InDel3 molecular marker tightly linked to cucumber leaf yellowing mutation and its application
By developing InDel3 molecular markers closely linked to cucumber leaf yellowing mutations, the problem of difficulty in quickly identifying cucumber leaf yellowing mutations and heterozygous sites in the prior art has been solved, and rapid identification and breeding efficiency have been achieved in the seed period.
Patent Information
- Application Number
- CN202211182732.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-27
- Publication Date
- 2025-05-09
- Estimated Expiration
- 2042-09-27
AI Technical Summary
The prior art is difficult to quickly and accurately identify the yellowing mutations and heterozygous sites of cucumber leaves, which limits the efficiency of cucumber breeding and the process of germplasm selection.
An InDel3 molecular marker closely linked to the yellowing mutation of cucumber leaves was developed. The band pattern of the product was amplified by PCR, which can accurately identify the yellowing and green phenotypes of cucumber leaves.
The phenotype of the cucumber plant leaf color is achieved in the seed period, which improves breeding efficiency, simplifies operations, and reduces breeding costs and cycles.
Smart Images
Figure CN115466798B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cucumber genetic breeding and molecular biology, and in particular to an InDel3 molecular marker closely linked to cucumber leaf color and an application thereof. Background Art
[0002] Leaves are important organs for plant photosynthesis and an important basis for plant morphology and fruit growth. Plants absorb light energy and carbon dioxide through photosynthesis, convert inorganic matter into organic matter for storage, and maintain the nutrient requirements necessary for plant growth and the need to stabilize the balance of the ecosystem. As the direct carrier of photosynthesis, leaves are particularly important for plant growth and energy conversion. Generally, plant leaves appear green because they contain a large amount of photosynthetic pigments, which maintain the progress of photosynthesis and energy conversion. Therefore, photosynthetic pigments are an important basis for photosynthesis, play a very important role in photosynthesis, and are a platform for light energy absorption and conversion.
[0003] Leaves are an important site for photosynthesis in plants, and chlorophyll content, chloroplast structure, and chloroplast-related gene expression are closely related to photosynthesis. Under certain conditions, chloroplast mutations in plants will change the chlorophyll content in leaves, resulting in different leaf color phenotypes. In scientific research, using leaf color mutants to mine genes and study the mechanism of photosynthesis is of great significance for clarifying the regulatory function of genes and the molecular mechanism of photosynthesis.
[0004] With the completion of cucumber genome sequencing, the localization reports of genes related to important agronomic traits of cucumber have increased. These studies have provided new ideas for the exploration of important trait regulatory genes and molecular breeding. Under natural conditions or artificial induction, cucumbers often undergo various mutations. One of the more common mutations is leaf color mutation, which is also a common mutation phenomenon in nature. These mutants are important materials for studying plant photosynthesis mechanisms, photomorphogenesis, chlorophyll metabolism, chloroplast structure and function, and genetic development regulation mechanisms. They are also ideal materials for analyzing and identifying gene functions and understanding gene interactions and expression regulation. In the breeding process, cucumber leaf color mutations can be used as trait markers in improved variety breeding and hybrid breeding, which can greatly improve the identification efficiency in the selection of hybrid generation. This is not only easy to operate, simple procedures and intuitive results, but also can greatly reduce breeding costs and breeding cycles. Summary of the invention
[0005] In view of this, the present invention relates to an InDel3 molecular marker closely linked to the yellowing mutation of cucumber leaves and its application. The marker is used to quickly detect the yellowing mutation and heterozygous loci of cucumber leaf color. Through the association with the InDel3 molecular marker, the phenotype of the leaf color of cucumber plants can be quickly determined at the seed stage, thereby realizing rapid assisted breeding and cucumber germplasm selection, screening heterozygous plants with recessive genes, and screening cucumber germplasm materials with specific photosynthesis characteristics, providing a theoretical basis and application guidance for cucumber assisted breeding.
[0006] To solve the problems existing in the prior art, the technical solution of the present invention is: an InDel3 molecular marker tightly linked to the yellowing mutation of cucumber leaves, characterized in that: the InDel3 molecular marker is located in the 18.6kb interval between the InDel21 and SSR20583 markers of chromosome 7 of the cucumber Gy14 genomic DNA, and the physical position on the chromosome is between 1245674-1226636bp.
[0007] The InDel3 molecular marker is developed based on a 5 bp base insertion at the 1239217 bp position of the cucumber Gy14 genomic DNA, and the inserted nucleotide sequence is CTGAG.
[0008] The InDel3 molecular marker primers include an upstream primer InDel3-F: 5'-CACCAC GCT CAG GAT TCAGAT G-3' and a downstream primer InDel3-R: 5'-TCTTAA ATC ATA CCC AGT AGA CCA GG-3'.
[0009] The molecular marker is used for rapid detection of yellowing and heterozygous sites of cucumber leaves.
[0010] The molecular markers are used in the rapid detection method of cucumber leaf yellowing and heterozygous sites, which is characterized by:
[0011] The whole genome DNA of the cucumber material to be tested was extracted; PCR amplification was performed according to the InDel3 molecular marker primer pair; a single characteristic band of 526 bp was produced by PCR amplification in a single plant with a yellow leaf phenotype; a single characteristic band of 531 bp was produced in a homozygous single plant with a green leaf phenotype; when the leaf color was normal green but the locus was a heterozygous genotype, two characteristic bands of 526 bp and 531 bp were obtained by PCR amplification.
[0012] Compared with the prior art, the advantages of the present invention are as follows:
[0013] 1. The present invention provides an InDel3 molecular marker that is closely linked to the yellowing mutation of cucumber leaves. The InDel3 molecular marker is located in the 18.6kb interval between the InDel21 and SSR20583 markers on chromosome 7 of cucumber Gy14 genomic DNA, and its physical position is between 1245674-1226636bp.
[0014] 2. The present invention can accurately identify the yellowing and green phenotypes of cucumber leaves through the banding of the PCR amplification product of the InDel3 molecular marker. A single plant with a yellow leaf phenotype can produce a single characteristic band of 526 bp through PCR amplification; a homozygous plant with a green leaf phenotype can produce a single characteristic band of 531 bp; in the case of a normal green leaf color heterozygous genotype, PCR amplification obtains two characteristic bands of 526 bp and 531 bp.
[0015] 3. The identification method of the present invention can be used for identification at the seed stage, is simple and convenient to operate, and can greatly improve breeding efficiency, providing a theoretical basis for further accelerating the application of molecular markers in breeding. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 This is the result of fine positioning of candidate genes for cucumber leaf yellowing.
[0017] Figure 2 The electrophoresis diagram of PCR amplification using molecular marker InDel3. 1 represents the green leaf inbred line Gy14, and 2 represents the yellow leaf mutant material. DETAILED DESCRIPTION
[0018] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
[0019] When the InDel3 molecular marker of the present invention is used for assisted breeding, there is no need for seedling identification, and seeds can be used for accurate and rapid identification, which can improve breeding efficiency, shorten identification time, and reduce workload and production costs. In particular, the detection method through molecular markers is accurate and reliable, and the operation is simple. It provides a technical basis for rapid identification of breeding materials and variety selection, and has a very far-reaching theoretical significance and important application value for accelerating the breeding process.
[0020] The present invention aims to develop an InDel3 molecular marker that is closely linked to the cucumber leaf yellowing mutation, and the method steps adopted are:
[0021] Step 1. Select the inbred line Gy14 with normal green leaf color of cucumber as the male parent; the mutant yf of cucumber leaf yellowing as the female parent, construct a segregation population, and investigate the leaf color segregation rules and finely locate the leaf color yellow mutation. It is determined that the candidate gene of the leaf color yellow mutation trait is located in the 18.6kb interval between InDel21 and SSR20583 markers on chromosome 7, and the physical position is between 1245674-1226636bp.
[0022] Step 2. Sequence analysis of the parents revealed that there was a 5 bp CTGAG sequence insertion at bp 1239217 of chromosome 7 in the DNA genome of Gy14;
[0023] Step 3. Design marker InDel3 according to the inserted sequence. The primers consist of a pair of upstream primer InDel3-F and downstream primer InDel3-R for PCR amplification, specifically: InDel3-F: 5'-CAC CAC GCT CAG GAT TCAGAT G-3', InDel3-R: 5'-TCT TAA ATC ATA CCC AGT AGA CCA GG-3';
[0024] Step 4. F2 plants can be distinguished by PCR amplification using marker InDel3. Plants with yellow leaf phenotype can produce a single characteristic band of 526 bp through PCR amplification; homozygous plants with green leaf phenotype can produce a single characteristic band of 531 bp; when the heterozygous genotype is normal green leaf color, two characteristic bands of 526 bp and 531 bp are obtained through PCR amplification.
[0025] The following are the specific steps for developing the InDel3 molecular marker that is tightly linked to the cucumber leaf yellowing mutation:
[0026] 1. Experimental operation
[0027] (1) DNA extraction
[0028] Tender leaves of two parents of cucumber inbred line Gy14 (green leaf color), cucumber yellow mutant material (yellow leaf color) and F2 segregation population obtained by hybridization of the two parents were collected, and total plant DNA was extracted using the CTAB method. The preparation method of CTAB is 20g CTAB, 81.9g NaCl, 7.46g EDTA Na2·2H2O, 0.8g NaCl, 12.14g Tris base and 2g PVP, dissolved to 1L. Collect about 1g of fresh plant tissue samples in a sterilized 2mL centrifuge tube, freeze and grind in liquid nitrogen, and use NanoDrop micro-ultraviolet spectrophotometer to measure the concentration after extraction.
[0029] (2) Observation of leaf color segregation: The leaf colors of the parents and the F1 and F2 generations were observed. 593 F2 plants were randomly selected to identify leaf color segregation using the chi-square test and a chi-square test was performed.
[0030] (3) Positioning: Using the extracted F2 population DNA as a template, PCR amplification was performed using the screened SSR primers. The total PCR reaction system was 10 μL, including 1 μL template DNA, 0.5 μL of each 10 ng / μL primer (upstream + downstream), 4 μL of ddH2O, and 4 μL of 2×TaqPCR Master Mix; PCR reaction program: 94°C denaturation for 5 min; 94°C denaturation for 30 sec, 55°C annealing for 30 sec, 72°C extension for 30 sec, 32 cycles; 72°C extension for 10 min; 4°C storage. 9% polyacrylamide gel was used to identify the amplified fragments of SSR markers. Joinmap software was used to calculate the recombination value and genetic distance between the linked marker and the mutant gene.
[0031] (4) Resequencing and sequence alignment
[0032] The yf mutant material was resequenced using the Illumina Hi-seq2000 high-throughput sequencing platform with a sequencing depth of >20×. The segment initially located in the published cucumber Gy14 whole genome sequence (http: / / cucurbitgenomics.org / organism / 16) was used as a reference sequence, and the resequenced sequence of the yf parent was compared with the Gy14 candidate segment using IGV_2.11.1 software to find the different fragments between the two parents in the candidate segment.
[0033] (5) Sequence alignment and determination of differential sequences
[0034] By comparing the difference sequences of the candidate segments, insertion or deletion sites with more than 3 base differences were screened.
[0035] (6) Design of InDel markers and polymorphism screening
[0036] According to the deletion and insertion of bases, 14 pairs of InDel molecular markers were designed and synthesized using Primer5 software. The polymorphism of the designed InDel primers was screened between the parents, and primers with stable and clear amplification bands with different sizes were selected.
[0037] (7) Verify the linkage relationship between InDel molecular markers and target traits in an expanded F2 population
[0038] The F2 mapping population was further expanded to 1,200 plants, and the population was screened using InDel8, SSR20583, and the newly added InDel3 marker between the two markers to find recombinant plants. The linkage relationship between the newly added InDel3 molecular marker and the target trait was detected.
[0039] (8) Determination of characteristic bands of the selected InDel3 markers in green-leaf and yellow-leaf plants. The selected InDel3 molecular markers were used to perform PCR amplification in the parents and F2 individuals to determine the characteristic bands of green-leaf and yellow-leaf plants.
[0040] 2. Experimental results and analysis
[0041] (1) Observation and preliminary positioning of leaf color segregation: The leaf colors of the parents and the F1 and F2 generations were observed. 593 F2 plants were randomly selected and identified by chi-square test. The identification results showed that among the 593 F2 plants, 452 plants had green leaf color phenotype and 141 plants had yellow leaf color phenotype. The chi-square test showed that the leaf color segregation of the two plants was 96.3%, 95.3%, 97.7%, 96.7%, 97.6%, 98.4%, 96.7%, 97.6%, 98.3%, 96.7%, 97.6%, 98.3%, 96.7%, 97.3%, 96.7 ... 2 =0.261, which is consistent with the separation ratio of 3:1. This indicates that the yellowing trait of cucumber leaves is controlled by a pair of recessive nuclear genes.
[0042] (2) Screening and preliminary localization of polymorphic molecular markers using the BSA method
[0043] The screening results found that 4 pairs of molecular markers showed polymorphism between the two pools. Using Joinmap software and calculating the recombination value and genetic distance between the linked marker and the mutant gene, the candidate gene was initially located in the 123.96 kb interval between InDel8 and SSR20583, with genetic distances of 6.4 cM and 2.9 cM, respectively (see Figure 2). Figure 1 shown).
[0044] (4) Resequencing of yf mutants and alignment of parental genomes
[0045] The segment between SSR markers SSR20583 and SSR10066 was used as the reference sequence, and the resequencing sequence of the mutant parent was compared with the Gy14 candidate segment using IGV_2.11.1 software to find the different fragments between the two parents in the candidate segment.
[0046] (5) Development of InDel molecular markers closely linked to the leaf yellowing gene yf
[0047] By comparing the difference sequences of the candidate segments, insertion or deletion sites with more than 3 base differences were selected, and 14 pairs of InDel molecular markers were designed and synthesized using Primer5 software.
[0048] The 14 InDel markers were screened for polymorphism between the parents, and the screening results showed that InDel3 had a stable and clear amplification band with different sizes between the two parents. The InDel3 molecular marker was designed based on the 5 bp nucleotide sequence CTGAG inserted at the 1239217 bp of cucumber Gy14 genomic DNA.
[0049] The labeled upstream and downstream primers are:
[0050] InDel3-F:5'-CACCACGCTCCAGGATTCAGATG-3'
[0051] InDel3-R:5'-TCTTAAATCATACCCAGTAGACCAGG-3'.
[0052] (6) Confirmation of the tight linkage relationship between InDel3 and target traits
[0053] The F2 mapping population was further expanded to 1000 plants, and the population was screened using SSR20583, SSR10066, and InDel3 markers to find recombinant plants. The analysis results showed that there were 4 recombinant plants between the SSR20583 molecular marker and the yf gene, 8 recombinant plants between the SSR10066 molecular marker and the yf gene, and no recombinant plants appeared between the InDel3 molecular marker and the yf gene. Figure 1 shown.
[0054] (7) Determination of characteristic bands in green-leaf and yellow-leaf plants using the InDel3 molecular marker
[0055] The amplified products of InDel3 in the two parents and the F2 population were detected by electrophoresis. Figure 2 When the molecular marker InDel3 was used to amplify the genomic DNA of the yellow leaf color single plant, a single tightly linked characteristic band of 526 bp was obtained; when the molecular marker InDel3 was used to locate the normal green leaf color homozygous genotype, PCR amplification obtained a tightly linked characteristic band of 531 bp; when the molecular marker InDel3 was used to locate the normal green leaf color heterozygous genotype, PCR amplification obtained two tightly linked characteristic bands of 526 bp and 531 bp.
Claims
1. A method for rapid detection of cucumber leaf yellowing using InDel3 molecular marker primer pairs, characterized in that: The whole genome DNA of the cucumber material to be tested was extracted; PCR amplification was performed using the InDel3 molecular marker primer pair; a single characteristic band of 526 bp was produced by PCR amplification in the individual plant with yellow leaf phenotype; a single characteristic band of 531 bp was produced in the homozygous individual plant with green leaf phenotype; when the normal green leaf color was heterozygous genotype, two characteristic bands of 526 bp and 531 bp were obtained by PCR amplification; The upstream primer InDel3-F of the InDel3 molecular marker primer pair is: 5'-CAC CAC GCT CAG GAT TCAGAT G-3' and the downstream primer InDel3-R is: 5'-TCT TAA ATC ATA CCC AGT AGA CCA GG-3'.