InDel molecular marker and method for identifying melon and fruit tumor morphology of bitter gourd and application of InDel molecular marker and method

Abstract

The invention provides an InDel molecular marker and a method for identifying the morphology of a melon fruit tumor and application. The nucleotide sequence of the InDel molecular marker is as shown in SEQ ID NO.1 or SEQ ID NO.2. The InDel molecular marker can be used for identifying the morphology of the melon fruit tumor. The molecular marker disclosed by the invention can be used for directly extracting DNA (deoxyribonucleic acid) of any tissue in the seedling stage of the bitter gourd and predicting and identifying the shape of the fruit tumor of the bitter gourd fruit, and has important application value in identification of the shape of the fruit tumor, molecular marker-assisted breeding and germplasm resource improvement; and an efficient and reliable technical support is provided for genetic improvement and excellent variety breeding of bitter gourds.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant breeding, and relates to InDel molecular markers, methods and applications for identifying the morphological characteristics of Momordica charantia fruit tubercles. Background Art

[0002] Momordica charantia L. is widely distributed in subtropical, tropical and temperate regions, and is an important horticultural crop of the Cucurbitaceae family that can be used both for food and medicine. The fruit of Momordica charantia is a pepo, with various shapes, including spindle-shaped, long rod-shaped or large top-shaped, etc. The morphological characteristics of the fruit tubercles show significant diversity. For example, the fruit tubercles can present continuous or discontinuous ridge-like structures, with different degrees of density, and can be smooth or serrated.

[0003] As a sensory quality trait, the epidermal characteristics of fruits are often affected by factors such as regional culture, consumption habits and cultivation measures, thus determining their acceptance in different markets. Therefore, growers usually select suitable varieties according to the needs of the target market. Among many phenotypic characteristics, the morphological characteristics of fruit tubercles are the key first sensory traits. By finely mapping genes related to the morphological characteristics of Momordica charantia fruit tubercles and developing molecular markers, an efficient, rapid and accurate screening tool can be provided for breeders. This not only helps to reduce the land and labor costs in the breeding process, but also significantly improves the efficiency and progress of Momordica charantia breeding.

[0004] Currently, the evaluation of Momordica charantia fruit traits mainly relies on naked-eye observation during the growth and development of fruits, which requires cultivation and careful management during the appropriate growing season until the fruits expand before trait evaluation can be carried out. In order to screen out plants with target traits, it is usually necessary to plant a large-scale population, thus increasing the labor and land costs. In addition, as a diploid plant, Momordica charantia can only generally identify the phenotypes of contemporary plants through naked-eye observation, and cannot identify the heterozygous situation of target trait loci. This limitation results in the inability to predict the trait segregation pattern of offspring (for example, serrated fruit tubercles are dominant over smooth fruit tubercles), so it is necessary to continue sowing the next generation to obtain plants with stable phenotypes. However, currently in Momordica charantia, no DNA molecular markers have been developed to detect whether the fruit tubercles are smooth or serrated. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides InDel molecular markers, methods and applications for identifying the morphological characteristics of Momordica charantia fruit tubercles, develops an InDel-type DNA molecular marker and applies it. By PCR and polyacrylamide gel electrophoresis (PAGE), it can effectively detect whether the fruit tubercles on the surface of Momordica charantia fruits are smooth or serrated. It can achieve rapid screening at the seedling stage and save the cost of seed selection and breeding.

[0006] The technical solution adopted by the present invention to achieve the technical purpose is as follows:

[0007] The present invention provides InDel molecular markers for identifying the morphological characteristics of Momordica charantia fruit tumors, and the nucleotide sequences of the InDel molecular markers are as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] The present invention also provides specific primers for amplifying the above-mentioned InDel molecular markers, and the nucleotide sequences of the specific primers are as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0009] The present invention also provides a kit for identifying the morphological characteristics of Momordica charantia fruit tumors, including the above-mentioned specific primers.

[0010] The present invention further provides a method for identifying the morphological characteristics of Momordica charantia fruit tumors, comprising: extracting genomic DNA at the seedling stage of Momordica charantia, using PCR to amplify the target DNA sequence to obtain an amplification product, and detecting the product fragment by PAGE electrophoresis. The nucleotide sequence length of the normal type is 91bp, and the corresponding fruit tumor morphology is a serrated shape; the nucleotide sequence length of the deletion type is 89bp, and the corresponding fruit tumor morphology is a smooth shape.

[0011] Preferably, the bands of the amplification product are divided into three types: a single upper band corresponds to the homozygous genotype of serrated fruit tumors; a single lower band corresponds to the homozygous genotype of smooth fruit tumors; and a double band corresponds to the heterozygous genotype of serrated fruit tumors.

[0012] Preferably, the target DNA sequence is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0013] Preferably, the nucleotide sequences of the PCR amplification primers are as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0014] Preferably, the reaction system for PCR amplification is: the total reaction volume is 10 μL, including 5 μL of 2×PCR mix, 0.2 μL of the upstream primer, 0.2 μL of the downstream primer, 3.6 μL of ddH2O, and 1 μL of the DNA template.

[0015] More preferably, the concentrations of both the upstream primer and the downstream primer are 10 μM, and the concentration of the DNA template is 30 ng / μL.

[0016] Preferably, the reaction program for PCR amplification is set as follows: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 20 seconds, annealing at 54°C for 25 seconds, extension at 72°C for 20 seconds, for 30 cycles; and finally extension at 72°C for 5 minutes to end the reaction.

[0017] The present invention also provides the application of the above-mentioned InDel molecular markers in the identification of Momordica charantia pericarp morphology, Momordica charantia molecular marker-assisted breeding, and the improvement of Momordica charantia germplasm resources.

[0018] Compared with the prior art, the present application has at least the following beneficial effects:

[0019] 1. Early detection in the seedling stage

[0020] Using the molecular markers disclosed in the present application, DNA can be directly extracted from any tissue in the seedling stage of Momordica charantia, and the fruit tubercle morphology (spiked or smooth) of Momordica charantia can be predicted and detected, realizing early identification and significantly shortening the breeding cycle.

[0021] 2. Precise identification of genetic status

[0022] This method can not only predict the phenotype of the target trait, but also identify the homozygous or heterozygous state of the genetic locus, thus providing a scientific basis for the precise prediction of the offspring phenotype.

[0023] 3. Broad application prospects

[0024] Through the accurate prediction and detection of the fruit tubercle morphology of Momordica charantia, this technology has important application value in fruit tubercle morphology identification, molecular marker-assisted breeding, and germplasm resource improvement, providing efficient and reliable technical support for the genetic improvement and elite variety selection of Momordica charantia.

[0025] In summary, compared with the existing methods, the molecular marker technology of the present application has significant advantages of early detection, precise prediction, and broad application, further expanding the potential of molecular marker technology in the research of Momordica charantia. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Showing the characteristics of the parental materials in the embodiments of the present invention and different fruit tubercle morphologies when constructing the F2 genetic segregation population.

[0027] Figure 2 Showing the QTL mapping results of the spiked fruit tubercle trait in the present invention using a high-density genetic map.

[0028] Figure 3 Showing the process of fine mapping of the spiked fruit tubercle gene using linkage genetic analysis with the F2 segregation population.

[0029] Figure 4 Showing 40 Momordica charantia with different fruit tubercle morphologies.

[0030] Figure 5 Showing the PAGE electrophoresis detection results of different samples. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The present invention will be described in detail below in conjunction with the specific embodiments. The following specific examples are helpful for those skilled in the art to further understand the present invention, but do not limit the present invention in any form.

[0032] Example

[0033] The embodiments of the present application have elaborated in detail the application of molecular marker technology in the morphological identification of bitter gourd fruit tumors, and verified and optimized its technical solutions through specific embodiments.

[0034] First, the present application constructs an F2 genetic segregation population by hybridizing the bitter gourd inbred lines "HNU025" and "HNU004" as parents to obtain the F1 generation, and then self-crossing the F1 plants to construct the F2 population. The fruit tumor morphology of "HNU025" is smooth, while that of "HNU004" is serrated, as shown in Figure 1 . By observing the fruit tumor morphology of the F1 and F2 populations, it is found that all the fruit tumors in the F1 generation are serrated, and the segregation ratio of serrated to smooth fruit tumors in the F2 population is 3:1. This indicates that the smooth fruit tumor of bitter gourd is a trait controlled by a single recessive gene. Through quantitative trait locus (QTL) analysis, the gene locus (fwt6.1) controlling the fruit tumor morphology is preliminarily located in the front-end region of chromosome 6. In the present application, the smooth fruit tumor variety HNU025 and the serrated fruit tumor variety HNU004 of the test materials are prior arts, such as Chen Hongrong. Construction of a high-density genetic map of bitter gourd and QTL mapping of main agronomic traits [D]. Hainan: Hainan University, 2021. Among them, HNU025 is hai09-1 in the article, and HNU004 is hai09-2 in the article.

[0035] Subsequently, further backcross the F1 generation with "HNU004" to obtain the BC1F1 population, and self-cross to construct the BC1F2 population. By expanding the population to screen for recombinant exchange single plants and combining the fruit tumor phenotype data, the fwt6.1 locus is finally finely mapped to an interval of 19.23 kb, and the process is shown in Figure 2 and 3 . Further analysis shows that there is a 2-bp base deletion ("GG") in this interval, which is closely linked to the smooth fruit tumor phenotype of bitter gourd. The detection results show that the nucleotide sequence length of the normal type is 91 bp, and the corresponding fruit tumor morphology is serrated; compared with the reference sequence, the "GG" base after the 67th site from the 5' end is deleted in the deletion type, and the sequence length is 89 bp, and the corresponding fruit tumor morphology is smooth.

[0036] To verify the reliability and applicability of the molecular marker, the present application selects 40 bitter gourd inbred lines or commercial seeds with different fruit tumor morphologies as planting materials, such as Figure 4 . After the seeds are germinated, they are sown in plug trays and conventional seedling management is carried out. After the first pair of true leaves grow, leaf tissues are taken for DNA extraction. The detection results show that among the 40 samples, 29 show smooth fruit tumors and 11 show serrated fruit tumors, and the genotype detection results are completely consistent with the phenotypes, as shown in Table 1.

[0037] Table 1 Genotyping Results and Phenotypes of 40 Samples

[0038]

[0039]

[0040] DNA extraction was completed using the conventional CTAB method. The extracted genomic DNA was diluted to 30 ng / μL for subsequent PCR reactions. The total volume of the PCR reaction system was 10 μL, including 5 μL of 2×PCR mix (Yugong Biotech Co., Ltd., product number EG21102), 0.2 μL (10 μM) of the upstream primer, 0.2 μL (10 μM) of the downstream primer, 3.6 μL of ddH2O, and 1 μL of DNA template (30 ng / μL). The reaction procedure was pre-denaturation at 94°C for 3 minutes; subsequently, denaturation at 94°C for 20 seconds, annealing at 54°C for 25 seconds, extension at 72°C for 20 seconds, for a total of 30 cycles; finally, extension at 72°C for 5 minutes to end the reaction.

[0041] PAGE electrophoresis detection: First, a 30% (w / v) solution was prepared by dissolving acrylamide and methylene bisacrylamide in water at a ratio of 29:1. Then, 1 L of 9% PAGE working solution was prepared by mixing 300 mL of the above solution, 100 mL of 10xTBE buffer, and 600 mL of water. When preparing the gel, the PCR products were analyzed by 9% polyacrylamide gel electrophoresis (PAGE). The sample loading volume per well was 1 μL. After electrophoresis at a constant voltage of 180 V for 1 hour and 40 minutes, the bands were observed after staining with 0.1% AgNO3 solution and developing with 1.5% NaOH formaldehyde solution.

[0042] The results showed that the bands of the amplification products were divided into three types: a single upper band (Type A, 91 bp) corresponding to the homozygous genotype of the serrated fruit tumor; a single lower band (Type B, 89 bp) corresponding to the homozygous genotype of the smooth fruit tumor; and a double band (Type H) corresponding to the heterozygous genotype of the serrated fruit tumor, as Figure 5 shown.

[0043] The embodiments of the present application disclose the applications of this molecular marker in the following aspects:

[0044] 1. Identification of Momordica charantia Peel Morphology

[0045] Using this molecular marker, the morphological characteristics of Momordica charantia fruit tumors can be quickly and accurately identified as serrated or smooth. This method provides an efficient tool for the molecular identification of Momordica charantia morphological phenotypes, reducing the time cost and environmental dependence of traditional methods.

[0046] 2. Molecular Marker-Assisted Breeding of Momordica charantia

[0047] In the process of bitter gourd breeding, this molecular marker can serve as an effective molecular tool to help screen bitter gourd varieties with the target fruit tumor morphology at the seedling stage. This technology can improve breeding efficiency and accelerate the breeding process of excellent varieties.

[0048] 3. Improvement of bitter gourd germplasm resources

[0049] This molecular marker has important application value in the improvement of bitter gourd germplasm resources. Through molecular marker analysis, the selection and utilization of germplasm resources can be optimized, laying a foundation for cultivating new bitter gourd varieties with high quality and diversity.

[0050] Obviously, the above-mentioned embodiments of the present invention are only examples for more clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to enumerate all the implementation methods here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. An InDel molecular marker for identifying the morphological characteristics of Momordica charantia fruit tubercles, characterized in that, The nucleotide sequence of the InDel molecular marker is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. The specific primer for amplifying the InDel molecular marker described in claim 1, characterized in that, The nucleotide sequences of the specific primers are shown in SEQ ID NO.3 and SEQ ID NO.

4.

3. A kit for identifying the fruit nodule morphology of bitter gourd, comprising the specific primers described in claim 2.

4. A method for identifying the morphological characteristics of Momordica charantia fruit protuberances, comprising: Extract genomic DNA during the seedling stage of bitter gourd, use PCR to amplify the target DNA sequence to obtain an amplification product, and detect the product fragment by PAGE electrophoresis. The nucleotide sequence length of the normal type is 91bp, and the corresponding fruit nodule morphology is a serrated morphology; the nucleotide sequence length of the deletion type is 89bp, and the corresponding fruit nodule morphology is a smooth morphology.

5. The method according to claim 4, characterized in that, The bands of the amplification product are divided into three types: a single upper band corresponds to the homozygous genotype of serrated fruit nodules; a single lower band corresponds to the homozygous genotype of smooth fruit nodules; and a double band corresponds to the heterozygous genotype of serrated fruit nodules.

6. The method according to claim 4, wherein The target DNA sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

7. The method according to claim 4, characterized in that, The nucleotide sequences of the PCR amplification primers are shown in SEQ ID NO.3 and SEQ ID NO.

4.

8. The method according to claim 4, characterized in that, The reaction system for PCR amplification is: the total reaction volume is 10 μL, including 5 μL of 2×PCR mix, 0.2 μL of the upstream primer, 0.2 μL of the downstream primer, 3.6 μL of ddH2O, and 1 μL of DNA template.

9. The method according to claim 4 or 8, characterized in that, The reaction program for PCR amplification is set as follows: pre-denaturation at 94°C for 3 minutes; then denaturation at 94°C for 20 seconds, annealing at 54°C for 25 seconds, extension at 72°C for 20 seconds, for 30 cycles; finally, extension at 72°C for 5 minutes to end the reaction.

10. Use of the InDel molecular marker described in claim 1 in the identification of bitter gourd pericarp morphology, molecular marker-assisted breeding of bitter gourd, and improvement of bitter gourd germplasm resources.

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