CN116103425A - Functional molecular marker CX-5-2 for identifying length of lateral branches of watermelons and application thereof - Google Patents

Functional molecular marker CX-5-2 for identifying length of lateral branches of watermelons and application thereof Download PDF

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CN116103425A
CN116103425A CN202211048664.6A CN202211048664A CN116103425A CN 116103425 A CN116103425 A CN 116103425A CN 202211048664 A CN202211048664 A CN 202211048664A CN 116103425 A CN116103425 A CN 116103425A
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刘秀杰
高美玲
郭宇
刘继秀
高越
赵文
尹贞
段雅如
包秀萍
袁成志
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Qiqihar Agricultural Technology Promotion Center
Qiqihar University
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Abstract

The invention discloses a functional molecular marker CX-5-2 for identifying the length of a watermelon lateral branch and application thereof, belonging to the technical field of molecular markers. The molecular marker is obtained by PCR amplification by using a forward primer shown as SEQ ID NO.1 and a reverse primer shown as SEQ ID NO.2 and using watermelon genome DNA as a template. The molecular marker is a functional molecular marker designed based on the genotype sequence of the short lateral branch variety, can be directly applied to molecular marker assisted breeding of the short lateral branch variety of the watermelon, can accurately and rapidly identify whether the watermelon variety has the short lateral branch character in the seedling stage, improves the breeding efficiency, accelerates the breeding process, and has good application value for breeding of the short lateral branch variety of the watermelon. The functional molecular marker disclosed by the invention effectively solves the problem that the existing molecular marker capable of identifying the short side branches of watermelons is lacking.

Description

Functional molecular marker CX-5-2 for identifying length of lateral branches of watermelons and application thereof
Technical Field
The invention relates to a functional molecular marker CX-5-2 for identifying the length of a lateral branch of a watermelon and application thereof. The invention belongs to the technical field of molecular markers.
Background
Watermelon (Citrullus lanatus) is an important cucurbitaceae crop that is widely planted worldwide. The branching of watermelons is a process of developing into shoots from axillary buds (axillary meristems) in leaf axils. Most of the existing watermelon varieties are branch varieties, a great amount of manpower is consumed for pruning the watermelon varieties in the cultivation process, and the investment of manpower can be reduced to a great extent by short side branches or materials with few branches, so that the method is more beneficial to the realization of light simplification and saving production. In addition, the leaf included angle of the short side branches or few branches becomes smaller, which is more beneficial to increasing the planting density in unit area.
Molecular markers are classified into two kinds of molecular markers, namely broad sense and narrow sense. Generalized: the sequence of the DNA or the protein can be inherited and detected. Narrow definition: specific DNA fragments that reflect a certain difference between individuals or populations of organisms. The functional molecular marker is designed according to the gene sequence, and can directly detect genes, distinguish and forecast alleles and relative characters thereof. Compared with the defect of longer breeding period of conventional crossbreeding, molecular marker assisted selective breeding can directly select on the gene level, and has higher accuracy and efficiency. The invention develops the functional molecular marker of the watermelon lateral branch length, can identify the watermelon lateral branch length in the seedling stage, realizes molecular marker assisted selective breeding, and accelerates the breeding process of the short lateral branch variety.
Disclosure of Invention
The invention aims to provide a functional molecular marker CX-5-2 for identifying the length of a watermelon lateral branch and application thereof.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention takes a watermelon long side branch strain GWAS-38 and a watermelon short side branch mutant slb as parents to obtain F 2 The group finds the candidate region of the target gene on chromosome 5, locates the candidate region into a 25.35kb section by genotyping, has 2 candidate genes in the section, compares the parent sequences, finds that a SNP locus exists on Cla97C05G088180 gene, develops a functional molecular marker according to the locus, and is named CX-5-2. The forward primer sequence is shown as SEQ ID NO.5, and the reverse primer sequence is shown as SEQ ID NO. 6. The invention discloses a functional molecular marker and a watermelon short side branch trait co-separated. On the molecular level, the phenotype of whether the watermelon plant has short side branches can be identified in the seed or seedling stage, the selection efficiency and accuracy are improved, and the breeding process can be shortened.
Based on the research, the invention provides a functional molecular marker for identifying the length of the lateral branches of watermelons, which is named CX-5-2, wherein the molecular marker is obtained by PCR amplification by using a forward primer shown as SEQ ID NO.5 and a reverse primer shown as SEQ ID NO.6 and using the genomic DNA of watermelons as a template.
Furthermore, the invention also provides application of the functional molecular marker CX-5-2 for identifying the length of the watermelon lateral branch in molecular breeding of watermelon lateral branch varieties.
Preferably, the functional molecular marker can be used for identifying or assisting in identifying whether the watermelon variety has the short side branch character in a seedling stage.
Still further, the invention also provides a pair of primers for amplifying the functional molecular marker, wherein the pair of primers consists of a forward primer shown in SEQ ID NO.5 and a reverse primer shown in SEQ ID NO. 6.
Still further, the invention also provides application of the primer pair in molecular breeding of watermelon short side branch varieties.
Preferably, the primer pair can identify or assist in identifying whether the watermelon variety has the short side branch character in the seedling stage.
Furthermore, the invention also provides a method for identifying the length of the lateral branches of the watermelons, which comprises the following steps:
(1) Separating and extracting total DNA from watermelon tissues;
(2) Using the DNA obtained in the step (1) as a template, and carrying out PCR amplification by using a forward primer shown in SEQ ID NO.5 and a reverse primer shown in SEQ ID NO. 6;
(3) Carrying out agarose gel electrophoresis detection on the PCR amplified product obtained in the step (2), and then carrying out product recovery;
(4) Performing a first generation sequencing on the PCR product recovered in the step (3): if a product with the size of 474bp is obtained, judging that the watermelon variety is a short lateral branch; if 475bp products are obtained, the watermelon variety with long lateral branches is judged.
Preferably, in the step (1), the total DNA of the watermelon tissues is extracted by using a modified CTAB method.
Preferably, in the step (1), the watermelon tissue is a true leaf blade of watermelon.
Preferably, in the step (2), the PCR amplification reaction system is: 1. Mu.l template DNA, 1. Mu.l upstream primer, 1. Mu.l downstream primer, 3. Mu.l ddH 2 O, 4. Mu.l 2X Es Taq Master Mix; the conditions for the PCR amplification reaction were: pre-denaturation at 94℃for 5min, denaturation at 94℃for 60s, annealing at 54℃for 45s, extension at 72℃for 60s, and final extension at 72℃for 10min for 35 cycles, and storage at 4 ℃.
Compared with the prior art, the invention has the beneficial effects that:
the functional molecular marker for identifying the short side branch character of the watermelon plant disclosed by the invention can be applied to auxiliary selective breeding of the short side branch molecular marker of the watermelon. The molecular marker is separated from the short side branch character of the watermelon, so that whether the watermelon plant has the phenotype of the short side branch can be identified in the seed or seedling stage, a large amount of manpower and material resources are saved, the selection efficiency and accuracy are improved, and the breeding period can be shortened.
Conventional functional molecular markers will choose to design CAPS or Indel type molecular markers. As the difference of candidate genes between parents only has 1bp, the candidate genes are designed into Indel marks, and the candidate genes cannot be detected by agarose gel or polypropylene electrophoresis. Meanwhile, the candidate gene can not be converted into CAPS, dCAPS or KASP markers because the difference between parents is continuous A bases. Therefore, the invention carries out the first generation sequencing of the PCR product, judges the genotype and the character by comparing the sizes of the sequencing products, and has accurate and reliable identification result.
Drawings
FIG. 1 is a physical diagram of a long side branch strain and a short side branch strain;
a: watermelon long side branch strain GWAS-38; b: a watermelon short side branch mutant slb;
FIG. 2 is a map of the gene mapping of the short side branch trait of watermelon:
FIG. 3 is a diagram of the first generation sequencing results of PCR products;
FIG. 4 shows the base changes at position 6,291,086 of Chr5 in mutant slb and parent and partial natural populations.
Detailed Description
The following examples of the present invention are described in detail, and are provided by taking the technical scheme of the present invention as a premise, and the detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the following examples. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products available commercially without the manufacturer's attention.
The technical scheme of the invention is not limited to the specific embodiments listed below, and also includes any combination of the specific embodiments.
Example 1: gene fine positioning and molecular marker development for regulating and controlling short side branch character of watermelon
1. Construction of materials and isolated genetic populations
Hybridization is carried out by taking watermelon long lateral branch material GWAS-38 as male parent and watermelon short lateral branch mutant slb as female parent (shown in figure 1) to obtain F 1 ，F 1 F is obtained after selfing 2 Seed generation. For F 2 The phenotype of each individual in the segregating population was identified, 336F 2 The group identification result is a long lateral branch 258 strain, a short lateral branch 78 strain, and the ratio of long lateral branch individuals to short lateral branch individuals accords with the separation ratio of 3:1, which indicates that the short lateral branch character is controlled by a recessive single gene.
2. Candidate gene localization
(1) Construction of a mixing pool
In the 357 strain F constructed 2 In the population, 30 plants are selected from the normal long side branch phenotype and the short side branch phenotype, tender true leaf blades of each plant are taken, the plants are respectively frozen, dried and ground, and then mixed in equal quantity to construct a DNA mixed pool, the numbers 38 and slb respectively represent parents, the numbers DuancezhiA and Changcezhia respectively represent the short side branch pool and the long side branch pool, and the parents and the mixed pool are sent to Huada genes for whole genome resequencing. GWAS-38 sequencing depth was 10×, slb, duancezhiA and ChangcezhiA depth was 30×.
(2) Gene localization and molecular marker development
Development of molecular markers by BSA-seq technology was combined with the development of 335 strain F 2 The population was genotyped and the gene Clslb regulating the short side shoots was located 1.63Mb on chromosome 5 (4,680,000-6,310,000, 97103v 2). Subsequently, 1010 strain F was expanded 2 The population was expanded, molecular markers were encrypted, and finally, candidate segments were reduced to 25.354kb (6,280,285-6,305,639, 97103v 2). There are two candidate genes in the segment, and the Cla97C05G088180.1 gene is presumed to be a candidate gene for regulating the short side branch character of the watermelon according to gene annotation and sequence comparison analysis (as shown in figure 2). The total length of the gene is 5253bp (the sequence is shown in SEQ ID NO. 3), and the total number of the gene is 4 exons and 3 introns. To amphiphilic Cla97C05G0Cloning and sequence alignment of 88180.1 gene full length revealed that in mutant slb, cla97C05G088180.1 gene second exon 6,291,086bp had a deletion of A base (sequence see SEQ ID NO.1, FIG. 3), and the mutation was found only in watermelon short side branch mutant slb (FIG. 4). Resulting in premature translation, the normal protein is 1131aa long (SEQ ID NO. 4), and 854aa long (SEQ ID NO. 2) after truncation. A functional marker is developed by utilizing the deletion of one A base at the position of 6,291,086bp, and a molecular marker CX-5-2 which is co-separated from Clslb is developed and used for molecular marker assisted selection. The primer sequences are as follows:
CX-5-2 forward primer: 5'-GGACGCCAATCTTTGTTGTT-3' (SEQ ID NO. 5)
CX-5-2 reverse primer: 5'-CCAACAATCTGACCCTCCAT-3' (SEQ ID NO. 6)
1) The PCR reaction system is shown in Table 1:
TABLE 1 PCR amplification reaction System
Figure BDA0003821257390000051
2) The conditions for the PCR amplification reaction were: pre-denaturation at 94℃for 5min, denaturation at 94℃for 60s, annealing at 54℃for 45s, extension at 72℃for 60s, and final extension at 72℃for 10min for 35 cycles, and storage at 4 ℃.
3) The PCR product obtained was detected on agarose at 1%. And (3) performing gel recovery by using a DC301 kit to obtain a PCR product.
4) The parental PCR products recovered in step 3) were sent to Shanghai Biotechnology for first generation sequencing.
5) Comparing the results of the parent sequencing, the result shows that the product of the long lateral branch strain GWAS-38 is 475bp, the product of the watermelon short lateral branch mutant slb is 474bp, and the difference between the two products is 1bp (figure 3). Therefore, when the molecular marker is applied, if a product with the size of 474bp is obtained, the watermelon variety with short lateral branches is judged; if 475bp products are obtained, the watermelon variety with long lateral branches is judged.
Example 2: identification of short side branch character of watermelon
1. Extraction of DNA from tissue of watermelon sample
Picking up tender true leaves around growing points, and extracting DNA of a watermelon sample tissue by adopting an improved CTAB method. Agarose gel with the concentration of 1% is selected for electrophoresis detection, and the concentration and purity of DNA are detected by a NanoDrop micro-spectrophotometer. Typically, the ratio of A260/280 of DNA is between 1.8 and 2.0, and DNA samples within this ratio range can be used.
2. Primer sequence:
CX-5-2 forward primer: 5'-GGACGCCAATCTTTGTTGTT-3' (SEQ ID NO. 5)
CX-5-2 reverse primer: 5'-CCAACAATCTGACCCTCCAT-3' (SEQ ID NO. 6)
3. The PCR reaction system is shown in Table 2:
TABLE 2 PCR amplification reaction System
Figure BDA0003821257390000061
The conditions for the PCR amplification reaction were: pre-denaturation at 94℃for 5min, denaturation at 94℃for 60s, annealing at 54℃for 45s, extension at 72℃for 60s, and final extension at 72℃for 10min for 35 cycles, and storage at 4 ℃.
4. The PCR product obtained was detected on agarose at 1%. And (3) performing gel recovery by using a DC301 kit to obtain a PCR product.
5. The parental PCR products recovered in step 4 were sent to Shanghai Biotechnology for first generation sequencing.
6. Sequencing result analysis
Comparing the results of the parent sequencing, the length of the sequencing product of the watermelon long side branch strain GWAS-38 is 475bp, the length of the sequencing product of the watermelon short side branch mutant slb is 474bp, and the difference of 1bp exists between the two products (figures 3 and 4). Therefore, when the molecular marker is applied, if a product with the size of 474bp is obtained, the variety is a short-side branch watermelon variety; if 475bp products are obtained, the variety is a long-side branch watermelon variety.

Claims (10)

1. A functional molecular marker for identifying the length of the lateral branches of watermelons is named CX-5-2, and is obtained by PCR amplification of a forward primer shown in SEQ ID No.5 and a reverse primer shown in SEQ ID No.6 by taking the genomic DNA of watermelons as a template.
2. The use of the functional molecular marker CX-5-2 for identifying the length of the lateral branches of watermelons according to claim 1 in molecular breeding of short lateral branch varieties of watermelons.
3. The use according to claim 2, wherein the functional molecular markers are used for identifying or assisting in identifying whether the watermelon variety has short side branch traits in the seedling stage.
4. A pair of primers for amplifying the functional molecular marker of claim 1, wherein the pair of primers consists of a forward primer shown in SEQ ID No.5 and a reverse primer shown in SEQ ID No. 6.
5. The use of the primer pair according to claim 4 in molecular breeding of short lateral branch varieties of watermelons.
6. The use according to claim 5, wherein the primer pair can be used for identifying or assisting in identifying whether the watermelon variety has a short side branch trait in the seedling stage.
7. A method for identifying the length of a lateral branch of a watermelon, comprising the steps of:
(1) Separating and extracting total DNA from watermelon tissues;
(2) Performing PCR amplification by using the DNA obtained in the step (1) as a template and adopting a forward primer shown in SEQ ID NO.5 and a reverse primer shown in SEQ ID NO. 6;
(3) Carrying out agarose gel electrophoresis detection on the PCR amplified product obtained in the step (2), and then carrying out product recovery;
(4) Performing a first generation sequencing on the PCR product recovered in the step (3): if a product with the size of 474bp is obtained, judging that the watermelon variety is a short lateral branch; if 475bp products are obtained, the watermelon variety with long lateral branches is judged.
8. The method of claim 7, wherein in step (1), the total DNA of the watermelon tissue is extracted using a modified CTAB method.
9. The method of claim 7, wherein in step (1), the watermelon tissue is watermelon true leaf blades.
10. The method of claim 7, wherein in step (2), the system of the PCR amplification reaction is: 1. Mu.l of template DNA, 1. Mu.l of upstream primer, 1. Mu.l of downstream primer, 3. Mu.l of ddH2O, 4. Mu.l of 2X Es Taq Master Mix; the conditions for the PCR amplification reaction were: pre-denaturation at 94℃for 5min, denaturation at 94℃for 60s, annealing at 54℃for 45s, extension at 72℃for 60s, and final extension at 72℃for 10min for 35 cycles, and storage at 4 ℃.
CN202211048664.6A 2022-08-29 2022-08-29 Functional molecular marker CX-5-2 for identifying length of lateral branches of watermelons and application thereof Pending CN116103425A (en)

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