Molecular marker of strawberry pulp flavor gene and application
Through multi-omic mining, FvC3H66, a key candidate gene for strawberry fruit quality was discovered, and KASP molecular markers were developed, which solved the problem of slow strawberry breeding process and achieved the improvement of high-quality breeding efficiency of strawberries.
Patent Information
- Application Number
- CN202510047966.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-13
- Publication Date
- 2025-04-11
AI Technical Summary
In the prior art, the strawberry breeding process is too slow, making it difficult to quickly cultivate new high-quality strawberry varieties.
Through multiomic mining, FvC3H66, a key candidate gene that regulates fruit quality was discovered, and KASP molecular markers were developed to conduct early screening of breeding materials, and individuals containing target genes were quickly identified.
The efficiency of high-quality molecular marking assisted breeding in strawberries has been significantly improved, and the cultivation process of excellent varieties has been accelerated through early screening.
Smart Images
Figure CN119799951A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant gene inheritance, in particular to molecular markers of strawberry pulp flavor genes and applications thereof. Background Art
[0002] Strawberry is a perennial evergreen herbaceous plant of the genus Fragaria in the family Rosaceae. Strawberry has a short production cycle, quick results and high returns. Strawberry berries are fragrant and juicy, sweet and sour, and have high nutritional value. They are known as the "Queen of Fruits" and are loved by consumers. According to data: During the period 2011-2022, China's strawberry planting area and output showed an overall upward trend, and it has become the world's largest strawberry producer and consumer. However, against the background of growing consumer demand, everyone requires strawberries to taste better and last longer. Because past breeding work focused on industry-friendly traits such as strawberry fruit size, appearance, yield and disease resistance, the cultivation and promotion of new strawberry varieties with comprehensive traits such as high quality is imminent. Based on this, this study discovered a key candidate gene that regulates fruit quality through multi-omics, and developed analytical markers for the candidate gene. The above results greatly promote high-quality molecular marker-assisted breeding of strawberries. Summary of the invention
[0003] The purpose of the present invention is to provide a molecular marker of a strawberry pulp flavor gene and its application, thereby solving the problem of slow cultivation process in the prior art.
[0004] To achieve the above object, the present invention provides the following technical solutions:
[0005] Molecular markers of strawberry pulp flavor genes, developing KASP markers through the non-synonymous mutation SNP site of FvC3H66;
[0006] Each pair of KASP primers consists of three DNA sequences, namely forward primer 1, forward primer 2, and reverse primer;
[0007] In addition, a FAM fluorescent group linker sequence was added to the 5′ end of each forward primer 1, the sequence was 5′-GAAGGTGACCAAGTTCATGCT-3′, and a HEX fluorescent group linker sequence was added to the 5′ end of each forward primer 2, the sequence was 5′-GAAGGTCGGAGTCAACGGATT-3′;
[0008] The genomic DNA of the parents P1_69 and P2_SX was extracted by Cetyl Trimethyl Ammonium Bromide (CTAB) method, and the DNA quality was tested by 1% agarose gel electrophoresis;
[0009] The above designed primers were used for PCR amplification and fluorescence signals were collected. The PCR reaction was carried out in a fluorescence quantitative PCR instrument (BioRad, CFX-96); the polymorphic KASP marker was used to perform genotyping on the F1 population (71 strains) constructed by P1_69×P2_SX, and the PCR reaction conditions were the same as above.
[0010] On the basis of the above technical solution, the present invention also provides the following optional technical solution:
[0011] In an alternative scheme: the PCR reaction system is 4.5 μL 1×KASP Mastermixture (LGCGenomics, Hoddeston, UK), 2 μL (50 ng / μL) genomic DNA, 2 μL KASP primer mix and 1.5 μL ddH2O.
[0012] In an optional scheme: the PCR reaction program is 95°C for 10 min; 95°C for 20 s, 61°C for 40 s, 10 cycles, with the annealing extension temperature decreasing by 0.6°C each cycle; 95°C for 20 s, 55°C for 40 s, 30 cycles; and insulation at 25°C.
[0013] In an optional scheme: in the 2 μL KASP primer mix: forward primer 1: forward primer 2: reverse primer: water = 6:6:15:23.
[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0015] This application discovered a key candidate gene that regulates fruit quality through multi-omics and developed analytical markers for the candidate gene, which greatly promoted the efficiency of high-quality molecular marker-assisted breeding of strawberries. The KASP marker was used for early screening of breeding materials, and individuals containing the target gene were quickly identified, thereby improving breeding efficiency and accelerating the breeding process of excellent varieties. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 The figures are the whole body and cross-section diagrams of Benihoppe (Hongyan), Parajatan (Tai Li) and ShuXing (Shu Xing) pulps according to the present invention.
[0017] Figure 2 For the present invention: (A) a representative hierarchical clustering tree; (B) a clustering diagram of module feature values; (C) a clustering diagram of module level; (D) a histogram of gene counts for each module.
[0018] Figure 3 The present invention shows: (A) heat map of sample expression patterns; (B) correlation diagram of trait association; (C) statistical diagram of transcription factors; (D) heat map of key candidate gene expression patterns.
[0019] Figure 4 The figure shows the validation result of the molecular marker KASP_FvC3H66 of the present invention in the F1 population (NTC is a non-template control; in the F1 population, the allele AG comes from the genotypes of the donor parent 'P1-69' and the recipient parent 'P2-SX', and AA and GG are the genotypes of the F1 offspring). DETAILED DESCRIPTION
[0020] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0021] This study used the strawberry varieties "Tai Li" (female parent) and "Hong Yan" (male parent) for hybridization. After years of identification and variety comparison tests and regional tests in different environments, an optimal strain was finally selected and named "Shu Xing". This variety has medium growth, a relatively open bead shape, strong continuous fruiting ability, high yield, high natural fruit setting rate, and few deformed fruits. The fruit is large, uniform in size, and conical in shape. The fruit surface is dark red and shiny, and the seeds are evenly distributed. The flesh is red and has a strong aroma. The maturity period is the same as Hong Yan, and it matures more than one week earlier than "Tai Li" ( Figure 1 ). Subsequently, 'Shuxing', 'Taili' and 'Hongyan' were planted in Taixing Town, Xindu District, Chengdu City, Sichuan Province (longitude: 104.231400; latitude: 30.787505; altitude 450m) and Dechang County, Liangshan Prefecture, Sichuan Province (longitude: 102°9'11"E; latitude 27°20'45"N; altitude 1494m). All experimental materials were planted in double rows on high ridges in the open field in late August 2023, with a plant spacing of 20cm×15cm, and routine field management. After the fruit matured, fresh strawberry pulp was collected from the two test points, with three biological replicates for each material, for a total of 18 samples. After quick freezing with liquid nitrogen, it was stored in a -80℃ low-temperature refrigerator for transcriptome and metabolome sequencing;
[0022] The FPKM values of 9292 differentially expressed genes and 14 quality indicators were used for weighted gene co-expression network analysis (WGCNA). First, we hierarchically clustered all samples according to the expression levels of all genes, treating each sample as a cluster and calculating the distance between each cluster ( Figure 2-A). Next, a gene clustering tree is constructed based on the correlation of gene expression, and gene modules are divided according to the clustering relationship between genes. Genes with similar expression patterns will be classified into the same module. The branches of the clustering tree are cut and distinguished to generate different modules. Each color represents a module, and gray represents genes that cannot be classified into any module ( Figure 2 -BC). The results showed that 9292 DEGs were divided into 21 modules ( Figure 3 -A). It is worth noting that the saddlebrown module had the highest correlation with Hardness, SFW, SSO, SSU, VC, SOAR, SUAR, PA, HA, T_Hex, Linalool, AF, and E_HB (P < 0.001) ( Figure 3 -B). Therefore, the saddlebrown module was subsequently analyzed, and the results showed that there were 406 DEGs in the saddlebrown module ( Figure 2 -D). Among them, there are 15 transcription factors ( Figure 3 -C). Next, we screened the 15 transcription factors in the saddlebrown module for key candidate genes according to the criteria of |K.in|>40 and |MM|>0.80. We finally screened a key candidate gene augustus_masked-Fvb3-3-processed-gene-253.2(FvC3H66)(|K.in|=48.39;|MM|=0.93)( Figure 3 -D);
[0023] Development of KASP molecular markers:
[0024] Through PolyMarker( http: / / www.polymarker.info / ) platform KASP markers were developed for the non-synonymous mutation SNP sites of FvC3H66. Each pair of KASP primers consisted of three DNA sequences, namely forward primer 1, forward primer 2, and reverse primer. In addition, a FAM fluorescent group linker sequence was added to the 5′ end of each forward primer 1, the sequence was 5′-GAAGGTGACCAAGTTCATGCT-3′, and a HEX fluorescent group linker sequence was added to the 5′ end of each forward primer 2, the sequence was 5′-GAAGGTCGGAGTCA ACGGATT-3′. The synthesis of the primers was completed by Beijing Qingke Biotechnology Co., Ltd. (Tables 1, 2).
[0025] The genomic DNA of parents P1_69 and P2_SX was extracted by Cetyl Trimethyl Ammonium Bromide (CTAB) method, and the DNA quality was detected by 1% agarose gel electrophoresis. The primers designed above were used for PCR amplification. The PCR reaction system was 4.5μL 1×KASP Mastermixture (LGC Genomics, Hoddeston, UK), 2μL (50ng / μL) genomic DNA, 2μL KASP primer mix (forward primer 1: forward primer 2: reverse primer: water = 6: 6: 15: 23) and 1.5μL ddH2O. The PCR reaction program was 95℃10min; 95℃20s, 61℃40s, 10 cycles, annealing extension temperature decreased by 0.6℃ each cycle; 95℃20s, 55℃40s, 30 cycles; 25℃ insulation, and fluorescence signal collection. PCR reaction was carried out in a fluorescence quantitative PCR instrument (BioRad, CFX-96). The polymorphic KASP marker was used to genotype the F1 population (71 strains) constructed by P1_69×P2_SX, and the PCR reaction conditions were the same as above.
[0026] The genetic analysis of 71 F1 populations constructed by P1_69×P2_SX using KASP-FvC3H66 showed that 25 materials were of AA (sweet) genotype, 18 materials were of GG (light) genotype, and 28 materials were of AG (fragrant) genotype (Table 3 and Figure 4 );
[0027] Table 1. SNP sequences involved in this study;
[0028]
[0029] Table 2. Primer sequences for KASP molecular markers designed in this paper;
[0030]
[0031] Table 3 Molecular marker development and application.
[0032]
[0033]
[0034]
[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A molecular marker for a strawberry pulp flavor gene, characterized in that: KASP markers were developed through the nonsynonymous mutation SNP site of FvC3H66; Each pair of KASP primers consists of three DNA sequences, namely forward primer 1, forward primer 2, and reverse primer; In addition, a FAM fluorescent group linker sequence was added to the 5′ end of each forward primer 1, the sequence was 5′-GAAGGTGACCAAGTTCATGCT-3′, and a HEX fluorescent group linker sequence was added to the 5′ end of each forward primer 2, the sequence was 5′-GAAGGTCGGAGTCAACGGATT-3′; The genomic DNA of the parents P1_69 and P2_SX was extracted by CTAB method, and the DNA quality was tested by 1% agarose gel electrophoresis; Perform PCR amplification using the above designed primers and collect fluorescence signals; The polymorphic KASP markers were used to genotype the F1 population constructed from P1_69×P2_SX.
2. The molecular marker of strawberry pulp flavor gene according to claim 1, characterized in that: The PCR reaction system consisted of 4.5 μL 1× KASP Mastermixture (LGC Genomics, Hoddeston, UK), 2 μL (50 ng / μL) genomic DNA, 2 μL KASP primer mix and 1.5 μL ddH2O.
3. The molecular marker of strawberry pulp flavor gene according to claim 1, characterized in that: The PCR reaction program was 95°C for 10 min; 95°C for 20 s, 61°C for 40 s, 10 cycles, with the annealing extension temperature decreasing by 0.6°C each cycle; 95°C for 20 s, 55°C for 40 s, 30 cycles; and insulation at 25°C.
4. The molecular marker of strawberry pulp flavor gene according to claim 1, characterized in that: PCR reactions were performed in a fluorescence quantitative PCR instrument, BioRad, CFX-96.
5. The molecular marker of strawberry pulp flavor gene according to claim 2, characterized in that: In the 2 μL KASP primer mix: forward primer 1: forward primer 2: reverse primer: water = 6:6:15:
23.
6. The molecular marker of strawberry pulp flavor gene according to claim 2, characterized in that: The F1 population consisted of 71 strains.
7. An application of a molecular marker of a strawberry pulp flavor gene according to any one of claims 1 to 5, characterized in that: The molecular marker of the strawberry pulp flavor gene is used to promote the application of high-quality molecular marker-assisted breeding of strawberries.