CN116121430A - Melon seed dormancy main effect QTL qsg5.1 closely linked molecular marker SNP53 and application thereof - Google Patents
Melon seed dormancy main effect QTL qsg5.1 closely linked molecular marker SNP53 and application thereof Download PDFInfo
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Abstract
The molecular marker SNP53 closely linked with the main effect QTLqsg5.1 of melon seed dormancy and application thereof belong to the field of plant molecular genetic breeding research. The molecular marker SNP53F: CACAGGAAAGCAGAACAAAGTG, SNP53R: TGTTCTACACAGCGGTAATGC. According to the invention, melon genome data is obtained according to a high-throughput sequencing technology, SNP molecular markers closely linked with a main effect QTL of melon seed dormancy are designed and developed, melon resources containing seed dormancy genes can be screened in a melon seedling stage by a molecular marker assisted selective breeding method, the seed dormancy gene-containing melon resources can be screened, the seed sowing time is planned, the breeding efficiency is improved, the seedling strengthening rate is improved, and the problem that whether offspring have seed dormancy characteristic varieties or not can be distinguished only after melon seeds are mature in the prior art is solved. The method is simple in operation and high in stability, and provides a new auxiliary selection method for melon molecular breeding.
Description
Technical Field
The invention belongs to the field of research of plant molecular genetic breeding, and particularly relates to a molecular marker SNP53 closely linked to a main effect QTL qsg5.1 of melon seed dormancy and application thereof, which can be used for screening potential genes of melon seed dormancy and marker-assisted breeding.
Background
Melon (Cucumis melo.l) is an annual vining herb of the genus cucurbitaceae, and is one of the important cash crops of cucurbitaceae. The phenomenon of seed dormancy refers to the phenomenon that the plant does not germinate or grows very slowly or temporarily stops under a proper growth environment, and is an active adaptation process of the plant to resist and adapt to a poor environment. The phenomenon of seed after-ripening is common in plants, that is, the dormancy of seeds can be broken after the seeds are dried for a period of time, but the regulation and control mechanisms in different plants are different. The dormancy of the melon seeds has great significance for large-scale production, not only affects the uniformity of germination, but also has different degrees of influence on the cultivation of strong seedlings, the yield and the quality. Through researching the dormancy traits of melon seeds, not only can the varieties with dormancy genes potentially exist be planned and planted and separately managed, thereby achieving the purposes of saving management cost and balanced supply in the market, but also the molecular markers can be used for screening the varieties with dormancy/non-dormancy of the seeds, so that the economic benefit brought to producers by early marketing of the melon is improved, and therefore, the dormancy trait genes of the melon seeds are positioned by constructing a genetic linkage map, the molecular markers with linked dormancy traits are obtained, theoretical basis is provided for auxiliary selection and breeding of the melon molecular markers, and foundation is provided for excavation and functional analysis of related genes of dormancy of the melon seeds.
The research of the QTL positioning of melon seed-related traits by researchers at home and abroad is mainly focused on hundred weight, seed length, width and the like, and the QTL positioning research of seed germination traits is less reported. The dormancy trait of crop seeds is a complex quantitative trait controlled by multiple genes, has a main gene effect and is greatly influenced by environmental factors. Compared with field crops, the dormancy traits of melon seeds are less researched, but yield loss and economic value reduction caused by dormancy are not ignored. Along with the rapid development of high-throughput sequencing technology and the perfection of melon genome sequencing, the feasibility of melon seed dormancy trait QTL analysis is further accelerated.
Disclosure of Invention
The invention aims to provide a molecular marker SNP53 closely linked with a main effect QTL qsg5.1 of melon seed dormancy and application thereof, wherein the molecular marker can be used for screening and rapidly identifying potential dormancy gene breeding materials and varieties of melons.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the primer sequence of the molecular marker SNP53 is as follows:
SNP53F：CACAGGAAAGCAGAACAAAGTG，
SNP53R：TGTTCTACACAGCGGTAATGC；
and selecting MseI by enzyme digestion, wherein the sequence is TTAA.
The PCR method is adopted to detect genome DNA of the melon to be detected by utilizing the specific primer pair provided by the invention, the molecular marker SNP53 is amplified, only one band is provided for dormant melon plants after restriction enzyme MseI is cut, and two bands are provided for non-dormant plant amplified bands after restriction enzyme MseI is cut, so that the identification of dormant melon genes contained in the melon can be accurately carried out through the molecular marker, the molecular marker is used for screening plants, and the breeding efficiency of the melon is greatly improved.
Application of the molecular marker SNP53 in identifying melon seeds with dormant major QTL qsg5.1 locus genes in seedling stage.
Further, the application is specifically:
(1) Extracting DNA of a sample to be detected, and carrying out PCR amplification by adopting a molecular marker SNP 53;
(2) The PCR reaction product is used for MseI digestion reaction, and the digestion method comprises the following steps: 10 mu L, mseI of PCR reaction products, 0.5 mu L of restriction enzyme, 1U/mu L of concentration, 10X Fast Digest buffer mu L of concentration and 7.5 mu L of deionized water, wherein the enzyme digestion reaction is carried out in a water bath kettle at 37 ℃ for 20min, 4 mu L of 6X loading buffer is added, then electrophoresis is carried out for 30min on 2% agarose gel 120U voltage, and photographing is carried out on a Tanon2500 gel imager;
(3) After PCR amplification and MseI enzyme digestion of the DNA sample to be detected, the electrophoresis mode can detect that the 111bp fragment is the seed dormant melon material, and the fragment size is 196bp, and the fragment size is the seed non-dormant melon material.
Further, the PCR reaction system is as follows: 30 ng/. Mu.L of LDNA 2. Mu.L, 0.2. Mu.L upstream and downstream of SNP53 primer, 1. Mu.L of 10 XPCR buffer, 0.3. Mu.L of 2.5mM dNTP, 0.1. Mu.L of Taq enzyme, and ddH 2 O 6.4μL。
Further, the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 20sec, annealing at 68 ℃ for 1min, extension at 72 ℃ for 30sec, 6 cycles of each cycle temperature reduced by 2 ℃; denaturation at 94℃for 20sec, annealing at 58℃for 1min, extension at 72℃for 30sec for 6 cycles, each cycle temperature reduced by 1 ℃; denaturation at 94℃for 20sec, annealing at 50℃for 30sec, extension at 72℃for 30sec for 20 cycles, and finally extension at 72℃for 5min.
An application of the molecular marker SNP53 in melon molecular marker assisted breeding.
Compared with the prior art, the invention has the beneficial effects that:
1. SNP markers are molecular markers that generate single nucleotide polymorphisms (single nucleotide polymorphism) at cleavage sites, and are characterized by a wide distribution range and high mutation stability, and therefore have become a new generation of markers for gene targeting in recent years. According to the invention, melon genome data is obtained according to a high-throughput sequencing technology, SNP molecular markers related to melon seed dormancy are designed and developed, and selection and breeding are assisted by the molecular markers, so that melon seed dormancy site screening can be performed in a melon seedling stage, the breeding period is saved, and the problem of low breeding efficiency caused by whether the breeding seeds have dormancy features or not is solved.
2. At present, no related report is made on the molecular marker closely linked with the melon seed dormancy main effect QTL qsg5.1 at home and abroad, and the molecular marker SNP53 is closely linked with the melon seed dormancy trait main effect QTL qsg 5.1.
3. The molecular marker has very important value in melon production practice and breeding.
4. The operation method is simple, the stability is strong, and a novel auxiliary selection method is provided for melon molecular breeding.
Drawings
FIG. 1 is a fine positioning map of the melon seed dormancy trait master QTL;
FIG. 2 shows that melon SNP53 is marked in F 2 Detecting an electrophoresis chart in the population, wherein M is marker, and the display site is 100bp; phenotype 1-21 is seed dormant plant, phenotype 22-33 is seed non-dormant plant, and phenotype 34-43 is intermediate plant. The seed dormant plants can amplify specific bands of 111bp, the intermediate type amplifies heterozygous bands of 111bp and 196bp, and the seed non-dormant plants amplify specific bands of 196 bp.
Detailed Description
The following description of the present invention refers to the accompanying drawings and examples, but is not limited to the same, and modifications and equivalents of the present invention can be made without departing from the spirit and scope of the present invention.
According to the invention, melon genome data is obtained according to a high-throughput sequencing technology, SNP molecular markers closely linked with a main effect QTL of melon seed dormancy are designed and developed, melon resources containing seed dormancy genes can be screened in a melon seedling stage by a molecular marker assisted selective breeding method, the seed dormancy gene-containing melon resources can be screened, the seed sowing time is planned, the breeding efficiency is improved, the seedling strengthening rate is improved, and the problem that whether offspring have seed dormancy characteristic varieties or not can be distinguished only after melon seeds are mature in the prior art is solved. The method is simple in operation and high in stability, and provides a new auxiliary selection method for melon molecular breeding.
The invention utilizes the seed dormancy material P5 as a female parent (thin-skin melon, which is easy to dormancy when the seed is harvested in the same year, and is at 28 ℃ C.) through the genetic mechanism of melon seed dormancy character and QTL positioning researchSeed soaking for 48h with germination rate of 0), the easy-germination material P10 is used as male parent (melon with thin skin and female parent P5, the seeds harvested in the current year are easy to germinate, seed soaking for 48h with germination rate of 100%) and hybridization combination is prepared to construct F 2 Segregating populations and F 2:3 Family, using F 2:3 Preliminary analysis is carried out on the control dormancy QTL in the families and in 2020 and 2021 respectively to obtain a two-year repetition site qsg 5.1.1, and F is reused 2 The group system carries out further fine positioning, analyzes the sequence difference of the P5 and P10 genes, digs the SNP locus difference, designs a primer, develops a linkage molecular marker which is closer to the QTL qsg5.1 distance for selecting the dormancy trait of the melon seeds and has higher selection efficiency.
The first embodiment is as follows: the embodiment provides a molecular marker SNP53 closely linked with a muskmelon seed dormancy main effect QTL qsg5.1, and the primer sequence is as follows:
SNP53F：CACAGGAAAGCAGAACAAAGTG，
SNP53R：TGTTCTACACAGCGGTAATGC。
and selecting MseI by enzyme digestion, wherein the sequence is TTAA.
The method for obtaining the molecular marker SNP53 comprises the following steps:
1. construction of melon genetic populations
Female parent P5, the seeds harvested in the current year are easy to dormancy, and the germination rate of the seeds soaked at 28 ℃ for 48 hours is 0; p10 male parent, the seeds harvested in the same year are easy to germinate, and the germination rate of the seeds immersed for 48 hours at 28 ℃ is 100%. Preparing hybridization combination to obtain F 1 Single plant, selfing to obtain F 2 Population and F 3 Family.
To parent F 1 F (F) 2 The group respectively harvest and air-dried seeds in 2020 and 2021, treat the seeds, and soak the seeds for 48 hours at 28 ℃ to count the germination rate. P (P) 1 、P 2 、F 1 F (F) 2 100 seeds were measured each time for the population, 3 replicates were taken, averaged, F 2 Single plant measurement of seed germination Rate (one fruit per single plant is left, 200 seeds per fruit are taken), F 3 The germination rate of seeds of each family is counted by the population, 10 single-plant fruits are taken from each family, 100 seeds are taken from each fruit for investigation and analysis, and QTL positioning analysis is carried out.
2. SLAF sequencing and seed dormancy trait preliminary localization
Sequencing F by SLAF 2 Single plant sequencing was performed in two years F 3 Family germination rate average value is F 2 And (3) constructing a melon gene map according to the phenotype data of the single plant, and preliminarily obtaining a seed dormancy character stable repeated candidate interval.
3. Caps molecular marker screening
The genome sequence information of two parents is obtained by carrying out high-throughput sequencing on P5 and P10, the difference sites between the two parents are analyzed and compared, the SNP site difference between the parents is analyzed, caps primers are designed by software Primer 5, polymorphic primers are screened on the female parent and the male parent by molecular marker Primer pairs in candidate regions, a high-density genetic map is constructed by SLAF sequencing technology, QTL site candidate intervals related to muskmelon dormancy are obtained, a molecular marker 13 pair is designed and developed altogether, molecular markers linked with the muskmelon seed dormancy trait are screened, and SNP53 is found to be closely linked with the muskmelon seed dormancy trait, as shown in figure 1.
The second embodiment is as follows: the embodiment provides a molecular marker SNP53 closely linked with a dormancy main effect QTL of muskmelon seeds for identifying the varieties of the muskmelon potentially containing dormancy genes in a seedling stage, which comprises the following specific steps:
(1) Extracting DNA of a sample to be detected, and carrying out PCR amplification by using a molecular marker SNP 53. The 10. Mu.L PCR reaction system was: 30 ng/. Mu.L of LDNA 2. Mu.L, 0.2. Mu.L of each of the SNP53 primer upstream and downstream, 1. Mu.L of 10 XPCR buffer, 0.3. Mu.L of 2.5mM dNTP, 0.1. Mu.L of Taq enzyme, and ddH 2 O6.4. Mu.L. The PCR amplification conditions were: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 20sec, annealing at 68 ℃ for 1min, extension at 72 ℃ for 30sec, 6 cycles of each cycle temperature reduced by 2 ℃; denaturation at 94℃for 20sec, annealing at 58℃for 1min, extension at 72℃for 30sec for 6 cycles, each cycle temperature reduced by 1 ℃; denaturation at 94℃for 20sec, annealing at 50℃for 30sec, extension at 72℃for 30sec for 20 cycles, and finally extension at 72℃for 5min.
(2) The PCR reaction product is used for MseI digestion reaction, and the digestion method comprises the following steps: the PCR reaction product 10 mu L, xhoI restriction enzyme 0.5 mu L, concentration 1U/. Mu.L, 10X Fast Digest buffer mu L, deionized water 7.5 mu L, enzyme digestion reaction in a 37 ℃ water bath for 20min, adding 4 mu L of 6X loading buffer, electrophoresis in a 2% agarose gel 120U voltage for 30min, and taking photos in a Tanon2500 gel imager.
(3) After PCR amplification and MseI digestion, the DNA sample to be detected can detect that the 111bp fragment is seed dormant melon material and the fragment size is 196bp is non-seed dormant melon material by electrophoresis (figures 1 and 2). Therefore, the different genotypes of the dormant sites of the seeds can be accurately distinguished through the amplification of the closely linked markers, and the purpose of auxiliary breeding is achieved.
Claims (6)
1. The melon seed dormancy major QTL qsg5.1 closely linked molecular marker SNP53 is characterized in that: the primer sequence of the molecular marker SNP53 is as follows:
SNP53F：CACAGGAAAGCAGAACAAAGTG，
SNP53R：TGTTCTACACAGCGGTAATGC。
2. an application of the molecular marker SNP53 in the identification of the gene with dormancy main effect QTL qsg5.1 locus in melon seeds in seedling stage.
3. The application of a molecular marker SNP53 in identifying melon seeds with dormant major QTL qsg5.1 locus genes in a seedling stage according to claim 2, which is characterized in that: the method is that
(1) Extracting DNA of a sample to be detected, and carrying out PCR amplification by adopting a molecular marker SNP 53;
(2) The PCR reaction product is used for MseI digestion reaction, and the digestion method comprises the following steps: 10 mu L, mseI of PCR reaction products, 0.5 mu L of restriction enzyme, 1U/mu L of concentration, 10X Fast Digest buffer mu L of concentration and 7.5 mu L of deionized water, wherein the enzyme digestion reaction is carried out in a water bath kettle at 37 ℃ for 20min, 4 mu L of 6X loading buffer is added, then electrophoresis is carried out for 30min on 2% agarose gel 120U voltage, and photographing is carried out on a Tanon2500 gel imager;
(3) After PCR amplification and MseI enzyme digestion of the DNA sample to be detected, the electrophoresis mode can detect that the 111bp fragment is the seed dormant melon material, and the fragment size is 196bp, and the fragment size is the seed non-dormant melon material.
4. The use of the molecular marker SNP53 according to claim 3 in the identification of melon seeds with dormant major QTL qsg5.1 locus gene in seedling stage, which is characterized in that: the PCR reaction system is as follows: 30 ng/. Mu.L of LDNA 2. Mu.L, 0.2. Mu.L of each of the SNP53 primer upstream and downstream, 1. Mu.L of 10 XPCR buffer, 0.3. Mu.L of 2.5mM dNTP, 0.1. Mu.L of Taq enzyme, and ddH 2 O 6.4μL。
5. The use of the molecular marker SNP53 according to claim 3 in the identification of melon seeds with dormant major QTL qsg5.1 locus gene in seedling stage, which is characterized in that: the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 2min, denaturation at 94 ℃ for 20sec, annealing at 68 ℃ for 1min, extension at 72 ℃ for 30sec, 6 cycles of each cycle temperature reduced by 2 ℃; denaturation at 94℃for 20sec, annealing at 58℃for 1min, extension at 72℃for 30sec for 6 cycles, each cycle temperature reduced by 1 ℃; denaturation at 94℃for 20sec, annealing at 50℃for 30sec, extension at 72℃for 30sec for 20 cycles, and finally extension at 72℃for 5min.
6. The application of the molecular marker SNP53 in melon molecular marker assisted breeding according to claim 1.
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| EP1800535A1 (en) * | 2005-12-21 | 2007-06-27 | De Ruiter Seeds R&D B.V. | Closterovirus-resistant melon plants |
| CN113151550A (en) * | 2021-04-16 | 2021-07-23 | 黑龙江八一农垦大学 | Molecular marker CmSSR02 closely linked with main effect QTL fft2 of early flowering characteristics of melons and application thereof |
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| EP1800535A1 (en) * | 2005-12-21 | 2007-06-27 | De Ruiter Seeds R&D B.V. | Closterovirus-resistant melon plants |
| CN113151550A (en) * | 2021-04-16 | 2021-07-23 | 黑龙江八一农垦大学 | Molecular marker CmSSR02 closely linked with main effect QTL fft2 of early flowering characteristics of melons and application thereof |
| CN114182032A (en) * | 2021-10-11 | 2022-03-15 | 中国农业科学院蔬菜花卉研究所 | SNP molecular marker for detecting seed coat color of muskmelon and application thereof |
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| CN117210596A (en) * | 2023-06-26 | 2023-12-12 | 青岛农业大学 | Melon SNP locus marker combination, SNP locus marker detection probe combination, liquid phase chip and application |
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