SNP Loci Associated with the Sex of Grape Flowers and Their Applications

Abstract

The present invention belongs to the technical field of genetic engineering, and specifically relates to SNP loci associated with grape flower sex and their applications. The present invention uses GWAS analysis based on a 200K chip to identify SNP loci and candidate gene CYP1 associated with flower sex. These loci can be used for molecular marker-assisted breeding of hermaphrodite grape materials. Since molecular markers have the advantages of simplicity, rapidity, and high throughput in the assisted breeding system, the SNP loci provided by the present invention have good application value in the cultivation of new hermaphrodite grape varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to an SNP locus associated with grape flower sex and its application. Background Art

[0002] The sex of grape flowers can be divided into male flowers, female-fertile flowers and hermaphrodite flowers. By predicting the sex of flowers at an early stage, excellent plants with hermaphrodite flowers can be screened out in advance, reducing cultivation costs and laying a foundation for grape breeding. Exploring genes related to grape sex and developing molecular markers closely related to grape sex, and using molecular marker-assisted selection to utilize new genes or aggregate them with known excellent genes are of great significance for cultivating excellent grape varieties. At present, the known flower sex control region of grapes is mainly in the SEX-DETERMINING REGION (SDR) of about 5M on chromosome 2 (Dalbó et al., 2000, Lowe and Walker, 2006, Riaz et al., 2006, Marguerit et al., 2009, Battilana et al., 2013). The genes controlling male flowers mainly include two genes, VviYABBY3 and VviSKU5. Eleven genes from TREHALOSE-6-PHOSPHATE PHOSPHATASE (TPP) to WRKY21 are located in the female flower region of grapes (Zou et al., 2020).

[0003] This study is based on single nucleotide polymorphism markers (Single nucleotide polymorphisms, SNPs) detected by a 200K SNP chip. Through GWAS, the correlation analysis between genotypes and phenotypic traits is carried out to screen out SNPs markers associated with target traits, and then candidate genes related to target traits are located. The inventors of this application have successfully completed the resequencing of 335 grape natural populations and the development and design of 200K chips for 463 materials of 4 F1 populations (the reciprocal cross populations of Beihong x ES7-11-49, Beifeng x Yan 73, and Beichun x Concord) in the early stage, obtaining a large number of high-quality SNPs. Among them, the SNPs of the F1 population can be used for the construction of genetic maps, providing an important guarantee for the identification of grape germplasm resources. The present invention uses GWAS analysis based on SNPs of 200K chips to identify that the natural variation of the candidate gene CYP1 (VIT_05s0094g01190) controlling grape flower sex is highly associated with grape flower sex. Then, we carried out a significance analysis of the significant correlation sites of this gene and traits and found that the base type of this site is significantly correlated with the sex of grape flowers. Summary of the Invention

[0004] The object of the present invention is to provide an SNP locus associated with grape flower sex and its application.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] An SNP locus associated with grape flower sex, the SNP locus is located at position 24,498,562 on chromosome 5 of the grape reference genome (PN40024, v2) (chr5: 24,498,562), or the corresponding locus on the homologous genomic fragment among its germplasm resources. The nucleobase of this locus is "GG", "TT" or "GT", and this locus is located in the intron region of the gene.

[0007] Through analysis, the gene associated with grape flower sex was found to be VIT_05s0094g01190, named CYP1.

[0008] The present invention can assist in identifying grape flower sex through the SNP locus associated with grape flower sex, and can predict and screen the flower sex of grapes at an early stage.

[0009] The present invention uses SNP (chr5: 24,498,562) to genotype the flower sex traits of 463 hybrid populations and 335 natural populations. It is found that there are 130 materials with the genotype of "GG", among which 109 are female-fertile individuals (84%), and the remaining 21 samples are hermaphrodite. There are 321 materials with the genotype of "TT", among which 312 are hermaphrodite individuals (97%), and 9 of them are female-fertile. There are 236 materials with the genotype of "GT", among which 214 are hermaphrodite individuals (91%), 21 are female-fertile, and 1 is male. These results show that this SNP (chr5: 24,498,562) can be well used to distinguish the flower sex.

[0010] In addition, the present invention also protects the application of the SNP locus associated with grape flower sex in grape assisted breeding, as well as its application in locating genes closely linked to grape flower sex.

[0011] In addition, the present invention also discloses a GWAS analysis method related to the SNP locus associated with grape traits and grape flower sex, specifically including the following steps:

[0012] 1) According to the sequencing results of 314 re-sequenced samples, perform raw data processing and SNP screening to obtain 500,724 high-quality SNPs for the design of 463 grape 200K chips.

[0013] 2) A total of 174,464 SNPs were finally obtained, and genome-wide association analysis of flower sex was performed on 463 total sample populations and three of the F1 populations (reciprocal crosses of Beihong x ES7-11-49, Beifeng x Yan 73, and Beichun x Concord); PLINK (Version v1.90b6.24) was used to screen data with missing < 10% and maf > 0.01. SNPs were screened for 463 total samples and the reciprocal cross populations of Beihong x ES7-11-49, Beifeng x Yan 73, and Beichun x Concord. Finally, 155,404, 130,917, 80,560, and 102,191 SNPs were used for genome-wide association analysis respectively.

[0014] 4) Further, gec was used to determine the significance threshold. The significance threshold for 463 samples was 6.0, the significance threshold for the Beihong x ES7-11-49 population was 5.7, the significance threshold for the Beifeng x Yan 73 population was 5.5, and the significance threshold for the reciprocal cross population of Beichun x Concord was 5.6;

[0015] 5) The GLM model of TASSEL was used for genome-wide association analysis; the results of the genome-wide association analysis showed that in addition to the known chromosome 2, a locus significantly related to flower sex was newly discovered on chromosome 5;

[0016] 6) We found that at position 24,498,562 on chromosome 5 (chr5: 24,498,562), when the base at this position was homozygous "GG", it was mostly female flowers, and when the base was "GT" or "TT", it was mostly bisexual flowers. The variation of the base at this position was significantly correlated with flower sex.

[0017] Advantages of the present invention:

[0018] The present invention identified an SNP locus associated with grape flower sex, laying a foundation for molecular marker breeding of grapes. By using the analysis of GWAS based on a 200K chip, the present invention identified an SNP locus and a candidate gene CYP1 associated with flower sex. This locus can be used for molecular marker-assisted breeding of grape bisexual flower materials. Since molecular markers have the advantages of simplicity, rapidity, and high throughput in the assisted breeding system, the SNP locus provided by the present invention has good application value in the cultivation of new grape varieties with bisexual flowers.

[0019] In addition, since molecular markers are of great significance for the final mapping of functional genes, identifying the structure of this gene helps us understand the evolution and biological functions of this gene, and at the same time lays a foundation for molecular marker-assisted breeding and breeding. Coupled with the advantages of simplicity, rapidity, and high throughput of SNP loci in the establishment of the molecular marker breeding system, the present application thus has very important application value and also has very important protection significance for the cultivation of new grape varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the development process of a 200K chip based on 314 re-sequenced samples;

[0021] Figure 2 is the genome-wide association analysis of grape flower sexuality based on a population of 463 samples, the Beihong x ES7-11-49 population, the Beifeng x Yan 73 population, and the reciprocal cross populations of Beichun x Concord;

[0022] Figure 3 is the analysis result of the significant difference in the sex of flowers and the bases at this position among 463 samples. 1 represents female-fertile flowers, and 0 represents hermaphrodite flowers;

[0023] Figure 4 is the analysis result of the significant difference in the sex of flowers and the bases at this position among 335 natural populations. 1 represents female-fertile flowers, 0 represents hermaphrodite flowers, and -1 represents male flowers; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] The present invention will be described in detail below through specific embodiments. These embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0025] As used throughout the specification and claims, the term "comprising" or "including" is an open-ended term and should be interpreted as "including but not limited to". The subsequent description in the specification is for the purpose of describing the preferred embodiments of implementing the present invention, but the description is for the general purpose of the specification and is not intended to limit the scope of the present invention. The protection scope of the present invention shall be defined by the appended claims.

[0026] The experimental reagents not specifically described in the present invention are all conventional reagents in the art, or are prepared by conventional methods in the art, can be commercially obtained, and the specifications are laboratory pure grade.

[0027] The biological materials used in the present invention are all materials disclosed in the prior art and are all preserved in the laboratory of the applicant's unit. They can be distributed to the public for verification tests within twenty years from the filing date, or the public can also obtain them by purchasing.

[0028] I. Experimental Materials

[0029] Young leaves of 463 grape samples were sampled, which were from Beihong x ES7-11-49 population, Beifeng x Yan 73 population, and F1 generations of reciprocal crosses of Beichun x Concord.

[0030] II. Flower sex investigation

[0031] During the grape flowering season every year, the flower sex was investigated and recorded.

[0032] III. DNA extraction from grape leaves

[0033] The collected samples were immediately put into liquid nitrogen and then stored in a -80 °C freezer. The DNA of grape leaves was extracted using the magnetic bead method.

[0034] IV. Design of a 200K SNP chip based on SNPs obtained from 314 re-sequenced samples.

[0035] V. Obtain a 200K SNP gene chip from Affymetrix.

[0036] VI. Perform GWAS analysis on flower sex

[0037] Based on SNPs, a genome-wide association analysis was performed. First, PLINK (Version v1.90b6.24) was used with a window of 50 SNPs and r 2 equal to 0.2 (Purcell et al., 2007) to screen SNPs. After obtaining high-quality SNPs, these SNPs were used with GEC (Li et al., 2012) to perform threshold correction calculations. The GLM model of TASSEL was used for GWAS of flower sex, and PCA and kinship were generated by TASSEL for GWAS analysis.

[0038] More specifically, the present invention obtained an SNP locus related to flower sex on chromosome 5 in the following manner:

[0039] 1) According to the sequencing results of 314 re-sequenced samples, the raw data was processed and SNPs were screened to obtain 500,724 high-quality SNPs for the design of a 200K chip for 463 grape samples;

[0040] 2) Further, 204,860 SNPs were preliminarily determined for the 200K chip through screening on the Affymetrix platform. Since grapes are highly heterozygous crops, 174,464 SNPs were finally actually obtained;

[0041] 3) Genome-wide association analysis of flower sex was performed on 463 total sample populations and four of the F1 populations among them (the reciprocal crosses of Beihong x ES7-11-49, Beifeng x Yan 73, and Beichun x Concord). Using PLINK (Version v1.90b6.24) (Purcell et al., 2007), data screening criteria of missing < 10% and maf > 0.01 were applied. SNPs were screened for 463 total samples and the reciprocal cross populations of Beihong x ES7-11-49, Beifeng x Yan 73, and Beichun x Concord. Finally, 155,404, 130,917, 80,560, and 102,191 SNPs were used for genome-wide association analysis respectively;

[0042] 4) Further, gec was used to determine the significance thresholds. The recommended threshold for 463 samples was 4.7 and the significance threshold was 6.0. The recommended threshold for the Beihong x ES7-11-49 population was 4.4 and the significance threshold was 5.7. The recommended threshold for the Beifeng x Yan 73 population was 4.2 and the significance threshold was 5.5. The recommended threshold for the reciprocal cross populations of Beichun x Concord was 4.3 and the significant threshold was 5.6.

[0043] 5) The GLM model of TASSEL was used for genome-wide association analysis. The results of genome-wide association analysis showed that in addition to the known chromosome 2, a locus significantly related to flower sex was newly discovered on chromosome 5. The -logP values of this locus in the 463 population, Beihong x ES7-11-49 population, Beifeng x Yan 73 population, and reciprocal cross populations of Beichun x Concord were 73.6, 11.6, 58.6, and 9.6 respectively.

[0044] 6) The present invention used 463 grape 200K chips to perform genome-wide association analysis on the sex of grape flowers. The results showed that in addition to the reported section about flower sex at a distance of approximately 4M on chromosome 2, an SNP locus related to flower sex was also found on chromosome 5. The -logP value of this locus was much higher than the significance threshold, indicating that the SNP at this locus was more likely to be significantly related to flower sex ( Figure 2 )

[0045] The present invention utilizes the SNP locus (chr5: 24,498,562) to genotype the flower sex traits of 463 hybrid populations (statistical results are shown in Table 1) and 335 natural populations (statistical results are shown in Table 2). It is found that there are 130 materials with the genotype of "GG", among which 109 are female-fertile individuals (84%), and the remaining 21 samples are hermaphrodite. There are 321 materials with the genotype of "TT", among which 312 are hermaphrodite individuals (97%), and 9 samples are female-fertile. There are 236 materials with the genotype of "GT", among which 214 are hermaphrodite individuals (91%), 21 are female-fertile, and 1 is male. These results indicate that this SNP (chr5: 24,498,562) can be used to better distinguish the sex of flowers, can be used to assist in identifying the flower sex of grapes, and has very important guiding significance for the breeding of hermaphrodite grapes.

[0046] Table 1 Relationship between SNP (chr5: 24,498,562) genotype and flower sex of GWAS population (463 hybrid populations)

[0047]

[0048] Table 2 Relationship between SNP (chr5: 24,498,562) genotype and flower sex of 335 natural populations

[0049]

[0050] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0051] References

[0052] Battilana, J., Lorenzi, S., Moreira, F.M., Moreno-Sanz, P., Failla, O., Emanuelli, F. and Grando, M.S. (2013) Linkage mapping and molecular diversity at me flower sex locus in wild and cultivated grapevine reveal a prominent SSR haplotype in hermaphrodite plants. Mol Biotechnol, 54, 1031-1037.

[0053] Dalbó, M.A., Ye, G.N., Weeden, N.F., Steinkellner, H., Sefc, K.M. and Reisch, B.i. (2000) A gene controlling sex in grapevines placed on a molecular marker-based genetic map. Genome, 43, 333-340.

[0054] Li M X, Yeung J M Y, Cherny S S, et al. Evaluating the effective numbers of independent ests and significant p-value thresholds in commercial genotyping arrays and public imputation reference datasets[J]. Human genetics, 2012, 131(5): 747-756.

[0055] Lowe, K.M. and Walker, M.A. (2006) Genetic linkage map of the interspecific grape rootstock cross Ramsey (Vitis champinii) x Riparia Gloire (Vitis riparia). Theor Appl Genet, 112, 1582-1592.

[0056] Marguerit, E., Boury, C., Manicki, A., Donnart, M., Butterlin, G., Némorin, A., Wiedemann-Merdinoglu, S., Merdinoglu, D., Ollat, N. and Decroocq, S. (2009) Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine. Theor Appl Genet, 118, 1261-1278.

[0057] Purcell, S. et al. (2007) PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559-575.

[0058] Riaz, S., Krivanek, A.F., Xu, K. and Walker, M.A. (2006) Refined mapping of the Pierce′s disease resistance locus, PdR1, and Sex on an extended genetic map of Vitis rupestris x V. arizona. Theor Appl Genet, 113, 1317-1329.

[0059] Zou, C., Karn, A., Reisch, B., Nguyen, A., Sun, Y., Bao, Y., Campbell, M.S., Church, D., Williams, S., Xu, X., Ledbetter, C.A., Patel, S., Fennell, A., Glaubitz, J.C., Clark, M., Ware, D., Londo, J.P., Snn, Q. and Cadle-Davidson, L. (2020) Haplotyping the Vitis Collinear core geaome with rhAmpSeq improves marker transferability in a diverse genus. Nat Commun, 11, 413.

Claims

1. Application of SNP loci associated with grape flower sex in assisting in identifying grape flower sex, characterized in that, The SNP locus is located at position 24,498,562 on chromosome 5 of the grape reference genome Pinot Noir PN40024, v2, and the genotypes of this locus are "GG", "TT", or "GT".

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