Carya illinoensis KASP primer combination, kit and application

Abstract

The invention discloses a carya illinoensis KASP primer combination, a kit and application of the carya illinoensis KASP primer combination. The primer combination is used for detecting a plurality of different SNP (Single Nucleotide Polymorphism) sites located at the 351th site of any sequence of SEQ ID NO: 1-23. The polymorphism information content of the detected SNP site is concentrated in the range of 0.3-0.4, the minimum allele frequency is mainly distributed in the range of 0.4-0.5, genetic variation in a group can be effectively reflected, and the genetic diversity of the group is represented. Meanwhile, based on the carya illinoensis KASP primer combination, identification, differentiation and genetic background analysis of carya illinoensis germplasm resources can be effectively realized, and the further screened core KASP primer combination only contains 9 groups of primers, so that the identification and analysis efficiency and economy can be greatly improved. On the other hand, a DNA fingerprint spectrum can be further constructed based on the carya illinoensis core KASP primer combination, and a new tool is provided for molecular breeding of carya illinoensis.

Description

Technical Field

[0001] This invention relates to thin-shelled pecans, and more particularly to a KASP primer combination, kit, and application for thin-shelled pecans. Background Technology

[0002] Thin-shelled pecans ( Carya illinoinensis (Wangenh.) K. Koch) is a woody plant belonging to the genus Hickory in the family Juglandaceae. Its kernels are rich in unsaturated fatty acids such as oleic acid and linoleic acid, as well as protein, polyphenols, flavonoids and various trace elements. It is highly nutritious and has health benefits such as improving brain function, delaying aging and preventing cardiovascular and cerebrovascular diseases.

[0003] Competitive allele-specific polymerase chain reaction (KASP) is a novel genotyping technology based on single nucleotide polymorphisms (SNPs). It achieves accurate genotyping of target SNP sites through specific primer design. It features low cost, high throughput, high accuracy and ease of operation, and provides an ideal tool for SNP genotyping of large-scale germplasm resources.

[0004] Currently, KASP is widely used in molecular marker-assisted breeding of various crops, but its application in pecans is relatively limited. Patent CN119979766A discloses a KASP primer for identifying different pecan varieties; however, it consists of 33 primer sets, resulting in high application costs. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a more economical KASP primer combination for thin-shelled pecans that can be used for multiple applications such as germplasm resource identification, genetic background analysis, and molecular breeding; the second purpose is to provide a kit containing this KASP primer combination for thin-shelled pecans; and the third purpose is to provide applications of the above-mentioned product.

[0006] Technical solution: The thin-shelled pecan KASP primer combination of the present invention can detect multiple different SNP sites, wherein the SNP sites are located at position 351 of any sequence of SEQ ID NO: 1~23.

[0007] Preferably, the primers in the primer combination use any one of SEQ ID NO: 1 to 23 as the design reference sequence, have a GC content between 45% and 55%, a melting temperature of 55 to 65°C, and a length of 60 to 120 bases.

[0008] Preferably, the primer combination consists of primers with sequences as shown in SEQ ID NO: 24~92.

[0009] Further preferably, in the primer combination, the primer with sequence as shown in SEQ ID NO: 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90 is modified with 6-carboxyfluorescein group at 5' end, and the primer with sequence as shown in SEQ ID NO: 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91 is modified with VIC phosphoramidite monomer at 5' end.

[0010] Preferably, the primer combination consists of primers with sequences as shown in SEQ ID NO: 33~35, SEQ ID NO: 36~38, SEQ ID NO: 45~47, SEQ ID NO: 48~50, SEQ ID NO: 51~53, SEQ ID NO: 60~62, SEQ ID NO: 66~68, SEQ ID NO: 72~74, SEQ ID NO: 84~86.

[0011] Further preferably, in the primer combination, the primer with sequence as shown in SEQ ID NO: 33, 36, 45, 48, 51, 60, 66, 72, 84 is modified with 6-carboxyfluorescein group at 5' end, and the primer with sequence as shown in SEQ ID NO: 34, 37, 46, 49, 52, 61, 67, 73, 85 is modified with VIC phosphoramidite monomer at 5' end.

[0012] The kit of the present application contains the aforementioned Carya illinoensis KASP primer combination.

[0013] The Carya illinoensis KASP primer combination or the kit of the present application is applied in Carya illinoensis germplasm resource identification.

[0014] The Carya illinoensis KASP primer combination or the kit of the present application is applied in Carya illinoensis genetic background analysis.

[0015] The Carya illinoensis KASP primer combination or the kit of the present application is applied in Carya illinoensis molecular breeding.

[0016] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: 1. The polymorphism information content of the SNP sites detected by the KASP primer combination for thin-shelled pecans is concentrated in the range of 0.3-0.4, and the minimum allele frequency is mainly distributed in the range of 0.4-0.5, indicating that the detected SNP sites can effectively reflect the genetic variation within the population and represent the genetic diversity of the population; 2. Based on the KASP primer combination for thin-shelled pecans, the identification, differentiation and genetic background analysis of thin-shelled pecan germplasm resources can be effectively realized. The core KASP primer combination for further screening contains only 9 primers and has the same identification effect, which can greatly improve the efficiency and economy of identification and analysis; 3. Based on the core KASP primer combination for thin-shelled pecans, DNA fingerprinting can be further constructed, providing a new tool for molecular breeding of thin-shelled pecans. Attached Figure Description

[0017] Figure 1 The graph shows the statistical results of genetic polymorphism analysis of 23 SNP loci, where A is the statistical results of polymorphism information content and B is the statistical results of minimum allele frequency. Figure 2 The figure shows the results of population structure analysis of 58 thin-shelled pecan samples based on KASP primer combinations, where A is the phylogenetic tree and B is the principal component analysis result. Figure 3 The following diagram shows the screening results of core SNP loci combinations. A represents the statistical results of SNP loci identification efficiency, B represents the genetic distance among 58 thin-shelled pecan varieties based on 23 SNP loci, C represents the genetic distance among 58 thin-shelled pecan varieties based on 9 core SNP loci, and D represents the correlation analysis results of genetic distance among 58 thin-shelled pecan varieties based on two sets of SNP loci. Figure 4 DNA fingerprinting of 58 thin-shelled pecan germplasm resources based on 9 core SNP loci. Detailed Implementation

[0018] The technical solution of the present invention will be further described below.

[0019] Example 1: Genomic data analysis and SNP site screening 1. Genomic data analysis and initial screening of SNP sites Simplified genome sequencing (GBS) data were downloaded from the Sequence Reading Archive (SRA) database of the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / sra). Detailed information on the 107 thin-shelled pecan germplasm resources used is shown in Table 1.

[0020] Table 1. Detailed Information on Thin-Shelled Pecan Germplasm Resources

[0021] (continued from previous table)

[0022] Based on the simplified genome sequencing (GBS) data, the Genome Analysis Toolkit 4.1 (GATK 4.1) software was used for quality-related filtering, and the filtering parameters were set as follows: variant quality depth ratio (QD) <2, variant quality score (Qual) <30, Fisher's exact test value (FS) >60, alignment quality mean (MQ) <40, alignment quality rank sum test (MQRankSum) <12.5, and read break position rank sum test (ReadPosRankSum) <-8.

[0023] The processed data was further filtered by VcfTools software, and the filtering parameter settings included: SNP integrity (miss-data) =1, Hardy-Weinberg equilibrium (hwe) =0.001, and minimum sequencing depth =15. The resulting results were screened for sites without other SNP variations within 150bp upstream and downstream, to avoid interference of neighboring SNPs in subsequent analysis. The polymorphism of 738 sites was calculated using R package vcfR, and 30 SNP sites were screened for pre-experiment considering factors such as variation quality (Qual) and chromosome distribution uniformity.

[0024] Further extraction of 350bp sequence upstream and downstream of each SNP site, a total of 701bp, was performed by Sanger sequencing for verification, and finally 23 SNP sites were screened and KASP markers were established.

[0025] 2. According to the 30 pre-experiment SNP sites obtained above, specific primers were designed using Primer Premier 5 software, and the primer design parameters were as follows: GC content 44%-72%, melting temperature (T m ) 56-68℃, length 19-25 bp, and the designed primer sequences are shown in Table 2, which were synthesized by Shanghai Shengong Bioengineering Technology Service Co., Ltd.

[0026] Table 2 Specific primers for pre-experiment SNP sites

[0027] (continued from previous table)

[0028] The obtained 30 primer sets were used as templates for verifying the accuracy of SNP sites by leaf DNA samples from four representative thin-shelled pecan varieties: Elliot (No. 15.11), Mahan (No. 3.23), Peruque (No. P), and Pawnee (No. 27.1). PCR amplification was performed using the Novizan 2 × Rapid Taq Master Mix kit (catalog number P222). The PCR reaction conditions were: 95℃, 5 min; 95℃, 30 sec, 56℃, 30 sec, 72℃, 1 min, 35 cycles; 72℃, 10 min. All PCR amplification products were separated by 1% agarose gel electrophoresis. After imaging with a Tanon-3500 gel imaging system, the PCR products were recovered to obtain the target fragment.

[0029] The target fragment was sequenced using a 3730 XL sequencer, and the sequencing data was analyzed using SnapGene software. Table 3 shows the KASP genotyping results of four samples (15.11, 3.23, P, 27.1) at 23 SNP loci. It was found that the genotypes (homozygous / heterozygous, allele type) of different samples were inconsistent at the same SNP locus, indicating that these samples have genetic polymorphism at these 23 SNP loci. The genotype of each sample was clear, and the results were highly reliable when combined with sequencing and SnapGene analysis.

[0030] Table 3 Sequencing data analysis results

[0031] (Continued from previous table)

[0032] Twenty-three high-quality SNP sites were finally screened and located at position 351 of any sequence of SEQ ID NO: 1 to 23, which were used for subsequent competitive allele-specific PCR (KASP) typing experiments.

[0033] The 23 high-quality SNP loci obtained by screening were statistically analyzed for polymorphism information content (PIC) and minimum allele frequency (MAF) using POPGENE32 (1.32) software to evaluate their application value in population genetics research.

[0034] PIC statistical analysis results are as follows Figure 1As shown in Table B, the SNPs are mainly distributed in the interval of 0.4-0.5, and the proportion of the SNPs in the interval is more than 60%, indicating that the screened SNP sites have a wide distribution of alleles in the population, can better represent the population genetic diversity, and further verify the effectiveness of the screened SNP markers in population genetic analysis.

[0035] The MAF statistical analysis results are shown in Table B. Figure 1 As shown in Table B, the SNPs are mainly distributed in the interval of 0.4-0.5, and the proportion of the SNPs in the interval is more than 60%, indicating that the screened SNP sites have a wide distribution of alleles in the population, can better represent the population genetic diversity, and further verify the effectiveness of the screened SNP markers in population genetic analysis.

[0036] Example 2: KASP genotyping experiment According to the specific sequences (as shown in SEQ ID NO: 1-23) of the upstream and downstream 350 bp of the SNP sites obtained in Example 1, KASP primers were designed and synthesized by Nanjing Jisihuiruan Biological Technology Co., Ltd., wherein for each SNP site (i.e. KASP target site), a universal reverse primer (Primer-R) and two allele-specific forward primers (Primer-F1, Primer-F2) were designed based on the flanking sequences. The 5' end of Primer-F1 is added with a linker sequence 5'-GAAGGTGACCAAGTTCATGCT-3' compatible with 6-carboxyfluorescein group (6-FAM); the 5' end of Primer-F2 is added with a linker sequence 5'-GAAGGTCGGAGTCAACGGATT-3' compatible with VIC amidite monomer (VIC Amidite), and the target SNP site is located at the 3' end of the primer.

[0037] The primer design parameters are as follows: GC content 45%-55%, Tm value of Primer-F1 and Primer-F2 55-62℃, Tm value of Primer-R 62-65℃, and length 60-120 bp; the specific information is shown in Table 4: m Table 4: SNP sites, alleles and primers for KASP genotyping

[0038] (Continue the previous table)

[0039] (Continue the previous table)

[0040] ​Wherein, the 5' end of primer Primer-F1 is modified with 6-carboxyfluorescein group (6-FAM); the 5' end of primer Primer-F2 is modified with VIC amidite monomer (VIC Amidite), and the target SNP site is located at the 3' end of the primer.

[0041] The KASP primers above were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd., and a KASP primer combination was obtained.

[0042] KASP detection was performed using a CFX Connect™ real-time fluorescent quantitative PCR system: for each KASP primer, the primers Primer-F1, Primer-F2 and Primer-R with a concentration of 10 mM were mixed at a volume ratio of 1:1:3 to prepare a primer mixture.

[0043] 58 Carya illinoensis germplasms were provided by the Carya illinoensis Germplasm Resource Bank of Jiangsu Academy of Agricultural Sciences, and the specific information is shown in Table 5.

[0044] Table 5 Information of Carya illinoensis germplasms

[0045] The DNAs of the leaf blades of the above-mentioned 58 Carya illinoensis germplasms were extracted using a Tian Gen Plant Genomic DNA Extraction Kit (item number DP305) as a template, KASP 2xPCR mix kit (purchased from Nanjing Jisihuixuan Biological Technology Co., Ltd., item number K001) was used for KASP reaction based on the KASP primer mixture of each SNP site, and the reaction conditions are shown in Table 6.

[0046] Table 6 KASP reaction conditions

[0047] After the fluorescence signals of the reaction were detected, the fluorescence signals were analyzed by Bio-Rad CFX Maestro software, the maximum likelihood method was used to construct an evolution tree by FastTree, and the cluster tree was optimized by MEGA7.0 software; the genetic distance matrix was calculated by GenALEX (6.51b2) software, two-dimensional principal coordinate analysis was performed based on the matrix, and the PCoA scatter plot of principal coordinate 1 and principal coordinate 2 was drawn.

[0048] The results are as follows Figure 2As shown, the phylogenetic tree divided 58 thin-shelled pecan varieties into two subgroups, Group I and Group II. Principal component analysis (PCA) further divided these 58 varieties into two subgroups, G1 and G2, and one unclassified species, NA (variety 0912). The division of G1 was consistent with Group I, while the division of G2, except for variety 0912, was consistent with Group II. This demonstrates a high degree of consistency between the cluster analysis and PCA results. The results indicate that the aforementioned KASP primer combination based on 23 highly polymorphic SNP loci can cluster different thin-shelled pecan varieties according to their phylogenetic relationships. Strong genetic differentiation exists between different subgroups, and the sample distribution within the same subgroup is more compact, reflecting a high degree of genetic similarity among individuals within the group.

[0049] Example 3: Optimization of KASP primer combinations Analysis of the genotyping results of 58 thin-shelled pecan germplasm resources showed no significant overlap between different genotype clusters, indicating high experimental specificity and preventing genotype misidentification, thus accurately distinguishing combinations of different alleles. Therefore, further analysis was conducted on the correlation between the number of molecular markers and identification efficiency, and the results are as follows: Figure 3 As shown in Figure A, the identification efficiency increases rapidly with the increase in the number of markers. When the number of SNP sites increases to 9 (including S03_795714, S03_39885209, S05_7364264, S06_23130711, S06_33910196, S09_8108940, S11_12488068, S12_22334916, and S13_30247900), the identification efficiency approaches and stabilizes at 100%, indicating that 9 core SNP sites are sufficient to achieve accurate identification of all samples.

[0050] Genetic distances to 58 thin-shelled pecan varieties were calculated for 23 SNP loci and 9 core SNP loci, respectively. The statistical results of genetic distances based on the 23 SNP loci are as follows: Figure 3 As shown in B, the pairwise genetic distances of the 58 thin-shelled pecan varieties ranged from 0.015 to 0.975, with an average genetic distance of 0.29, indicating a 100% distinguishing efficiency for the 58 thin-shelled pecan varieties. The statistical results of genetic distances based on the 9 core SNP loci are as follows: Figure 3 As shown in C, the pairwise genetic distances of the 58 thin-shelled pecan varieties ranged from 0.032 to 1.1712, with an average genetic distance of 0.33. The differentiation efficiency for the 58 thin-shelled pecan varieties was also 100%.

[0051] Further linear fitting analysis was performed on the genetic distance of thin-shelled pecans based on 9 core SNP loci and the genetic distance of thin-shelled pecans based on 23 SNP loci. The results are as follows:Figure 3 The correlation coefficient of the two groups of genetic distance was 76.2%, showing a significant positive correlation (P<0.01), as shown in Figure D.

[0052] The above results show that the 9 core SNP sites can effectively replace the 23 SNP sites, that is, the KASP primer combination based on S03_795714, S03_39885209, S05_7364264, S06_23130711, S06_33910196, S09_8108940, S11_12488068, S12_22334916, S13_30247900 can be used for variety identification.

[0053] Based on the above 9 core SNP sites, a specific DNA fingerprint of the genotyping results of 58 Carya illinoensis varieties was further constructed.

[0054] The results are shown in Figure Figure 4 The color richness of different markers is different, which can reflect the differentiation of polymorphism between markers and has strong variety identification ability.

Claims

1. A KASP primer combination for thin-shelled pecans, characterized in that, The primer combination detects multiple different SNP sites, which are located at position 351 of any sequence of SEQ ID NO: 1 to 23.

2. The thin-shelled pecan KASP primer combination according to claim 1, characterized in that, The primers in the primer combination use any one of SEQ ID NO: 1 to 23 as the design reference sequence, have a GC content between 45% and 55%, a melting temperature of 55 to 65°C, and a length of 60 to 120 bases.

3. The thin-shelled pecan KASP primer combination according to claim 2, characterized in that, The primer combination consists of primers with sequences as shown in SEQ ID NO: 24~92.

4. The thin-shelled pecan KASP primer combination according to claim 3, characterized in that, In the primer combinations, primers with sequences as shown in SEQ ID NO: 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90 have a 5' end modified with a 6-carboxyfluorescein group, and primers with sequences as shown in SEQ ID NO: 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91 have a 5' end modified with a VIC phosphorusamide monomer.

5. The thin-shelled pecan KASP primer combination according to claim 2, characterized in that, The primer combination consists of primers with sequences as shown in SEQ ID NO: 33~35, SEQ ID NO: 36~38, SEQ ID NO: 45~47, SEQ ID NO: 48~50, SEQ ID NO: 51~53, SEQ ID NO: 60~62, SEQ ID NO: 66~68, SEQ ID NO: 72~74, and SEQ ID NO: 84~86.

6. The thin-shelled pecan KASP primer combination according to claim 5, characterized in that, In the primer combinations, primers with sequences such as SEQ ID NO: 33, 36, 45, 48, 51, 60, 66, 72, 84 have a 5' end modified with a 6-carboxyfluorescein group, and primers with sequences such as SEQ ID NO: 34, 37, 46, 49, 52, 61, 67, 73, 85 have a 5' end modified with a VIC phosphorusamide monomer.

7. A reagent kit, characterized in that, The kit contains the thin-shelled pecan KASP primer combination as described in any one of claims 1-6.

8. The application of the thin-shelled pecan KASP primer combination according to any one of claims 1-6, or the kit according to claim 7, in the identification of thin-shelled pecan germplasm resources.

9. The application of the thin-shelled pecan KASP primer combination according to any one of claims 1-6, or the kit according to claim 7, in the genetic background analysis of thin-shelled pecans.

10. The application of the thin-shelled pecan KASP primer combination according to any one of claims 1-6, or the kit according to claim 7, in the molecular breeding of thin-shelled pecans.

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