0040] Genetic diversity analysis of SSR markers

[0041] Thirty pairs of polymorphic SSR primers were selected and used for PCR amplification of DNA from 115 hyacinth bean germplasm resources. Genetic diversity of the population was analyzed. The results showed that the 30 primer pairs amplified a total of 124 polymorphic bands, with an average of 4.13 bands per primer pair in the population. A total of 101 alleles were detected, with an average of 3.37 alleles per locus. The gene diversity index ranged from 0.05 to 0.74, with an average diversity index of 0.45. The polymorphism information content ranged from 0.05 to 0.69, with four primer pairs having a polymorphism information content greater than 0.5, indicating high polymorphism (Table 3). These results demonstrate that 30 primer pairs can effectively distinguish the genetic diversity of 115 hyacinth bean resources.

[0042] Table 3. Genetic diversity in hyacinth bean populations

[0043]

[0044]

[0045] Genetic similarity coefficients were calculated using NTsys 2.10e, and MEGA 7.0 software was used to analyze 115 hyacinth bean germplasm resources. A UPGMA phylogenetic tree based on SSR markers was then constructed. Figure 3 The results showed that the 115 hyacinth bean germplasm resources were divided into two subgroups and some individuals not clustered at a correlation coefficient of 0.135. Hyacinth bean resources from different sources were present in each subgroup. This indicates that there was no significant difference in genetic diversity among materials from different sources, suggesting possible gene flow between hyacinth bean resources from different regions, or that hyacinth beans from different regions originated from the same region and were not isolated over a long period.

[0046] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A primer composition for SSR markers developed using the lentil genome, characterized in that, The SSR marker primer composition consists of forward and reverse primers corresponding to the following 30 markers: 。 2. The application of the SSR marker primer composition according to claim 1 in the genetic diversity analysis of hyacinth bean germplasm resources.

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