Functional molecular marker cx-5-2 for identifying length of lateral branch of watermelon and application thereof

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application relates to a functional molecular marker CX-5-2 for identifying the length of lateral branches of watermelon and its application. BACKGROUND

[0002] Watermelon (Citrullus lanatus) is an important Cucurbitaceae crop and is widely planted worldwide. Branching of watermelon is a process in which axillary buds (axillary meristems) in leaf axils develop into branches. Most existing watermelon varieties are branching varieties, and during cultivation, a large amount of labor is consumed for pruning. Short lateral branches or materials with few lateral branches can greatly reduce labor input and are more conducive to the realization of light, simple, and economical production. In addition, short lateral branches or materials with small leaf angles are more conducive to increasing planting density per unit area.

[0003] Molecular markers are divided into broad and narrow molecular markers. Broad: sequences or proteins that can be inherited and detected. Narrow: specific DNA fragments that can reflect differences between individuals or populations. Functional molecular markers are molecular markers designed based on gene sequences, which can directly detect genes, distinguish and predict alleles and their relative traits. Compared with conventional hybrid breeding, which has a long breeding cycle, molecular marker-assisted selection breeding can directly select at the gene level, with high accuracy and efficiency. The present application develops a functional molecular marker for the length of lateral branches of watermelon, which can identify the length of lateral branches of watermelon at the seedling stage, realize molecular marker-assisted selection breeding, and accelerate the breeding process of short lateral branch varieties. SUMMARY

[0004] The present application aims to provide a functional molecular marker CX-5-2 for identifying the length of lateral branches of watermelon and its application.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0006] The application takes watermelon long lateral branch line GWAS-38 and watermelon short lateral branch mutant slb as parents, obtains F2 population, finds a candidate region of the target gene on chromosome 5, locates the candidate interval to a 25.35 kb segment through genotyping, and finds that there is a SNP site on the Cla97C05G088180 gene after comparing the sequences of the parents. A functional molecular marker is developed according to the site and named as CX-5-2. The sequence of the forward primer is shown as SEQ ID NO. 5, and the sequence of the reverse primer is shown as SEQ ID NO. 6. The functional molecular marker disclosed by the application is co-segregated with the watermelon short lateral branch trait. At the molecular level, whether the watermelon plant has the short lateral branch phenotype can be identified at the seed or seedling stage, the selection efficiency and accuracy are improved, and the breeding process can be shortened.

[0007] On the basis of the above research, the application provides a functional molecular marker for identifying the length of lateral branches of watermelon, which is named as CX-5-2, and the molecular marker is obtained by PCR amplification with the forward primer shown as SEQ ID NO. 5 and the reverse primer shown as SEQ ID NO. 6 as templates and watermelon genomic DNA.

[0008] Further, the application also provides the application of the functional molecular marker CX-5-2 for identifying the length of lateral branches of watermelon in the molecular breeding of watermelon short lateral branch varieties.

[0009] Preferably, the functional molecular marker can be used for identifying or assisting in identifying whether the watermelon variety has the short lateral branch trait at the seedling stage.

[0010] Further, the application also provides a primer pair for amplifying the functional molecular marker, which consists of the forward primer shown as SEQ ID NO. 5 and the reverse primer shown as SEQ ID NO. 6.

[0011] Further, the application also provides the application of the primer pair in the molecular breeding of watermelon short lateral branch varieties.

[0012] Preferably, the primer pair can be used for identifying or assisting in identifying whether the watermelon variety has the short lateral branch trait at the seedling stage.

[0013] Further, the application also provides a method for identifying the length of lateral branches of watermelon, which comprises the following steps:

[0014] (1) extracting total DNA from watermelon tissue;

[0015] (2) using the DNA obtained in step (1) as a template, PCR amplification is carried out by using a forward primer shown in SEQ ID NO. 5 and a reverse primer shown in SEQ ID NO. 6;

[0016] (3) the PCR amplification product obtained in step (2) is detected by agarose gel electrophoresis and then the product is recovered;

[0017] (4) the PCR product recovered in step (3) is sequenced: if a product of 474 bp is obtained, it is determined that the watermelon variety is short vining; if a product of 475 bp is obtained, it is determined that the watermelon variety is long vining.

[0018] Preferably, in step (1), the total DNA of the watermelon tissue is extracted by using a modified CTAB method.

[0019] Preferably, in step (1), the watermelon tissue is a watermelon true leaf blade.

[0020] Preferably, in step (2), the PCR amplification reaction system is: 1 μl of template DNA, 1 μl of upstream primer, 1 μl of downstream primer, 3 μl of ddH2O, and 4 μl of 2 × Es Taq Master Mix; the PCR amplification reaction conditions are: 94 ℃ pre-denaturation for 5 min, 94 ℃ denaturation for 60 s, 54 ℃ annealing for 45 s, 72 ℃ extension for 60 s, a total of 35 cycles, and finally 72 ℃ extension for 10 min, and 4 ℃ preservation.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] The functional molecular marker for identifying the short vining trait of a watermelon plant disclosed in the present application can be applied to the molecular marker assisted selection breeding of a watermelon short vining trait. The molecular marker in the present application is co-segregated with the watermelon short vining trait, so that whether the watermelon plant has a short vining phenotype can be identified at 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.

[0023] A conventional functional molecular marker is usually a CAPS or Indel type molecular marker. Since the difference between the candidate gene in the present application and the parent is only 1 bp, the design of an Indel marker cannot be detected by agarose gel electrophoresis or polyacrylamide electrophoresis. Meanwhile, since the difference between the candidate gene in the present application and the parent is continuous A bases, it cannot be converted into a CAPS, dCAPS or KASP marker. Therefore, the PCR product is sequenced in the present application, the size of the sequencing product is compared, the genotype and trait are judged, and the identification result is accurate and reliable. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Figure 1 is a physical map of long and short side branch lines;

[0025] A: Watermelon long side branch line GWAS-38; B: Watermelon short side branch mutant slb;

[0026] Figure 2 Figure 2 is a map of the watermelon short side branch trait gene location;

[0027] Figure 3 Figure 3 is a map of PCR product first sequencing results;

[0028] Figure 4 Figure 4 is a map of base changes of the mutant slb and parents and part of natural population at Chr5:6,291,086 sites. DETAILED DESCRIPTION

[0029] The following detailed description of the embodiments of the present application is given under the premise of the technical solutions of the present application, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present application is not limited to the following examples. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased in the market.

[0030] The technical solutions of the present application are not limited to the following specific embodiments, and also include any combination of the specific embodiments.

[0031] Example 1: Fine mapping of watermelon short side branch trait regulating gene and development of molecular markers

[0032] 1. Materials and construction of separated genetic population

[0033] The long side branch material GWAS-38 of watermelon is used as the father, and the short side branch mutant slb of watermelon is used as the mother (for example, GWAS-38 is used as the father and slb is used as the mother), and F1 is obtained by hybridization, and F2 generation seeds are obtained by selfing of F1. The phenotype of each single plant in the F2 separation population is identified, and the identification results of 336 F2 populations are 258 long side branch plants and 78 short side branch plants, the ratio of long side branch individuals to short side branch individuals is consistent with the separation ratio of 3:1, which indicates that the short side branch trait is controlled by a recessive single gene. Figure 1

[0034] 2. Candidate gene location

[0035] (1) Construction of pool

[0036] ​In the constructed 357 F2 population, 30 strains of normal long lateral branch phenotype and 30 strains of short lateral branch phenotype were selected respectively, and the young true leaves of each plant were freeze-dried and ground to mix in equal amounts to construct DNA pools. No. 38 and slb represent the parents, and Duancezhi A and Changcezhi A represent the short lateral branch pool and the long lateral branch pool respectively. The parents and the pool were sent to Huada Gene for whole genome resequencing. The sequencing depth of GWAS-38 was 10x, and the depth of slb, Duancezhi A and Changcezhi A was 30x.

[0037] (2) Gene mapping and development of molecular markers

[0038] Using BSA-seq technology combined with the development of molecular markers, the genotypes of 335 F2 populations were identified, and the gene Clslb regulating short lateral branch was located on chromosome 5 at 1.63 Mb (4,680,000-6,310,000, 97103v2). Subsequently, 1010 F2 expanded populations were expanded, and molecular markers were encrypted. Finally, the candidate segment was narrowed down to 25.354 kb (6,280,285-6,305,639, 97103v2). There are two candidate genes in this segment. According to gene annotation and sequence alignment analysis, it is speculated that the Cla97C05G088180.1 gene is the candidate gene regulating the short lateral branch trait of watermelon (as shown in Figure 2 ). The full length of the gene is 5253 bp (the sequence is shown in SEQ ID NO. 3), and there are 4 exons and 3 introns. The full length of the parent Cla97C05G088180.1 gene was cloned and sequenced, and it was found that in the mutant slb, there was a deletion of an A base at 6,291,086 bp of the second exon of Cla97C05G088180.1 gene (the sequence is shown in SEQ ID NO. 1, as shown in Figure 3 ). This mutation only exists in the short lateral branch mutant slb (as shown in Figure 4 ). It causes the translation to terminate early, and the normal protein length is 1131 aa (the sequence is shown in SEQ ID NO. 4), and the length after truncation is 854 aa (the sequence is shown in SEQ ID NO. 2). A functional marker was developed using the deletion of an A base at 6,291,086 bp, and a molecular marker CX-5-2 co-segregated with Clslb was developed for molecular marker assisted selection. The primer sequence is:

[0039] CX-5-2 forward primer: 5'-GGACGCCAATCTTTGTTGTT-3' (shown in SEQ ID NO. 5)

[0040] CX-5-2 reverse primer: 5'-CCAACAATCTGACCCTCCAT-3' (shown in SEQ ID NO. 6)

[0041] 1) The PCR reaction system is shown in Table 1:

[0042] Table 1 PCR amplification reaction system

[0043]

[0044] 2) The PCR amplification reaction conditions are as follows: 5 min of pre-denaturation at 94°C, 60 s of denaturation at 94°C, 45 s of annealing at 54°C, 60 s of extension at 72°C, a total of 35 cycles, and then 10 min of re-extension at 72°C, and storage at 4°C.

[0045] 3) The obtained PCR product is detected on a 1% agarose gel. The DC301 kit is used for gel recovery to obtain the PCR product.

[0046] 4) The double-parent PCR product recovered from the gel in step 3) is sent to Shanghai Shengong Bioengineering Company for first-generation sequencing.

[0047] 5) The double-parent sequencing results are compared, and it is found that the product of the long lateral branch line GWAS-38 is 475 bp, and the product of the short lateral branch mutant slb of watermelon is 474 bp, and there is a 1 bp difference between the two products. Figure 3 Therefore, when this molecular marker is applied, if a product of 474 bp size is obtained, it is determined to be a short lateral branch watermelon variety; if a product of 475 bp size is obtained, it is determined to be a long lateral branch watermelon variety.

[0048] Example 2: Identification of the short lateral branch trait of watermelon

[0049] 1. Extraction of DNA from watermelon sample tissue

[0050] The tender true leaves around the growth point are picked, and the modified CTAB method is used to extract the DNA from the watermelon sample tissue. A 1% agarose gel is selected for electrophoresis detection, and the DNA concentration and purity are detected by a NanoDrop microspectrophotometer. Generally, the A260 / 280 ratio of the DNA is between 1.8 and 2.0, and the DNA sample within this ratio range can be used.

[0051] 2. Primer sequence:

[0052] CX-5-2 forward primer: 5'-GGACGCCAATCTTTGTTGTT-3' (shown in SEQ ID NO. 5)

[0053] CX-5-2 reverse primer: 5'-CCAACAATCTGACCCTCCAT-3' (shown in SEQ ID NO. 6)

[0054] 3. The PCR reaction system is shown in Table 2:

[0055] Table 2 PCR amplification reaction system

[0056]

[0057] The PCR amplification reaction conditions are: 94°C pre-denaturation for 5 min, 94°C denaturation for 60 s, 54°C annealing for 45 s, 72°C extension for 60 s, a total of 35 cycles, and finally 72°C extension for 10 min, and 4°C storage.

[0058] 4. The obtained PCR product is detected on 1% agarose gel. The DC301 kit is used for gel recovery to obtain the PCR product.

[0059] 5. The double parent PCR product recovered from step 4 is sent to Shanghai Shengong Bioengineering Company for first-generation sequencing.

[0060] 6. Sequencing result analysis

[0061] By comparing the sequencing results of the parents, it is found that the sequencing product length of the long lateral branch watermelon line GWAS-38 is 475 bp, the sequencing product length of the short lateral branch mutant slb is 474 bp, and there is a 1 bp difference between the two products. Figure 3 、 Figure 4 Therefore, when the molecular marker is applied, if a product of 474 bp size is obtained, the variety is a short lateral branch watermelon variety; if a product of 475 bp size is obtained, the variety is a long lateral branch watermelon variety.

Claims

1. The application of a primer pair for amplifying the functional molecular marker CX-5-2 for watermelon lateral branch length in molecular breeding of watermelon varieties with short lateral branches, characterized in that, The primer pair consists of the forward primer shown in SEQ ID NO.5 and the reverse primer shown in SEQ ID NO.

6. The watermelon variety is the watermelon short lateral branch mutant slb, the watermelon long lateral branch material GWAS-38, or the hybrid offspring of the watermelon long lateral branch material GWAS-38 and the watermelon short lateral branch mutant slb. If a product of 474 bp is obtained after amplification, it is determined to be a watermelon variety with short lateral branches; if a product of 475 bp is obtained after amplification, it is determined to be a watermelon variety with long lateral branches.

2. The application as described in claim 1, characterized in that, The primer pairs are used to identify or assist in identifying whether a watermelon variety has the short lateral branch trait during the seedling stage.

3. A method for determining the length of lateral branches of a watermelon, characterized in that, Includes the following steps: (1) Total DNA was isolated and extracted from watermelon short lateral branch mutant slb or from F2 generation watermelon tissue obtained by crossing watermelon long lateral branch material GWAS-38 with watermelon short lateral branch mutant slb; (2) Using the DNA obtained in step (1) as a template, PCR amplification was performed using the forward primer shown in SEQ ID NO.5 and the reverse primer shown in SEQ ID NO.6; (3) The PCR amplification products obtained in step (2) were detected by agarose gel electrophoresis and then the products were recovered. (4) Perform first-generation sequencing on the PCR products recovered in step (3): if a product of size 474bp is obtained, it is determined to be a watermelon variety with short lateral branches; if a product of size 475bp is obtained, it is determined to be a watermelon variety with long lateral branches.

4. The method as described in claim 3, characterized in that, In step (1), total DNA was extracted from watermelon tissue using a modified CTAB method.

5. The method as described in claim 3, characterized in that, In step (1), the watermelon tissue is the true leaf of a watermelon.

6. The method as described in claim 3, characterized in that, In step (2), the PCR amplification reaction system is as follows: 1 μl template DNA, 1 μl upstream primer, 1 μl downstream primer, 3 μl ddH2O, 4 μl 2×Es Taq Master Mix; the PCR amplification reaction conditions are: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 60 s, 54℃ annealing for 45 s, 72℃ extension for 60 s, for a total of 35 cycles, and finally 72℃ extension for 10 min, and stored at 4℃.

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