SNP (Single Nucleotide Polymorphism) molecular marker linked with sucrose content of peanut seed kernels, KASP primer group and application of SNP molecular marker and KASP primer group

Abstract

The invention discloses an SNP molecular marker linked with the sucrose content of peanut seed kernels, a KASP primer group and application of the SNP molecular marker and the KASP primer group, and belongs to the technical field of molecular genetic breeding. The molecular marker SucB06 related to the sucrose content of the peanut seed kernels is screened, a KASP primer group is developed according to the molecular marker and the SucA06 molecular marker, a peanut variety with the high sucrose content of the seed kernels can be accurately distinguished from a peanut variety with the low sucrose content of the seed kernels by utilizing a KASP genetic typing technology, and the peanut variety with the low sucrose content of the seed kernels can be accurately distinguished. The method can be used for early identification and screening of peanut materials, the peanut breeding period can be shortened, the genetic resource improvement process is accelerated, and new genetic resources and theoretical basis are provided for molecular assisted breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular genetic breeding, in particular to a SNP molecular marker linked to the sucrose content of peanut kernels, a KASP primer set and applications thereof. Background Art

[0002] Flavor is a key quality indicator of edible peanuts. The carbohydrate content, particularly sucrose, in the kernel is significantly positively correlated with sweetness and taste. The sucrose content of peanut kernels typically ranges from 2% to 8%, with a noticeable sweetness when the content exceeds 5%. Therefore, sucrose content has become a key selection criterion for edible peanut varieties.

[0003] In recent years, systematic studies have been conducted on the genetic analysis of sucrose content in peanut kernels. Analysis of genotype variation showed that genotype could explain 64.9% of the total variation in sucrose content. Qin Li et al. found that genetic analysis showed that this trait was co-regulated by two major effect genes and multiple minor effect genes. Through genome-wide association analysis (GWAS), 22 significantly associated QTLs for sucrose content have been identified. Guo et al. detected four QTLs on chromosomes A03 and A06 based on cluster segregation analysis sequencing (BSA-seq). Among them, the stable QTL qSUCA06 in the 115.37-115.66Mb interval on chromosome A06 could explain 31.95%-41.05% of the phenotypic variation. Li Weitao et al. identified two sucrose-related regions, qSUC-A08a (10.88-42.07 Mb) on chromosome A08 and qSUC-B06.2 (20.60-30.22 Mb) on chromosome B06, using QTL-seq. The major QTL, qSUC-A08.2, explained 5.43%-17.84% of the phenotypic variation across five environments. Furthermore, Wang et al. detected five new QTLs on chromosomes A07, B06, and B09 using QTL-seq. The newly discovered major QTL, qSCB09, explained 21.51%-33.58% of the phenotypic variation across three environments. Two major QTLs (qSCA06.2 and qSCB06.2) were identified, explaining 31.41% and 24.13% of the phenotypic contribution, respectively. Huai et al., using BSA-seq and fine mapping, discovered a QTL in the 115.79-115.81 Mb interval on chromosome A06 that explained 47.5% of the phenotypic variation. These findings lay an important foundation for the analysis of sucrose-regulating genes in peanut kernels.

[0004] However, the QTLs located in different populations have different chromosomal locations, reflecting the complexity of the sucrose accumulation regulatory network. Marker-assisted selection (MAS) can screen target trait germplasm in the early developmental stage without being affected by complex environments. To achieve the breeding goals of MAS, the development of practical molecular markers has become a current research focus. Zhang Xiaojun used BSA-seq to locate a major QTL related to soluble sugar content on peanut chromosome B06, and developed molecular markers related to soluble sugar content on both sides of the QTL. Lei Yong et al. used Zhonghua 16 and Rwandan peanut varieties to establish a population and located the major QTL sites qSCA06.2 and qSCB06.2 for peanut sucrose content, and developed InDel molecular markers linked to these two sites. Liu Lifeng et al. discovered a significant association site on chromosome B03 through genome-wide association analysis and developed the KASP marker.

[0005] Previous researchers used different populations, and located different intervals and markers. This shows that sucrose content is a complex trait. Fully exploring the variation sites related to sucrose content and developing related molecular markers and detection methods can provide a theoretical basis for subsequent exploration of genes related to sugar content and molecular-assisted breeding. Summary of the Invention

[0006] The present invention aims to provide a single-nucleotide polymorphism (SNP) molecular marker linked to sucrose content in peanut kernels, a KASP primer set, and their use to address the aforementioned problems in the prior art. The present invention screened for a molecular marker, SucB06, associated with sucrose content in peanut kernels. Based on this marker and the SucA06 marker, a KASP primer set was developed. Using KASP genotyping technology, the KASP primer set can accurately distinguish peanut varieties with high kernel sucrose content from those with low kernel sucrose content. This can be used for early identification and screening of peanut materials, shortening the peanut breeding cycle and accelerating the improvement of genetic resources. This provides new genetic resources and a theoretical basis for molecular-assisted breeding.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a SNP molecular marker SucB06 linked to the sucrose content of peanut kernels, wherein the nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.2;

[0009] There is a C / A mutation at base 461 of the sequence shown in SEQ ID NO.2.

[0010] The present invention also provides the use of the above-mentioned SNP molecular marker SucB06 in any of the following:

[0011] A1. Screening or identification of peanut kernel sucrose content phenotype;

[0012] A2. Early screening or identification of peanut varieties with high sucrose content in the kernels;

[0013] A3. Molecular genetic breeding of peanut.

[0014] The present invention also provides a KASP primer set for screening or identifying the sucrose content phenotype of peanut kernels, wherein the KASP primer set includes a SucA06 primer set and a SucB06 primer set;

[0015] The SucA06 primer set includes an upstream primer A as shown in SEQ ID NO.3, an upstream primer B as shown in SEQ ID NO.4, and a downstream primer as shown in SEQ ID NO.5; the SucA06 primer set includes an upstream primer A as shown in SEQ ID NO.6, an upstream primer B as shown in SEQ ID NO.7, and a downstream primer as shown in SEQ ID NO.8;

[0016] The SucA06 primer set is used to detect the SucA06 molecular marker; the nucleotide sequence of the SucA06 molecular marker is shown in SEQ ID NO.1, and there is a C / A mutation at the 110th base;

[0017] The SucB06 primer set is used to detect the above-mentioned SNP molecular marker SucB06.

[0018] The present invention also provides a reagent for screening or identifying the sucrose content phenotype of peanut kernels, wherein the reagent comprises the KASP primer set.

[0019] The present invention also provides a kit for screening or identifying the sucrose content phenotype of peanut kernels, wherein the kit comprises the KASP primer set.

[0020] The present invention also provides use of the KASP primer set, the reagent, or the kit in any of the following:

[0021] B1. Screening or identification of peanut kernel sucrose content phenotype;

[0022] B2. Early screening or identification of peanut varieties with high sucrose content in the kernel;

[0023] B3. Molecular genetic breeding of peanut.

[0024] The present invention also provides a method for early screening or identifying peanut varieties with high sucrose content in kernels, comprising the following steps:

[0025] Using the DNA of the sample to be tested as a template, the above-mentioned KASP primer set is used for PCR amplification to genotype the SucA06 molecular marker and the above-mentioned SNP molecular marker SucB06 site of the sample to be tested;

[0026] The sucrose content in the kernels of samples with AACC typing results for the SucA06 molecular marker and the above-mentioned SNP molecular marker SucB06 locus was higher than that of samples with CCCC and AAAA typing results.

[0027] Furthermore, the PCR amplification reaction system comprises: 2.5 μL template DNA, 2.5 μL 2×KASP MasterMix, 0.07 μL Primer Mix;

[0028] The Primer Mix includes: 12 μL of 100 μM upstream primer A, 12 μL of 100 μM upstream primer B, 30 μL of 100 μM downstream primer, and ddH2O to 100 μL.

[0029] Furthermore, the reaction procedure of the PCR amplification is: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing at 61°C for 60 seconds, 10 cycles, with the annealing temperature decreasing by 0.6°C each cycle; denaturation at 94°C for 20 seconds, annealing at 55°C for 60 seconds, 26 cycles.

[0030] The present invention discloses the following technical effects:

[0031] The present invention has screened a molecular marker, SucB06, associated with sucrose content in peanut kernels. This marker is located at locus Arahy.16_148167024 on chromosome B06 and contains a C / A single-nucleotide polymorphism (SNP). Based on the SucB06 and SucA06 molecular markers, the present invention developed a KASP primer set. Using KASP genotyping technology, the set can accurately distinguish peanut varieties with high kernel sucrose content from those with low kernel sucrose content. Using this KASP primer set for early identification of peanut materials and genetic breeding or genetic resource improvement can shorten the peanut breeding cycle and accelerate the genetic resource improvement process, providing new genetic resources and a theoretical basis for molecular-assisted breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 The results of sucrose phenotypic variation analysis of the F2 genetic population;

[0034] Figure 2 This is the result of fine mapping of QTL for sucrose content on peanut chromosome B06;

[0035] Figure 3 are the KASP typing results of SucA06 and SucB06 molecular markers in peanut; among them, A is the KASP typing result of SucA06 molecular marker, B is the KASP typing result of SucB06 molecular marker, C is the KASP typing and sucrose content analysis results in the F2 genetic population, and D is the KASP typing and sucrose content analysis results in the natural population. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] The peanut varieties JMT, Fushun Silihong and natural populations involved in the following embodiments of the present invention are all provided by the Germplasm Resources Institute of Shandong Academy of Agricultural Sciences and are available to the public here.

[0042] The experimental methods in the following examples of the present invention are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0043] Example 1 Screening of molecular markers

[0044] The sucrose content of high-sugar peanut germplasm JMT reaches 8.22%, while the sucrose content of multi-grain peanut farmer variety Fushun Silihong (FSH) is lower (4.79%). In order to explore the genetic basis of peanut sucrose content, this example constructed an F2 genetic population containing 274 strains by hybridizing JMT and FSH. Field trials were carried out at the Jiyang Experimental Station of Shandong Academy of Agricultural Sciences (Jinan, Shandong) in the spring of 2020-2022. After harvest, near-infrared spectroscopy was used to quantitatively determine the sucrose content of the grains. The sucrose content of the F2 population varied from 0.53% to 10.90%, showing a continuous frequency distribution characteristic ( Figure 1 ), consistent with a quantitative trait inheritance model. Histogram analysis revealed significant infra-segregation in the population, and the Shapiro-Wilk normality test (P < 0.05) confirmed a non-normal distribution. In summary, this suggests that sucrose accumulation in peanut may be regulated by a polygenic inheritance model.

[0045] To locate genomic regions associated with sucrose content, this example uses segregant group analysis (BSA) combined with whole-genome sequencing technology to construct two extreme sucrose content pools: a low sucrose content pool (BLS) and a high sucrose content pool (BHS). The BLS consists of 21 F2 plants with sucrose content ranging from 1.30% to 2.94%, and the BHS consists of 21 F2 plants with sucrose content ranging from 4.80% to 6.14%. Whole-genome resequencing was performed on both parents and the two extreme pools using the Illumina HiSeq PE150 platform. A total of 373,352 polymorphic sites were detected throughout the genome. By calculating the ΔSNP-index values ​​of the two extreme pools, a genomic region of 2.36 Mb [reference genome Tifrunner (Arachis hypogaea) position: 146.37-148.73 Mb] was found on chromosome B06 to be significantly associated with sucrose content. The candidate region contains 165 genes and 691 variant sites (including 456 SNPs and 135 InDels), of which 24 large-effect variants (LEVs) involve 19 genes.

[0046] To precisely locate the sucrose content-related region on chromosome B06, this example developed 26 SNP markers within the 2.36Mb candidate interval (146.37-148.73Mb) (Table 1). By typing the polymorphic markers in the F2 population, a high-resolution genetic map (total map distance 22.23cM) was constructed ( Figure 2 Using the composite interval mapping (CIM) method, the QTL qSucB06 was precisely anchored within the genomic interval defined by markers PB07 and PA15, explaining 41.83% of the phenotypic variation (PVE). These results confirm that the B06 chromosomal region is the primary genetic regulatory locus for sucrose content in peanut. Fine-mapping ultimately narrowed the critical interval to 131 kb (148.03-148.16 Mb). Within this fine-mapping interval, only Arahy.3URM83 exhibited variation in the exon region, and expression was differential in young seeds of the two parents. Therefore, the SNP variant (148,167,024 bp) located in the exon of this gene is likely closely linked to sucrose content.

[0047] Table 1 Variation information of QTL fine mapping markers

[0048]

[0049]

[0050] Example 2 Application of molecular markers

[0051] Previous studies have shown that there is a C→A base substitution 110bp downstream of the start codon of the Arahy.42CAD1 gene in the high-sugar peanut variety Zhonghuatian No. 1 (ZHT1), namely the SucA06 molecular marker. This nonsense mutation causes premature termination of Arahy.42CAD1 translation, thereby promoting sucrose accumulation in peanut seeds (Huai et al. 2024). In the present invention, sequence alignment revealed the presence of a C / A SNP variation at the Arahy.16_148167024 site on chromosome B06, namely the SucB06 molecular marker. This site is located in the Arahy.3URM83 gene, resulting in the replacement of threonine (Thr) with asparagine (Asn). Arahy.3URM83 is a homologous gene of Arahy.42CAD1. Therefore, these two sites were selected to develop KASP molecular markers. The nucleotide sequences of the two molecular markers are as follows:

[0052] SucA06 molecular marker:

[0053]

[0054] SucB06 molecular marker:

[0055]

[0056] Based on the locations of the two mutation sites, KASP allelic differential primers were designed, as shown in Table 2. The SNP differential sequence was located at the 3' end of the primer, while the 5' end was linked to a universal fluorescent probe sequence labeled with a FAM or HEX fluorophore, respectively. High-throughput PCR was performed using the primers listed in Table 2. The PCR reaction system consisted of: 2.5 μL template DNA (20 ng / μL), 2.5 μL 2× KASP Master Mix, and 0.07 μL Primer Mix. The Primer Mix consisted of 12 μL 100 μM A-FAM, 12 μL 100 μM B-HEX, and 30 μL 100 μM C-Common, and the mixture was made up to 100 μL with ddH2O. The amplification program was run on an LGC water bath PCR instrument: first, pre-denaturation at 94°C for 15 minutes; followed by 10 cycles of touchdown PCR (denaturation at 94°C for 20 seconds → annealing at 61°C for 60 seconds, with the annealing temperature decreasing by 0.6°C per cycle); and finally 26 cycles (denaturation at 94°C for 20 seconds → annealing at 55°C for 60 seconds).

[0057] Fluorescence results such as Figure 3 A and Figure 3 As shown in Figure B, the blue fluorescent plant has an AA genotype, the red fluorescent plant has a CC genotype, and the pink fluorescent plant failed the test due to poor DNA quality. The gray color represents the NTC blank control.

[0058] Table 2 KASP primer sequences of SucA06 and SucB06

[0059]

[0060] The results of strain typing of 274 F2 genetic populations showed that ( Figure 3 All lines with SucA06 markers were of AA genotype, while lines with SucB06 markers of C:C genotype had an average sucrose content of 5.01%, lines with C:A genotype had an average sucrose content of 3.71%, and lines with A:A genotype had an average sucrose content of 2.93%. KASP typing of SucA06 and SucB06 markers was performed on 42 peanut varieties in a natural population. The results showed (Table 3, Figure 3 D), 5 accessions had the AACC genotype, with an average sucrose content of 8.44% and a minimum of 6.95%. Three accessions had the AAAA genotype, with an average sucrose content of 4.65%. 34 accessions had the CCCC genotype, with an average sucrose content of 4.02%. These results indicate that when these two loci are AACC, the accession can be identified as a high-sucrose material.

[0061] The above results indicate that the molecular markers and KASP primers developed based on the Arahy.06_115805462 and Arahy.16_148167024 loci have high efficiency in identifying the sucrose content phenotype of peanut kernels, and have significant application potential in molecular breeding for improving the sucrose content of peanuts.

[0062] Table 3 Genotyping results and sucrose content phenotypes in natural populations

[0063]

[0064]

[0065] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A SNP molecular marker SucB06 linked to the sucrose content of peanut kernels, characterized by: The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.2; There is a C / A mutation at base 461 of the sequence shown in SEQ ID NO.

2.

2. Use of the SNP molecular marker SucB06 according to claim 1 in any of the following: A1. Screening or identification of peanut kernel sucrose content phenotype; A2. Early screening or identification of peanut varieties with high sucrose content in the kernels; A3. Molecular genetic breeding of peanut.

3. A KASP primer set for screening or identifying the sucrose content phenotype of peanut kernels, characterized in that: The KASP primer set includes a SucA06 primer set and a SucB06 primer set; The SucA06 primer set includes an upstream primer A as shown in SEQ ID NO.3, an upstream primer B as shown in SEQ ID NO.4, and a downstream primer as shown in SEQ ID NO.5; the SucA06 primer set includes an upstream primer A as shown in SEQ ID NO.6, an upstream primer B as shown in SEQ ID NO.7, and a downstream primer as shown in SEQ ID NO.8; The SucA06 primer set is used to detect the SucA06 molecular marker; the nucleotide sequence of the SucA06 molecular marker is shown in SEQ ID NO.1, and there is a C / A mutation at the 110th base; The SucB06 primer set is used to detect the SNP molecular marker SucB06 according to claim 1.

4. A reagent for screening or identifying the sucrose content phenotype of peanut kernels, characterized in that: The reagents include the KASP primer set according to claim 3.

5. A kit for screening or identifying the sucrose content phenotype of peanut kernels, characterized in that: The kit comprises the KASP primer set according to claim 3.

6. Use of the KASP primer set according to claim 3, the reagent according to claim 4, or the kit according to claim 5 in any of the following: B1. Screening or identification of peanut kernel sucrose content phenotype; B2. Early screening or identification of peanut varieties with high sucrose content in the kernel; B3. Molecular genetic breeding of peanut.

7. A method for early screening or identification of peanut varieties with high sucrose content in kernels, characterized in that: The steps include: Using the DNA of the sample to be tested as a template, PCR amplification is performed using the KASP primer set described in claim 3 to genotype the SucA06 molecular marker of the sample to be tested and the SNP molecular marker SucB06 site described in claim 1; The sucrose content in the seed kernels of samples whose typing results for the SucA06 molecular marker and the SNP molecular marker SucB06 site according to claim 1 are AACC is higher than that of samples whose typing results are CCCC and AAAA.

8. The method according to claim 7, characterized in that The PCR amplification reaction system comprises: 2.5 μL template DNA, 2.5 μL 2×KASP Master Mix, 0.07 μL Primer Mix; The Primer Mix includes: 12 μL of 100 μM upstream primer A, 12 μL of 100 μM upstream primer B, 30 μL of 100 μM downstream primer, and ddH2O to 100 μL.

9. The method according to claim 7, characterized in that The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 15 minutes; denaturation at 94°C for 20 seconds, annealing at 61°C for 60 seconds, for 10 cycles, with the annealing temperature decreasing by 0.6°C each cycle; denaturation at 94°C for 20 seconds, annealing at 55°C for 60 seconds, for 26 cycles.

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