SSR marker-based germplasm genetic diversity analysis and fingerprint spectrum construction method for balsam pear

Abstract

The invention belongs to the field of vegetable molecular technology breeding, and particularly relates to a method for analyzing germplasm genetic diversity and constructing a fingerprint spectrum of balsam pear based on a simple sequence repeat (SSR) marker. By screening high-polymorphism SSR core markers, genetic diversity analysis is carried out on 26 germplasm parts of the balsam pear, and an SSR molecular fingerprint spectrum is constructed. Core SSR markers (MC0569594, MC0450530, MC0687314 and MC10146038) are used for carrying out PCR (Polymerase Chain Reaction) amplification and banding pattern analysis, the polymorphism information content (PIC) is calculated, and the germplasm of the balsam pear is clustered into three main groups through clustering analysis. By combining a fingerprint database, efficient identification and information storage of germplasm of the balsam pear are realized. The method is easy and convenient to operate and high in accuracy, and important technical support is provided for protection, utilization and breeding of germplasm resources of the balsam pear. By means of the method, the variety of the large-top bitter gourds on the market can be rapidly identified, materials with excellent characters can be effectively screened out in the breeding process, the breeding efficiency is improved, and the method has wide application prospects.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of vegetable molecular breeding technology, and particularly relates to a method for genetic diversity analysis and fingerprint construction of Momordica charantia L. var. abbreviata based on simple sequence repeat (SSR) markers. BACKGROUND

[0002] Momordica charantia L. is an important melon vegetable, and is mainly cultivated and consumed in southern China. The fruit of Momordica charantia L. var. abbreviata is conical, and is a characteristic Momordica charantia L. type in southern China. The genetic background of Momordica charantia L. var. abbreviata is relatively narrow, and it is difficult to identify the authenticity of Momordica charantia L. var. abbreviata. At present, there are few studies on genetic diversity analysis and molecular fingerprint construction of Momordica charantia L. var. abbreviata germplasm, and an effective method is urgently needed for identification and protection of Momordica charantia L. var. abbreviata germplasm.

[0003] At present, with the continuous change of agricultural modernization and market demand, the cultivation area of Momordica charantia L. is gradually expanding, and the area of facility cultivation in the north is also increasing. At the same time, the implementation of the new Seed Law puts forward higher requirements for the protection of germplasm resources. How to accurately identify the authenticity of Momordica charantia L. varieties, protect germplasm resources and promote their rational use has become an important task of breeding work. Therefore, developing an efficient and accurate method for genetic diversity analysis and fingerprint construction of Momordica charantia L. var. abbreviata germplasm not only can meet the needs of scientific research and breeding, but also has important significance for improving the protection and utilization level of Momordica charantia L. germplasm resources. SUMMARY

[0004] The application provides a method for genetic diversity analysis and fingerprint construction of Momordica charantia L. var. abbreviata germplasm based on SSR (simple sequence repeat) markers, which aims to solve the problems of high difficulty in identification of Momordica charantia L. var. abbreviata germplasm and insufficient protection of varieties in the prior art. By screening and using high polymorphic SSR markers, efficient identification and fingerprint construction of Momordica charantia L. var. abbreviata germplasm are realized, which provides important technical support for protection, utilization and breeding of Momordica charantia L. var. abbreviata germplasm.

[0005] The specific scheme adopted by the application is as follows: A method for genetic diversity analysis of Momordica charantia L. var. abbreviata germplasm based on SSR markers, characterized in that the method comprises the following steps: a. selecting 26 Momordica charantia L. var. abbreviata germplasms including main cultivars; b. screening high polymorphic SSR markers for PCR amplification; c. analyzing and screening core markers by using polymorphic information content (PIC); d. using core SSR markers for genetic diversity analysis and fingerprint construction.

[0006] The method according to claim 1, wherein the SSR marker comprises single nucleotide, di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide and hexa-nucleotide types.

[0007] A primer pair for amplifying the SSR marker according to claim 1, wherein the primer pair comprises: MC05_69594-forward primer: 5'-TGTGTAGTGATGGGACAAGACTTT-3'; MC05_69594-reverse primer: 5'-TGGATAGCTTCATACCGTGTCA-3'; MC04_50530-forward primer: 5'-TGCAATACTCTACCCCACACA-3'; MC04_50530-reverse primer: 5'-CGCCCCACCACACTAGAATA-3'; MC06_87314-forward primer: 5'-CCAAGAAGGAAGCCAACAGA-3'; MC06_87314-reverse primer: 5'-AGGAGAAATCCCACGAAACT-3'; MC10_146038-forward primer: 5'-GGCCTGCGACAAATAAAAGA-3'; MC10_146038-reverse primer: 5'-TCTTCACCCATCTCAAAGGG-3'.

[0008] A method for identifying a Momordica charantia var. bispinosa germplasm, comprising the following steps: S1. extracting genomic DNA from young true leaf tissue of the Momordica charantia var. bispinosa; S2. using the primer pair according to claim 3 to perform PCR amplification with the genomic DNA of the young true leaf tissue of the Momordica charantia var. bispinosa as a template, and performing electrophoresis detection; S3. using the electrophoresis result bands, judging the Momordica charantia var. bispinosa germplasm according to the detection result.

[0009] The method for identifying the Momordica charantia var. bispinosa germplasm according to claim 4, wherein if there is a specific band combination in the electrophoresis result, it is determined as a specific Momordica charantia var. bispinosa germplasm.

[0010] A kit for identifying the Momordica charantia var. bispinosa germplasm, comprising the primer pair according to claim 3.

[0011] The method of claim 1 or the primer pair of claim 3 is applied in any one of (A)-(D) as follows: (A) for identifying Momordica charantia L. germplasm; (B) for breeding or assisting breeding of new Momordica charantia L. varieties; (C) for preparing products for identifying Momordica charantia L. germplasm; (D) for preparing products for breeding or assisting breeding of new Momordica charantia L. varieties.

[0012] Compared with the prior art, the present application has the following beneficial effects:

[0013] 1. The present application realizes efficient identification and fingerprint construction of Momordica charantia L. germplasm for the first time by screening high polymorphic SSR core markers, and analyzing the genetic diversity of 26 Momordica charantia L. germplasms including main cultivars, thereby providing technical support for Momordica charantia L. germplasm protection and utilization.

[0014] 2. The SSR core markers screened in the present application have high polymorphism, and the average polymorphic information content (PIC) value reaches 0.37, wherein the PIC value of 11 core markers is greater than 0.50, and these core markers can be used as reference markers for identification of Momordica charantia L. germplasm, and have high practical value and application prospect.

[0015] 3. The present application constructs the fingerprint of Momordica charantia L. germplasm by using core SSR markers (MC05_69594, MC04_50530, MC06_87314 and MC10_146038), and generates the QR code fingerprint of each germplasm by QR Code Generator tool, thereby realizing rapid coding and storage of Momordica charantia L. germplasm information, and being convenient and fast.

[0016] 4. The method of the present application is simple in steps, and the genetic diversity analysis and fingerprint construction can be completed by operations such as PCR amplification, polyacrylamide gel electrophoresis and silver staining development, thereby providing a simple and accurate technical means for researchers, and being conducive to systematic management and protection of Momordica charantia L. germplasm resources.

[0017] 5. The method of the present application is not only suitable for identification and protection of existing Momordica charantia L. germplasm, but also can be widely applied to identification and evaluation of newly introduced germplasm, and through establishment of a fingerprint database, systematic management and genetic relationship analysis of Momordica charantia L. germplasm resources are realized, thereby providing theoretical support for genetic improvement and variety breeding. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1Figure 2 is a cluster tree of SSR markers of Momordica charantia germplasm; the figure shows the results of cluster analysis of 28 Momordica charantia germplasm using 40 SSR markers. At a genetic similarity coefficient of 0.80, 26 Momordica charantia germplasm were clustered into three main groups. The blue line represents group I, the red line represents group II, the yellow line represents group III, and the black line represents the outgroup. The right side of the line is a representative fruit of the fruit shape in the group.

[0019] Figure 2 Figure 3 is a band pattern map of 4 core SSR markers in 28 Momordica charantia germplasm; the figure shows the band type difference of core SSR markers MC05_69594, MC04_50530, MC06_87314, and MC10_146038 in different germplasm. N indicates no amplification. The figure directly shows the polymorphism of the core markers in different Momordica charantia germplasm, providing data support for fingerprint construction. DETAILED DESCRIPTION

[0020] The following examples are further illustrations of the present application and are not intended to limit the present application.

[0021] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present application are conventional reagents, methods, and equipment in the technical field.

[0022] Example 1

[0023] (I) Material preparation

[0024] Six Momordica charantia materials with obvious phenotypic differences were selected, including the oil Momordica charantia types 'Dali-11' and 'A008', the pearl Momordica charantia type 'S015', the large top Momordica charantia type 'Tanbian Dadizhong', the local variety 'Fenxi L', and the wild type 'M3'. Twenty-six large top Momordica charantia germplasms were collected, covering the main large top Momordica charantia varieties on the market, including 'Donglaidadizhong', 'No. 1 Dadizhong', 'No. 3 Dadizhong', etc.

[0025] (II) SSR marker screening

[0026] A total of 180 SSR markers of single nucleotide, di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide, and hexa-nucleotide were selected from the Momordica charantia whole genome. Through PCR amplification and polymorphism analysis, 40 SSR markers with higher polymorphism were screened for further analysis. The information of the 40 polymorphic SSR markers of Momordica charantia is shown in Table 1.

[0027] Table 1: Information of 40 polymorphic SSR markers of Momordica charantia

[0028] (Three) PCR amplification

[0029] (1) The PCR amplification reaction system (20 μL) was configured: 50 ng of template DNA, 1 μL of upstream and downstream primers (1 μmol / L), 0.2 μL of dNTPs (10 mmol / L), 0.2 μL of Taq enzyme (5 U / μL), 2.0 μL of 10×Taq buffer (containing Mg 2+ ), and ddH2O was added to 20 μL.

[0030] (2) 94°C pre-denaturation for 5 min.

[0031] (3) 30 cycles (94°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extension for 1 min per cycle).

[0032] (4) Final extension at 72°C for 10 min.

[0033] (5) The amplified product was detected by 6% polyacrylamide gel electrophoresis, electrophoresis was performed at 180 V for 2 hours, and the band type was recorded after development with silver staining solution.

[0034] (Four) polymorphism analysis

[0035] (1) According to the amplification results, the amplification rate and polymorphism rate of the SSR marker were calculated.

[0036] (2) The polymorphism information content (PIC) was calculated using PowerMarker software, and 11 core markers with PIC values greater than 0.50 were screened out.

[0037] (Five) Cluster analysis

[0038] According to the "0-1" matrix of the amplification results, UPGMA method in NTSYS-pc software was used for cluster analysis of Momordica charantia var. abel var. abel germplasm. At a genetic similarity coefficient of 0.80, the 26 Momordica charantia var. abel var. abel germplasms were clustered into three main groups (see Figure 1 ).

[0039] (Six) Fingerprint construction

[0040] From the selected core SSR markers, the markers with higher PIC and easier to read band type (MC05_69594, MC04_50530, MC06_87314, MC10_146038) were preferentially selected for fingerprint construction. According to the PCR amplification band type of 4 high-efficiency SSR core markers in 26 Momordica charantia var. abel var. abel germplasms, the fingerprint information was represented in the form of digital coding ( Figure 2). After encoding, the DNA banding fingerprint code of each material has 4 digits, and each digit represents the banding of each corresponding marker amplification (Table 2). For example, the 'Donglai' fingerprint code is "1-1-1-1", which means that 'Donglai' contains 1 band of primer MC05_69594, 1 band of primer MC04_50530, 1 band of primer MC06_87314, and 1 band of primer MC10_146038.

[0041] Table 2: SSR banding fingerprint code of Momordica charantia L. germplasm

[0042] The present application provides a method for genetic diversity analysis and fingerprint construction of Momordica charantia L. germplasm based on SSR markers. By screening and using high polymorphic SSR markers, efficient identification and fingerprint construction of Momordica charantia L. germplasm are achieved, which provides important technical support for the protection, utilization and breeding of Momordica charantia L. germplasm. The detailed examples demonstrate the specific application of the present application in genetic diversity analysis, variety identification, breeding material screening, germplasm resource protection and parthenocarpy breeding, which provides strong support for related research and application.

[0043] It should be noted that the above-mentioned embodiments should be understood as illustrative, rather than limiting the scope of protection of the present application, and the scope of protection of the present application is subject to the claims. For those skilled in the art, some non-essential improvements and adjustments of the present application without departing from the spirit and scope of the present application still belong to the protection scope of the present application.

Claims

1. A method for analyzing the genetic diversity of bitter gourd germplasm based on SSR markers, characterized in that: The method comprises the following steps: a. selecting 26 Momordica charantia germplasms including main cultivars; b. screening SSR markers with high polymorphism for PCR amplification; c. analyzing and screening core markers using polymorphism information content (PIC); d. using core SSR markers for genetic diversity analysis and fingerprint construction.

2. The method of claim 1, wherein, The SSR markers include single nucleotide, double nucleotide, triple nucleotide, quadruple nucleotide, quintuple nucleotide and sextuple nucleotide types.

3. A primer pair for amplifying the SSR marker of claim 1, wherein the primer pair comprises a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO:

2. The primer pairs comprise: MC05_69594-forward primer: 5'-TGTGTAGTGATGGGACAAGACTTT-3'; MC05_69594-reverse primer: 5'-TGGATAGCTTCATACCGTGTCA-3'; MC04_50530-forward primer: 5'-TGCAATACTCTACCCCACACA-3'; MC04_50530-reverse primer: 5'-CGCCCCACCACACTAGAATA-3'; MC06_87314-forward primer: 5'-CCAAGAAGGAAGCCAACAGA-3'; MC06_87314-reverse primer: 5'-AGGAGAAATCCCACGAAACT-3'; MC10_146038-forward primer: 5'-GGCCTGCGACAAATAAAAGA-3'; MC10_146038-reverse primer: 5'-TCTTCACCCATCTCAAAGGG-3'.

4. A method for identifying a large monordica charantia germplasm, characterized by, The method comprises the following steps: S1. extracting genomic DNA of Momordica charantia young true leaf tissue; S2. using the primer pairs of claim 3 for PCR amplification with the genomic DNA of Momordica charantia young true leaf tissue as a template, and performing electrophoresis detection; S3. using the electrophoresis result bands, judging the Momordica charantia germplasm according to the detection results.

5. The method of identifying Momordica charantia L. germplasm according to claim 4, characterized in that, If there is a specific band combination in the electrophoresis results, it is determined as a specific Momordica charantia germplasm.

6. A kit for identifying Momordica charantia germplasm, characterized in that, The kit comprises the primer pairs of claim 3.

7. The method of claim 1 or the primer pairs of claim 3 are used in any one of the following (A)-(D): (A) for identifying Momordica charantia germplasms; (B) for breeding or assisting breeding of new Momordica charantia varieties; (C) for preparing products for identifying Momordica charantia germplasms; (D) for preparing products for breeding or assisting breeding of new Momordica charantia varieties.

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