Calabash pericarp color mutation site, primer group, pericarp color detection method and application

Abstract

The invention provides a calabash pericarp color mutation site, a primer group, a pericarp color detection method and application, and belongs to the technical field of plant breeding. According to the invention, two parents (flower color pericarp and white pericarp) and the reciprocal cross F1 thereof are subjected to pericarp color identification after fruits are aged, the white pericarp of the calabash is found to be recessive inheritance, then the two parents are subjected to re-sequencing, and differential sites are screened. After the fruits of the F2 group are ripe, investigating the color of the pericarp, constructing a white pericarp and flower-color pericarp mixing pool according to the color of the pericarp, detecting the mixing pool by utilizing difference sites among parents, positioning by utilizing an SNP Gprime method, combining bioinformatics analysis, screening pericarp color related mutation sites, and identifying the pericarp color related mutation sites through natural group verification. It is found that the calabash peel color mutation site is the 1310809th nucleotide of the 7th exon of the Lsi05G000660 gene of the calabash chromosome 5, wherein the 1310809th nucleotide of the Lsi05G000660 gene is mutated from G to A.

Description

Technical Field

[0001] The present invention relates to the technical field of plant breeding, and particularly relates to a mutant locus of gourd pericarp color, a primer set, a method for detecting pericarp color and applications thereof. Background Art

[0002] Gourd [Lagenaria siceraria (Molina) Standl.] (2n = 2x = 22), also known as night-blooming cucumber, bottle gourd, white gourd, and field squash, originated in Africa and is an annual vine herb of the Cucurbitaceae family. It is widely cultivated around the world and can be used as a rootstock for grafting melons, vegetables, daily necessities, cultural and art crafts, etc., with rich germplasm and genetic resources.

[0003] The color of the gourd pericarp is one of the important manifestations of variety characteristics, and has an important impact on the cultural and art, ornamental attributes of gourds, and the commerciality of edible bottle gourds. Gourds with bright, uniform, and moist colors are often more popular among consumers and have higher prices.

[0004] The colors of gourd pericarps are mainly solid colors and variegated colors, among which green and white are more common. Through color identification, different varieties can be distinguished, and then targeted breeding and improvement work can be carried out. Moreover, the breeding of excellent varieties is often accompanied by the optimization of pericarp color. Therefore, color identification has also become an important link in the variety breeding process. At present, the screening of gourd pericarp color mainly relies on visual observation after the fruits are mature in the field. However, this method takes a long time, has a high cost, and there are errors in screening and observation due to the influence of the pericarp color by leaves and other factors. Therefore, there is an urgent need for a method for identifying gourd pericarp color with high accuracy, simple method, and short cycle.

[0005] Molecular marker-assisted breeding is one of the most widely used technologies in the field of agricultural science and technology breeding. By accurately identifying genetic markers of target traits, it significantly improves the efficiency and controllability of breeding, providing unprecedented technical support for crop improvement. This technology does not require a complex field screening process, significantly shortens the breeding cycle, reduces the cost of field screening, breaks through the bottleneck of traditional breeding methods, can perform genotype identification at the seedling stage, is fast, highly accurate, low-cost, and is not affected by the environment, which can significantly improve the breeding efficiency and accuracy, providing technical support for variety improvement. However, there are currently no relevant molecular marker loci that can be used for screening gourd pericarp color in the existing technology. Therefore, it is of great significance to develop relevant molecular markers for gourd pericarp color identification and related variety breeding. Summary of the Invention

[0006] The object of the present invention is to provide a mutation site of gourd pericarp color, a primer set, a method for detecting pericarp color and an application thereof. By detecting the gourd pericarp color through molecular markers, it overcomes the defects in the existing breeding that the breeding efficiency of gourd pericarp color-related varieties is low, the cost is high, the period of related purity identification is long, the accuracy is easily affected by the environment, and the related genes are insufficient.

[0007] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0008] The present invention provides a mutation site of gourd pericarp color, and the mutation site of gourd pericarp color is located on chromosome 5 of the gourd.

[0009] Furthermore, the mutation site of gourd pericarp color is a mutation of nucleotide G→A at position 1310809 in exon 7 of the Lsi05G000660 gene on chromosome 5 of the gourd.

[0010] The present invention also provides a KASP marker primer set for detecting gourd pericarp color, including primers with the following sequences:

[0011] White pericarp site binding primer - F1 - SEQ ID NO.1:

[0012] 5’-GAAGGTGACCAAGTTCATGCTTCAAAGATGGAAATGATTAATGTTTGTG-3’;

[0013] Flower-colored pericarp site binding primer - F2 - SEQ ID NO.2:

[0014] 5’-GAAGGTGACCAAGTTCATGCTTCAAAGATGGAAATGATTAATGTTTGTA-3’;

[0015] Downstream primer - R1 - SEQ ID NO.3:

[0016] 5’-AAACCCCTAAAACACTTCCAATGA-3’.

[0017] Furthermore, the 5’ ends of the white pericarp site binding primer and the flower-colored pericarp site binding primer are respectively labeled with different fluorescent groups.

[0018] The present invention also provides an application of the KASP marker primer set for detecting gourd pericarp color in gourd breeding.

[0019] The present invention also provides a method for detecting gourd pericarp color, including the following steps:

[0020] (1) Extract the DNA of the gourd material to be identified;

[0021] (2) Use SEQ ID NO.1 and SEQ ID NO.2 with different fluorescent groups, and the primers shown in SEQ ID NO.3 to perform PCR amplification on the DNA of the gourd material to be identified;

[0022] (3) Analyze the results of the PCR amplification.

[0023] Furthermore, in step (2), the reaction system for the PCR amplification is as follows:

[0024] 2 - 4 μL of 2x Taq DNA Polymerase Mix, 1 - 2 μL of 4x SNP Primer Mix, and 2 - 4 μL of DNA sample.

[0025] Furthermore, the PRIMER mix includes raw materials with the following volume ratios:

[0026] F1:F2:R1:pure water = 0.5 - 2:0.5 - 2:1 - 4:2 - 8; the concentrations of F1, F2, and R1 are all 90 - 110 μm / mL.

[0027] Furthermore, in step (2), the reaction program for the PCR amplification is as follows:

[0028] Table 2 PCR Amplification Program

[0029]

[0030] The present invention also provides an application of a method for detecting the color of gourd peel in gourd breeding.

[0031] The beneficial effects of the present invention compared with the prior art are as follows:

[0032] (2) After the fruits of two parents (flower - colored peel and white peel) and their reciprocal cross F1 are mature, the present invention identifies the peel color and finds that the white peel of gourd is recessive inheritance. Then, the two parents are re - sequenced to screen for differential sites. After the fruits of the F2 population are mature, the peel color is examined. According to the peel color, a mixed pool of white - peel and flower - colored - peel is constructed, and the differential sites between the parents are used to detect the mixed pool. The SNP Gprime method is used for mapping, combined with bioinformatics analysis to screen for mutation sites related to peel color. Through verification in natural populations, it is found that the mutation site of the gourd peel color is the nucleotide mutation of G→A at the 1310809th position of the 7th exon of the Lsi05G000660 gene on chromosome 5 of the gourd.

[0033] (2) The present invention designs a KASP marker primer set SEQ ID NO.1 - SEQ ID NO.3 for detecting the nucleotide G→A mutation at position 1310809 of the 7th exon of the Lsi05G000660 gene on chromosome 5 of bottle gourd, which can be used to detect the peel color of bottle gourd. This KASP primer set has the characteristics of high efficiency, high accuracy, and no pollution, overcomes the defects of low breeding efficiency, high cost, long cycle for purity identification, easy susceptibility of accuracy to the environment, and insufficient related genes in the existing breeding for bottle gourd peel color, and accelerates the breeding and purity identification of related varieties. Detailed Embodiments

[0034] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0035] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0037] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0038] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0039] The present invention provides a method for detecting the peel color of bottle gourd, comprising the following steps:

[0040] (1) Extract the DNA of the gourd material to be identified;

[0041] (2) Use the primers shown in SEQ ID NO.1 to SEQ ID NO.3 to perform PCR amplification on the DNA of the gourd material to be identified;

[0042] (3) Analyze the results of the PCR amplification. Among them, the method for analyzing the results of the PCR amplification includes:

[0043] If the detected genotype is G, it is a pure white peel gourd material;

[0044] Or if the detected genotype is A, it is a pure flower peel gourd material;

[0045] Or if the detected genotype is G / A, it is a heterozygous flower peel gourd material.

[0046] Mutation sequence SEQ ID NO.4:

[0047] Note: In the sequence, the first row R is the mutation site, representing G or A

[0048] >chr05 chr05:1310764..1320889(+strand)class=mRNAlength=10126

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] Example 1

[0058] In Example 1 of the present invention, a KASP marker primer set for detecting the gourd peel color was designed, and the specific steps are as follows:

[0059] (1) Using a gourd material resistant to gourd fusarium wilt

[0060] Using one flower peel gourd material Y4 and one white peel material HGZ as parents to perform reciprocal crosses to obtain the offspring F1, and the F2 population obtained by self-crossing the offspring F1.

[0061] (2) Soak the seeds of Y4, HGZ, F1, and F2 for seed soaking and germination promotion. After sowing in plug trays, field planting is carried out at the one-leaf and one-heart stage. Prune the plants with single vines, let the side vines bear fruits, and strictly perform self-pollination. Let each plant bear 2 fruits to avoid environmental impacts such as fruit rot or sunlight exposure.

[0062] Examine the fruit peel color after 55 days of fruit development. It is found that the F1 of reciprocal crosses is all variegated skin, indicating that the white fruit peel of gourd is recessive inheritance. And there are 71 variegated skin gourds and 25 white skin gourds in the F2 generation, conforming to 3:1, indicating that the peel color is a qualitative trait.

[0063] Sample the leaves of individual plants of the parents, F1, and F2. Use the conventional genomic DNA extraction kit (containing RNaseA) of TransGen Biotech Co., Ltd. to extract DNA. Resequence the two parents to detect the differential sites between the two parents. According to the fruit peel color, construct extreme bulks in the F2 population. Using BSA sequencing, a functional mutation of G→A at the 1310809th nucleotide of the 7th exon of the gene Lsi05G000660 homologous to Cytochrome P450 on chromosome 5 is detected by the Gprime method (the sequence is shown in SEQ ID NO.4). Therefore, it is preliminarily determined that this gene is the gene related to the fruit peel color of gourd. According to the sequence of this mutation site, design KASP molecular markers, and its sequences are as follows:

[0064] Forward primer: White fruit peel locus binding primer - F1 - SEQ ID NO.1:

[0065] 5’-GAAGGTGACCAAGTTCATGCTTCAAAGATGGAAATGATTAATGTTTGTG-3’

[0066] Forward primer: Variegated fruit peel locus binding primer - F2 - SEQ ID NO.2:

[0067] 5’-GAAGGTGACCAAGTTCATGCTTCAAAGATGGGAAATGATTAATGTTTGTA-3’

[0068] Reverse primer R1 - SEQ ID NO.3:

[0069] 5’-AAACCCCTAAAACACTTCCAATGA-3’

[0070] Example 2

[0071] In Example 2 of the present invention, the detection effect of the KASP molecular marker primer set shown in SEQ ID NO.1~SEQ ID NO.3 in Example 1 is detected, and the specific steps are as follows:

[0072] Table 1 Source of Samples

[0073] Sample Number Source Sample Number Source Sample Number Source Z063 Shandong HN1 Hainan Z0618 Shandong JL082 Jilin J03 Japan Z078 Shandong Z077 Shandong JTT Japan Z0620 Shandong SD0810 Shandong JZS Japan YH2 Ningbo J081 Japan KZ Shandong JTK01 Japan C2002 Japan T2002 Japan JX0812 Beijing DP-1 Taizhou Y4 Japan JX0815 Beijing FH1 Japan Y1 Japan TB10 Japan HGZ Japan Z062 Shandong Shengtongli Japan

[0074] Note: The above germplasm resource materials can be obtained from the applicant by the public within twenty years from the application date and are only used for repeating the experiments related to this invention.

[0075] (1) Sow the 27 materials in Table 1 and grow them to the one-leaf and one-heart stage, then take leaf samples. Extract genomic DNA from the leaves, measure the concentration using NanoDrop 2000, and dilute each DNA stock solution to 20 ng / μL. Use the molecular markers described in Example 1 to amplify and detect the extracted genomic DNA. At the same time, plant these 27 gourd germplasm resource materials in the field. Prune the plants to a single vine, let the side vines bear fruits, and strictly self-pollinate. Let each plant bear 2 fruits to avoid environmental influences such as fruit rot or sun exposure. Examine the fruit peel color 55 days after fruit development.

[0076] (2) Configure the PCR reaction system as follows: 2 μL of 2x Taq DNA Polymerase Mix, 1 μL of SNP PrimerMix(4x), and 2 μL of DNA sample.

[0077] (3) Perform PCR amplification according to the procedure shown in Table 2:

[0078] Table 2 PCR Amplification Procedure

[0079]

[0080] (4) Take the mixture after the amplification is completed and perform SNP locus detection. Fluorophores FAM and VIC are used to distinguish two isogenic loci in SNP locus detection, and FAM and VIC are respectively labeled on primers F1 and F2. The passive reference dye ROX (passive reference dye ROX) is used to correct the signal differences caused by reaction volume errors between wells. The relevant excitation and emission wavelengths are shown in Table 3. The reading software is the oemga device of LGC.

[0081] Table 3 Relevant Excitation and Emission Wavelengths

[0082] Fluorescent Group Excitation Light (nm) Emission Light (nm) FAM 485 520 VIC 535 556 ROX 575 610

[0083] (5) Analyze the result data of PCR amplification using the genotype reading software (Kluster Caller) of LGC_OMEGA. Obtain the relative fluorescence values corresponding to VIC and FAM for each PCR reaction well. Cluster and classify the samples based on the relative fluorescence values, and further determine the genotype according to the sample clusters and fluorescence types. The genotype and phenotype results of the tested samples are shown in Table 4.

[0084] Table 4 Genotype of Samples and Peel Color

[0085] Sample Number Genotype Skin Color Sample Number Genotype Skin Color Sample Number Genotype Skin Color Z063 A / A Spotted Skin HN1 A / A Spotted Skin Z0618 G / G White Skin JL082 G / G White Skin J03 G / G White Skin Z078 G / G White Skin Z077 G / G White Skin JTT A / A Spotted Skin Z0620 A / A Spotted Skin SD0810 G / G White Skin JZS G / G White Skin YH2 G / G White Skin J081 G / G White Skin KZ A / A Spotted Skin JTK01 G / G White Skin C2002 A / A Spotted Skin T2002 G / G White Skin JX0812 A / A Spotted Skin DP-1 G / G White Skin Y4 A / A Spotted Skin JX0815 A / A Spotted Skin FH1 A / A Spotted Skin Y1 G / G White Skin TB10 G / G White Skin HGZ G / G White Skin Z062 G / G White Skin Shengtongli G / G White Skin

[0086] As can be seen from Table 4, the result genotype is consistent with the phenotype result. The genotypes of 10 flower skin germplasm resources are all A / A, and the genotypes of 17 white skin germplasm resources are all G / G. It shows that this marker can significantly distinguish flower skin and white skin gourd materials and can be used for the creation of gourd germplasm resources related to peel color and variety breeding.

[0087] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A gourd peel color mutation site, characterized in that: The gourd peel color mutation site is located on chromosome 5 of the gourd.

2. The cucurbit peel color mutation site according to claim 1, characterized in that: The gourd peel color mutation site is the nucleotide G→→A mutation at position 1310809 of exon 7 of the Lsi05G000660 gene on chromosome 5 of gourd.

3. A KASP marker primer set for detecting the color of gourd peel, characterized in that: The primers included the following sequences: White peel site binding primer-F1-SEQ ID NO.1: 5'-GAAGGTGACCAAGTTCATGCTTCAAAGATGGAAATGATTAATGTTTGTG-3'; Flower color pericarp site binding primer-F2-SEQ ID NO.2: 5'-GAAGGTGACCAAGTTCATGCTTCAAAGATGGAAATGATTAATGTTTGTA-3'; Downstream primer-R1-SEQ ID NO.3: 5'-AAACCCCTAAAACACTTCCAATGA-3'.

4. The KASP marker primer set for detecting the color of cucurbit peel according to claim 3, characterized in that: The 5' ends of the white pericarp site binding primer and the flower-colored pericarp site binding primer are respectively labeled with different fluorescent groups.

5. Use of the KASP marker primer set capable of detecting the color of gourd peel according to any one of claims 3 to 4 in gourd breeding.

6. A method for detecting the color of gourd peel, characterized in that: The steps include: (1) extracting DNA from the gourd material to be identified; (2) using primers shown in SEQ ID NO.1 and SEQ ID NO.2 and SEQ ID NO.3 with different fluorescent groups to perform PCR amplification on the DNA of the gourd material to be identified; (3) Analyze the results of PCR amplification.

7. The method for detecting the color of the gourd peel according to claim 6, characterized in that: In step (2), the reaction system of the PCR amplification is: 2x Taq DNA Polymerase Mix 2~4μL, 4x SNP Primer Mix 1~2μL, DNA sample 2~4μL.

8. The method for detecting the color of the gourd peel according to claim 7, characterized in that: The PRIMERmix comprises the following raw materials in volume ratios: F1:F2:R1:pure water=0.5-2:0.5-2:1-4:2-8; the concentrations of F1, F2 and R1 are all 90-110 μm / mL.

9. The method for detecting the color of the gourd peel according to claim 6, characterized in that: In step (2), the reaction procedure of the PCR amplification is:

10. Use of the method for detecting the color of gourd peel according to any one of claims 6 to 9 in gourd breeding.

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