CN117802262A - SNP molecular marker closely linked with pumpkin soluble sugar character and application thereof - Google Patents
SNP molecular marker closely linked with pumpkin soluble sugar character and application thereof Download PDFInfo
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Abstract
The invention relates to the field of plant molecular genetic breeding, in particular to a SNP molecular marker closely linked with pumpkin soluble sugar characters and application thereof. The nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and a T/C base mutation is arranged at the 23 bp. The invention also provides a specific primer for detecting the SNP molecular marker and a method for identifying the content of soluble sugar in pumpkin pulp, wherein the specific primer is shown as SEQ ID NO. 2-4. The SNP molecular marker is applied to screening and identifying the content character of the soluble sugar of pumpkin fruits, has the advantages of high accuracy, high efficiency and low cost, and greatly promotes the breeding process of pumpkin nutrition quality.
Description
Technical Field
The invention relates to the field of plant molecular genetic breeding, in particular to a SNP molecular marker closely linked with pumpkin soluble sugar characters and application thereof.
Background
The whole Genome association analysis (GWAS, genome-wide Association Study) performs association analysis based on variation information existing in Genome in the population and the traits, and is helpful for mining new regulatory sites related to the traits, analyzing new biological regulatory mechanisms and promoting precise molecular breeding. The single nucleotide polymorphism (SNP, single Nucleotide Polymorphism) is widely applied to the construction of molecular marker linkage maps or the gene location of whole genome association analysis due to high repeatability and abundant quantity, thereby promoting the development of cucurbitaceae crop genetic location research. Competitive allele-specific polymerase chain reaction (KASP, kompetitive Allele Specific PCR, KASP) is a novel genotyping technique based on Single Nucleotide Polymorphisms (SNPs), which can accurately judge SNPs and InDel polymorphisms (indels) at the genomic level, and has been widely used in the fields of life sciences research and medicine.
Sugar is a major product of photosynthesis, not only an important source of carbon in plants, but also functions as a signaling molecule capable of comprehensively responding to signals from the environment, development and metabolism. Furthermore, as soluble sugars are transported and accumulate in vacuoles, they become critical role players in regulating fruit quality and development of stress tolerance. Thus, soluble sugars play an important role in plant growth, crop yield and quality, and response to abiotic stress.
Pumpkin is a vegetable and grain dual-purpose crop of cucurbitaceae widely cultivated worldwide, and contains abundant carbohydrate. With the development of economy, the living standard of people is obviously improved, vegetables are an indispensable part of healthy diet, the demand is gradually changed from quantity to quality, and consumers are increasingly focusing on the nutrition and taste of the vegetables. The soluble sugar is an important quality character of fruits and vegetables, and the content of the soluble sugar directly influences the acceptance degree of pumpkin by markets and consumers. In addition, soluble sugar is quantitative character controlled by multiple genes, there is interaction between genotype and environment, character expression is greatly affected by environment, and conventional breeding technology based on phenotype identification is difficult to effectively screen variety resources. Therefore, analyzing the regulation and control mechanism of the soluble sugar at the genetic level, and breeding the pumpkin variety with high soluble sugar meeting the market demand is important.
However, so far, there have been few studies on the development of genes and molecular markers for soluble sugar of pumpkin. Therefore, it is necessary to find a molecular marker related to the pumpkin soluble sugar, so as to realize accurate and efficient identification of the pumpkin fruit soluble sugar character.
Disclosure of Invention
The invention aims to provide an SNP molecular marker closely linked with pumpkin soluble sugar characters and application thereof, so as to solve the problems in the prior art. The molecular marker and the pumpkin fruit soluble sugar are highly tightly linked, and SNP typing is obtained by using a KASP technology, so that the pumpkin fruit soluble sugar character can be accurately and efficiently identified. Meanwhile, the molecular marker can be used for pumpkin molecular marker assisted selective breeding, and can accelerate the breeding process of pumpkin nutrition quality.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides an SNP molecular marker closely linked with pumpkin soluble sugar character, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and the 23 rd position of the nucleotide sequence has a T/C base mutation.
The invention provides a KASP primer group for detecting the SNP molecular marker, which comprises an upstream primer F1 with a nucleotide sequence shown as SEQ ID NO.2, an upstream primer F2 with a nucleotide sequence shown as SEQ ID NO.3 and a downstream primer R with a nucleotide sequence shown as SEQ ID NO.4.
The invention provides a detection reagent or a detection kit of the SNP molecular marker, which comprises the KASP primer set.
The invention provides application of the SNP molecular marker, the KASP primer set or the detection reagent or the detection kit in identifying the content of soluble sugar in pumpkin pulp.
The invention provides a method for identifying the content of soluble sugar in pumpkin pulp, which comprises the following steps:
taking a pumpkin sample genome to be detected as a template, performing KASP PCR amplification on the template by using the KASP primer group, and genotyping according to the amplification result.
Preferably, if the genotyping result is TT, judging that the pumpkin to be detected is a variety with low soluble sugar content; if the genotyping result is CC, judging that the pumpkin to be detected is a variety with high soluble sugar content.
Preferably, the KASP PCR amplification system comprises, in 10. Mu.L, 0.1. Mu.L of the upstream primer F1, 0.1. Mu.L of the upstream primer F2, 0.2. Mu.L of the downstream primer R, 5. Mu.L of 2 XPRMS Master Mix,80ng of genomic DNA and the balance of water;
the KASPPCR amplification procedure was: 94 ℃ for 15min; gradient annealing at 94 ℃ for 20s at 65-57 ℃ for 1min,10 cycles; 94℃for 20s,57℃for 1min,30 cycles.
The invention provides application of the SNP molecular marker, the KASP primer group or the detection reagent or the detection kit in screening or predicting pumpkin varieties with high soluble sugar content.
The invention provides application of the SNP molecular marker, the KASP primer group or the detection reagent or the detection kit in improving pumpkin germplasm resources.
The invention provides application of the SNP molecular marker, the KASP primer group or the detection reagent or the detection kit in pumpkin breeding.
The invention discloses the following technical effects:
according to the invention, 268 parts of pumpkin materials collected in a laboratory are used as natural population materials, and a high-throughput sequencing technology is utilized to perform whole genome association analysis on pumpkin soluble sugar, so that SNP molecular markers closely linked with the content of the soluble sugar are developed, and the 23 rd position of the molecular markers has a T/C base mutation. The molecular marker can be used for establishing an auxiliary selective breeding system for pumpkin soluble sugar characters, and accelerating the breeding process of pumpkin nutritional quality. The invention discloses SNP molecular markers closely linked with pumpkin soluble total sugar for the first time, the molecular markers can carry out genotype identification on pumpkin fruits, pumpkin seeds or seedlings, and pumpkin varieties with high soluble sugar can be accurately and efficiently screened through genotypes.
In addition, when the SNP molecular marker provided by the invention is applied to screening and identifying the soluble sugar content character of pumpkin fruits, the SNP molecular marker has the advantages of high accuracy, high efficiency and low cost, and greatly promotes the breeding process of pumpkin nutrition quality.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing a genome-wide association analysis of pumpkin soluble sugar traits, and a Manhattan diagram of soluble sugar on the left side: physical location on chromosome represented by abscissa, smaller value of-log 10P, P represented by ordinate, larger value of ordinate represents stronger correlation; the right side is a Quantile-Quantile graph of the correlation analysis of the soluble sugar;
FIG. 2 shows the result of fluorescence signal genotyping of SNP molecular markers in 70 materials with extreme differences in soluble sugar content, wherein green is genotype HEX and blue is genotype FAM.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 acquisition of SNP molecular markers closely linked to the soluble sugar content of pumpkin
1. Construction of genetic populations
(1) Test materials:
268 parts of pumpkin natural population germplasm used in the research (including high-generation inbred line, homozygous farmyard species and F 1 Hybrids) are obtained by years of collection and purification from 11 countries, respectively, china, thailand, india, pakistan, japan, united states, spanish, russia, etc. Wherein, about 214 parts of germplasm resources come from China and are distributed in 22 provinces. The 268 pumpkin germplasm resources were planted in Bai Yunji field test field of the national institute of agriculture, academy of agricultural sciences, guangdong in the first half of 2019.
(2) Determination of soluble sugar content of pumpkin fruits:
the individual pumpkin fruits in 268 parts of natural populations are collected, smashed by a stirrer, then quickly precooled by liquid nitrogen, placed in a freeze dryer for freeze-drying, ground into powder by grinding freeze-dried samples, the composition and the content of soluble sugar are quantitatively detected by using an HPLC-RI system, a chromatographic column is Waters X bridge BEHAmide (4.6X250 mm,5 mu m), and an extracting solution is 50% acetonitrile (the detection result of the content of the soluble sugar of partial natural populations is shown in table 1).
2. Genome-wide association analysis of soluble sugar content
(1) Extraction of pumpkin genome DNA:
the genome DNA of 268 parts of natural population is extracted by adopting a modified cetyl trimethyl ammonium bromide method (CTAB, cetyltrimethylammonium bromide), the quality and the concentration of pumpkin DNA are detected by 2% agarose gel electrophoresis, and the pumpkin DNA is diluted to 80 ng/. Mu.L for standby.
(2) Analysis of resequencing data
Based on a second-generation resequencing platform Illumina Hi-Seq, through data quality control, comparison of reference genome, quality inspection after comparison, mutation detection based on different algorithms, and filtering of mutation detection results, clean ready data of 1452.15Gb is obtained, the average of 5.37Gb of each sample is obtained, and the maximum sequencing depth is: 30.6, the lowest depth is: 9.28, average sequencing depth for genome reached: 16.58×. The average coverage (one base coverage at least) for all sample alignments is: 96.38%, the lowest coverage is: 68.78%, the highest coverage is: 99.37%, and 1,157,313 high-quality SNP loci are obtained through filtration.
(3) Genome-wide association analysis of soluble sugar content
The invention utilizes GEMMA analysis software to screen potential candidate SNPs, and the threshold condition of the GEMMA is set as follows: p is less than or equal to 10 -6 (-log 10 p More than or equal to 6) and p is less than or equal to 10 -7 (-log 10 p 7) resulted in 25 significant SNPs sites (FIG. 1) associated with soluble sugar content, wherein the site with the highest contribution on chromosome 4 was defined as Sug-SNP1, the polymorphism of this site was T/C, the high soluble sugar content was C, and the low soluble sugar content was T.
EXAMPLE 2KASP technique for detecting genotype of SNP site related to soluble sugar content trait of pumpkin
Based on the genome sequence information of the pumpkin and the correlation analysis result of the whole genome of the soluble sugar, selecting a sequence SEQ ID NO.1 at the periphery of a Sug-SNP1 locus, specifically CTTACCATTGGGCTCGTATTGTMTCGGTCATTTGTGAAGGGTTGTTCCAGTTGTATGACT CATCTGTGTATGTTCCCCACCGG, wherein M represents that the base at the locus is T or C, and the sequence is used as an SNP molecular marker.
The KASP primer is designed according to the sequence shown in SEQ ID NO.1, and the forward primer sequence consists of a general adapter sequence, a common amplification primer sequence and a typing site sequence, and is specifically shown as follows:
upstream primer F1: GAAGGTGACCAAGTTCATGCTCTTACCATTGGGCTCGTATTGTT, SEQ ID No.2, wherein the italic part is FAM tag sequence and the black bolded part is the differential Sug-SNP1 site;
the upstream primer F2: GAAGGTCGGAGTCAACGGATTCTTACCATTGGGCTCGTATTGTC, SEQ ID No.3, in italics, HEX tag sequence, in black bolded, differential Sug-SNP1 locus;
the downstream primer R: CCGGTGGGGAACATACACAG, SEQ ID No.4.
The KASPPCR amplification reaction system comprises: 0.2. Mu.L of upstream primer (10. Mu.M, F1 in 0.1. Mu.M, F2 in 0.1. Mu.M), 0.2. Mu.L of downstream primer (10. Mu.M), 5. Mu.L of 2 XPRMS Master Mix,80ng of genomic DNA, water make up 10. Mu.L;
the reaction procedure for the PCR amplification of KASP was: 94 ℃ for 15min; gradient annealing at 94 ℃ for 20s at 65-57 ℃ for 1min,10 cycles; 94℃for 20s,57℃for 1min,30 cycles;
after PCR was completed, the fluorescent signal was read using a TECAN index M1000 microplate reader and then resolved using online software snpdecoder (http:// www.snpway.com/snpdecoder /). When the base of Sug-SNP1 is T, the detection sample is combined with a specific FAM detection primer and releases a blue fluorescent group, and the blue fluorescent signal is enhanced along with the increase of the number of PCR reaction cycles, so that the material with low soluble sugar can be judged by the fluorescent color (KASP typing result is defined as FAM); when the base of Sug-SNP1 is C, the detection sample binds to a specific HEX detection primer and releases a green fluorescent group, and as the number of PCR reaction cycles increases, the green fluorescent signal is enhanced, so that a material with high solubility sugar can be judged by the fluorescent color (KASP typing result is defined as: HEX). Although the judgment method is based on fluorescence color, it is needless to say that the same object can be achieved by detecting the base at the 23bp position of SEQ ID NO.1 by other known methods to determine the SNP mutation condition.
Of 268 parts of natural population materials, 70 parts of materials with extremely different soluble contents are selected, and the soluble sugar content and genotype are shown in table 1: wherein, 35 parts of materials with high soluble sugar content and 35 parts of materials with low soluble sugar content are subjected to KASP typing analysis by adopting the molecular marker fluorescence signal provided by the invention, so as to prove the detection accuracy of the molecular marker developed by the invention. The typing results of the molecular markers on 70 parts of soluble sugar content extreme materials are shown in the accompanying figure 2 and the table 1: individuals with extremely low soluble sugar content aggregate to the position of the abscissa axis, and individuals with extremely high soluble sugar aggregate to the position of the ordinate axis. The molecular markers developed by the invention are tightly linked with the content character of the soluble sugar.
Table 170 parts of soluble sugar character of pumpkin and genotype corresponding to Sug-SNP1 marker
The same object can be achieved by detecting the molecular marker Sug-SNP1 by other known methods and determining the SNP condition.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. A SNP molecular marker closely linked with pumpkin soluble sugar character is characterized in that the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and the 23 rd position of the nucleotide sequence has a T/C base mutation.
2. The KASP primer group for detecting the SNP molecular markers as set forth in claim 1, characterized in that the KASP primer group comprises an upstream primer F1 having a nucleotide sequence shown as SEQ ID No.2, an upstream primer F2 having a nucleotide sequence shown as SEQ ID No.3, and a downstream primer R having a nucleotide sequence shown as SEQ ID No.4.
3. A SNP molecular marker detection reagent or detection kit according to claim 1, comprising the KASP primer set according to claim 2.
4. Use of the SNP molecular marker of claim 1, the KASP primer set of claim 2, or the detection reagent or detection kit of claim 3 for identifying the soluble sugar content of pumpkin pulp.
5. A method for identifying the level of soluble sugar in pumpkin pulp, comprising:
taking a pumpkin sample genome to be detected as a template, performing KASP PCR amplification on the template by using the KASP primer group according to claim 2, and genotyping according to the amplification result.
6. The method of claim 5, wherein if the genotyping result is TT, determining that the pumpkin to be tested is a low soluble sugar variety; if the genotyping result is CC, judging that the pumpkin to be detected is a variety with high soluble sugar content.
7. The method of claim 5, wherein the KASP PCR amplification system comprises, in 10 μl, 0.1 μl of upstream primer F1, 0.1 μl of upstream primer F2, 0.2 μl of downstream primer R, 5 μl of 2×parms Master Mix,80ng of genomic DNA, and the balance water;
the KASPPCR amplification procedure was: 94 ℃ for 15min; gradient annealing at 94 ℃ for 20s at 65-57 ℃ for 1min,10 cycles; 94℃for 20s,57℃for 1min,30 cycles.
8. Use of the SNP molecular marker of claim 1, the KASP primer set of claim 2, or the detection reagent or detection kit of claim 3 for screening or predicting a variety of pumpkin with high soluble sugar content.
9. Use of the SNP molecular marker of claim 1, the KASP primer set of claim 2, or the detection reagent or detection kit of claim 3 for improving pumpkin germplasm resources.
10. Use of the SNP molecular marker of claim 1, the KASP primer set of claim 2, or the detection reagent or detection kit of claim 3 in pumpkin breeding.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN118166155A (en) * | 2024-04-25 | 2024-06-11 | 广东省农业科学院蔬菜研究所 | SNP molecular marker related to pumpkin pulp thickness, amplification primer set and application thereof |
| CN118186141A (en) * | 2024-04-26 | 2024-06-14 | 广东省农业科学院蔬菜研究所 | SNP locus closely linked with pumpkin handle length major QTL, molecular marker, primer pair and application thereof |
| CN118563011A (en) * | 2024-06-24 | 2024-08-30 | 浙江省农业科学院 | Application of group of SNP molecular markers as detection targets in identification of pumpkin germplasm resources |
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