Linked molecular marker of major QTL (quantitative trait loci) uq2 of peanut legume constriction character as well as identification method and application of linked molecular marker

Abstract

The invention discloses a linked molecular marker of a major QTL (quantitative trait loci) uq2 of a peanut legume singulation character as well as an identification method and application of the linked molecular marker. The molecular marker and a major QTL site uq2 of the peanut legume singulation character are co-localized on a second chromosome of a peanut genome, and the molecular marker comprises molecular markers SNP4 and SNP57. The physical position of the molecular marker SNP4 is the 11591222th base position of the second chromosome of a cultivated peanut genome, and the polymorphism is C / T; the physical position of the molecular marker SNP57 is the 11894442 base position of the second chromosome of the cultivated peanut genome, and the polymorphism is A / T. The linked molecular marker is obtained by using a primer group. The nucleotide sequences of the primer group are respectively as shown in SEQ ID No. 1 to SEQ ID No. 6. The major QTL locus uq2 of the peanut legume constriction character as well as the linked molecular marker and the identification method of the major QTL locus uq2 are suitable for gene localization of the peanut legume constriction character and molecular marker-assisted selective breeding.

Description

Technical Field

[0001] The present invention belongs to the field of peanut molecular biology and genetic breeding technology, and specifically relates to a major effect QTL locus of peanut pod constriction trait uq2 Linked molecular markers and identification methods and applications. Background Art

[0002] my country is a major producer and consumer of peanuts. Peanut pods are the harvested organs of peanuts, consisting of kernels and shells. Peanuts have the characteristic of "underground fruiting". When ripe, the shell gradually hardens in the soil to form a deep or shallow constriction, called the fruit waist. The fruit waist is an important agronomic trait and variety-specific indicator of peanuts. It not only determines the commercial value of pod products, but also affects the peanut drop rate, clay content and breakage rate during processing. Despite its important research value, the genetic mechanism of peanut pod constriction trait is still unclear and lacks linked molecular markers, which seriously limits the genetic improvement of this trait.

[0003] The degree of peanut pod constriction varies widely, with different grades such as presence or absence, shallow, medium, deep and extremely deep. In traditional peanut pod waist breeding and improvement, the selection of offspring mainly relies on manual phenotypic observation, which often leads to low selection efficiency, poor accuracy and high labor costs. In recent years, some stable major effect QTLs have been reported for peanut pod size and weight traits, but there are few reports on stable major effect loci related to peanut pod constriction. By locating stable QTL loci related to the degree of pod constriction and developing efficient molecular marker-assisted genotype selection, the prediction, verification and aggregation of target traits can be effectively achieved.

[0004] PARMS (Penta-primer Amplification Refractory Mutation System) technology is a new SNP typing technology. It adds two primers labeled with 6-carboxyfluorescein (FAM) and hexachloroflurescein (HEX) on the basis of ARMS-PCR. Compared with traditional SSR and InDel marker typing, PARMS technology has the advantages of simple operation, short time consumption and low cost. Therefore, the development of PARMS markers linked to the peanut pod constriction trait is of great practical significance for accelerating the cloning of related genes and cultivating new peanut varieties suitable for mechanized production. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a major effect QTL locus for peanut pod constriction trait uq2 Linked molecular markers and identification methods and applications.

[0006] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions: The present invention provides a major effect QTL locus for peanut pod constriction trait uq2 Linked molecular markers, wherein the molecular markers are linked to the main QTL loci of peanut pod constriction traits uq2 Co-localized on chromosome 2 of the cultivated peanut genome, including molecular markers SNP_4 and SNP_57, the major effect QTL locus uq2 Located between base positions 11591222-11894442 on chromosome 2 of the peanut genome.

[0007] Furthermore, the physical position of the molecular marker SNP_4 is the 11591222 base position of chromosome 2 of the peanut genome; the physical position of the molecular marker SNP_57 is the 11894442 base position of chromosome 2 of the peanut genome.

[0008] Furthermore, the allele variation base of the molecular marker SNP_4 is C / T, and the allele variation base of SNP_57 is A / T.

[0009] The present invention also provides a PARMS primer combination for detecting the linked molecular marker, the primer combination includes 6 primers; the nucleotide sequence of the primer combination for detecting the SNP_4 is shown in SEQ ID No.1-SEQ ID No.3; the nucleotide sequence of the primer combination for detecting the SNP_57 is shown in SEQ ID No.4-SEQ ID No.6.

[0010] The present invention also provides a main effect QTL locus for peanut pod constriction trait uq2 The identification method comprises the following steps: S1, using the CTAB method to extract the genomic DNA of the peanut material to be identified; S2, using the extracted genomic DNA as a template, and using two sets of PARMS primer combinations shown in SEQ ID No.1-SEQ ID No.3 and SEQ ID No.4-SEQ ID No.6 to perform touchdown PCR amplification; S3, based on the fluorescence signal detected by the Touchdown PCR amplification reaction, determine the polymorphic type and site of the two molecular markers uq2 Allele type: If the HEX signal is mainly detected, the base of the molecular marker SNP_4 is determined to be C, and the base of SNP_57 is A, that is, hh type; if the FAM signal is mainly detected, the base of the molecular marker SNP_4 is determined to be T, and the base of SNP_57 is T, that is, HH type.

[0011] Furthermore, the base of the molecular marker SNP_4 of the variety Luhua No. 11 is C, and the base of the molecular marker SNP_4 of the variety 06B16 is T; the base of the molecular marker SNP_57 of the variety Luhua No. 11 is A, and the base of the molecular marker SNP_57 of the variety 06B16 is T.

[0012] Furthermore, the reaction system for the Touchdown PCR amplification is 10 μL, specifically including 0.5 μL of 2~5 ng DNA template, 5 μL of 2×PARMS master mix, 0.15 μL of forward primer 1, 0.15 μL of 10 mM forward primer 2, 0.4 μL of 10mM reverse primer, and 3.8 μL of ddH2O; the sequence of the forward primer 1 is shown in SEQ ID NO.1 and SEQ ID NO.4; the sequence of the forward primer 2 is shown in SEQ ID NO.2 and SEQ ID NO.5; the sequence of the reverse primer is shown in SEQ ID NO.3 and SEQ ID NO.6.

[0013] Furthermore, the reaction procedure of the Touchdown PCR amplification is: pre-denaturation at 94°C for 15 minutes; first-stage amplification reaction, 94°C for 20 seconds, annealing and extension at 65°C~57°C for 1 minute, 10 cycles, and the annealing and extension temperature is reduced by 0.8°C in each cycle; second-stage amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 57°C for 60 seconds, and 30 cycles.

[0014] The present invention also provides a major effect QTL locus uq2 Application in regulating peanut pod constriction traits, the main effect QTL locus uq2 The physical location of the major QTL site is between base positions 11591222-11894442 of chromosome 2 of the peanut genome, which can be detected by the primer set shown in SEQ ID No.1-SEQ ID No.6; uq2 The reduced-effect allele can reduce the degree of peanut pod constriction.

[0015] The present invention also provides the application of the linkage molecular marker or the primer combination in breeding peanut varieties or strains with low degree of pod constriction.

[0016] The present invention also provides the application of the linkage molecular marker or the primer combination in peanut molecular breeding, cultivating transgenic peanuts or improving peanut germplasm resources.

[0017] Compared with the prior art, the present invention has the following beneficial effects and advantages: the present invention discloses for the first time a major effect QTL locus of peanut pod constriction trait uq2It is located between two molecular markers SNP_4 and SNP_57, and between base positions 11591222-11894442 on chromosome 2 of the peanut genome. Its different genotypes have different regulatory effects on the peanut pod constriction trait, and have high utilization value for breeding peanut varieties or lines with low degrees of pod constriction.

[0018] The present invention screened and disclosed the main effect QTL locus of pod constriction trait for the first time uq2 The two linked molecular markers SNP_4 and SNP_57, as well as the primer combination and molecular marker method for detecting the linked molecular markers, can be effectively applied to the map-based cloning of the peanut pod constriction trait gene, assist in the selection of peanut pod constriction trait offspring, and help improve breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the evaluation index related to the degree of pod constriction in the present invention, wherein Front_D is the front constriction depth, Back_D is the back constriction depth, PW is the pod width, Front_DI is the front constriction depth index, and Back_DI is the back constriction depth index; Figure 2 The main QTL locus of peanut pod constriction trait in the present invention uq2 In the localization map of chromosome 2, QD stands for Qingdao and WH stands for Weihai; Figure 3 This is the genotyping diagram of 90 individual plants detected using linked molecular markers in the present invention, in which orange is uq2 The allele is hh type peanut family, indigo is uq2 The allele is the HH type peanut family, and the light grey is the negative control. DETAILED DESCRIPTION

[0020] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] Example 1 Test materials: Peanut variety Luhua No. 11 was used as the female parent and peanut variety 06B16 was used as the male parent for hybridization. The recombinant inbred line (RIL) population F9 (2022) and F10 (2023) generations containing 256 families were obtained through single-seed descent. The degree of pod constriction of the female parent Luhua No. 11 was not obvious, while the degree of pod constriction of the male parent 06B16 was more obvious. Both varieties can be purchased from Shandong Peanut Research Institute.

[0023] 1. Phenotypic identification: The test materials were planted in the test base in Qingdao (36.82°N, 120.51°E) from 2022 to 2023; and in the test base in Weihai (37.18°N, 121.99°E) in 2023; the planting method was ridge-covered film planting, with a ridge spacing of 0.3 meters, 10 plants planted in each row, and a plant spacing of 0.2 meters; a completely randomized block design was set up with 3 repetitions. The fertility of the test base is uniform, conventional field management is carried out, and pests and diseases are controlled in time. The mature pods of the 8 single plants in the middle of each row were harvested. More than 30 pods were taken from each family and placed on the platform for photography. Based on the pod image, the constriction on the same side as the fruit mouth is defined as the front constriction, the constriction on the opposite side of the fruit mouth is defined as the rear constriction, and the width of the minimum circumscribed rectangle of the pod is the pod width. Photoshop software was used to measure the anterior constriction depth, posterior constriction depth and pod width ( Figure 1 The front constriction depth index and the rear constriction depth index were calculated using the formula (constriction depth index = constriction depth / pod width).

[0024] The experimental results showed that in three different environments (2022 Qingdao, 2022 Weihai, and 2023 Qingdao), the variation ranges of pre-pod constriction in the RIL population were 0-0.395, 0-0.361, and 0-0.342, respectively, and the variation ranges of post-pod constriction were 0.070-0.306, 0.060-0.303, and 0.057-0.290, respectively, all showing super-parent inheritance (Table 1), indicating that this population is suitable for QTL analysis of peanut pod constriction traits.

[0025] Table 1 Phenotypic data of pod constriction in RIL population under three environments

[0026] Note: * and ** indicate significant differences at P<0.05 and P<0.01, respectively.

[0027] 2. Construction of genetic map: The genomic DNA of variety Luhua No. 11, variety 06B16 and 256 RIL families were extracted using Tiangen DNA extraction kit.

[0028] SNP typing: Whole genome resequencing and SNP typing were completed by Hangzhou Lianchuan Biotechnology Co., Ltd. The specific process is as follows: Sequencing libraries were constructed according to the resequencing library construction process. After the library quality inspection was qualified, it was sequenced using the IlluminaNova6000 platform. The sequencing depth of the parents was more than 20×, and the sequencing depth of the RIL family was more than 3×. After filtering to remove sequencing adapters and low-quality reads, high-quality reads were aligned to the cultivated peanut reference genome (https: / / data.legumeinfo.org / Arachis / hypogaea / genomes / Tifrunner. gnm2.J5K5 / ). SNP detection was performed using SAMtools v0.1.19 and GATK v3.3.0 software toolkits. Only SNPs that were polymorphic and homozygous in the parents, had a missing frequency and heterozygous frequency of less than 10% in the RIL family, and had a sequencing depth of more than 3× were used for subsequent gene analysis and genetic map construction.

[0029] Genetic map construction: Based on the genotype information of the parents, the SNP markers were binned using the hidden Markov model method of the SNPBinner software. The 3692 bin genotypes obtained were used to construct a genetic linkage map using the QTL ICIMapping V4.2 software. The linkage map was constructed using the 2-opt algorithm in the software, and rippling local optimization was performed with a window size of 5.

[0030] Genetic linkage map information: a total of 3692 bin markers (including 82292 SNP markers), distributed in 20 linkage groups, corresponding to 20 chromosomes of the cultivated peanut genome, covering a genetic map distance of 981.65 cM.

[0031] 3. QTL localization analysis: Using the genetic map constructed above and the phenotypic data of the three environments, a co-localized QTL interval was detected in the second linkage group using the complete interval mapping method of QTLIciMapping V4.1 software, which contained 6 additive QTLs ( qFront2.2a , qFront2.2b , qFront2.2c , qBack2a , qBack2b , qBack2c ).

[0032] BioMercator 2.1 software was further used to integrate the six additive QTLs into one main effect locus based on meta-analysis. uq2 ( Figure 2 ), uq2It can be stably expressed in three environments, and its contribution to the degree of pod constriction varies from 20.15% to 26.20%. Its reduced-effect allele comes from the variety Luhua No. 11, and its physical location is between base positions 11591222 and 11894442 on chromosome 2 of the peanut genome.

[0033] Further densification of the map to obtain the main effect QTL loci uq2 The tightly linked molecular markers are: molecular markers SNP_4 and SNP_57.

[0034] The physical position of the molecular marker SNP_4 is the 11591222 base position of chromosome 2 of the peanut genome, and its allele variation base is C / T; the physical position of the molecular marker SNP_57 is the 11894442 base position of chromosome 2 of the peanut genome, and its allele variation base is A / T.

[0035] Example 2 Using the major QTL locus for peanut pod constriction uq2 The two linked molecular marker primer combinations were used to perform Touchdown PCR amplification on the genomic DNA of 90 families, variety Luhua No. 11 and variety 06B16 randomly selected from the above RIL population. The fluorescent signals detected by the Touchdown PCR amplification reaction were used to determine the polymorphisms of the two molecular marker sequences in the RIL families, variety Luhua No. 11 and variety 06B16.

[0036] Amplification of the major QTL loci for peanut pod constriction uq2 The primer composition of the linked molecular marker SNP_4 includes: Forward primer 4-1 sequence 5'-GAAGGTGACCAAGTTCATGCTTTTTTCACAATCCCAAATCATGA-3' (as shown in SEQ ID NO. 1); Forward primer 4-2 sequence 5'-GAAGGTCGGAGTCAACGGATTTTTTCACAATCCCAAATCATGG-3' (as shown in SEQ ID NO.2); The reverse primer 4-3 sequence is 5'-ACTCAAAAAATGATGCTTTCTTCAT-3' (as shown in SEQ ID NO.3).

[0037] The target base of the molecular marker SNP_4 of the variety Luhua No. 11 is C, and the target base of the molecular marker SNP_4 of the variety 06B16 is T.

[0038] Amplification of the major QTL loci for peanut pod constriction uq2The primer composition of the linked molecular marker SNP_4 includes: Forward primer 57-1 sequence 5'-GAAGGTGACCAAGTTCATGCTTGTGAGTTGAATTGTTTGATTTTCAT-3' (as shown in SEQ ID NO.4); Forward primer 57-2 sequence 5'-GAAGGTCGGAGTCAACGGATTTGTGAGTTGAATTGTTTGATTTTCAA-3' (as shown in SEQ ID NO. 5); The reverse primer 57-3 sequence is 5'-GACTGGACCGATTAGTTCGTCTG-3' (as shown in SEQ ID NO.6).

[0039] When amplifying molecular markers, the reaction system of the Touchdown PCR amplification is 10 μL, specifically 0.5 μL of 2-5 ng DNA template, 5 μL of 2×PARMS master mix, 0.15 μL of forward primer 1 (SEQ ID NO.1 and SEQ ID NO.4), 0.15 μL of 10 mM forward primer 2 (SEQ ID NO.2 and SEQ ID NO.5), 0.4 μL of 10 mM reverse primer (SEQ ID NO.3 and SEQ ID NO.6), and 3.8 μL of ddH2O.

[0040] The reaction procedure of the Touchdown PCR amplification is as follows: pre-denaturation at 94°C for 15 minutes; first-stage amplification reaction, 94°C for 20 seconds, annealing and extension at 65°C-57°C for 1 minute, 10 cycles, with the annealing and extension temperature decreasing by 0.8°C in each cycle; second-stage amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 57°C for 60 seconds, 30 cycles.

[0041] According to the fluorescence signal detected by the Touchdown PCR amplification reaction, the polymorphic type and site of the two molecular markers are determined uq2 Allele type: If the HEX signal is mainly detected, the base of the molecular marker SNP_4 is determined to be C and the base of SNP_57 is A, that is, uq2 The locus allele comes from the variety Luhua No. 11, recorded as hh type; if the FAM signal is mainly detected, the base of the molecular marker SNP_4 is determined to be T, and the base of SNP_57 is T, that is, uq2 The locus allele comes from variety 06B16 and is recorded as HH type. 90 families were randomly selected from the RIL population, and the genotype analysis of SNP_4 and SNP_57 of the families was performed. The results showed that 43 of the 90 RIL families were HH type and 47 were hh type.Figure 2 ).

[0042] A total of 90 RIL families (43 HH types and 47 hh types) were planted in the Qingdao (36.82° N, 120.51° E) test base in 2024. The planting method was ridge-covered planting with a ridge spacing of 0.3 meters, 10 plants per row, and a plant spacing of 0.2 meters. The randomized block design was set up with 3 replications. The test base had uniform fertility, routine field management, and timely pest and disease control. The mature pods of the middle 8 plants in each row were harvested, and the front constriction depth index and the rear constriction depth index of each plant were calculated.

[0043] The results showed (Table 2) that the average values ​​of the anterior and posterior constriction depth indexes of the HH type families were 0.099 and 0.144, respectively, while the average values ​​of the anterior and posterior constriction depth indexes of the HH type families were 0.167 and 0.177, respectively. There were extremely significant differences in the anterior and posterior constriction depth indexes between the HH type and HH type families, indicating that uq2 Different genotypes can have different regulatory effects on the degree of peanut pod constriction. The two linked molecular markers have a good predictive effect on the peanut pod constriction trait and are suitable for peanut molecular marker-assisted selection breeding.

[0044] Table 2 Different uq2 Statistics of pod constriction degree among genotype families

[0045] Note: ** indicates that there is a significant difference between HH type families and hh type families at the p < 0.01 level.

[0046] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. Major QTL loci for peanut pod constriction trait uq2 A linked molecular marker, characterized in that The linkage molecular marker and the main QTL locus of peanut pod constriction trait uq2 Co-localized on chromosome 2 of the peanut genome, including molecular markers SNP_4 and SNP_57, the major effect QTL locus uq2 Located between base positions 11591222-11894442 on chromosome 2 of the peanut genome.

2. The linked molecular marker according to claim 1, characterized in that The physical position of the molecular marker SNP_4 is the 11591222 base position of chromosome 2 of the peanut genome; the physical position of the molecular marker SNP_57 is the 11894442 base position of chromosome 2 of the cultivated peanut genome.

3. The linked molecular marker according to claim 2, characterized in that The allele variation base of the molecular marker SNP_4 is C / T, and the allele variation base of the SNP_57 is A / T.

4. A PARMS primer composition for detecting the linked molecular marker according to claim 1, characterized in that: The primer composition includes 6 primers; the nucleotide sequence of the primer composition for detecting the SNP_4 is shown in SEQ ID No.1-SEQ ID No.3; The nucleotide sequences of the primer compositions used to detect the SNP_57 are shown in SEQ ID No.4-SEQ ID No.

6.

5. A major QTL locus for peanut pod constriction trait uq2 The identification method is characterized in that The identification method comprises the following steps: S1, extracting genomic DNA of peanut materials to be identified; S2, using the extracted genomic DNA as a template, and using two sets of PARMS primer combinations shown in SEQ ID No.1-SEQ ID No.3 and SEQ ID No.4-SEQ ID No.6 to perform touchdown PCR amplification; S3, based on the fluorescence signal detected by the Touchdown PCR amplification reaction, determine the polymorphic type and site of the two molecular markers uq2 Allele type: If the HEX signal is mainly detected, the base of the molecular marker SNP_4 is determined to be C, and the base of SNP_57 is A, that is, hh type; if the FAM signal is mainly detected, the base of the molecular marker SNP_4 is determined to be T, and the base of SNP_57 is T, that is, HH type.

6. The identification method according to claim 5, characterized in that: The reaction system of the Touchdown PCR amplification is 10 μL, specifically including 0.5 μL of 2-5 ng DNA template, 5 μL of 2×PARMS master mix, 0.15 μL of forward primer 1, 0.15 μL of 10 mM forward primer 2, 0.4 μL of 10mM reverse primer, and 3.8 μL of ddH2O; the sequence of the forward primer 1 is shown in SEQ ID NO.1 and SEQ ID NO.4; the sequence of the forward primer 2 is shown in SEQ ID NO.2 and SEQ ID NO.5; the sequence of the reverse primer is shown in SEQ ID NO.3 and SEQ ID NO.

6.

7. The identification method according to claim 5, characterized in that: The reaction procedure of the Touchdown PCR amplification is as follows: pre-denaturation at 94°C for 15 minutes; first-stage amplification reaction, 94°C for 20 seconds, annealing and extension at 65°C-57°C for 1 minute, 10 cycles, with the annealing and extension temperature decreasing by 0.8°C in each cycle; second-stage amplification reaction, denaturation at 94°C for 20 seconds, annealing and extension at 57°C for 60 seconds, 30 cycles.

8. The major QTL locus according to claim 1 uq2 The application in regulating the constriction trait of peanut pods is characterized in that: The major QTL loci uq2 can be detected by the primer set shown in SEQ ID No.1-SEQ ID No.6; the main effect QTL site uq2 The reduced-effect allele can reduce the degree of peanut pod constriction.

9. Use of the linked molecular marker according to any one of claims 1 to 3 or the primer combination according to claim 4 in breeding peanut varieties or lines with low degree of pod constriction.

10. Use of the linked molecular marker according to any one of claims 1 to 3 or the primer combination according to claim 4 in peanut molecular breeding, cultivation of transgenic peanuts or improvement of peanut germplasm resources.

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