Molecular markers associated with pecan nut weight and their application

Abstract

This invention discloses a set of molecular markers related to the nut weight of thin-shelled pecans and their applications, relating to the field of biotechnology. The nucleotide sequences of the molecular markers are shown in SEQ ID NO. 1-2. The molecular markers of this invention, used for marker-assisted selection and genomic selection of thin-shelled pecans, can accelerate the genetic improvement of thin-shelled pecan varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a molecular marker related to the weight of thin-shelled pecan nuts and its application. Background Technology

[0002] Nut weight is a core economic trait of the thin-shelled pecan (Carya illinoensis), directly determining fruit grade and yield per unit area. Nut weight quality is currently a key objective in targeted variety improvement.

[0003] Currently, there are significant technical bottlenecks in the accurate assessment of nut weight of thin-shelled pecans and the targeted breeding of large-fruited varieties: Traditional nut weight identification can only be performed after the plants have matured and been harvested and dried. Nut weight-related genotypes cannot be determined by phenotype during the seedling stage. Breeding requires the mass cultivation of a large number of individual plants and the selection of target superior plants after fruiting, resulting in a serious waste of breeding resources such as land, manpower, and time.

[0004] Traditional breeding relies on direct selection based on field phenotypic patterns. Nut reselection requires multiple generations of planting observation and multiple rounds of validation, resulting in long breeding cycles and low efficiency, failing to meet the industry's urgent need for superior new varieties with large fruits. Molecular marker-assisted breeding (MAS) and genomic selection (GS) are core technologies for crop genetic improvement. SNP molecular markers, due to their wide genomic distribution, high polymorphism, accurate detection, and suitability for high-throughput detection, have become the preferred markers for forestry molecular breeding. Existing research has developed some polymorphic molecular markers for thin-shelled pecans, but currently there are no specific SNP markers closely linked and stably associated with key nut traits. There is a lack of biomarkers and supporting detection systems that can be directly applied to breeding practices. Existing markers cannot support nut retargeting breeding, and GS models, lacking core trait marker support, have insufficient predictive accuracy, severely hindering the genetic improvement and new variety breeding process of large-fruited thin-shelled pecans.

[0005] Currently, key industry scenarios such as breeding and selection, seedling identification, and germplasm resource evaluation lack rapid and accurate early assessment technologies for nut weight. This results in low efficiency in screening high-quality large-fruited seedlings and insufficient utilization of superior germplasm, hindering the high-quality development of the industry towards improved varieties. Therefore, developing SNP molecular markers specifically associated with nut weight in thin-shelled pecans, constructing a supporting efficient detection system, and applying them to MAS breeding and GS model construction will enable accurate prediction of nut weight at the seedling stage and targeted selection of superior large-fruited plants. This has significant industrial application value and practical significance for reducing breeding costs, shortening the breeding cycle, accelerating the cultivation and promotion of large-fruited varieties, and promoting the high-quality development of the thin-shelled pecan industry. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention has developed a set of molecular markers associated with the nut weight of thin-shelled pecans. Specifically, SNP molecular markers associated with the nut weight of thin-shelled pecans were developed using high-throughput sequencing technology and validated in genetic and natural populations. These markers can be used to predict and identify the nut weight trait of thin-shelled pecans, and their application in MAS breeding and GS model construction can accelerate the genetic improvement of thin-shelled pecan varieties.

[0007] The primary objective of this invention is to identify molecular markers associated with the heavy pecan phenotype, located on the pecan genome, with specific SNP information shown in Table 1:

[0008] Table 1 SNP locus information

[0009]

[0010] The sequence of the molecular marker includes at least one of SEQ ID NO.1 to SEQ ID NO.2, wherein the polymorphic site of SEQ ID NO.1 corresponds to the C>A mutation at position 301 of SEQ ID NO.1, wherein CC and / or CA are genotypes with high nut weight and AA are genotypes with low nut weight; wherein the polymorphic site of SEQ ID NO.2 corresponds to the G>A mutation at position 301 of SEQ ID NO.2, wherein GG is genotypes with high nut weight and GA are genotypes with low nut weight.

[0011] The sequence is a single copy within the genome, and the specific sequence is as follows:

[0012] SEQ ID NO.1

[0013] AGAAGTTCCTGCTTAATATTTGGCAGGGCTGACTTGCAGCCTCCGCCATTAACGAAGGTCTTTTAATGTCCTCTTCAACAATAACAAATTTCCCTTTTTTAGTGAATTGGGCTCTAAATAGCCATCATCTGCATCTAATAGCCTATGTATAAAACTAAGACTCAACATAATAGTTGAATTGTTACCTTTGCTATGGGATCACCTCTTCCATTTGCCCTGGTTAGCCTTTCCCTTATCCGATCTACCATTATTCATCCTCTCTTTTGTAACTTTTTGTAGTTCTTGATAGCTAACATTGTAMTAAGCTTCATCATTTTTTGTTTGACTCATATTGGGCCTATAGCCCACAGTTTCAATGACTAAGTCCATAACCCCTTTGTCACAATGTTTGACTGTCCCATATTCACTTCACCACCTTCATCCACATCGTCACAATTTTCAAATCCCATTTCCTTGTCTACCTCTAATTTCAAATTTGTAATTTTATTCTAAATTATTTTGATCCTTTATTTACCTAGTTTCCTTCTGATTCCTAAGATTTTTTAATACAAAATTTGAATTTCATTGATCCACAGCTTTCGTTTCACTTCTGCTAGACATC

[0014] SEQ ID NO.2

[0015] AGACTCTGCTACCTTCCTAAGGTGATAAGCAAAACCCTCCCATCCACAGCCCTTATCTCCCTCGGGTATGACCAGTGAGCCTTTTCTTCTATCATTGCTAAATTCCTCCAACAAGAGATAACATCCCCTTGAGTTTTTGAGGAGCTGCAG AATGAGAACTCCGCTGCCTAACCTCAATTTTCTGAAGAAACCTTCTCGCCGACGAGAAACAGTGGCCTCCATTACCATTCTGACCACCCATGCAGCTGACGTCCCGTTAATGATTAAATATAAGGTCATACGTCTGTTTCTTTTGGTAAG RCGAAAGAGGTTGGCACCCACCTTTGCGAAGAAGAAAGTTTTTGATTCAATCGGAAAACGCCTAACCACATCCATCATAAACAGGAAGAGCAAAAACTGGCGAGAACAAACCACTCGAAAAACCCAGAAAATGCTGGCTGAAAACATACC AAACCGATGACCCAATGCCGACAGAAGCAACCACTCGTGTGAATCCAGAACCCACTGGTCTGAGAACCCAGAAAAACTAGGCTGAAAACAGACCAGTCCGATGACCCCAGGAAGCACTAGTCTGAAAACAGACAGTGCCGATGAACCCAA

[0016] To achieve efficient and accurate detection of the aforementioned molecular markers, this invention provides a set of adaptive specific primers, which contains two primer pairs, wherein SEQ ID NO.3~SEQ ID NO.4 are used to detect molecular marker SNP1, and SEQ ID NO.5~SEQ ID NO.6 are used to detect molecular marker SNP2.

[0017] SEQ ID NO.3

[0018] ACCTCTTCCATTTGCCCTGG

[0019] SEQ ID NO.4

[0020] TGACGATGTGGATGAAGGTGG

[0021] SEQ ID NO.5

[0022] AGAATGAGAACTCCGCTGCC

[0023] SEQ ID NO.6

[0024] TCTGTCGGCATTGGGTCATC.

[0025] The primers mentioned above can be used for first-generation or second-generation sequencing.

[0026] The present invention also provides a kit for detecting the above-mentioned molecular markers, the core component of which is the above-mentioned specific primer set, and also includes a special mix (containing Taq enzyme, dNTP, and reaction buffer), sterile water, and standard controls (one tube each of standard genotype templates, each with a concentration of 50 ng / μL).

[0027] This invention also provides a method for genetically improving thin-shelled pecan nuts, the method comprising the following steps:

[0028] 1) Determine the genotypes of molecular marker sites associated with the nut weight traits of thin-shelled pecans in the pecan resource population, wherein the molecular marker sites are the aforementioned SNP sites.

[0029] 2) Make appropriate selections based on the genotype of the molecular marker and the breeding objectives.

[0030] Step 1) includes the following steps:

[0031] 1.1) Extract genomic DNA from the thin-shelled pecans to be tested;

[0032] 1.2) Genotyping of the thin-shelled pecans to be tested;

[0033] 1.3) Based on the detection results, determine the genotypes of the thin-shelled pecans to be tested at the SNP1 site on chromosome 11 and the SNP2 site on chromosome 6 of the reference genome;

[0034] Step 2) includes at least one of the following steps:

[0035] 2.1) In the aforementioned thin-shelled pecan resource population, individuals with the AA type at the SNP1 locus of thin-shelled pecans were eliminated to progressively increase the frequency of the CC and / or CA genotypes at this locus, thereby increasing the nut weight of the offspring thin-shelled pecans;

[0036] 2.2) In the thin-shelled pecan resource population, individuals with the GA type at the SNP2 locus of thin-shelled pecans were eliminated to increase the frequency of the GG genotype at this locus generation by generation, thereby increasing the nut weight of the offspring thin-shelled pecans.

[0037] Preferably, in step 2.1), individuals with the CA and AA types at the SNP1 locus of thin-shelled pecans are eliminated to increase the frequency of the CC genotype at this locus generation by generation, thereby increasing the nut weight of the offspring thin-shelled pecans.

[0038] Preferably, in order to perform genetic probability breeding more accurately, step 2) includes the following steps:

[0039] In the thin-shelled pecan resource population, individuals with the SNP1 and SNP2 combination genotypes CC_GG and / or CA_GG were retained, thereby increasing the nut weight of the offspring thin-shelled pecans.

[0040] Preferably, in order to conduct genetic breeding more accurately, step 2) is as follows:

[0041] In the thin-shelled pecan resource population, individuals with the genotype CC_GG on chromosomes 11 and 6 of the thin-shelled pecan were retained, thereby increasing the nut weight of the offspring thin-shelled pecans.

[0042] The accuracy of the prediction results has been verified to be high through population testing, and can provide a reliable basis for seedling transactions.

[0043] The present invention further provides a method for evaluating the weight of thin-shelled pecan nuts, the method comprising the following steps:

[0044] 1) Determine the genotypes of molecular marker sites associated with the nut weight traits of thin-shelled pecans in the pecan resource population, wherein the molecular marker sites are the aforementioned SNP sites.

[0045] 2) Determine the nut weight of the thin-shelled pecan sample based on the genotype of the molecular marker.

[0046] Step 1) includes the following steps:

[0047] 1.1) Extract genomic DNA from the thin-shelled pecans to be tested;

[0048] 1.2) Genotyping of the thin-shelled pecans to be tested;

[0049] 1.3) Based on the detection results, determine the genotypes of the thin-shelled pecans to be tested at the SNP1 site on chromosome 11 and the SNP2 site on chromosome 6 of the reference genome;

[0050] Step 2) determining the nut weight of the thin-shelled pecan sample based on the genotype of the molecular marker includes at least one of the following steps:

[0051] 2.1) In the thin-shelled pecan population, if the genotype of the thin-shelled pecan sample at the SNP1 locus is CC or CA, then the sample is determined to be a high-nut-weight sample.

[0052] 2.2) In the thin-shelled pecan population, if the genotype of the thin-shelled pecan sample at the SNP2 locus is GG, then the sample is determined to be a high-nut-weight sample.

[0053] Preferably, in step 2.1), if the genotype of the thin-shelled pecan sample at the SNP1 locus is CC, then the sample is determined to be a high-nut-weight sample.

[0054] Preferably, in order to more accurately reassess the nuts, step 2) includes the following steps:

[0055] In the thin-shelled pecan population, if the combined genotype of the thin-shelled pecan sample at SNP1 and SNP2 loci is CC_GG and / or CA_GG, then the sample is determined to be a high-nut-weight sample.

[0056] Preferably, in order to perform more accurate nut reassessment, step 2) is as follows:

[0057] In a population of thin-shelled pecans, if the combined genotype of the thin-shelled pecan sample at SNP1 and SNP2 loci is CC_GG, then the sample is determined to be a sample with high nut weight.

[0058] The beneficial effects of this invention are as follows: The SNP molecular markers of this invention have significant application value in breeding for the pecan nut weight correlation trait or in predicting the pecan nut weight. Applying them to MAS breeding enables precise seedling selection, eliminating individuals with non-target genotypes, and reducing breeding costs. Integrating them into the GS model can improve the predictive accuracy of multi-trait aggregation breeding and accelerate the process of variety genetic improvement. Simultaneously, the primer set or kit of this invention also has significant application advantages in breeding for the pecan nut weight correlation trait or in predicting the pecan nut weight, exhibiting high specificity and stable detection results. Attached Figure Description

[0059] The method of the present invention and its beneficial effects will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0060] Figure 1 The results are statistical results of nut weights of different genotypes at the SNP1 locus. Different lowercase letters indicate significant differences, and different uppercase letters indicate extremely significant differences.

[0061] Figure 2 The results are statistical results of nut weights of different genotypes at the SNP2 locus, with different capital letters indicating highly significant differences.

[0062] Figure 3This is a heatmap showing the significance of differences in nut weight among different genotype combinations. The values ​​represent the p-values ​​of the significance test between the genotype combinations on the x-axis and y-axis. P ≤ 0.05 indicates that there is a significant difference in nut weight between the genotype combinations on the x-axis and y-axis. Detailed Implementation

[0063] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0064] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0065] Example

[0066] Example 1: Development of SNPs and QTL Localization

[0067] 1. Experimental Materials

[0068] 112 thin-shelled pecan germplasm resource samples were collected.

[0069] 2. Sample DNA extraction, library construction, and sequencing

[0070] First, the leaves of each sample were preserved using liquid nitrogen. Genomic DNA was extracted from samples 1 to 112 using a kit. A total of 994 Gb of raw data was obtained from sequencing, and the sequencing results were 150 bp of paired-end data.

[0071] Specific experimental steps:

[0072] Library construction was initiated with 1 μg of DNA;

[0073] High-quality genomic DNA was extracted using the CTAB method;

[0074] 0.75% agarose gel electrophoresis was used to detect DNA fragment size and the degree of DNA degradation.

[0075] The NanoDrop One spectrophotometer (Thermo Fisher Scientific) was used to detect DNA purity, with an OD260 / 280 ratio between 1.8 and 2.2, indicating no protein or visible contaminants.

[0076] The Qubit 3.0 fluorescence analyzer (Life Technologies, Carlsbad, CA, USA) was used to detect DNA concentrations greater than 50 ng / μl and total amounts greater than 2 μg.

[0077] After DNA was broken down by sonication with a Covaris M220, magnetic beads were used for fragment selection, resulting in sample bands concentrated between 200-400 bp.

[0078] The qualified libraries were then put into the sequencing machine for 2*150bp sequencing.

[0079] 3. Data quality control

[0080] (1) Remove the adapter sequence from the sequence;

[0081] (2) Remove polyG and polyX from the end of the reads (minimum length is 10bp);

[0082] (3) The average quality value of the bases in the window is calculated by using a sliding window method. Low-quality sliding windows are clipped out. Its function is similar to Trimmomatic.

[0083] (4) Remove N reads that are greater than 5;

[0084] (5) Remove reads with a base content of less than 15 that is higher than 40%;

[0085] (6) Remove reads with a length of less than 15bp after filtering.

[0086] 4. Data comparison and SNP development

[0087] In this invention, we use the genome of thin-shelled pecan as a reference genome, use BWA alignment software to align the sequencing fragments back to the reference genome, and then use Picard-tools to remove the sequencing fragments generated by PCR-duplication.

[0088] For each sample, a bwa (version: 0.7.17; parameter: mem) alignment analysis was performed. The filtered cleanreads were then aligned to the reference genome, and the alignment results were statistically analyzed. The specific analysis steps are as follows:

[0089] (1) Use bwa alignment software (parameter: mem-R, other parameters are the software default parameters) to align the clean reads of all samples with the reference genome;

[0090] (2) Use samtools (parameter: sort) to convert the alignment results from the sam (Sequence Alignment / MAP) file to a sorted bam (binary Alignment / Map) file;

[0091] (3) Use samtools (parameter: markdup -r) to remove duplicates from the sorted alignment results for subsequent analysis;

[0092] (4) Develop SNPs using GATK software.

[0093] 5. Genome-wide association analysis

[0094] The SNPs obtained in step 4 above were used to perform GWAS correlation analysis with the weight of each sample nut (nuts refer to shelled fruits that have been fully ripened and had their green skin removed, and after harvesting, they are dried and weighed after drying to constant weight). The GWAS analysis of this invention uses the lm and lmm models of GEMMA software, the GLM and MLM models of rMVP software, and the FarmCPU model of rMVP software.

[0095] Ultimately, one locus was found on chromosomes 11 and 6, totaling two loci that significantly affected nut weight. Figures 1-3 As shown in Table 2:

[0096] Table 2. Associated SNP sites

[0097]

[0098] Note: Columns 4-8 show the significance P-values ​​of the association results between SNP sites and nut weight traits for different models.

[0099] The molecular marker SNP1 C>A is located at position 301 of the sequence in SEQ ID NO.1, and the molecular marker SNP2 G>A is located at position 301 of SEQ ID NO.2. The sequences are single-copy sequences within the genome.

[0100] SEQ ID NO.1

[0101] AGAAGTTCCTGCTTAATATTTGGCAGGGCTGACTTGCAGCCTCCGCCATTAACGAAGGTCTTTTAATGTCCTCTTCAACAATAACAAATTTCCCTTTTTTAGTGAATTGGGCTCTAAATAGCCATCATCTGCATCTAATAGCCTATGTATAAAACTAAGACTCAACATAATAGTTGAATTGTTACCTTTGCTATGGGATCACCTCTTCCATTTGCCCTGGTTAGCCTTTCCCTTATCCGATCTACCATTATTCATCCTCTCTTTTGTAACTTTTTGTAGTTCTTGATAGCTAACATTGTAMTAAGCTTCATCATTTTTTGTTTGACTCATATTGGGCCTATAGCCCACAGTTTCAATGACTAAGTCCATAACCCCTTTGTCACAATGTTTGACTGTCCCATATTCACTTCACCACCTTCATCCACATCGTCACAATTTTCAAATCCCATTTCCTTGTCTACCTCTAATTTCAAATTTGTAATTTTATTCTAAATTATTTTGATCCTTTATTTACCTAGTTTCCTTCTGATTCCTAAGATTTTTTAATACAAAATTTGAATTTCATTGATCCACAGCTTTCGTTTCACTTCTGCTAGACATC

[0102] SEQ ID NO.2

[0103] AGACTCTGCTACCTTCCTAAGGTGATAAGCAAAACCCTCCCATCCACAGCCCTTATCTCCCTCGGGTATGACCAGTGAGCCTTTTCTTCTATCATTGCTAAATTCCTCCAACAAGAGATAACATCCCCTTGAGTTTTTGAGGAGCTGCAG AATGAGAACTCCGCTGCCTAACCTCAATTTTCTGAAGAAACCTTCTCGCCGACGAGAAACAGTGGCCTCCATTACCATTCTGACCACCCATGCAGCTGACGTCCCGTTAATGATTAAATATAAGGTCATACGTCTGTTTCTTTTGGTAAG RCGAAAGAGGTTGGCACCCACCTTTGCGAAGAAGAAAGTTTTTGATTCAATCGGAAAACGCCTAACCACATCCATCATAAACAGGAAGAGCAAAAACTGGCGAGAACAAACCACTCGAAAAACCCAGAAAATGCTGGCTGAAAACATACC AAACCGATGACCCAATGCCGACAGAAGCAACCACTCGTGTGAATCCAGAACCCACTGGTCTGAGAACCCAGAAAAACTAGGCTGAAAACAGACCAGTCCGATGACCCCAGGAAGCACTAGTCTGAAAACAGACAGTGCCGATGAACCCAAA

[0104] Example 2: Correlation analysis between phenotype and genotype in a genetic population

[0105] Two pairs of primers were designed and used for PCR amplification to construct a high-throughput sequencing library. The primer sequences are shown in SEQ ID NO.3~SEQ ID NO.6.

[0106] Table 3 Primers for SNP site detection

[0107]

[0108] Sequencing was performed on the two loci mentioned above in 112 thin-shelled pecan samples. Nut weight (detection method as in Example 1) of individuals with different genotypes was statistically analyzed and subjected to Tukey's test. The results are shown in Table 4 and... Figures 1-2 As shown:

[0109] Table 4 Mutation sites and nut weight traits

[0110]

[0111] Note: Different lowercase letters indicate significant differences between groups (P≤0.05), different uppercase letters indicate extremely significant differences between groups (P≤0.01), and the same uppercase or lowercase letter indicates no significant difference; the statistical results exclude individuals whose genotypes were not detected, and the nut weight is the average value.

[0112] Table 4 shows that there was no significant difference between the CC and CA genotypes at SNP1, but both were significantly higher than the A / A genotypes. At SNP2, the GG genotype had a significantly higher nut weight than the GA genotype.

[0113] Further statistical analysis and Tukey's test were performed on the nut weights of different genotype combinations at the two loci. The results are shown in Table 5. Figure 3 As shown:

[0114] Table 5 Nut weights of different genotype combinations

[0115]

[0116] From Table 5 and Figure 3 It can be seen that the CC_GG genotype had the highest nut weight in the SNP1_SNP2 combination, which was significantly higher than that of the CC_GA, CA_GA and AA_GA combinations, but not significantly different from that of CA_GG (P=0.1287); CA_GG was in the middle, which was significantly higher than that of AA_GA; there was no significant difference among CC_GA, CA_GA and AA_GA.

[0117] Example 3: Method for genetic improvement of thin-shelled pecan nuts

[0118] A method for genetically improving thin-shelled pecan nuts, the method comprising the following steps:

[0119] 1) Determine the genotypes of molecular marker sites related to the nut weight traits of thin-shelled pecans in the pecan resource population, wherein the molecular marker sites are the sites in the aforementioned embodiments.

[0120] 2) Make appropriate selections based on the genotype of the molecular marker and the breeding objectives.

[0121] Step 1) includes the following steps:

[0122] 1.1) Extract genomic DNA from the thin-shelled pecans to be tested;

[0123] 1.2) Genotyping of the thin-shelled pecans to be tested;

[0124] 1.3) Based on the detection results, determine the genotypes of the thin-shelled pecans to be tested at the SNP1 site on chromosome 11 and the SNP2 site on chromosome 6 of the reference genome;

[0125] Step 2) includes at least one of the following steps:

[0126] 2.1) In the thin-shelled pecan resource population, individuals with the AA type at the SNP1 locus of thin-shelled pecans were eliminated to increase the frequency of the CC and / or CA genotypes at this locus generation by generation, thereby increasing the nut weight of the offspring thin-shelled pecans.

[0127] 2.2) In the thin-shelled pecan resource population, individuals with the GA type at the SNP2 locus of thin-shelled pecans were eliminated to increase the frequency of the GG genotype at this locus generation by generation, thereby increasing the nut weight of the offspring thin-shelled pecans.

[0128] Preferably, in step 2.1), individuals with the CA and AA types at the SNP1 locus of thin-shelled pecans are eliminated to increase the frequency of the CC genotype at this locus generation by generation, thereby increasing the nut weight of the offspring thin-shelled pecans.

[0129] Preferably, in order to perform genetic probability breeding more accurately, step 2) includes the following steps:

[0130] In the thin-shelled pecan resource population, individuals with the SNP1 and SNP2 combination genotypes CC_GG and / or CA_GG were retained, thereby increasing the nut weight of the offspring thin-shelled pecans.

[0131] Preferably, in order to conduct genetic breeding more accurately, step 2) is as follows:

[0132] In the thin-shelled pecan resource population, individuals with the genotype CC_GG on chromosomes 11 and 6 of the thin-shelled pecan were retained, thereby increasing the nut weight of the offspring thin-shelled pecans.

[0133] Example 4: Method for evaluating the weight of thin-shelled pecan nuts

[0134] A method for assessing the weight of thin-shelled pecan nuts, the method comprising the following steps:

[0135] 1) Determine the genotypes of molecular marker sites related to the nut weight traits of thin-shelled pecans in the pecan resource population, wherein the molecular marker sites are the sites in the aforementioned embodiments.

[0136] 2) Determine the nut weight of the thin-shelled pecan sample based on the genotype of the molecular marker.

[0137] Step 1) includes the following steps:

[0138] 1.1) Extract genomic DNA from the thin-shelled pecans to be tested;

[0139] 1.2) Genotyping of the thin-shelled pecans to be tested;

[0140] 1.3) Based on the detection results, determine the genotypes of the thin-shelled pecans to be tested at the SNP1 site on chromosome 11 and the SNP2 site on chromosome 6 of the reference genome;

[0141] Step 2) determining the nut weight of the thin-shelled pecan sample based on the genotype of the molecular marker includes at least one of the following steps:

[0142] 2.1) In the thin-shelled pecan population, if the genotype of the thin-shelled pecan sample at the SNP1 locus is CC or CA, then the sample is determined to be a high-nut-weight sample.

[0143] 2.2) In the thin-shelled pecan population, if the genotype of the thin-shelled pecan sample at the SNP2 locus is GG, then the sample is determined to be a high-nut-weight sample.

[0144] Preferably, in step 2.1), if the genotype of the thin-shelled pecan sample at the SNP1 locus is CC, then the sample is determined to be a high-nut-weight sample.

[0145] Preferably, in order to more accurately reassess the nuts, step 2) includes the following steps:

[0146] In the thin-shelled pecan population, if the combined genotype of the thin-shelled pecan sample at SNP1 and SNP2 loci is CC_GG and / or CA_GG, then the sample is determined to be a high-nut-weight sample.

[0147] Preferably, in order to perform more accurate nut reassessment, step 2) is as follows:

[0148] In a population of thin-shelled pecans, if the combined genotype of the thin-shelled pecan sample at SNP1 and SNP2 loci is CC_GG, then the sample is determined to be a sample with high nut weight.

[0149] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to the above embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Molecular markers associated with the weight of thin-shelled pecan nuts, characterized in that, The molecular markers include at least one of SNP1 or SNP2; the polymorphic site of SNP1 corresponds to the C>A mutation at position 301 of SEQ ID NO.1, wherein CC and / or CA are genotypes with high nut weight and AA are genotypes with low nut weight; the polymorphic site of SNP2 corresponds to the G>A mutation at position 301 of SEQ ID NO.2, wherein GG is genotypes with high nut weight and GA is genotypes with low nut weight.

2. A primer set for detecting molecular markers, characterized in that, The nucleotide sequences of the primer set are shown in SEQ ID NO.3~SEQ ID NO.6, wherein SEQ ID NO.3~SEQ ID NO.4 are used to detect molecular marker SNP1, and SEQ ID NO.5~SEQ ID NO.6 are used to detect molecular marker SNP2. SNP1 corresponds to the C>A mutation at position 301 of SEQ ID NO.1, where CC and / or CA are genotypes with high nut weight and AA is a genotype with low nut weight; SNP2 corresponds to the G>A mutation at position 301 of SEQ ID NO.2, where GG is a genotype with high nut weight and GA is a genotype with low nut weight.

3. A kit for detecting molecular markers, characterized in that, The kit comprises the primer set as described in claim 2.

4. A method for evaluating the weight traits of thin-shelled pecan nuts, characterized in that, The method includes the following steps: (1) Extract genomic DNA from the thin-shelled pecan samples to be tested; (2) The genotype of the thin-shelled pecan to be tested is detected using the primer set described in claim 2 or the kit described in claim 3; (3) Based on the test results, determine the genotype of the thin-shelled pecan to be tested; (4) The weight of thin-shelled pecan nuts was assessed based on the genotype results obtained from the test. The assessment criteria were that individuals with the following genotypes at the following loci were individuals with high nut weight: individuals with the CC or CA genotype at the SNP1 locus and individuals with the GG genotype at the SNP2 locus. The SNP1 corresponds to the C>A mutation at position 301 of SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.

1. The SNP2 corresponds to the G>A mutation at position 301 of SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.

2.

5. A genetic breeding method for improving the nut traits of thin-shelled pecans, characterized in that, The method includes the following steps: (1) Extract genomic DNA from the thin-shelled pecan resource population to be tested; (2) The genotype of the thin-shelled pecan to be tested is detected using the primer set described in claim 2 or the kit described in claim 3; (3) Based on the test results, determine the genotype of the thin-shelled pecan to be tested; (4) Select individuals with different genotypes as parents according to the breeding goal. When the breeding goal is to breed varieties with high nut weight, select individuals with the following genotypes as parents: Individuals with the genotype CC or CA at SNP1 and / or the genotype GG at SNP2, wherein SNP1 corresponds to the C>A mutation at position 301 of SEQ ID NO.1 and its nucleotide sequence is shown in SEQ ID NO.1, and SNP2 corresponds to the G>A mutation at position 301 of SEQ ID NO.2 and its nucleotide sequence is shown in SEQ ID NO.

2.

6. The application of the primer set of claim 2 or the kit of claim 3 in breeding related to the pecan nut weight trait or in predicting the pecan nut weight.

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