CN114574629A - SNP molecular marker related to papaya fruit weight and application thereof - Google Patents
SNP molecular marker related to papaya fruit weight and application thereof Download PDFInfo
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Abstract
The invention relates to the field of molecular biology, in particular to an SNP molecular marker related to papaya fruit weight and application thereof, and the SNP molecular marker related to papaya fruit weight is found based on a GWAS analysis method, is located in a Cpa09g016360 gene CDs region on a Chr09 chromosome and is named as a molecular marker Cpa09g016360:1846, and determines a candidate gene Cpa09g016360 related to papaya fruit weight. The SNP molecular marker can be used for detecting papaya breeding materials with high fruit weight, assisting in cross breeding, shortening the breeding period of new varieties, and is low in detection cost, free of environmental limitation, high in detection result accuracy and easy to repeat.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to an SNP molecular marker related to the weight of papaya fruits and application thereof.
Background
Papaya is a tropical evergreen fruit tree, a perennial fleshy herbaceous plant of the genus papaya of the family Caricaceae, also known as cushaw, rock-melon, papaya, wax gourd, papaya, and lotus seed fruit. The strain is originally produced in Mexico and Central America, is widely cultivated in tropical and subtropical regions all over the world, is introduced into China from the 17 th century, is mainly produced in Guangdong, Hainan, Guangxi, Yunnan, Taiwan, Fujian, and is also cultivated in Sichuan Wenchang and Jiangxi. The fresh papaya has beautiful appearance, thin skin and thick meat, multiple tastes and sweetness, fragrant and sweet smell and rich nutrition. The ripe papaya flesh is yellow or red, and the carotene and lycopene are rich, so that the papaya flesh has excellent health-care effect and important edible value and industrial value. The development of papaya production greatly stimulates the development of related industries such as food processing, medicine and health, beauty and health care, breeding and the like, and has important significance.
Molecular breeding facilitates selection of a desired trait and then breeding of a new variety. With the development of genetics, genetic markers can be used as an aid to help people to select and improve the accuracy of breeding (Sunde et al, 2006, seeds, 25(12): 54-57). As for the antiviral breeding research of papaya, the genetic engineering technology has been greatly developed with the continuous improvement of tissue culture technology. In the research of Fitch et al (1990), transgenic papaya plants were developed by particle gun method, and then further cultured and successfully obtained callus with stable genetic characteristics. Zhongpeng et al (1997, journal of tropical crops, 16(2): 66-69) studied the bivalent gene of PRV-CP-SN into papaya and successfully obtained transgenic plants. The transgenic plants obtained by Zhongpeng and Zheng's office (1996, the Collection of tropical crops, 17(2): 84-87) by the Agrobacterium-mediated method can successfully express the gene of the Sm strain CP. Zhao Shi Ying et al (1998, the report on tropical crops, 19(2): 20-26) successfully obtained transgenic plants by transferring the ribozyme-related gene of PRV RNA into papaya using genetic engineering techniques, and also carried out the corresponding challenge study by artificial inoculation. Transgenic papaya plants have also been successfully obtained by a number of studies in units such as the national institute of Life sciences of Zhongshan university (Mei et al, 2001). Kumar et al (2018) cultured papaya with CP and GFP fusion gene transferred based on sequence homology analysis of transgenic sources, and used for improving transgenic papaya strain to resist PRSV infection. The transgenic papaya strain produced by the experiments of Jia et al (2017) has certain application value to PRSV. The gene engineering method can be used as a better method for preventing and controlling the disease-resistant breeding in the breeding of the papaya, and the method makes the papaya capable of resisting various virus diseases (such as ringspot virus diseases) possible; the papaya tissue culture and rapid propagation technology (Chuan Yixingchun and Laishongong, 2003) not only provides an effective way for preserving papaya germplasm resources, but also successfully maintains the genetic stability of excellent varieties (disease-resistant, high-quality and high-yield), and simultaneously provides convenience for international germplasm exchange.
Single Nucleotide Polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by variation of a single nucleotide at the genome level. Compared with molecular markers such as RAPD, AFLP, SSR and the like in the early stage, the SNP molecular marker has the advantages of wide distribution and more quantity on an individual genome, easiness in genotyping (binary SNP), suitability for rapid and large-scale screening and the like. Genome-wide association analysis (GWAS) is a method for mining candidate genes related to traits based on the relationship between high-throughput SNP molecular markers and the traits, and the basic principle of the method is the phenomenon of Linkage Disequilibrium (LD) among genes in a population. GWAS was first applied in human disease research, plays an important role in genetic basic analysis of complex quantitative traits, and has absolute advantages in mining real major genes or key mutation sites. Until 2010, the popularization of the second-generation sequencing technology and the large-scale development of high-throughput molecular markers, and the whole-genome association analysis method are applied to the research of complex traits of crops on a large scale, and have complete use methods and experiences in rice, wheat, corn, rape and cotton.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides an SNP molecular marker related to the weight of papaya fruits and application thereof.
The technical scheme adopted by the invention is as follows:
the SNP molecular marker related to the weight of the papaya fruit is positioned on a nucleotide sequence shown in SEQ ID No.1, and the 144 th base from the 5' end of the sequence is an SNP locus. The marker is located at the 25329094 th base of the chromosome Chr09 (Cpa 09g016360 gene CDs region). The SNP molecular marker is significantly related to the Cpa09g016360 gene of papaya for regulating the fruit size (P < 0.01). With the A/G polymorphism, the G allele is very significantly associated with high fruit weight. Can be used for detecting the breeding material of the papaya with high fruit weight.
The SNP molecular marker can be used for detecting the papaya breeding material with high fruit weight.
The SNP molecular marker can be used for detecting or predicting the weight of papaya fruits.
The SNP molecular marker can be used for selecting high fruit weight varieties of papaya.
The SNP molecular marker can be used for papaya molecular marker assisted breeding.
The SNP molecular marker can be used in the breeding process of high fruit weight of papaya.
The invention also provides a method for preparing the molecular marker, which comprises the steps of designing a primer pair by using the nucleotide sequence position basic sequence containing the SNP marker, carrying out PCR amplification by using papaya genomic DNA as a template, and converting the SNP marker into a 226bp molecular marker, wherein an upstream primer of the primer is as follows: cttcctatca caattcaaag, the downstream primer is: agtatgggag caagcagccc t are provided.
The invention discovers an SNP molecular marker Cpa09g016360:1846 related to the weight of papaya fruits based on a GWAS analysis method, and determines a candidate gene Cpa09g016360 related to the weight of papaya fruits. The SNP molecular marker can be used for detecting a papaya breeding material with high fruit weight, assisting in hybridization breeding, shortening the breeding period of a new variety, and has the advantages of low detection cost, no environmental limitation, high detection result accuracy and easiness in repetition.
Drawings
FIG. 1 Manhattan chart of SNP markers of the present invention which are very significantly related to papaya fruit weight
FIG. 2 is a Manhattan diagram of a local interval of Chr09 chromosome where SNP markers which are significantly related to the weight of papaya fruits are located and a linkage disequilibrium haplotype block diagram, wherein red markers in the interval are molecular markers screened by the invention.
FIG. 3 is a comparison of fruit weight differences between different genotype subgroups of SNP markers of the present invention that are significantly correlated with papaya fruit weight, with the G allele being significantly correlated with high fruit weight.
FIG. 4 is an agarose gel electrophoresis of the primers of the present invention, wherein lane M: DNA Marker DL 1000; the right side of the lane M is provided with 6 AA genotypes and 6 GG genotypes in turn.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Papaya whole genome SNP marker development
340 parts of papaya collected from Mexico, south Africa, Guangxi China, Hainan China and the like are sown in Wenchang base of Chinese tropical agricultural science institute, soil fertility is moderate, diseases and insect pests do not exist, the papaya is planted for many years at multiple points, phenotypic character data are collected, and the phenotypic character data are processed by Excel2016 and then used for subsequent analysis.
Taking 1-2g of fresh young leaves from a papaya plant, grinding by liquid nitrogen, and extracting papaya material DNA according to a selected rootstock genome DNA extraction kit (DP 305). The quality and concentration of a DNA sample are detected by using an ultramicro spectrophotometer and 1% agarose gel electrophoresis, and the DNA sample with clear electrophoresis band, no obvious protein residue in a gel hole, OD260/OD280 between 1.8 and 1.9 and concentration more than 100 ng/mu L is selected for library construction and sequencing.
The DNA is randomly broken into fragments of about 300bp by adopting an ultrasonic wave breaking (or enzyme digestion) method, and the construction of a sequencing library is completed by the DNA fragments through end repair, 3' end adding A, sequencing joint coupling adding, purification and PCR amplification. The library is qualified by quality inspection and then sequenced by an illumina platform. After the sequencing data are downloaded, the quality control of original data (Raw reads) is required according to a certain standard, and the filtering standard is as follows: (1) removing sequences with a linker (adapter), (2) removing a pair of sequences with a single-ended sequence nitrogen content >10%, and (3) removing a pair of sequences with a low-mass base number exceeding 50%. And removing the low-quality sequence, the linker sequence and the inaccurate sequence to carry out the next sequence alignment. Selecting a newly assembled papaya fruit material 'kamiya' genome from the subject group by referring to the genome, comparing Clean reads with a reference genome sequence by using BWA-mem software, sequencing results by using samtools software, removing PCR repetitive sequences by using GATK4.0 software, performing mutation detection, filtering a variation set by using hard standards of QD > 2.0, QUAL > 30.0, FS < 60.0 and MQ > 40.0, and reserving a variation site data set with statistical significance. And (4) filtering the mutation sites again by taking MAF (minor allele frequency) > = 0.05 and miss (deletion rate) < =0.2 as standards to obtain a high-quality mutation site collection.
Analysis of papaya fruit weight association SNP sites
And annotating the variation sites by using the genome DNA interval feature description file, and respectively counting the number of the variation sites which fall in a gene coding region, a non-coding region, an intergenic region, non-synonymous mutation and the like. After a population genetic relationship matrix is obtained through population structure analysis, genome-wide association analysis is carried out by combining phenotypic character data, a linkage disequilibrium region which is significantly related to the weight of papaya fruits (-log 10 (P-value) =8.52 and is higher than a significant correlation threshold value 6) and has the size of 6kb is detected on a Chr09 chromosome, and a candidate gene Cpa09g016360 related to the weight of papaya fruits is determined.
Screening candidate markers
Based on SNP markers in the related regions in GWAS analysis, allele frequency, character difference and gene expression quantity difference are compared, and nonsynonymous mutation SNP loci Cpa09G016360:1846 (A/G) which are obviously related to the weight of papaya fruits are screened in the Cpa09G016360 gene CDs region.
TABLE 1 number of individuals of different genotypes and average fruit weight (g) in the population
After a 226bp DNA fragment is obtained by using the SNP marker primer through common PCR reaction, a sequencing result is compared with a related gene fragment SEQ ID No.1 of the papaya and analyzed, and the genotype carried by the 144 th SNP site of the sequence is detected, so that the fruit weight of a papaya variety can be detected or predicted, the papaya variety with high fruit weight can be effectively selected, and the breeding process of the papaya variety with high fruit weight is accelerated.
In order to verify the practicability of the SNP marker, 30 papayas (excluding 340 papayas for SNP marker development) are randomly selected from the papayas growing areas of Wenchang base of the institute of biotechnology of Chinese tropical agrology academy, and are subjected to genotyping and papaya cavity size character investigation after sequencing.
Table 230 papaya strains different genotypes and fruit Cavity sizes at Cpa09g016360:1846
Table 330 papaya strains in Cpa09g016360:1846 different genotypes and fruit cavity sizes
As can be seen from tables 2 and 3, the G allele at Cpa09G016360:1846 is very significantly related to fruit weight.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.
Sequence listing
<110> three colleges of tropical agricultural sciences in China
<120> SNP molecular marker related to papaya fruit weight and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 226
<212> DNA
<213> Artificial Sequence (cttcctatca caattcaaag attactagct gatttcttgg 40 tgatgattaa taggatttac tgttatgaga gactttggaa 80 actttagagc ttgcaatact acgaatggtg aagcaactcc 120 atcaaccggt aggctaaatg accra/gtatgag tttagcatcc 160 ggatcaaggt ttattatgcc tcagattgct gaaatagaga 200 atgaaagtat gggagcaagc agccct 226)
<400> 1
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence (cttcctatca caattcaaag 20)
<400> 2
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence (Agtatgggag caagcagccc t 21)
<400> 3
Claims (9)
1. SNP molecular marker related to the weight of papaya fruits is characterized in that: the molecular marker is located at the 25329094 th base of the chromosome of Chr09 and is named as molecular marker Cpa09g016360:1846, and the SNP molecular marker is located at the 144 th base of the nucleotide sequence shown in SEQ ID No.1 respectively.
2. The SNP molecular marker according to claim 1, wherein: the molecular marker is very obviously related to the Cpa09g016360 gene of papaya in regulating fruit size, and the P is less than 0.01.
3. Use of the SNP molecular marker according to any one of claims 1 to 2, wherein: can be used for detecting the breeding material of the papaya with high fruit weight.
4. Use of the SNP molecular marker according to any one of claims 1 to 2, wherein: can be used for detecting or predicting the weight of papaya fruits.
5. Use of the SNP molecular marker according to any one of claims 1 to 2, wherein: can be used for selecting varieties with larger fruit weight of papaya.
6. Use of the SNP molecular marker according to any one of claims 1 to 2, wherein: can be used for papaya molecular marker assisted breeding.
7. Use of the SNP molecular marker according to any one of claims 1 to 2, wherein: can be used in the breeding process of varieties with heavy papaya fruit weight.
8. A method of preparing the molecular marker of claim 1, wherein: designing a primer pair by using a nucleotide sequence position basic sequence containing the SNP marker, and carrying out PCR amplification by using papaya genome DNA as a template to convert the SNP marker into a 226bp molecular marker.
9. The method of claim 8, wherein: the upstream primer of the primer is as follows: cttcctatca caattcaaag, the downstream primer is: agtatgggag caagcagccc t are provided.
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Cited By (3)
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| CN117144055A (en) * | 2023-10-31 | 2023-12-01 | 中国热带农业科学院三亚研究院 | Application of haplotype molecular marker related to regulation and control of papaya fruit length |
| CN117144056A (en) * | 2023-10-31 | 2023-12-01 | 中国热带农业科学院三亚研究院 | Application of haplotype molecular marker related to papaya fruit fructose accumulation |
| CN117512203A (en) * | 2024-01-04 | 2024-02-06 | 中国热带农业科学院三亚研究院 | Molecular marker related to papaya fruit width, method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117144055A (en) * | 2023-10-31 | 2023-12-01 | 中国热带农业科学院三亚研究院 | Application of haplotype molecular marker related to regulation and control of papaya fruit length |
| CN117144056A (en) * | 2023-10-31 | 2023-12-01 | 中国热带农业科学院三亚研究院 | Application of haplotype molecular marker related to papaya fruit fructose accumulation |
| CN117144055B (en) * | 2023-10-31 | 2024-03-22 | 中国热带农业科学院三亚研究院 | Application of haplotype molecular marker related to regulation and control of papaya fruit length |
| CN117144056B (en) * | 2023-10-31 | 2024-03-22 | 中国热带农业科学院三亚研究院 | Application of haplotype molecular marker related to papaya fruit fructose accumulation |
| CN117512203A (en) * | 2024-01-04 | 2024-02-06 | 中国热带农业科学院三亚研究院 | Molecular marker related to papaya fruit width, method and application |
| CN117512203B (en) * | 2024-01-04 | 2024-03-22 | 中国热带农业科学院三亚研究院 | Molecular marker related to papaya fruit width, method and application |
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