Indel molecular marker primers for identifying the density types of epidermal hairs in Wucai and their application

Abstract

The present invention provides an Indel molecular marker primer for identifying the type of epidermal hair density of Wucai and an application thereof, which relates to the field of molecular marker technology, and comprises an Indel molecular marker primer for identifying the type of epidermal hair density of Wucai, wherein the Indel molecular marker primer comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2; Wucai DNA is obtained, PCR amplification of the Wucai DNA is performed using the aforementioned Indel molecular marker primer, gel electrophoresis is performed on the amplification result, and the type of epidermal hair density of Wucai is determined according to the detection result; the present application can quickly determine the type of epidermal hair density of Wucai, and is helpful for the genetic breeding of Wucai.

Description

Technical Field

[0001] The invention relates to the technical field of molecular markers, and in particular to an Indel molecular marker primer for identifying the density type of epidermal hairs of Wucai and an application thereof. Background Art

[0002] Wucai (Brassica campestris L.var.rosularis) is one of the main vegetable crops in autumn and winter, and has an important market position. Traditional Wucai varieties are mainly for open-field cultivation, and are difficult to meet the rapidly developing facility vegetable planting system in terms of resistance to biological and abiotic stresses. Therefore, the market is in urgent need of new Wucai varieties with outstanding cold resistance, heat resistance, salt resistance, and disease and insect resistance.

[0003] Epidermal hairs have been shown to play an important role in plant resistance to various biological and abiotic stresses. Therefore, cultivating new varieties with more epidermal hairs has great potential to improve Wucai's ability to resist stress, diseases and pests, and help solve the problem of Wucai facility cultivation.

[0004] Traditionally, the methods for breeding new varieties of multi-skinned hairy cabbage are mostly based on traditional breeding models or first- and second-generation molecular marker technologies. The former is often time-consuming and labor-intensive, while the latter, such as restriction fragment length polymorphism marker (RFLP) and simple sequence repeat marker (SSR) technology, is not only relatively cumbersome, but also requires a large amount of DNA, is difficult to develop, and has high experimental costs.

[0005] Therefore, there is an urgent need to provide a method for quickly determining the density type of the epidermal hairs of Wucai to assist in the genetic breeding of Wucai, so as to solve the problem that it takes a long time to cultivate Wucai varieties with multiple epidermal hairs in the prior art. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides an Indel molecular marker primer for identifying the density type of epidermal hairs of Wucai and its application, so as to solve the problem that the cultivation of multi-epidermal hairy Wucai varieties in the prior art takes a long time.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] In a first aspect of the present invention, an Indel molecular marker primer for identifying the density type of epidermal hairs of Wucai is provided, wherein the Indel molecular marker primer comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2.

[0009] The second aspect of the present invention also provides a kit for identifying or assisting in identifying the type of density of epidermal hairs of Wucai, the kit comprising the above-mentioned Indel molecular marker primers.

[0010] Furthermore, the reagents required for PCR reaction and / or electrophoresis are also included.

[0011] The third aspect of the present invention also provides an application of the above-mentioned Indel molecular marker primer or kit in identifying or assisting in identifying the density type of epidermal hairs of Wucai.

[0012] The fourth aspect of the present invention also provides a method for identifying the density type of epidermal hairs of Wucai, which comprises using the above-mentioned Indel molecular marker primers or kit.

[0013] Further, the method comprises:

[0014] Obtaining DNA from Wucai leaves;

[0015] PCR amplification was performed using the above-mentioned Indel molecular marker primers and the DNA of Wucai leaves;

[0016] The amplification results were detected by gel electrophoresis;

[0017] The density type of the epidermal hairs of the corresponding Wucai was determined according to the gel electrophoresis test results; when the gel electrophoresis result had only one specific band with a length of 281bp, the corresponding Wucai variety was a homozygous Wucai variety with more epidermal hairs; when the gel electrophoresis result had two specific bands with lengths of 281bp and 238bp respectively, the corresponding Wucai variety was a heterozygous Wucai variety with more epidermal hairs; when the gel electrophoresis result had only one specific band with a length of 238bp, the corresponding Wucai variety was a homozygous Wucai variety with less epidermal hairs.

[0018] The Indel molecular marker primers and applications thereof for identifying the density type of epidermal hairs of Wucai provided by the present invention have the following beneficial effects compared with the prior art:

[0019] The provided Indel molecular marker primers can be used to quickly determine the density type of the corresponding epidermal hairs of Wucai, which improves the judgment efficiency, helps to cultivate new varieties of Wucai with more epidermal hairs, reduces the cultivation time, and is of great significance to the genetic breeding of Wucai. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is a leaf image of the multi-epidermal hairy black cabbage parent material W7-1 and the less-epidermal hairy black cabbage parent material W7-2 used to construct the population in Example 1; in the figure, the upper left is an enlarged image of the upper surface of the leaf of the multi-epidermal hairy black cabbage parent material W7-1, the upper right is an enlarged image of the lower surface of the leaf of the multi-epidermal hairy black cabbage parent material W7-1, the lower left is an enlarged image of the upper surface of the leaf of the less-epidermal hairy black cabbage parent material W7-2, and the lower right is an enlarged image of the lower surface of the leaf of the less-epidermal hairy black cabbage parent material W7-2;

[0022] Figure 2 This is a schematic diagram of the genotyping of the molecular marker Indel37355 in Example 1 in the F2 population;

[0023] Figure 3 This is a schematic diagram of the genotyping of the molecular marker Indel37355 in Example 2 in Wucai varieties with multiple epidermal hair density types;

[0024] Figure 4 Schematic diagram of the screening process of 1710 F2 plant populations with more epidermal hairs and less epidermal hairs based on Indel molecular markers of BSA-Seq in Example 1. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are 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 creative work are within the scope of protection of the present invention.

[0026] In order to solve the problem that it takes a long time to cultivate new varieties of multi-epidermal hairy cabbage in the prior art, the main idea of ​​this application is:

[0027] Provided is an Indel molecular marker primer for identifying the type of Wucai epidermal hair density, which is used for detecting or assisting in detecting the type of Wucai epidermal hair density, and a corresponding identification method is provided.

[0028] With the help of the above-mentioned Indel molecular marker primers related to the density type of epidermal hairs of Wucai, it can be used to quickly determine the corresponding epidermal hair density type of Wucai, which helps to improve the efficiency of cultivating multi-epidermal hair Wucai varieties and is of great significance to the genetic breeding of Wucai.

[0029] In order to have a clearer understanding of the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification.

[0030] It should be noted that most wucai are hairless or have a certain amount of epidermal hairs on the leaf margins, veins, and petioles; their epidermal hairs are single-celled, unbranched, and do not have glandular stings. According to the density of epidermal hairs, they can be divided into more epidermal hairy wucai or less epidermal hairy wucai.

[0031] According to the results of the previous trait survey, in this application, the black vegetable varieties with more than or equal to 15 epidermal hairs per square centimeter of leaf margin, veins, and petiole are classified as more epidermal hairy black vegetable varieties, while the ones with less than 15 are classified as less epidermal hairy black vegetable varieties.

[0032] Example 1

[0033] This embodiment provides a method for developing molecular markers for identifying the density type of epidermal hairs of Wucai, the method comprising:

[0034] A hybrid of a Wucai variety with more epidermal hair, W7-1, as the female parent and a Wucai variety with less epidermal hair, W7-2, as the male parent, was used to produce the F1 generation, which was then self-pollinated to obtain the F2 generation plants. The leaf epidermal hair conditions of W7-1 and W7-2 were referenced to Figure 1 .

[0035] Phenotypic identification was performed on 2224 F2 generation plants, and it was found that 1709 plants had more epidermal hairs and 515 plants had less epidermal hairs, with a ratio of 3.32:1. The chi-square test (chi-square value = 2.067 < 3.841, P > 0.05, not significant) was consistent with the genetic segregation ratio of 3:1, indicating that the more epidermal hairs trait of Wucai is a dominant trait controlled by a single gene.

[0036] On this basis, 73 strains with more epidermal hairs and 112 strains with less epidermal hairs from the F2 population, as well as 14 strains with less epidermal hairs and 16 strains with more epidermal hairs from the parents were selected to construct mixed pools, and bulked-segregant analysis combined with whole genome resequencing (BSA-seq) was performed. The candidate gene was preliminarily located in the 19.13Mb (8457953-27595719) interval of chromosome A06.

[0037] Based on BSA-seq, we further developed 26 Indel molecular markers for the homozygous insertion / deletion sites of W7-1 and W7-2, and conducted genotype-phenotype linkage analysis on 1710 F2 population lines, narrowing the interval where the candidate genes are located to 120Kb. By comparing with the homologous genes of Brassica rapa and the annotation information, we found that 120Kb contained 21 genes, and only one gene, BrCPR5 (CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5), had a homozygous mutation. Figure 4 .

[0038] We further cloned the CDS sequence of BrCPR5. The results of sequence comparison showed that the less epidermal variety W7-2 had a 43 bp Indel (named Indel37355) in its 5'UTR region compared with the more epidermal variety W7-1. The corresponding nucleotide sequence is shown in SEQ ID NO.3.

[0039] Corresponding Indel37355 molecular marker primers were developed for the above-mentioned Indel37355. The Indel molecular marker primers include an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2.

[0040] The above-mentioned Indel37355 molecular marker primers were used to perform PCR amplification on the aforementioned Wucai material DNA, and the amplification results were statistically analyzed.

[0041] After statistics, it was found that the multi-epidermal hairy black cabbage contained a complete 281bp (SEQ ID NO.4, 84bp-126bp in the nucleotide sequence were the aforementioned 43bp content) nucleotide sequence, while the less epidermal hairy black cabbage lacked the aforementioned 43bp nucleotide sequence, and the corresponding amplification result was only one band and the nucleotide sequence length of the band was 238bp (SEQ ID NO.5) (reference Figure 2 When the gel electrophoresis result shows only one specific band of 281bp in length, the corresponding genotype of the Wucai variety is a homozygous genotype with more epidermal hairs; when the gel electrophoresis result shows two specific bands of 281bp and 238bp in length respectively, the corresponding genotype of the Wucai variety is a heterozygous genotype with more epidermal hairs; when the gel electrophoresis result shows only one specific band of 238bp in length, the corresponding Wucai variety is a homozygous genotype with less epidermal hairs).

[0042] Based on the above characteristics, the present application proposes a method for identifying the type of density of epidermal hairs of Wucai, as shown in Example 2.

[0043] Example 2

[0044] This embodiment provides a method for identifying the type of hair density on the surface of Wucai, the method comprising:

[0045] (1) Obtaining DNA from Wucai leaves;

[0046] Using the DNA of leaves of a single plant of Wuling in the seedling stage as a template, Indel molecular marker primers were designed to amplify the molecular marker Indel37355.

[0047] Wherein, the Indel molecular marker primers include an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2;

[0048] SEQ ID NO.1: 5'-AAGTTCCAAATTGACCCCATAAG-3';

[0049] SEQ ID NO.2: 5'-CGAGTAGAATTGAGATTAGAGGCAG-3';

[0050] (2) PCR reaction system: total volume 10 μl, components as shown in Table 1:

[0051] Table 1

[0052]

[0053] PCR amplification procedure:

[0054] 1 = 95.0°C FOR 3:00;

[0055] 2 = 95.0 °C FOR 0:15;

[0056] 3 = 60.0 °C FOR 0:15;

[0057] 4 = 72.0 °C FOR 0:30;

[0058] 5=GO TO 2,28×

[0059] 6=72.0℃FOR 5:00

[0060] 7=4.0℃FOREVER

[0061] 8=END

[0062] (3) 8% polyacrylamide gel electrophoresis detection

[0063] 1) Preparation of 8% polyacrylamide gel: 5×TBE: 37.5ml; 40%AB (29:1): 36ml; TEMED: 150μl; H2O: 106.5ml. Pour the mother liquid into the beaker twice, add 40ml mother liquid to the base gel, add 300μLAPS (10%), pour into the glass plate, and let it stand for 30min. After the base gel solidifies, take 110ml mother liquid, add 825μl APS (10%), pour into the glass plate, insert the comb, and let it stand for 90min.

[0064] 2) Vertical electrophoresis: 175V, 90min;

[0065] 3) Fixation: 600 ml H2O, 75 ml anhydrous ethanol, 4.5 ml glacial acetic acid, shake on a horizontal decolorizing shaker for 6 minutes;

[0066] 4) Silver staining: 0.6 g silver nitrate, 600 ml H2O, shake on a horizontal decolorizing shaker for 5 min;

[0067] 5) Color development: 9g sodium hydroxide, 600ml H2O, 1ml formaldehyde, shake on a horizontal decolorizing shaker for 5-15min;

[0068] 6) Recording: Place the polyacrylamide gel on a white light plate and take a photo with a camera to record the band positions;

[0069] (4) 2.5% agarose gel electrophoresis;

[0070] 1) Preparation of 2.5% agarose gel: Agarose 1g, 0.5×TBE 40ml, microwave for three times, add nucleic acid dye, pour into mold, insert comb, and let stand for 30 minutes;

[0071] 2) Horizontal electrophoresis: 180V, 20min;

[0072] 3) Gel imaging: record the band types on a gel imager.

[0073] This example provides a functional verification of the aforementioned Indel molecular marker primers using 2.5% agarose gel electrophoresis. The verification process is as follows:

[0074] (1) DNA from four varieties of hairy Chinese cabbage with more epidermis, W7-1, W7-1♀×W7-2, HY7-2 and SW-4, one variety of hairy Chinese cabbage, SW-1, and three varieties of hairy Chinese cabbage with less epidermis, W7-2, W16-19-5 and W18-1, were used as templates.

[0075] (2) PCR amplification was performed using the following Indel molecular marker primers (the amplification system is as described above).

[0076] SEQ ID NO.1: 5'-AAGTTCCAAATTGACCCCATAAG-3';

[0077] SEQ ID NO. 2: 5'-CGAGTAGAATTGAGATTAGAGGCAG-3'.

[0078] (3) Detection was performed using 2.5% agarose gel electrophoresis (the detection process and method are described above). The method is as shown above.

[0079] (4) After gel imaging, Figure 3 There are two specific bands of 238bp and 281bp. The variety with only one specific band of 281bp is a homozygous Wucai variety with more epidermal hairs. The variety with both specific bands of 238bp and 281bp is a heterozygous Wucai variety with more epidermal hairs. The variety with only one specific band of 238bp is a homozygous Wucai variety with less epidermal hairs.

[0080] That is, when the amplification result contains a specific band of 281 bp, the corresponding type of density of the epidermal hairs of the black vegetable is a variety with more epidermal hairs, while when it only has a specific band of 238 bp, the corresponding type of density of the epidermal hairs of the black vegetable is a variety with less epidermal hairs.

[0081] In summary, the Indel molecular marker related to the density type of epidermal hairs of Wucai and its application provided in this application have the following beneficial effects compared with the prior art:

[0082] 1. It can be used to quickly identify the corresponding epidermal hair density type of Wucai varieties, improving the identification efficiency;

[0083] 2. It is of great significance to cultivate new varieties of multi-epidermal hairy cabbage and facilitate genetic breeding.

[0084] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0085] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An application of an Indel molecular marker primer in identifying or assisting in identifying the density type of epidermal hairs of Wucai, characterized in that: The Indel molecular marker primers include an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

2.

2. A kit for use in identifying or assisting in identifying the type of hair density on the epidermis of Wucai, characterized in that: The kit comprises the Indel molecular marker primers described in claim 1.

3. The use of a kit according to claim 2 in identifying or assisting in identifying the type of hair density on the epidermis of Wucai, characterized in that: The kit also includes reagents required for PCR reaction and electrophoresis.

4. A method for identifying the density type of Wucai epidermal hairs, characterized in that: The method is carried out using the Indel molecular marker primer described in claim 1 or the kit described in claim 2 or 3.

5. The method according to claim 4, characterized in that The method comprises: Obtaining DNA from Wucai leaves; Using the Indel molecular marker primers described in claim 1 to perform PCR amplification on the DNA of Wucai leaves; The amplification results were detected by gel electrophoresis; The density type of epidermal hairs of the corresponding Wucai was determined according to the gel electrophoresis test results; when the gel electrophoresis results contained a specific band with a length of 281bp, the corresponding Wucai variety was a Wucai variety with more epidermal hairs; when the gel electrophoresis results contained only one specific band with a length of 238bp, the corresponding Wucai variety was a Wucai variety with less epidermal hairs.

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