Application of KASP molecular marker primer related to peanut seed size

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a KASP molecular marker primer related to the size of peanut seeds. The SNP molecular marker related to the peanut seed size is located in an Arahy.07 chromosome and comprises three molecular marker sites. According to the invention, multi-environment data of a genetic population and 499 natural populations comprising American and domestic peanut germplasm resource materials, breeding parents and peanut varieties are utilized to screen and verify functions of SNP markers and KASP detection primers on molecular assisted selection of peanut seed size characters. When the SNP molecular marker and the KASP detection primer are applied to the identification of the peanut seed size character, the breeding process of peanut varieties can be accelerated, the breeding efficiency is remarkably improved, and the healthy development of the peanut planting industry is promoted.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the application of a KASP molecular marker primer related to peanut seed size. Background Technology

[0002] Peanuts (Arachis hypogaea L.) are important oil crops and cash crops. The size of peanut kernels is a direct factor in peanut yield. Numerous studies have found that increasing seed size makes an extremely important contribution to increasing crop yield. Improving peanut kernel size is of great significance to ensuring national oil security and increasing the benefits of the peanut industry, and it is also the direction for the development of the peanut industry.

[0003] In recent years, several key genes and important pathways regulating seed size have been identified in model crops Arabidopsis thaliana and rice through forward and reverse genetics. However, due to the narrow genetic variation background of peanuts and their unique physiological characteristics of "flowering above ground and fruiting underground," research progress on important genes and metabolic pathways related to peanut kernel size has been slow. Therefore, fine mapping of quantitative trait loci (QTLs) related to peanut kernel size and identification of candidate genes are crucial for improving peanut kernel size.

[0004] Hundred-kernel weight (HKW) is a core indicator for evaluating peanut kernel size. Due to the narrow genetic background of peanuts, this project utilized F2 segregating populations, F6 RIL populations, and BCF2 fine-mapped populations constructed from Luhua 6 and its small-kernel mutant T34. Simultaneously, combined with genome-wide association analysis results based on natural population resequencing, we conducted fine mapping of QTLs and candidate gene mining for the peanut HKW trait. Fine mapping and candidate gene identification of major-effect QTLs for peanut HKW are urgently needed to advance peanut synergistic breeding technology. Summary of the Invention

[0005] The purpose of this invention is to provide a KASP molecular marker primer related to peanut seed size and its application.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an application of SNP molecular markers related to peanut seed size in identifying peanut seed size traits, wherein the SNP molecular markers are one or more of WTA7-2661184, WTA7-2776237, and WTA7-3951865;

[0008] The WTA7-2661184 is located at 2661184bp on chromosome 7 of the peanut genome, and its bases are C / T.

[0009] The WTA7-2776237 is located at 2776237bp on chromosome 7 of the peanut genome, and its bases are C / G.

[0010] The WTA7-3951865 is located at 3951865bp on chromosome 7 of the peanut genome, and its bases are G / A.

[0011] Furthermore, the peanut genome is Tifrunner.gnm1.KYV3.

[0012] A primer pair for detecting the KASP molecular marker, the primer pair comprising one or more of SEQ ID NO.1-3, SEQ ID NO.4-6, and SEQ ID NO.7-9.

[0013] This invention also provides a method for identifying SNP molecular markers of QTLs related to peanut seed size, comprising the following steps:

[0014] (1) Extract genomic DNA from the peanut material to be identified and use the genomic DNA as a template;

[0015] (2) PCR amplification of the template is performed using primers as shown in SEQ ID NO.1~3, SEQ ID NO.4~6, and SEQ ID NO.7~9. The amplification products are sequenced and analyzed to determine the polymorphism of the molecular marker based on the sequencing results.

[0016] Preferably, the PCR amplification system is as follows:

[0017] Table 1

[0018] Element volume Final concentration 2×PARMS master mix 5μL 1× Allele X primer (10μM) 0.15μL 150nM Allele Y primer (10μM) 0.15μL 150nM Common primer (10μM) 0.4μL 400nM DNA template 10-100ng 10-100nM <![CDATA[ddH2O]]> Add to 10 μL

[0019] Preferably, the PCR amplification procedure is as follows:

[0020] Table 2

[0021]

[0022]

[0023] This invention also provides the application of the aforementioned molecular markers or identification methods in the breeding of large-grained peanut varieties or lines.

[0024] This invention also provides the application of the aforementioned molecular markers or identification methods in peanut molecular breeding, the cultivation of transgenic peanuts, or the improvement of peanut germplasm resources.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] To investigate the genetic mechanism of peanut seed size traits and finely map the QTL and candidate genes for seed size traits on chromosome Arahy.07, this invention previously used the F2 segregating population and F6 RIL population bred from Luhua 6 and its small-grain mutant material T34 to identify a QTL related to the trait of 100 kernel weight at the 5' end of chromosome Arahy.07—qHKW-chr07.

[0027] To overcome the narrow sources of peanut genetic variation and explore the distribution of superior alleles in natural peanut populations, this invention utilizes resequencing data from 499 natural populations comprising peanut germplasm resources, breeding parents, and peanut varieties from both the United States and China. The study screens and verifies the function of SNP markers and KASP detection primers in molecularly assisted selection of peanut seed size traits. Applying the SNP molecular markers and KASP detection primers of this invention to the identification of peanut seed size traits can accelerate the peanut variety breeding process, significantly improve breeding efficiency, and promote the healthy development of the peanut planting industry. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present 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 only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0029] Figure 1 Luhua 6 and mutant materials T34, T371 and T372.

[0030] Figure 2 Whole-genome distribution of .BSA-Seq sequencing fragments.

[0031] Figure 3 Distribution of ΔSNP-index and ΔInDel-index association values ​​on chromosomes.

[0032] Figure 4 .qHKW-chr07 KASP molecular markers for different genotypes of Bai Ren heavy t test.

[0033] Figure 5 Distribution of KASP molecular marker P-values.

[0034] Figure 6 Frequency distribution of phenotypic data of the .T34 F6 RIL population.

[0035] Figure 7 Identification of different genotypes at three loci. Detailed Implementation

[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1

[0038] 1. Mining of genes and molecular marker sites related to peanut seed size

[0039] 1.1 Population mapping of T34 mutants and QTLs related to the heavy trait in peanut kernels

[0040] Luhua No. 6 (77g of 100 kernels) 60 After Co-γ ray mutagenesis and screening over several generations (M2-M7), a genetically stable peanut seed-smalling mutant, T34, with a seed weight of approximately 40g, was obtained. Figure 1 These materials are important germplasm materials for the localization and identification of peanut seed size genes. The above materials are preserved in the Shandong Provincial Peanut Germplasm Resource Mid-term Bank and are routinely cultivated in the field at the National Peanut Seed Propagation Base in Laixi City. BSA-seq analysis was performed using the F2 population (T34 F2 population) constructed from the cross between T34 and Luhua 6. After years of self-pollination, the population has reached F6, constituting a recombinant inbred line population (T34 RIL population). Figure 1 ).

[0041] The T34 F2 population (T34 × Luhua 6) has been constructed. BSA-seq sequencing analysis was performed on T34, Luhua 6, large-particle mixed pool, and small-particle mixed pool (30 lines each). The clean bases obtained after sequencing filtering were 311.61 Gbp, with a Q30 of 80% and an average sequencing depth of 14.11 × per sample. The average alignment efficiency between the samples and the reference genome was 99.44%, the average depth was 27.25 ×, and the genome coverage was 98.50% (Table 3). Figure 2 A total of 427,188 SNPs were obtained among the parents and 93,202 SNPs were obtained among the mixed pools; a total of 145,558 Small InDels were obtained among the parents and 39,716 Small InDels were obtained among the mixed pools.

[0042] Table 3. Statistics of BSA-Seq sequencing results

[0043]

[0044]

[0045] Compared with the reference genome (Tifrunner Version 1, gnm1), SNP sites with multiple genotypes, SNP sites with read support less than 4, SNP sites with identical genotypes across pools, and SNP sites where recessive pool genes did not originate from recessive parents were filtered out, ultimately yielding 302,735 high-quality, reliable polymorphic SNP sites. Following the same criteria, 67,016 high-quality, reliable InDel polymorphic sites were obtained.

[0046] Marker association analysis was performed on the differences in genotype frequencies among pooled samples using the SNP-index and InDel-index methods to identify significant differences in genotype frequencies and to calculate ΔSNP-index. Figure 3 a) and ΔInDel-index Figure 3 b) Information. The closer the ΔSNP-index and ΔInDel-index values ​​are to 1, the stronger the association between the variant site and the trait difference.

[0047] The 99th percentile of the ΔSNP-index identified 48 regions with a total length of 22.93 Mb, containing 1,558 genes, including 82 genes with nonsynonymous mutations. The 99th percentile of the ΔInDel-index identified 19 regions with a total length of 14.42 Mb, containing 1,050 genes, including 7 genes with frameshift mutations. The intersection of the ΔSNP-index and ΔInDel-index results identified 5 chromosomal segments (Table 4), containing a total of 869 genes.

[0048] Table 4. Statistical Table of Related Areas

[0049]

[0050] 1.2 BSA-seq Localization Interval Validation

[0051] Based on BSA-seq sequencing data, and according to the criteria of homozygous and differentially genotypes among parents, large-grain mixed pool with the same genotype as Luhua 6, small-grain mixed pool with the same genotype as T34, and sequencing depth of variant sites exceeding 10X, SNP and InDel marker sites were developed in the 0.21-4.08 Mb region of Arahy.07 chromosome and the 133.62-141.65 Mb region of Arahy.16 chromosome. SNP primers were designed to genotype the T34 population. T-tests were used to analyze the phenotypic data of 100 kernel weight and 100 fruit weight of different genotypes in the population. Figure 4The phenotypic data of seed weight phenotype with different markers within the Arahy.07 interval showed significant differences (p<0.05), while the differences on the Arahy.16 chromosome t-test did not reach a significant level (p>0.05). This indicates that the 0.21-4.08 Mb interval of chromosome Arahy.07 is the QTL for the seed size-related trait of seed weight, named qHKW-chr07. The t-test results of the seed weight phenotype data showed that the differences were more significant closer to the edge of chromosome Arahy.07. Figure 5 The results indicate that the 0.15-2.64Mb region of chromosome Arahy.07 is more closely linked to the seed size trait, and the candidate gene may be located in the 0.15-2.64Mb region, which will be the focus of future research.

[0052] Statistical analysis of the 100g kernel weight phenotype in the T34 RIL population revealed two peaks in the frequency of 100g kernel weight among the 604 lines of the F6 RIL population, around 43g and 60g. Figure 6 This indicates the possible existence of a major QTL for peanut seed size. Based on the BSA-seq and T-test results, qHKW-chr07 is identified as the major QTL.

[0053] A natural population containing 499 peanut germplasm accessions (see Xu, J., Jiang, X., Yin, X. et al. Genome-wide association analysis in peanut accessions uncovers the genetic basis regulating oil and fatty acid variation. BMC Plant Biol) 25,651(2025).https: / / doi.org / 10.1186 / s12870-025-06690-9) From 2017 to 2020, BLUP analysis was performed on the phenotypic traits of peanut seed size in 10 different environments in Qingdao, Yantai, and Weihai. Simultaneously, genotyping of natural populations was conducted using 15 primer pairs (amplification system and procedures are shown in Tables 1 and 2). A T-test was performed based on the BLUP values ​​of the peanut seed size trait. It was found that the differences in seed size traits among germplasm materials with different genotypes at three loci (7-266, 7-27762, and 7-39518, Table 5) were extremely significant (p<0.001), indicating that molecular marker-assisted selection of peanut seed size traits can be performed. Figure 7 )

[0054] Table 5. Information on SNP molecular markers and SNP detection primers

[0055]

[0056]

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of SNP molecular markers in identifying peanut seed size traits, characterized in that, The SNP molecular marker is one or more of WTA7-2661184, WTA7-2776237 and WTA7-3951865; The WTA7-2661184 is located at 2661184bp of chromosome 7 of peanut genome, the base is C / T, and the corresponding phenotype is small grain / large grain; The WTA7-2776237 is located at 2776237bp of chromosome 7 of peanut genome, the base is C / G, and the corresponding phenotype is small grain / large grain; The WTA7-3951865 is located at 3951865bp of chromosome 7 of peanut genome, the base is G / A, and the corresponding phenotype is small grain / large grain; The peanut genome is Tifrunner.gnm1.KYV3.

2. A primer pair for detecting the KASP molecular marker of claim 1, characterized in that, The primer pair comprises one or more groups of SEQ ID NO. 1-3, SEQ ID NO. 4-6 and SEQ ID NO. 7-9.

3. A method of identifying KASP molecular marker primers for a peanut seed size associated SNP, characterized in that, The method comprises the following steps: (1) extracting the genomic DNA of the peanut material to be identified, and taking the genomic DNA as a template; (2) using the primer pair shown in SEQ ID NO. 1-9 to perform PCR amplification on the template, performing sequencing analysis on the amplification product, and judging the polymorphism of the molecular marker according to the sequencing result.

4. The method of claim 3, wherein, The PCR amplification system is as follows: Table 1 5. The method of claim 3, wherein the step of identifying is characterized by, The PCR amplification program is as follows: Table 2 6. The molecular marker of claim 1 or the identification method of any one of claims 3-5 in the breeding of large-grain peanut varieties or lines.

7. The molecular marker of claim 1 or the identification method of any one of claims 3-5 in the molecular breeding of peanuts, the breeding of transgenic peanuts or the improvement of peanut germplasm resources.

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