InDel molecular marker for identifying fruit gourd morphology, method and application thereof

Abstract

The application provides an InDel molecular marker for identifying the fruit tubercle morphology of Momordica charantia, a method and application, and the nucleotide sequence of the InDel molecular marker is shown in SEQ ID NO. 1 or SEQ ID NO. 2. By using the molecular marker disclosed in the application, the DNA of any tissue of the Momordica charantia seedling stage can be directly extracted, the fruit tubercle morphology of the Momordica charantia is predicted and identified, the application has important application value in fruit tubercle morphology identification, molecular marker assisted breeding and germplasm resource improvement, and provides efficient and reliable technical support for genetic improvement and excellent variety breeding of the Momordica charantia.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant breeding, and relates to an InDel molecular marker for identifying the fruit nodule shape of Momordica charantia, a method and application. BACKGROUND

[0002] Momordica charantia L. is widely distributed in subtropical, tropical and temperate regions and is an important food and medicinal type of Cucurbitaceae horticultural crops. The fruit of Momordica charantia L. is a pepo, and the shape is various, including spindle, long rod or large top, and the fruit nodule shape shows significant diversity, for example, the fruit nodule can present continuous or discontinuous ridge structure, and the density is different, and can present smooth or serrated characteristics.

[0003] The epidermal characteristics of the fruit as a sensory quality trait are often affected by factors such as regional culture, consumption habits and cultivation measures, thereby determining its acceptance in different markets. Therefore, growers usually select suitable varieties according to the needs of the target market. Among the various phenotypic characteristics, the fruit nodule shape is the key first sensory trait. Through fine mapping of the fruit nodule shape related gene of Momordica charantia L. and development of a molecular marker, an efficient, rapid and accurate screening tool can be provided for breeders. This not only helps to reduce the cost of land and labor in the breeding process, but also significantly improves the efficiency and process of Momordica charantia L. breeding.

[0004] At present, the evaluation of fruit traits of Momordica charantia L. mainly depends on visual observation during the growth and development of the fruit, which needs to be cultivated and carefully managed in the appropriate growing season, and the trait evaluation can be carried out only after the fruit is swollen. In order to select plants with target traits, a large-scale population usually needs to be planted, thereby increasing the cost of labor and land. In addition, as a diploid plant, only visual observation can generally identify the phenotype of the current generation of Momordica charantia L., and cannot identify the heterozygosity of the target trait locus. This limitation leads to the inability to predict the trait segregation pattern of the offspring (for example, serrated fruit nodule is dominant over smooth fruit nodule), so the next generation needs to be sown to obtain plants with stable phenotype. However, at present, no DNA molecular marker has been developed to detect the smooth or serrated DNA molecular marker of Momordica charantia L. SUMMARY

[0005] In order to solve the above technical problems, the application provides an InDel molecular marker for identifying the fruit nodule shape of Momordica charantia L., a method and application, develops an InDel type DNA molecular marker and applies it, and can effectively detect whether the fruit nodule of Momordica charantia L. is smooth or serrated through PCR and polyacrylamide gel electrophoresis (PAGE). The rapid screening is realized at the seedling stage, and the breeding cost is saved.

[0006] The technical scheme adopted by the application to achieve the technical purpose is that:

[0007] The application provides an InDel molecular marker for identifying the fruit nodule morphology of Momordica charantia, wherein the nucleotide sequence of the InDel molecular marker is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

[0008] The application also provides specific primers for amplifying the above InDel molecular marker, wherein the nucleotide sequences of the specific primers are shown as SEQ ID NO. 3 and SEQ ID NO. 4.

[0009] The application also provides a kit for identifying the fruit nodule morphology of Momordica charantia, comprising the above specific primers.

[0010] The application also provides a method for identifying the fruit nodule morphology of Momordica charantia, comprising the following steps: extracting genomic DNA at the seedling stage of Momordica charantia, using PCR to amplify a target DNA sequence to obtain an amplification product, and detecting the length of the product by PAGE electrophoresis, wherein the normal type of nucleotide sequence has a length of 91 bp, and the corresponding fruit nodule morphology is the sharp-toothed type; the deletion type of nucleotide sequence has a length of 89 bp, and the corresponding fruit nodule morphology is the smooth type.

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

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

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

[0014] Preferably, the reaction system of PCR amplification is as follows: a total reaction volume of 10 μL, comprising 5 μL of 2x PCR mix, 0.2 μL of an upstream primer, 0.2 μL of a downstream primer, 3.6 μL of ddH2O and 1 μL of a DNA template.

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

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

[0017] The application also provides the application of the above InDel molecular marker in the identification of the fruit skin morphology of Momordica charantia, the molecular marker assisted breeding of Momordica charantia and the improvement of the germplasm resources of Momordica charantia.

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

[0019] 1. Early detection in seedling stage

[0020] By using the molecular marker disclosed in the application, the DNA of any tissue of Momordica charantia seedling stage can be directly extracted, the fruit nodule morphology (tapered or smooth) of Momordica charantia can be predicted and detected, early identification can be realized, and the breeding cycle can be significantly shortened.

[0021] 2. Accurate identification of genetic state

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

[0023] 3. Wide application prospect

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

[0025] In summary, the molecular marker technology of the application has the significant advantages of early detection, accurate prediction and wide application compared with the prior art, and further expands the potential of molecular marker technology in Momordica charantia research. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The figure shows the characteristics of the parent materials of the embodiments of the application and the different fruit nodule morphologies when constructing F2 genetic separation population.

[0027] Figure 2 The figure shows the QTL positioning results of the tapered fruit nodule trait in the application using high-density genetic map.

[0028] Figure 3 The figure shows the process of fine positioning of the tapered fruit nodule gene using F2 separation population for linkage genetic analysis.

[0029] Figure 4 The figure shows 40 Momordica charantia with different fruit nodule morphologies.

[0030] Figure 5 The figure shows the PAGE electrophoresis detection results of different samples. DETAILED DESCRIPTION

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

[0032] Embodiments

[0033] The embodiments of the present application detail the application of molecular marker technology in the identification of balsam pear fruit tubercle morphology, and verify and optimize the technical solutions through specific embodiments.

[0034] Firstly, the present application constructs an F2 genetic separation population, using balsam pear inbred lines "HNU025" and "HNU004" as parents to cross to obtain F1 generation, and then using F1 plants to self-cross to construct F2 population. The fruit tubercle morphology of "HNU025" is smooth type, while that of "HNU004" is sharp tooth type, as shown in Figure 1 . By observing the fruit tubercle morphology of F1 and F2 populations, it is found that the fruit tubercles of F1 generation are all sharp tooth type, and the segregation ratio of sharp tooth type to smooth type fruit tubercles in F2 population is 3:1. This indicates that the smooth type fruit tubercle of balsam pear is a single recessive gene controlled trait. Through quantitative trait locus (QTL) analysis, the gene locus (fwt6.1) controlling fruit tubercle morphology is preliminarily located in the front end region of chromosome 6. In the present application, the smooth type fruit tubercle variety HNU025 and the sharp tooth type fruit tubercle variety HNU004 are prior art, as Chen Hongrong. Construction of high-density genetic map of balsam pear and QTL mapping of main agronomic traits [D]. Hainan: Hainan University, 2021. Among them, HNU025 is hai09-1 in the text, and HNU004 is hai09-2 in the text.

[0035] Subsequently, further backcrossing F1 generation with "HNU004" to obtain BC1F1 population, and self-crossing to construct BC1F2 population. By expanding the population to screen recombinant single plants, combined with fruit tubercle phenotype data, the fwt6.1 locus is finally fine-mapped to a 19.23kb interval, as shown in Figure 2 and 3 . Further analysis shows that there is a 2bp base deletion ("GG") in the interval, which is tightly linked to the smooth type fruit tubercle phenotype of balsam pear. The detection results show that the normal nucleotide sequence length is 91bp, and the corresponding fruit tubercle morphology is sharp tooth type; compared with the reference sequence, the "GG" base is deleted from the 67th site of the 5' end, the sequence length is 89bp, and the corresponding fruit tubercle morphology is smooth type.

[0036] To verify the reliability and applicability of the molecular marker, the present application selects 40 balsam pear inbred lines or commercial seeds with different fruit tubercle morphologies as planting materials, as shown in Figure 4 . After seed germination, the seeds are sown in the plug tray and subjected to routine seedling stage management. After the first pair of true leaves grows, leaf tissue is taken for DNA extraction. The detection results show that among the 40 samples, 29 exhibit smooth type fruit tubercle, and 11 exhibit sharp tooth type fruit tubercle, and the genotype detection results are completely consistent with the phenotype, as shown in Table 1.

[0037] Table 1. Genotype detection results of 40 samples and phenotypes

[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 2x PCR mix (Yigong Biological Co., Ltd., item number EG21102), 0.2 μL (10 μM) upstream primer, 0.2 μL (10 μM) downstream primer, 3.6 μL ddH2O, and 1 μL DNA template (30 ng / μL). The reaction program was 94°C pre-denaturation for 3 minutes; followed by 94°C denaturation for 20 seconds, 54°C annealing for 25 seconds, 72°C extension for 20 seconds, a total of 30 cycles; and finally 72°C extension for 5 minutes to end the reaction.

[0041] PAGE electrophoresis detection: first, acrylamide and methylenebisacrylamide were dissolved in water at a ratio of 29:1 to prepare a 30% (w / v) solution. Then 300 mL of the above solution, 100 mL of 10x TBE buffer, and 600 mL of water were used to prepare 9% PAGE working solution 1 L. When preparing the gel, the PCR products were analyzed by 9% polyacrylamide gel electrophoresis (PAGE). The amount of sample loaded 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: single upper band (type A, 91 bp) corresponding to the homozygous genotype of sharp-toothed tubercle; single lower band (type B, 89 bp) corresponding to the homozygous genotype of smooth tubercle; and double bands (type H) corresponding to the heterozygous genotype of sharp-toothed tubercle, as shown in Figure 5

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

[0044] 1. Identification of the peel morphology of Momordica charantia

[0045] Using the molecular marker, the morphological characteristics of Momordica charantia tubercle can be quickly and accurately identified as sharp-toothed morphology or smooth morphology. This method provides an efficient tool for the molecular identification of the morphology phenotype of Momordica charantia, and reduces the time cost and environmental dependence of the traditional method.

[0046] 2. Molecular marker-assisted breeding of Momordica charantia

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

[0048] 3. Improvement of Momordica charantia germplasm resources

[0049] The molecular marker has important application value in the improvement of Momordica charantia germplasm resources. Through molecular marker analysis, the selection and utilization of germplasm resources can be optimized, laying a foundation for cultivating high-quality and diversified new Momordica charantia varieties.

[0050] Obviously, the above embodiments of the present application are only examples for more clearly illustrating the present application, and are not intended to limit the embodiments of the present application. For ordinary skilled in the art, on the basis of the above description, other different forms of changes or variations can also be made. Here, all the embodiments cannot be exhausted, and any changes or variations derived from the technical solutions of the present application still fall within the protection scope of the present application.

Claims

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

2.

2. A specific primer for amplifying the InDel molecular marker of claim 1, characterized in that, The nucleotide sequence of the specific primer is shown as SEQ ID NO. 3 and SEQ ID NO.

4.

3. A kit for identifying the fruit nodule morphology of Momordica charantia, comprising the specific primer of claim 2.

4. A method of identifying the gynaceous morphology of a Momordica charantia plant, comprising: Genomic DNA is extracted from the seedling stage of Momordica charantia, the target DNA sequence is amplified by PCR to obtain an amplification product, and the product fragment is detected by PAGE electrophoresis, the normal nucleotide sequence length is 91 bp, and the corresponding fruit nodule morphology is the sharp-toothed morphology; the deletion type nucleotide sequence length is 89 bp, and the corresponding fruit nodule morphology is the smooth morphology, the target DNA sequence is shown as SEQ ID NO. 1 or SEQ ID NO. 2, and the nucleotide sequence of the PCR amplification primer is shown as SEQ ID NO. 3 and SEQ ID NO.

4.

5. The method of claim 4, wherein, The band of the amplification product is divided into three types: a single upper band corresponds to the homozygous genotype of the sharp-toothed fruit nodule; a single lower band corresponds to the homozygous genotype of the smooth fruit nodule; and double bands correspond to the heterozygous genotype of the sharp-toothed fruit nodule.

6. The method of claim 4, wherein, The reaction system of PCR amplification is 10 μL in total, including 5 μL of 2x PCR mix, 0.2 μL of an upstream primer, 0.2 μL of a downstream primer, 3.6 μL of ddH2O and 1 μL of a DNA template.

7. The method according to claim 4 or 6, characterized in that, The reaction program of PCR amplification is set as: 94℃ pre-denaturation for 3 minutes; then 94℃ denaturation for 20 seconds, 54℃ annealing for 25 seconds, 72℃ extension for 20 seconds, and cycling for 30 times; finally, 72℃ extension for 5 minutes to end the reaction.

8. The InDel molecular marker of claim 1 is applied to the identification of the fruit nodule morphology of Momordica charantia, the molecular marker assisted breeding of Momordica charantia and the improvement of Momordica charantia germplasm resources.

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