Molecular marker related to powdery mildew resistance of cucurbit as well as KASP primer combination and application thereof

Abstract

The invention discloses a molecular marker related to powdery mildew resistance of calabash as well as a KASP primer combination and application thereof, and belongs to the technical field of plant resistance breeding. A mutation site of the molecular marker related to the powdery mildew resistance of the calabash is located on a chromosome 11 of a calabash chromosome, and 24091441st nucleotide is mutated from G to C; the molecular marker related to the powdery mildew resistance of the cucurbit is applied to screening of powdery mildew resistant materials of the cucurbit or breeding of powdery mildew resistance of the cucurbit; the invention further discloses a KASP primer combination for detecting the molecular marker related to the powdery mildew resistance of the cucurbit. The sequence of the KASP primer combination is as follows: a disease-resistant site combined upstream primer is as shown in SEQ ID NO.1; the combination of the susceptible site and the upstream primer is as shown in SEQ ID NO. 2; the downstream primer is as shown in SEQ ID NO.3; the cucurbit powdery mildew resistance related molecular marker and the KASP primer combination thereof can remarkably distinguish a cucurbit powdery mildew resistant material from a cucurbit powdery mildew susceptible material, and can be applied to cucurbit powdery mildew resistant material screening or cucurbit powdery mildew resistant breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of plant resistance breeding, and in particular relates to a cucurbit powdery mildew resistance-related molecular marker and a KASP primer combination and application thereof. Background Art

[0002] The gourd (Lagenaria siceraria (Molina) Standl.) (2n=2x=22), also known as night-blooming gourd, gourd, gourd, and ground cattail, originated in Africa and is an annual climbing herb in the Cucurbitaceae family. It is widely cultivated worldwide and used as a rootstock for melon grafting, as a vegetable, as a daily necessity, and as a decorative craft. It boasts a rich germplasm and genetic resource. Powdery mildew, primarily caused by the powdery fungus Podosphaera xanthii (Sphaerotheca fuliginea), is a common and significant disease of all cucurbit crops in the Cucurbitaceae family. It is widely distributed and easily spreads, affecting the entire growth period of cucurbits from seedling to fruit maturity. Disease can occur in the hypocotyl, stem, leaf, and fruit, severely impacting the development of the cucurbit industry. Currently, production methods primarily address this problem through planting resistant varieties, controlling temperature and humidity, applying fungicides, and implementing crop rotation. However, these control measures are ineffective, often causing environmental pollution and food safety concerns due to pesticide residues.

[0003] However, currently, the selection of cucurbit varieties resistant to powdery mildew is primarily based on conventional breeding techniques, relying on natural field occurrence and laboratory inoculation to identify resistance levels, screen for disease-resistant materials, and select relevant varieties. This process is time-consuming, costly, and lacks high accuracy. Molecular marker-assisted breeding, which could significantly improve breeding efficiency and accuracy, has yet to be applied. Therefore, the development of molecular markers to improve cucurbit powdery mildew resistance is of great significance. Summary of the Invention

[0004] The present invention aims to provide a molecular marker related to powdery mildew resistance of cucurbit and its KASP primer combination and application, aiming to solve the problems of long identification cycle of powdery mildew resistance of cucurbit, low efficiency of breeding of resistant varieties, high cost and low accuracy. The present invention overcomes the defects of low efficiency, high cost and insufficient relevant genes in the existing breeding of powdery mildew resistant varieties of cucurbit. Through genotyping and phenotypic identification, the genome of Bottle gourd (USVL1VR-Ls) published in the article 'Wu Set al. The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papayaringspot virus resistance locus. The Plant Journal, 2017, 92 (5): 963-975' was used as a reference genome to dig out a CTP synthase gene mutation site located on chromosome 11. CTP synthase is a key enzyme in pyrimidine nucleotide metabolism, directly involved in immune regulation mechanism, and may be related to powdery mildew resistance of cucurbit. Therefore, a molecular marker related to powdery mildew resistance of cucurbit was developed based on this mutation site.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A molecular marker associated with powdery mildew resistance in cucurbits, wherein the mutation site of the molecular marker is located on chromosome 11 of the cucurbit genome, and is a G→C mutation at nucleotide position 24091441. When nucleotide position 24091441 is G, the genotype is G, and the corresponding phenotype of the G genotype is powdery mildew susceptibility; when nucleotide position 24091441 is C, the genotype is C, and the corresponding phenotype of the C genotype is powdery mildew resistance.

[0007] The bottle gourd genome is the Bottle gourd (USVL1VR-Ls) genome.

[0008] The genome Bottle gourd (USVL1VR-Ls) is the genome Bottle gourd (USVL1VR-Ls) published in the article 'Wu S et al. The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ringspot virus resistance locus. The Plant Journal, 2017, 92(5): 963-975'.

[0009] The application of the above-mentioned cucurbit powdery mildew resistance-related molecular markers in the screening of cucurbit powdery mildew-resistant materials or the breeding of cucurbit powdery mildew-resistant varieties.

[0010] Preferably, the gourd includes at least one of a powdery mildew resistant gourd and a powdery mildew susceptible gourd.

[0011] A KASP primer combination for detecting molecular markers associated with powdery mildew resistance in cucurbits is designed for the above-mentioned molecular markers associated with powdery mildew resistance in cucurbits. The sequence of the KASP primer combination is as follows:

[0012] The upstream primer of the disease resistance site is shown in SEQ ID NO.1:

[0013] 5'-GAAGGTCGGAGTCAACGGATTAATACTCACCCAATTGTATTTGTAATTC-3';

[0014] The susceptible site binding upstream primer is shown in SEQ ID NO.2:

[0015] 5'-GAAGGTGACCAAGTTCATGCTAATACTCACCCAATTGTATTTGTAATTG-3';

[0016] The downstream primer is shown in SEQ ID NO.3:

[0017] 5'-GAGGAAGAAGAACTCACTTCAACA-3'.

[0018] A kit for detecting powdery mildew resistance of cucurbit comprises the KASP primer combination for detecting molecular markers related to powdery mildew resistance of cucurbit.

[0019] Application of the KASP primer combination for detecting molecular markers related to cucurbit powdery mildew resistance in screening cucurbit powdery mildew-resistant materials or breeding cucurbit powdery mildew-resistant varieties.

[0020] A method for screening cucurbit powdery mildew-resistant materials, using the KASP primer combination for detecting molecular markers related to cucurbit powdery mildew resistance, comprises the following steps:

[0021] (1) extracting genomic DNA from the cucurbit material to be screened;

[0022] (2) Using the KASP primer combination for detecting molecular markers related to powdery mildew resistance in cucurbits, the genomic DNA of cucurbits was amplified and detected by fluorescence quantitative PCR instrument;

[0023] (3) The powdery mildew resistance of the gourd to be screened is determined by the amplification test results. If the mutation site detection results of the molecular marker are all C, the gourd material to be screened is a gourd homozygous disease-resistant material; if the mutation site detection results of the molecular marker are all G, the gourd material to be screened is a gourd homozygous disease-susceptible material; if the mutation site detection results of the molecular marker are G and C, the gourd material to be screened is a gourd heterozygous disease-susceptible material.

[0024] Preferably, in step (3), the determination of the powdery mildew resistance of the cucurbit to be screened by amplification detection results is specifically as follows:

[0025] If the color of the fluorescent signal of the PCR amplification result is consistent with the color of the fluorescent linker of the disease-resistant site bound to the upstream primer (SEQ ID NO.2), the gourd material to be screened is a gourd homozygous disease-resistant material; if the color of the fluorescent signal of the PCR amplification result is consistent with the color of the fluorescent linker of the disease-susceptible site bound to the upstream primer (SEQ ID NO.3), the gourd material to be screened is a gourd homozygous disease-susceptible material; otherwise, the gourd material to be screened is a gourd heterozygous disease-susceptible material.

[0026] Preferably, the parts of the gourd material to be screened in step (1) include leaves, roots, stems and other detection parts.

[0027] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0028] 1. The KASP primer combination for detecting molecular markers associated with powdery mildew resistance in cucurbits of the present invention is based on seedling inoculation of two parents (one resistant and one susceptible) and their reciprocal cross F1 with powdery spores. It was found that powdery mildew resistance in cucurbits is recessive. The two parents were then resequenced to screen for differentially expressed sites. After seedling inoculation of the F2 population, a mixed pool of resistant and susceptible lines was constructed based on the powdery mildew resistance performance. The mixed pool was tested using differentially expressed sites between the parents, and resistance was mapped using the SNPGprime method. Combined with bioinformatics analysis, mutation sites associated with resistance were screened. The obtained KASP primers were then validated in natural populations to identify sites associated with powdery mildew resistance in cucurbits.

[0029] 2. After searching the mutation site of the present invention, no identical cucurbit powdery mildew resistance site was found in the literature.

[0030] 3. The KASP primers developed by the present invention are used for screening cucurbit powdery mildew-resistant materials and variety breeding, and are characterized by high efficiency, high accuracy and no pollution. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0032] At present, the identification of cucurbit powdery mildew resistance has problems such as long cycle, low efficiency of breeding resistant varieties, high cost, and low accuracy. In order to solve the above technical problems, the present invention proposes a cucurbit powdery mildew resistance-related molecular marker and its KASP primer combination and application.

[0033] Example 1:

[0034] Test materials: one powdery mildew-resistant gourd material Z063, one susceptible material Z0612, and their reciprocal cross F1, and an F2 population obtained by self-pollination of the F1. The two parental materials are described in the article "Xing Nailin, Fu Yujing, Wang Yinger, et al. Identification of low-light tolerance of gourd rootstock germplasm resources at the seedling stage [J]. Journal of Jiangxi Agricultural Sciences, 2019, 31(5):11-16". The public may obtain this gourd material from the applicant within 20 years from the date of application for the sole purpose of repeating experiments related to the present invention.

[0035] Test steps: soak and germinate the seeds of Z063, Z0612, F1, and F2, sow in hole trays, and identify the powdery mildew resistance of seedlings at the one-leaf, one-heart stage. The identification method is as follows: (1) collect powdery mildew spores from leaves that have been infected for 7 days, add 4℃ sterile water, stir at high speed (12000r / min) for 3-5 minutes, examine under a microscope and count the bacterial content of the suspension using a hemocytometer; (2) add water to prepare a spore suspension of 100 spores / mL, and limit the preparation of the suspension to inoculation within 2 hours (the disease is most likely to occur if the suspension is placed within 0.5 hours); (3) at the one true leaf stage (plants are sown and cultured in advance to the one true leaf stage), brush the spore suspension evenly (leaf brush inoculation method) onto the leaves of the plant, and place them in a biological artificial climate chamber in the dark for 24 hours (covered with a moisturizing plastic bag) after inoculation, and then carry out normal light and dark alternation management; (4) control the daytime temperature at around 25℃ and the relative humidity at 70%-80%; the nighttime temperature at around 16℃ and the relative humidity at 85%-95%, and investigate the disease situation 15 days after inoculation. It was found that the powdery mildew resistance of cucurbit is recessive. Leaves of individual parents, F1, and F2 plants were sampled and DNA was extracted. The parents were resequenced to detect differential loci between the parents. An extreme hybrid pool was constructed for the F2 population based on powdery mildew resistance. Using the Bottle gourd genome (USVL1VR-Ls) published in the article 'Wu S et al. The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ringspot virus resistance locus. The Plant Journal, 2017, 92(5): 963-975' as the reference genome, BSA sequencing was used to detect a variation within the CTP synthase gene on chromosome 11 using the Gprime method. The CTP synthase gene is associated with immunity to viruses, bacteria, and other diseases. Based on this mutation, a molecular marker associated with powdery mildew resistance in cucurbits was obtained. The sequence fragment where the molecular marker site is located is shown in SEQ ID NO.4 (>chr11 chr11:24088019..24093723 (-strand) class=mRNA length=5705), where n is G / C. When n is G, the phenotype corresponding to the G genotype is susceptible to powdery mildew; when n is C, the phenotype corresponding to the C genotype is resistant to powdery mildew. Therefore, this gene was preliminarily identified as a gene associated with powdery mildew resistance in cucurbits. Based on the mutation site sequence of the molecular marker associated with powdery mildew resistance in cucurbits, the KASP molecular marker was designed, and its sequence is as follows:

[0036] The upstream primer for the disease resistance site is shown in SEQ ID NO. 1: 5'-GAAGGTCGGAGTCAACGGATTAATACTCACCCAATTGTATTTGTAATTC-3'

[0037] The upstream primer of the susceptible site is shown in SEQ ID NO.2: 5'-GAAGGTGACCAAGTTCATGCTAATACTCACCCAATTGTATTTGTAATTG-3'

[0038] The downstream primer is shown in SEQ ID NO.3:

[0039] 5'-GAGGAAGAAGAACTCACTTCAACA-3'

[0040] Example 2:

[0041] Experimental materials: 27 gourd germplasm resources, recorded in the article "Xing Nailin, Fu Yujing, Wang Yinger, et al. Identification of weak light tolerance of gourd rootstock germplasm resources at the seedling stage [J]. Journal of Jiangxi Agricultural Sciences, 2019, 31(5): 11-16". The public can obtain the gourd materials from the applicant within 20 years from the date of application, only for repeating experiments related to the present invention.

[0042] These 27 materials were sown, grown to the one-leaf, one-heart stage, and then inoculated with powdery mildew. The identification method is as follows: (1) collect powdery mildew spores from leaves that have been infected for 5 days, add 4℃ sterile water, stir at high speed (12000r / min) for 3-5min, examine under a microscope and use a hemocytometer to measure the bacterial content of the suspension; (2) add water to prepare a 150 spore / mL spore suspension, and limit the preparation of the bacterial suspension to the completion of inoculation within 2h (the disease is most likely to occur if the suspension is placed within 0.5h); (3) at the stage of one true leaf (the plant is sown and cultured in advance to the stage of one true leaf), pour the spore suspension into a spray bottle and spray it evenly on the leaves of the plant. After inoculation, place it in an artificial climate chamber in the dark and moisturize for 24h (covered with a moisturizing plastic bag), and then carry out normal light and dark alternation management; (4) control the daytime temperature at around 25℃ and the relative humidity at 70%-85%; control the nighttime temperature at around 16℃ and the relative humidity at 80%-95%, and investigate the disease situation 15d after inoculation. Genomic DNA was extracted from leaves and the concentration was measured using a NanoDrop 2000. Each DNA stock solution was diluted to 20 ng / ul. The extracted genomic DNA was amplified and detected using the molecular markers described in Example 1.

[0043] The PCR reaction system was prepared as follows: 2 μL of 2xTaq DNA Polymerase Mix, 1 μL of SNP Primer Mix (4x), and 2 μL of DNA sample.

[0044] PCR steps are shown in Table 1.

[0045] Table 1 PCR steps

[0046]

[0047] SNP detection uses the fluorophores FAM and VIC to distinguish between isogenic loci. The passive reference dye ROX is used to correct for signal variability between wells due to reaction volume errors. The relevant excitation and emission wavelengths are shown in Table 2. The reader software is OEM software from LGC.

[0048] Table 2 Related excitation and emission wavelengths

[0049]

[0050] PCR amplification data were analyzed using LGC_OMEGA's genotype caller software (Kluster Caller). Relative fluorescence values ​​corresponding to VIC and FAM were obtained for each PCR reaction well. Samples were clustered based on relative fluorescence values, and genotypes were further determined based on sample clusters and fluorescence patterns. The genotypes and phenotypes of the samples are shown in Table 3.

[0051] Table 3 Sample genotypes and phenotypes

[0052]

[0053] The results were generally consistent with the phenotypic results. The genotypes of all three powdery mildew-resistant accessions were C / C; of the 16 susceptible accessions, 12 had G / G genotypes and 4 had G / C genotypes. Of the 8 moderately resistant accessions, 3 had C / C genotypes, 3 had G / C genotypes, and 2 had G / G genotypes. This suggests that although other genes associated with powdery mildew resistance exist in cucurbits besides the genes associated with this marker, this marker can significantly distinguish between extremely resistant and susceptible accessions, making it a promising candidate for the development of powdery mildew-resistant cucurbit germplasm resources and variety selection.

[0054] Example 3

[0055] Experimental materials: 1 sample of gourd germplasm material TB10, recorded in the article “Xing Nailin, Fu Yujing, Wang Yinger, et al. Identification of weak light tolerance of gourd rootstock germplasm resources at the seedling stage [J]. Journal of Jiangxi Agricultural Sciences, 2019, 31(5):11-16”, and 1 sample of susceptible gourd germplasm material HX. The public can obtain this gourd material from the applicant within 20 years from the date of application, and it is only used for repeating experiments related to the present invention.

[0056] Using the KASP molecular marker designed by the present invention, TB10 and HX were identified, revealing that the loci were C / C and G / C, respectively. Combined with conventional breeding, F2 populations derived from TB10 and HX were inoculated and identified at the seedling stage. The TB10 population was screened for the most disease-resistant plants, and systematic breeding was conducted in conjunction with other traits. After homozygosity, the homozygous inbred line 'TB-33-12-6-4-7-5-3' was obtained as the female parent. HX was used as the seedling disease-resistant material. Plants with the T / T genotype were selected from the disease-resistant plants in the HX population, and systematic breeding was conducted in conjunction with other traits. After homozygosity, the homozygous inbred line 'HX-25-17-11-5-6-4-2' was obtained as the male parent. Pairing was performed with the other parents, and after comparison and demonstration, this combination was selected and named 'Green Warrior'. This variety has been promoted and applied in multiple provinces across China.

[0057] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0058] 1. The KASP primer combination for detecting molecular markers associated with powdery mildew resistance in cucurbits of the present invention is based on seedling inoculation of two parents (one resistant and one susceptible) and their reciprocal cross F1 with powdery spores. It was found that powdery mildew resistance in cucurbits is recessive. The two parents were then resequenced to screen for differentially expressed sites. After seedling inoculation of the F2 population, a mixed pool of resistant and susceptible lines was constructed based on the powdery mildew resistance performance. The mixed pool was tested using differentially expressed sites between the parents, and resistance was mapped using the SNPGprime method. Combined with bioinformatics analysis, mutation sites associated with resistance were screened. The obtained KASP primers were then validated in natural populations to identify sites associated with powdery mildew resistance in cucurbits.

[0059] 2. After searching the mutation site of the present invention, no identical powdery mildew resistance site for cucurbit was found in the public.

[0060] 3. The KASP primers developed by the present invention are used for screening cucurbit powdery mildew-resistant materials and variety breeding, and are characterized by high efficiency, high accuracy and no pollution.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A molecular marker related to cucurbit powdery mildew resistance, characterized in that: The mutation site of the molecular marker is located on chromosome 11 of the gourd genome, and is a mutation of nucleotide G→C at position 24091441.

2. The molecular marker associated with cucurbit powdery mildew resistance according to claim 1, characterized in that: The bottle gourd genome is the Bottle gourd genome (USVL1VR-Ls).

3. Use of the cucurbit powdery mildew resistance-related molecular markers according to claim 1 in screening cucurbit powdery mildew resistance materials or cucurbit powdery mildew resistance breeding.

4. The use of the cucurbit powdery mildew resistance-related molecular marker according to claim 1 in screening cucurbit powdery mildew resistance materials or cucurbit powdery mildew resistance breeding, characterized in that: The gourd includes at least one of a powdery mildew resistant gourd and a powdery mildew susceptible gourd.

5. A KASP primer combination for detecting molecular markers related to powdery mildew resistance in cucurbits, characterized in that: The molecular markers related to the powdery mildew resistance of cucurbits according to claim 1 are designed and obtained, and the primer combination sequence of KASP is as follows: The disease resistance site binding upstream primer is shown in SEQ ID NO.2: 5'-GAAGGTCGGAGTCAACGGATTAATACTCACCCAATTGTATTTGTAATTC-3'; The susceptible site binding upstream primer is shown in SEQ ID NO.3: 5'-GAAGGTGACCAAGTTCATGCTAATACTCACCCAATTGTATTTGTAATTG-3'; The downstream primer is shown in SEQ ID NO.4: 5'-GAGGAAGAAGAACTCACTTCAACA-3'.

6. A kit for detecting the powdery mildew resistance of cucurbit, characterized in that: The invention comprises the KASP primer combination for detecting molecular markers related to powdery mildew resistance of cucurbit as described in claim 5.

7. Use of the KASP primer combination for detecting molecular markers associated with powdery mildew resistance in cucurbit according to claim 5 in screening cucurbit powdery mildew resistant materials or cucurbit powdery mildew resistant breeding.

8. A method for screening cucurbit powdery mildew resistant materials, characterized in that: The use of the KASP primer combination for detecting molecular markers related to powdery mildew resistance in cucurbits as described in claim 5 comprises the following steps: (1) extracting genomic DNA from the parts of the gourd material to be screened; (2) using the KASP primer combination for detecting molecular markers related to powdery mildew resistance of cucurbits according to claim 4, amplifying and detecting cucurbit genomic DNA by using a fluorescent quantitative PCR instrument; (3) The powdery mildew resistance of the gourd to be screened is determined by the amplification test results. If the mutation site detection results of the molecular marker are all C, the gourd material to be screened is a homozygous disease-resistant material of the gourd; if the mutation site detection results of the molecular marker are all G, the gourd material to be screened is a homozygous disease-susceptible material of the gourd; if the mutation site detection results of the molecular marker are G and C, the gourd material to be screened is a heterozygous disease-susceptible material of the gourd.

9. The method for screening cucurbit powdery mildew resistant materials according to claim 8, characterized in that: In step (3), the powdery mildew resistance of the cucurbit to be screened is determined by amplification detection results, specifically: If the color of the fluorescent signal of the PCR amplification result is consistent with the color of the fluorescent linker of the disease-resistant site combined with the upstream primer (SEQ ID NO.2), the gourd material to be screened is a gourd homozygous disease-resistant material; if the color of the fluorescent signal of the PCR amplification result is consistent with the color of the fluorescent linker of the disease-susceptible site combined with the upstream primer (SEQ ID NO.3), the gourd material to be screened is a gourd homozygous disease-susceptible material; otherwise, the gourd material to be screened is a gourd heterozygous disease-susceptible material.

10. The method for screening cucurbit powdery mildew resistant materials according to claim 8, characterized in that: The parts of the gourd material to be screened in step (1) include detection parts of leaves, roots or stems.

Images