KASP molecular markers, primers and their application for identifying watermelon flesh firmness
By developing the KASP molecular marker for identifying watermelon flesh hardness and combining it with BSR-Seq and GWAS fine positioning, the problem of rapid identification of flesh hardness in watermelon breeding was solved, achieving a rapid and accurate improvement in breeding efficiency.
Patent Information
- Application Number
- CN202310107874.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Priority Date
- 2022-09-20
- Filing Date
- 2023-02-13
- Publication Date
- 2025-09-26
- Estimated Expiration
- 2043-02-13
AI Technical Summary
Existing technologies make it difficult to quickly and accurately identify the hardness of watermelon flesh, resulting in slow selection and long breeding time during the breeding process, making it difficult to cultivate hard-fleshed watermelon varieties that meet market demand.
A KASP molecular marker for identifying watermelon flesh firmness was developed. A primer set was designed using base 12831389 on chromosome 6 of the watermelon reference genome version 97103V2. Rapid genotyping analysis was achieved through PCR amplification and fluorescence scanning. The target gene was precisely located using BSR-Seq and GWAS, and a KASP molecular marker tightly linked to flesh firmness was developed.
It realizes the accurate and rapid identification of watermelon flesh hardness at the seedling stage, significantly shortens the phenotypic identification cycle, improves breeding efficiency, reduces workload and production costs, and is suitable for molecular marker-assisted selection breeding of watermelon.
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Figure CN116219057B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of gene technology, and particularly relates to a KASP molecular marker, a primer and an application thereof for identifying the firmness of watermelon flesh. Background Art
[0002] Marker-assisted selection (MAS) is an auxiliary method for crop variety improvement using molecular markers. It primarily involves foreground selection for target traits and background selection for genetic material. It can rapidly detect target genes or loci closely linked to target trait genes, enabling selection for these traits. It is fast, accurate, and unaffected by environmental factors. It can significantly accelerate selection and shorten breeding time.
[0003] Flesh hardness, a key sensory quality attribute of watermelon, is a crucial indicator of its commercial value. Flesh hardness affects taste. Too firm flesh results in less juice and a poorer flavor. Too loose flesh results in a softer texture, reduced refreshing flavor, and poor storage resistance, resulting in a shorter shelf life. The market currently favors watermelon varieties with firmer flesh, which are durable in storage and transportation and easier to slice and compote. However, most traditionally cultivated varieties have a crunchy flesh. Therefore, effectively cultivating firmer flesh varieties is a hot topic in watermelon breeding. Summary of the Invention
[0004] The purpose of the present invention is to provide a KASP molecular marker for identifying watermelon flesh hardness, which can accurately and quickly identify watermelon flesh hardness in the seedling stage, significantly shorten the phenotypic identification cycle, and thus improve breeding efficiency.
[0005] To achieve the above objectives, the present invention provides a KASP molecular marker for identifying watermelon flesh hardness. The KASP molecular marker corresponds to base 12831389 on chromosome 6 of the watermelon reference genome version 97103V2. In hard-fleshed watermelon fruits, base 12831389 on chromosome 6 is G; in non-hard-fleshed watermelon fruits, base 12831389 on chromosome 6 is A.
[0006] Specifically, if the plant genotype is AA, the watermelon fruit has non-hard flesh; if the plant genotype is GG or GA, the watermelon fruit has hard flesh; the hardness of the non-hard flesh watermelon fruit is less than or equal to 2.0 kg / cm 2 The hardness of the hard-fleshed watermelon fruit is greater than 2.0 kg / cm 2 .
[0007] The present invention further seeks to protect the application of the KASP molecular marker in watermelon molecular marker-assisted breeding.
[0008] The present invention also provides a primer set for identifying the KASP molecular marker, which comprises a FAM primer, a VIC primer and a COM primer. The FAM primer sequence is shown in SEQ ID NO.1, the VIC primer sequence is shown in SEQ ID NO.2, and the COM primer sequence is shown in SEQ ID NO.3.
[0009] The present invention further seeks to protect the application of the primer set in watermelon molecular marker-assisted breeding.
[0010] The present invention also provides a kit for identifying or assisting in identifying watermelon flesh hardness, the kit comprising a FAM primer, a VIC primer and a COM primer, the FAM primer sequence being shown in SEQ ID NO.1, the VIC primer sequence being shown in SEQ ID NO.2, and the COM primer sequence being shown in SEQ ID NO.3.
[0011] Another object of the present invention is to provide a method for identifying or assisting in the identification of watermelon varieties, comprising the following steps: extracting DNA from a watermelon tissue sample to be tested as a template, performing PCR amplification on the DNA template using the primer set described in claim 5 to obtain a PCR amplification product as shown in the sequence of SEQ ID NO.4 and / or SEQ ID NO.5, performing fluorescence scanning on the PCR amplification product, and performing genotype analysis. If the genotype is AA, it is a non-hard-fleshed watermelon variety; if the genotype is GG or GA, it is a hard-fleshed watermelon variety.
[0012] By implementing the technical solution of the present invention, the following beneficial effects can be achieved:
[0013] (1) The present invention uses group segregation analysis with resequencing (BSR-Seq) to identify the gene interval that controls watermelon flesh firmness. Through transcriptome sequencing and genome-wide association analysis (GWAS) of natural populations, the target gene is precisely located. A KASP molecular marker tightly linked to the target trait is developed, which can be used in watermelon molecular marker-assisted selection breeding, improving the accuracy of selection for flesh firmness and accelerating the breeding process.
[0014] (2) The KASP molecular marker detection method of the present invention is reliable, easy to operate, and has high throughput. It can be applied on a large scale in watermelon breeding and production. It can accurately and quickly identify watermelon flesh hardness at the seedling stage, significantly shortening the phenotypic identification cycle, thereby improving breeding efficiency. It also has the advantages of low workload and low production cost. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is a diagram showing the genotype identification results of some F2 strains in Example 3. DETAILED DESCRIPTION
[0016] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.
[0017] Example 1
[0018] This example obtained a KASP molecular marker that is closely linked to watermelon flesh hardness.
[0019] (1) Determination of watermelon flesh hardness
[0020] Twenty plants were planted for each material. During the ripening period of watermelon, five watermelons with the same node position and shape were selected. The watermelon flesh hardness was measured using a fruit hardness meter (FT011) using the five-point method (stem end, flower scar end, shady side, sunny side, and center). The average value of the five locations of the five single watermelons represented the flesh hardness of the material. Based on the phenotypic data of watermelon flesh hardness, the watermelon flesh hardness was measured at 2.0 kg / cm 2 The limit is that the flesh hardness exceeds 2.0kg / cm 2 The definition of hard flesh is that the flesh hardness is less than 2.0kg / cm 2 is defined as non-tough meat.
[0021] (2) BSR-Seq analysis of hybrid populations
[0022] Using a hard-fleshed watermelon (PI595203, flesh hardness 5.9 kg / cm 2 ) and a non-hard flesh watermelon (Zhongguo Jingxin, flesh hardness 1.24kg / cm 2 ) homozygous inbred lines were hybridized to obtain F1 (flesh hardness 3.68kg / cm 2 ), F1 was self-pollinated to obtain an F2 segregating population, and 30 hard-fleshed watermelons and 30 non-hard-fleshed watermelons were selected from the F2 generation segregating population. After RNA was extracted from the pulp of mature watermelons, the RNA of the 30 hard-fleshed watermelons and the RNA of the 30 non-hard-fleshed watermelons were mixed together.
[0023] Transcriptome sequencing was performed using an Illumina HiSeq™ sequencer and aligned to the watermelon reference genome 97103 (http: / / cucurbitgenomics.org / organism / 21). A total of 20,636 single-nucleotide polymorphisms (SNPs) were identified. Bioinformatics analysis was performed on these SNPs, and pooled SNP-index and ED values were calculated. The results of the two association analysis methods were intersected, ultimately anchoring candidate intervals controlling watermelon flesh firmness between nucleotides 35,657,184 and 35,667,086 on chromosome 2, nucleotides 35,688,059 and 37,794,383 on chromosome 2, and nucleotides 10,266,228 and 22,864,361 on chromosome 6. These three QTLs encompass a total of 547 genes, 133 of which have functional annotations, and 29 of these annotated genes harbor nonsynonymous mutations.
[0024] (3) Transcriptome sequencing analysis of hard-fleshed and non-hard-fleshed watermelon fruits
[0025] Transcriptome sequencing analysis was performed on the mature fruits of a hard-fleshed watermelon (PI595203) and a non-hard-fleshed watermelon (Zhongguo Jingxin) homozygous inbred line to screen for differentially expressed genes. Gene enrichment analysis revealed that genes in the plant hormone signal transduction pathway were the most significant in the KEGG pathway, suggesting that genes in this pathway may be involved in the regulation of watermelon flesh hardness.
[0026] (4) GWAS screening of SNP loci associated with watermelon flesh firmness
[0027] A total of 214 natural watermelon accessions were selected and planted for three consecutive years. The average of the three-year watermelon flesh firmness values was used to represent the final flesh firmness of each accession. The results are shown in Table 1. A GWAS analysis of the 214 natural watermelon accessions was performed using a mixed linear model in GEMMA 0.98.1 software, with the Q matrix and kinship matrix as covariates, using Tassel V5.2.43 and TASSEL 3.0 software. Manhattan plots were generated using R software. Significance thresholds were calculated using a Bonferroni correction, with -log10(P)0.1 / Ne (Ne = number of effective SNPs) and -log10(P)0.01 / Ne set as the two thresholds for screening significant SNPs. Thirty-six SNPs were detected across 11 chromosomes. Among these SNPs, one significant SNP on chromosome 6 was consistent with the BSR mapping results.
[0028] Table 1214 Natural Population Material Pulp Hardness
[0029]
[0030]
[0031]
[0032]
[0033] (5) Determination of candidate genes and SNPs for watermelon flesh firmness
[0034] Combining BSR and GWAS data, two shared genes were identified within the candidate QTL interval on chromosome 6: Cla97C06G118710, which encodes a kinase protein, and Cla97C06G118630, which encodes an auxin response protein (AUX / IAA). Transcriptome analysis revealed that Cla97C06G118710 was not differentially expressed between firm-fleshed and non-firm-fleshed watermelons, while Cla97C06G118630 was significantly upregulated in firm-fleshed watermelon flesh.
[0035] (6) Obtaining tightly linked KASP molecular markers
[0036] Based on the SNP site Cla97C06G118630 (base 12831389 on chromosome 6), a KASP marker tightly linked to watermelon flesh firmness was developed. Primers were designed based on the upstream and downstream sequences of nucleotides 12831389 on chromosome 6 of the watermelon genome. The FAM primers of the primer set were designed based on the upstream sequences of nucleotides 12831389 on chromosome 6 of the watermelon genome 97103. The VIC primers were designed based on the mutation and upstream sequences of nucleotides 12831389 on chromosome 6 of the watermelon genome 97103. The COM primers were designed based on the downstream sequences of nucleotides 12831389 on chromosome 6 of the watermelon genome 97103. Primers were synthesized by a biotechnology company. The primer sequences are shown in Table 2.
[0037] Table 2 Primer sequences
[0038]
[0039] Example 2
[0040] This example uses KASP molecular markers to identify watermelon flesh firmness
[0041] (1) DNA extraction
[0042] DNA was extracted from young watermelon leaves using the standard CTAB method, and RNA was removed. The DNA sample volume should be at least 50 μL. The OD values of the DNA sample at 260 nm and 280 nm were measured using a UV spectrophotometer to calculate the DNA content and the OD 260 / 280 ratio. The DNA sample purity should be between 1.8 and 2.0. The sample was diluted to a concentration of 100 ng / μL for later use. The working solution was diluted to a concentration of 5-50 ng / μL.
[0043] (2) PCR reaction system and detection
[0044] Detection was performed using the KASP platform produced by LGC. The PCR reaction system consisted of 1.5 μL DNA (2-10 ng / μL), 0.75 μL 2× Master Mix, 0.0417 μL Primer Mix, and 0.75 μL ddH₂O. The PCR procedure was as follows: 94°C pre-denaturation for 15 min; 94°C denaturation for 20 s, followed by 61-55°C annealing / extension for 1 min (gradient annealing, decreasing 0.6°C each cycle) for 10 cycles; and 94°C denaturation for 20 s, followed by 55°C annealing / extension for 1 min for 26 cycles. The PCR amplified sequence is shown in SEQ ID NO. 4 or SEQ ID NO. 5.
[0045] After the PCR reaction, the 384-well plate was placed on an Omega fluorescence signal reader to convert the fluorescence signal into analyzable values. Genotyping was then performed using Kraken™ analysis software provided by LGC. If the genotype is AA, the watermelon fruit has non-hard flesh. If the genotype is GG or GA, the fruit has hard flesh.
[0046] Example 3
[0047] This example verifies the KASP molecular marker for the identification of hybrid segregating populations
[0048] Using hard-fleshed watermelon (PI595203) and non-hard-fleshed watermelon (Zhongguo Jingxin) as parents, an F2 population was configured. The F2 population had a total of 216 plants, including 58 non-hard-fleshed watermelons and 158 hard-fleshed watermelons. The genotypes of the 216 F2 plants were identified using the KASP molecular markers on chromosome 6 of watermelon obtained in Example 1, which were related to flesh hardness. The specific identification method was referred to Example 2. There were 53 plants with a genotype of GG, 105 plants with GA, and 58 plants with AA. The results showed that the phenotype of the F2 plantlets matched the genotype identified by the KASP molecular markers at a 100% consistency rate. The genotype identification results of some strains are shown in Figure 2. Figure 1 shown.
[0049] Figure 1 The sample genotype that is clustered near the top of the Y axis is GG (see Figure 1 A), the middle sample genotype is GA (see Figure 1 B), the genotype of the samples clustered close to the right of the X-axis is AA (see Figure 1 D), the sample at the junction of the X-axis and Y-axis in the lower left corner is the negative control (see Figure 1 Middle C).
[0050] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope disclosed herein, in accordance with the principles of the present invention. Applications of the essential features may be made within the scope of the following claims.
Claims
1. Detection of the application of KASP molecular marker reagent in molecular marker-assisted breeding of watermelon flesh firmness, characterized in that: The KASP molecular marker corresponds to the 12831389th base of chromosome 6 of the watermelon reference genome version 97103V2, and the base is G or A; If the plant's genotype is AA, the watermelon fruit will have non-hard flesh; if the plant's genotype is GG or GA, the watermelon fruit will have hard flesh. Among them, the hardness of non-hard flesh watermelon fruit is less than or equal to 2.0kg / cm 2 The hardness of hard-fleshed watermelon fruit is greater than 2.0kg / cm 2 .
2. The use according to claim 1, characterized in that The KASP molecular marker detection reagent is a primer set, which includes a FAM primer, a VIC primer and a COM primer. The FAM primer sequence is shown in SEQ ID NO. 1, the VIC primer sequence is shown in SEQ ID NO. 2, and the COM primer sequence is shown in SEQ ID NO.
3.
3. A method for identifying or assisting in identifying watermelon varieties, characterized in that: The method comprises the following steps: extracting DNA from a watermelon tissue sample to be tested as a template, performing PCR amplification on the DNA template using a primer set to obtain a PCR amplification product as shown in the sequence of SEQ ID NO. 4 and / or SEQ ID NO. 5, performing fluorescence scanning on the PCR amplification product and performing genotype analysis; if the genotype is AA, it is a non-hard-fleshed watermelon variety; if the genotype is GG or GA, it is a hard-fleshed watermelon variety; The fruit hardness of the non-hard flesh watermelon variety is less than or equal to 2.0 kg / cm 2 The fruit hardness of the hard-fleshed watermelon variety is greater than 2.0 kg / cm 2 ; The primer set includes a FAM primer, a VIC primer and a COM primer. The FAM primer sequence is shown in SEQ ID NO. 1, the VIC primer sequence is shown in SEQ ID NO. 2, and the COM primer sequence is shown in SEQ ID NO. 3.