An indel marker closely linked to wild bitter gourd powdery mildew resistance and application thereof

Abstract

This invention discloses an InDel marker closely linked to powdery mildew resistance in wild bitter gourd and its application. This invention obtains highly resistant wild germplasm resources to powdery mildew through screening, with resistance controlled by a dominant single gene. Through forward genetic mapping, a molecular marker PM869 closely linked to the resistance gene was successfully developed. Using the PM869 marker of this invention to identify powdery mildew resistance in bitter gourd is characterized by its simplicity, high specificity, and high stability. It can accurately and efficiently detect the resistance and susceptibility of tested bitter gourd plants to powdery mildew, with a 99% concordance rate with field results. Using this method to assist in the identification of powdery mildew resistance in bitter gourd allows for the screening of resistant individual plants at the seedling stage, enabling the screening of large batches of samples, greatly shortening the bitter gourd breeding cycle and improving breeding efficiency.

Description

Technical Field

[0001] This invention relates to the field of biomolecular marker technology, and in particular to an InDel marker closely linked to powdery mildew resistance in wild bitter gourd and its application. Background Technology

[0002] Momordica charantia( Momordica charantia L. Bitter melon, also known as bitter gourd or bitter grape, is an annual plant belonging to the genus *Momordica* of the Cucurbitaceae family. It is widely cultivated in East China, South China, and Southwest my country. Rich in saponins, polypeptides, alkaloids, polysaccharides, flavonoids, and other substances, bitter melon possesses various medicinal properties, including lowering blood pressure, lowering blood sugar, anti-tumor effects, and enhancing immunity. Therefore, as a vegetable that is both food and medicine, it is widely loved by the public.

[0003] Powdery mildew (PM) is one of the most serious diseases affecting bitter gourd production, generally caused by infection with obligate parasitic powdery mildew fungi. When infected, the host plant's leaves are covered with a thick layer of white powder, gradually turning yellow and withering. The plant ages prematurely due to loss of photosynthetic capacity, ultimately causing severe economic losses. Powdery mildew thrives in hot and humid climates; in hot and humid southern regions, especially in greenhouse cultivation, the infection rate can reach almost 100% in severe cases.

[0004] The use of chemical pesticides is currently the main method for controlling powdery mildew in bitter gourd production. However, long-term use may induce drug resistance in pathogens, reducing their effectiveness, and may also lead to excessive pesticide residues, affecting the food safety of bitter gourd. Screening for disease-resistant germplasm resources and breeding disease-resistant bitter gourd varieties can not only reduce pesticide use and thus lower production costs, but also reduce pesticide residues and ensure food safety. Therefore, it is the most economical, effective, and environmentally friendly means to solve the problem of powdery mildew in bitter gourd. Traditional disease-resistant breeding relies on pathogen inoculation and phenotypic observation and identification at the seedling and mature plant stages in the laboratory or field. This process is not only time-consuming but also easily affected by environmental factors, resulting in unstable experimental results. In addition, the scarcity of superior powdery mildew resistance sources is also one of the main bottlenecks limiting disease-resistant breeding of bitter gourd.

[0005] Marker-assisted selection (MART) is a technique that uses DNA molecular markers closely linked to target traits to screen for superior genotypes, thereby accelerating genetic improvement. InDel (Insertion / Deletion) refers to the phenomenon of insertions or deletions of different nucleotide fragment sizes at the same locus in the genome of closely related species or different individuals of the same species. Markers designed based on PCR amplification using specific sequences flanking the insertion / deletion site are called InDel markers. InDel markers are characterized by their wide distribution across the genome, high density, co-dominance, ease of operation, and high stability, and are widely used in population genetic analysis of plants and animals, as well as molecular-assisted breeding. Summary of the Invention

[0006] To overcome the defects and shortcomings of the prior art, the purpose of this invention is to provide an InDel marker closely linked to powdery mildew resistance in wild bitter gourd and its application.

[0007] The technical solution provided by this invention is as follows: A PM869 primer set related to powdery mildew resistance in wild bitter gourd, wherein the bitter gourd is obtained by hybridization of a highly powdery mildew-resistant wild bitter gourd Y as the female parent and a highly powdery mildew-susceptible bitter gourd material K24 as the male parent to obtain F1, and the F1 plants are then self-pollinated to obtain F2 generation; the PM869 primer set includes: PM869-F: 5'-AGCAGCCAGAAATAACAAGTGT-3'; PM869-R: 5'-TTGACACCTCATCGTCCACT-3'.

[0008] The present invention further discloses a kit for identifying powdery mildew resistance in bitter gourd, screening powdery mildew-resistant single plants of bitter gourd, or breeding disease-resistant bitter gourd, the kit containing the above-mentioned PM869 primer set.

[0009] This invention further discloses the method for identifying powdery mildew resistance in bitter gourd using the PM869 molecular marker, the method comprising the following steps: (1) Using the genomic DNA of the sample to be tested as a template, PCR amplification reaction was performed using the PM869 primer set described in claim 1 to obtain the amplification product; (2) Detect and analyze the amplification products.

[0010] Preferably, in step (1), the total volume of the PCR reaction system is 10 μL, including 5 μL of 2×PCR Master mix, 0.5 μL of Primer-F (10 μM), 0.5 μL of Primer-R (10 μM), 0.5 μL of template DNA (30 ng / μL), and 3.5 μL of ddH2O; The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 60℃ annealing for 15 s; 72℃ extension for 30 s, for a total of 32 cycles; 72℃ extension for 5 min.

[0011] Preferably, in step (2), a product of size 305 bp is amplified in wild bitter gourd Y, a product of size 253 bp is amplified in K24, and a heterozygous product of sizes 305 bp and 253 bp is amplified simultaneously in F1; a product of size 253 bp is amplified in the susceptible single plant of F2, and a product of size 305 bp or a heterozygous product of sizes 253 bp and 305 bp is amplified in the resistant single plant of F2.

[0012] This invention overcomes the shortcomings of existing technologies by providing an InDel marker closely linked to powdery mildew resistance in wild bitter gourd and its application. Addressing the current lack of powdery mildew resistance sources and insufficient development of closely linked molecular markers, this invention, through extensive collection and identification work, screened a wild germplasm resource highly resistant to powdery mildew, with resistance controlled by a dominant single gene, possessing significant application value in breeding. Through forward genetic mapping, this invention developed a molecular marker, PM869, closely linked to the resistance gene.

[0013] The beneficial effects of this invention after adopting the above technical solution are as follows: (1) The PM869 marker of the present invention is used to identify the powdery mildew resistance of bitter gourd. It is simple to operate, highly specific and stable. It can detect the resistance and susceptibility of bitter gourd plants to powdery mildew very accurately and efficiently. The consistency between the detection results and the phenotypic identification reaches 99%. The method of the present invention can be used to assist in the identification of powdery mildew resistance of bitter gourd. Resistant single plants can be screened in the seedling stage. It can be used to screen a large number of samples, which greatly shortens the bitter gourd breeding cycle and improves the breeding efficiency.

[0014] (2) This invention has developed a molecular marker that is closely linked to the resistance to powdery mildew of wild bitter gourd. This marker can be used to detect whether other breeding materials or hybrids contain the disease resistance gene of wild bitter gourd, thereby protecting the disease resistance gene of wild bitter gourd and resolving some commercial disputes caused by unclear division of responsibilities. Therefore, the molecular marker in this invention also has certain application value for the healthy development of the bitter gourd industry. Attached Figure Description

[0015] Figure 1 Phenotypes of wild bitter gourd Y, which is highly resistant to powdery mildew, and bitter gourd inbred line K24, which is highly susceptible to powdery mildew, after inoculation with powdery mildew; the left side is wild bitter gourd Y, and the right side is susceptible inbred line K24. Figure 2 The disease severity standards for powdery mildew on bitter gourd leaves are as follows: 0, 1, and 3 indicate resistance, while 5, 7, and 9 indicate susceptibility. Figure 3 The results of fine mapping of the powdery mildew resistance gene in wild bitter gourd; Figure 4The results of polyacrylamide gel electrophoresis of PM869 markers in the parents and F2 population are shown in the figure. In the figure, M is the marker, P1 is the resistant wild bitter gourd Y, P2 is the susceptible bitter gourd inbred line K24, F1 is the first generation of hybrids, S single plant is the powdery mildew susceptible single plant in the F2 population, and R single plant is the powdery mildew resistant single plant in the F2 population. Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention.

[0017] I. Construction of Genetic Segregating Populations The wild bitter gourd Y, highly resistant to powdery mildew, was collected and preserved by the Vegetable Research Institute of Jiangsu Academy of Agricultural Sciences. The bitter gourd material K24, highly susceptible to powdery mildew, is a high-generation inbred line preserved by the Vegetable Research Institute of Jiangsu Academy of Agricultural Sciences.

[0018] The resistance of Y and K24 after powdery mildew inoculation is as follows: Figure 1 As shown, Y is on the left and K24 is on the right. Y was used as the female parent and K24 as the male parent to obtain F1, and the F1 plants were self-pollinated to obtain the F2 segregating population.

[0019] II. Analysis of the Inheritance Patterns of Powdery Mildew Resistance in Bitter Gourd Plump seeds from both parents, F1, and F2 populations were sown in 50-cell trays. When the seedlings reached the four-leaf stage, the bitter gourd was artificially inoculated with powdery mildew. The pathogen used for inoculation was a single-spore strain of *Erysiphe cucumeris* collected from the Liuhe Experimental Base of the Vegetable Research Institute of Jiangsu Academy of Agricultural Sciences, which had been identified, isolated, and preserved. During inoculation, fresh spores from the diseased leaves were brushed into sterile distilled water, and the spore concentration was adjusted to 1×10⁻⁶ using a hemocytometer. 6 Specimen / mL. Use a spray bottle to evenly spray the prepared spore solution onto each leaf until liquid drips from the leaf margins. After inoculation, place the bitter gourd seedlings in an artificial climate chamber (day / night temperature 28 ℃ / 18 ℃, photoperiod 16 h / 8 h). Observe the powdery mildew symptoms on the leaf surface 14 days after inoculation. Determine the disease severity based on the area of ​​powdery mildew spots on the leaf surface, according to the following specific criteria (…). Figure 2 ): Grade 0: No blemishes on the surface; Grade 1: Scattered white spots on the leaf surface, covering no more than 1 / 3 of the leaf area; Level 3: Powdery mildew spots cover 1 / 3 to 2 / 3 of the leaf area; Level 5: Powdery mildew spots cover more than 2 / 3 of the leaf area; Level 7: Powdery mildew spots cover more than 2 / 3 of the leaf area and large areas of the leaves turn yellow; Level 9: Powdery mildew spots cover more than 2 / 3 of the leaf area; leaves turn brown, dry out, and wilt. Fourteen days after powdery mildew inoculation, the leaves of Y plants showed no lesions, indicating resistance; K24 plants had a leaf disease grade of 7, indicating susceptibility; F1 plants had a leaf disease grade of 1, indicating resistance; in the F2 population, there were 202 resistant plants (≤3 grade) and 60 susceptible plants (≥5 grade). The chi-square test (χ²) was used to determine the resistant plants. 2 =3.16 (χ) 2 0.05 =3.84, P >0.05), which conforms to the Mendelian segregation ratio of 3:1, proving that the powdery mildew resistance of Y is controlled by a dominant single gene.

[0020] III. Fine mapping of the powdery mildew resistance gene in bitter gourd Thirty resistant and 30 susceptible progeny plants were selected from the F2 population, and leaf tissue DNA was extracted using the CTAB method. Equal amounts of DNA were collected from each plant and mixed to construct R and S pools, respectively. Additionally, five plants each of Y and K24 were selected, and leaf tissue DNA was extracted and mixed in equal amounts to construct P1 and P2 pools, respectively. DNA libraries were constructed from all four pools, and sequencing was performed using an Illumina HiSeq™ PE150 platform at a sequencing depth of 30×. Using the unpublished genome of wild bitter gourd Y assembled by our research group as a reference sequence, differential loci were compared between the parents and the resistant / susceptible extreme pools using the BWA tool. By calculating the Δ(SNP-index) value between the R and S pools, the bitter gourd powdery mildew resistance gene was preliminarily located in the 21.82–24.75 Mb region at the end of chromosome 8 of bitter gourd. Figure 3 ).

[0021] To further narrow down the candidate region, the resequencing data of both parents were aligned to the third-generation sequencing reference genome of wild bitter gourd Y chromosome. DNA sequence variations between the parents were analyzed, InDel markers were developed, and markers showing polymorphism between the parents were screened. These markers were used to genotype the F2 population (395 individual plants) to identify the exchanged plants. Based on the bitter gourd powdery mildew resistance phenotype data and the identified genotypes of the exchanged plants, the resistance gene was located within the region of 24184069–24268492 bp on chromosome 8 of bitter gourd. Figure 3 ).

[0022] IV. Development of Molecular Markers Linked to Bitter Melon Powdery Mildew Resistance Genes Based on a finely mapped 80 kb candidate region, insertion / deletion sites with variations between parents were searched. Corresponding InDel markers were developed and validated between parents. The results revealed a variation at 24249668 bp on chromosome 8, where resistant plants had a 52 bp nucleotide sequence inserted compared to susceptible plants (sequence shown in SEQ ID NO.3). An InDel marker, PM869, was designed based on this variation, with the following nucleotide sequence: PM869-F: 5'-AGCAGCCAGAAATAACAAGTGT (SEQ ID NO: 1); PM869-R: 5'-TTGACACCTCATCGTCCACT (SEQ ID NO: 2); The primer amplifies 305 bp in wild bitter gourd Y (disease-resistant material) and 253 bp in K24 (disease-susceptible material). Both fragments can be amplified simultaneously in F1.

[0023] V. Validation of the correlation between PM869 marker and powdery mildew resistance in bitter gourd (1) Extraction of genomic DNA Genomic DNA was extracted from the leaves of disease-resistant and susceptible individual plants in populations Y, K24, F1, and F2 using a modified CTAB (hexadecyl trimethyl ammonium bromide) method. The steps are as follows: Take a thumb-sized amount of tender bitter gourd leaves and put them into a 2 mL centrifuge tube, then add 3-4 small steel balls; Add 500 μL of CTAB lysis buffer (100 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1.4 M NaCl, 20 μL β-mercaptoethanol) preheated at 65℃, and pulverize the sample using a tissue homogenizer. Place the centrifuge tubes in a 65 ℃ water bath for 20 min, gently shaking them up and down once every 5 min; cool to room temperature, add an equal volume of chloroform / isoamyl alcohol (24:1), mix well for 3 min to fully emulsify, and centrifuge at 12,000 rpm for 15 min. Transfer the supernatant to a 1.5 mL centrifuge tube, add an equal volume of pre-cooled isopropanol, mix well, and place in a -20℃ refrigerator for 1~2 h. Centrifuge at 12,000 rpm for 15 min and discard the supernatant. The precipitate was washed twice with 500 μL of pre-cooled 75% ethanol; After air-drying at room temperature, add 200 μL of sterile ddH2O, mix well, and set aside.

[0024] (2) Identification of PM869 marker resistance to powdery mildew in bitter gourd The total volume of the PCR reaction system was 10 μL, including 5 μL of 2×PCR Master mix, 0.5 μL of Primer-F (10 μM), 0.5 μL of Primer-R (10 μM), 0.5 μL of template DNA (30 ng / μL), and 3.5 μL of ddH2O.

[0025] The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 60℃ annealing for 15 s; 72℃ extension for 30 s, for a total of 32 cycles; 72℃ extension for 5 min.

[0026] PCR products were detected by electrophoresis using an 8% non-denaturing polyacrylamide gel (PAGE). The PAGE gel was prepared according to the following formulation: 12 mL Acr-Bis (40%, 29:1), 6 mL 10×TBE, 42 mL ddH₂O, 500 μL APS (10%, w / v), and 50 μL TEMED. Electrophoresis was performed at 180 V for 70 min using 0.5×TBE buffer. Silver staining was performed after electrophoresis using the following method: Fixation: Remove the glass plate and place the adhesive into the fixative (900 mL distilled water + 100 mL anhydrous ethanol + 5 mL glacial acetic acid), then place it on a shaker and shake gently for 12 min; Silver staining: Discard the fixative and stain in silver staining solution (1000 mL distilled water + 2 g silver nitrate) for 12 min; Washing: Discard the silver staining solution and wash with 1000 mL of distilled water for 30 seconds; discard the distilled water, add 1000 mL of distilled water and 240 μL of sodium thiosulfate (10%, W / V) solution, and rinse for 30 seconds. Color development: Develop the colorimetric solution (1000 mL distilled water + 15 g sodium hydroxide + 3 mL formaldehyde) for 8-10 minutes until the bands are clear; Cleaning: Discard the colorimetric solution and rinse with 1000 mL of distilled water.

[0027] Observation of the banding patterns of the amplified products revealed that a 305 bp product could be amplified from wild bitter gourd Y (disease-resistant material), and a 253 bp product could be amplified from K24 (disease-susceptible material). Both fragments could be amplified simultaneously in F1, indicating that the marker is a co-dominant marker.

[0028] When PM869 was used for F2 population amplification, it was found that all susceptible plants could only amplify a product of 253 bp, while resistant plants could amplify a homozygous band of 305 bp or a heterozygous band of 253 bp and 305 bp. Figure 4 The marker PM869 developed in this invention showed over 99% genotypic and phenotype similarity in the F2 segregating population, indicating that the marker is closely linked to powdery mildew resistance. It can be used for early screening and identification of powdery mildew resistance in bitter gourd breeding materials, greatly improving the efficiency of bitter gourd disease resistance breeding.

[0029] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A PM869 primer set associated with powdery mildew resistance in wild bitter gourd, characterized in that, The bitter gourd was obtained by hybridization of wild bitter gourd Y (highly resistant to powdery mildew) as the female parent and bitter gourd material K24 (highly susceptible to powdery mildew) as the male parent to obtain F1, and then self-pollinating the F1 plants to obtain F2 generation; the PM869 primer set includes: PM869-F: 5'-AGCAGCCAGAAATAACAAGTGT-3'; PM869-R: 5'-TTGACACCTCATCGTCCACT-3'.

2. A kit for identifying powdery mildew resistance in bitter gourd, screening powdery mildew-resistant individual plants of bitter gourd, or for breeding disease-resistant bitter gourd, characterized in that: The kit contains the PM869 primer set as described in claim 1.

3. A method for identifying powdery mildew resistance in bitter gourd using the PM869 molecular marker, characterized in that, The method includes the following steps: (1) Using the genomic DNA of the sample to be tested as a template, PCR amplification reaction was performed using the PM869 primer set described in claim 1 to obtain the amplification product; (2) Detect and analyze the amplification products.

4. The method as described in claim 3, characterized in that, In step (1), the total volume of the PCR reaction system is 10 μL, including 5 μL of 2×PCR Master mix, 0.5 μL of Primer-F (10 μM), 0.5 μL of Primer-R (10 μM), 0.5 μL of template DNA (30 ng / μL), and 3.5 μL of ddH2O; The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 60℃ annealing for 15 s; 72℃ extension for 30 s, for a total of 32 cycles; 72℃ extension for 5 min.

5. The method as described in claim 3, characterized in that, In step (2), a product of size 305 bp is amplified in wild bitter gourd Y, a product of size 253 bp is amplified in K24, and a heterozygous product of sizes 305 bp and 253 bp is amplified simultaneously in F1; a product of size 253 bp is amplified in the susceptible single plant of F2, and a product of size 305 bp or a heterozygous product of sizes 253 bp and 305 bp is amplified in the resistant single plant of F2.

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