阅读说明：本技术 一种用于波棱瓜性别鉴定的特异性scar分子标记、引物组、试剂盒、鉴定方法及其应用 (Specific SCAR molecular marker, primer group, kit, identification method and application thereof for sex identification of pedunculate herpetospermum ) 是由 赵琦 谢坤 王宇成 赵锋 詹文瑶 于 2021-01-28 设计创作，主要内容包括：本发明公开了一种用于波棱瓜性别鉴定的特异性SCAR分子标记、引物组、试剂盒、鉴定方法及其应用,属于植物分子遗传学技术领域,波棱瓜是以种子为药用材料的雌雄异株植物,波棱瓜雌株的利用价值更大,在开花前采用形态学和生理学方法很难分辨性别,本发明利用RAPD和SCAR分子标记技术研究与波棱瓜性别研究相关的分子标记；在确立波棱瓜RAPD扩增最佳反应体系的基础上,找到了雌性相关的分子鉴定标记,该SCAR分子标记仅在雌性单株稳定扩增一条大小为423bp的特异条带；本发明获得的1个与雌性性别相关的SCAR分子标记实现了对不同性别波棱瓜的快速、准确的鉴定,将用于鉴定苗期性别未知的植株以及大规模种质材料的检测。(The invention discloses a specific SCAR molecular marker, a primer group, a kit, an identification method and application thereof for sex identification of herpetospermum pedunculosum, belonging to the technical field of plant molecular genetics.A herpetospermum pedunculosum is a female and male heterotrophic plant taking seeds as medicinal materials, the utilization value of female herpetospermum pedunculosum is higher, and the sex is difficult to distinguish by adopting morphological and physiological methods before flowering; on the basis of establishing an optimal reaction system for amplification of the herpetospermum pedunculosum RAPD, a molecule identification mark related to females is found, and the SCAR molecule mark only stably amplifies a specific band with the size of 423bp in a female single plant; the 1 SCAR molecular marker related to female sex obtained by the invention realizes the rapid and accurate identification of the herpetospermum pedunculosum of different sexes, and can be used for identifying plants with unknown sex at the seedling stage and detecting large-scale germplasm materials.)

1. A specific SCAR molecular marker for sex identification of the herpetospermum pedunculosum is characterized by being a DNA molecule with a nucleotide sequence shown in SEQ ID.NO1.

2. A primer group for amplifying the molecular marker of claim 1 and identifying the sex of the herpetospermum pedunculosum, wherein the primer group comprises a forward primer with a nucleotide sequence shown as SEQ ID.NO2 and a reverse primer with a nucleotide sequence shown as SEQ ID.NO3.

3. A kit for sex determination of pedunculate herpetospermum which comprises the primer set according to claim 2.

4. The identification method of the specific SCAR molecular marker for sex identification of the herpetospermum pedunculosum is characterized by comprising the following steps:

1) extracting genome DNA of a leaf of the pedunculate herpetospermum fruit to be detected;

2) taking the genomic DNA of the leaf of the cantaloupe as a template, and carrying out RAPD-SCAR-PCR amplification reaction by using a primer group with a nucleotide sequence shown as SEQ.ID.NO2 and SEQ.ID.NO3 to obtain an amplification product;

3) carrying out gel electrophoresis detection on the amplification product;

4) observing an electrophoresis result, and if a target band of 423bp exists, determining the detected pedunculate herpetospermum fruit to be a female plant; if the target band of 423bp does not exist, the detected herpetospermum pedunculosum is a male plant.

5. The method for identifying the specific SCAR molecular marker for sex identification of the herpetospermum pedunculosum according to claim 4, wherein the reaction system of RAPD-SCAR-PCR amplification reaction comprises every 20 μ L: 10 x buffer2 mul, forward primer 0.75 mul with concentration of 10 mul mol/L, reverse primer 0.75 mul with concentration of 10 mul mol/L, template DNA2 mul with concentration of 1.25-20 ng/mul, Taq DNA polymerase 4 mul with concentration of 5U/mul, dNTPs 1.6 mul with concentration of 2.5mmol/L, and ddH2O for the rest.

6. The method for identifying the specific SCAR molecular marker for sex identification of the herpetospermum pedunculosum according to claim 4, wherein the RAPD-SCAR-PCR amplification procedure is as follows: pre-denaturation at 95 ℃ for 3 min; the following cycle was then repeated 35 times: denaturation at 95 deg.C for 30s, renaturation at 35 deg.C for 30s, and extension at 72 deg.C for 1 min; finally, extension is carried out for 5min at 72 ℃ to obtain an amplification product.

7. The method for identifying the specific SCAR molecular marker for sex identification of the herpetospermum pedunculosum according to claim 4, wherein the detected herpetospermum pedunculosum leaves are selected from fresh herpetospermum pedunculosum young leaves without insect spots and disease spots.

8. The method for identifying the specific SCAR molecular marker for sex identification of the herpetospermum pedunculosum according to claim 4, wherein the step 3) comprises the following steps: separating the amplification product obtained in the step 2) by using 8% polyacrylamide gel electrophoresis, and observing the amplification product by using a gel imaging analyzer after staining the amplification product by using ethidium bromide.

9. Use of a specific SCAR molecular marker as defined in claim 1 or a primer set as defined in claim 2 or a kit as defined in claim 3 or an identification method as defined in any one of claims 4 to 8 for sex identification of a herpetospermum pedunculosum or breeding.

Technical Field

The invention belongs to the technical field of plant molecular genetics, and particularly relates to a specific SCAR molecular marker, a primer group, a kit, an identification method and application thereof for sex identification of pedunculate herpetospermum.

Background

The Herpetospermum pedunculosum (Ser.) C.B.Clarke is named as Ergelsmi in Tibetan language, belongs to the cucurbitaceae Herpetospermum, belongs to annual climbing herbaceous plants, mainly produces high altitude areas with elevation of 2300 and 3500m, such as Sichuan, Yunnan, Tibet and the like in the eastern part of the Qinghai-Tibet plateau, and dry and mature seeds of the Herpetospermum pedunculosum contain rich fatty acid, lignan, polysaccharide, amino acid and trace elements, have the functions of clearing heat, detoxifying and softening liver, particularly contain lignan compounds which can obviously reduce immunological liver injury, and are mainly applied to the treatment of hepatitis B in modern medicine. In view of the unique curative effect of the pedunculate herpetospermum fruit in treating liver diseases, research on the application of the pedunculate herpetospermum fruit is increasing at home and abroad, and the previous research on the pedunculate herpetospermum fruit mainly focuses on the aspects of tissue culture, cultivation technology, extraction, separation and purification of specific active ingredients, medicinal function research and the like. The herpetospermum pedunculosum is a hermaphrodite plant taking seeds as medicinal materials, in the production practice, in order to improve the utilization rate of herpetospermum pedunculosum resources, save resources such as manpower, material resources, land and the like, and ensure the yield of seeds, the economic value of a female plant is higher than that of a male plant, but the sex of the female plant before flowering is difficult to distinguish, and the identification system of the sex of the female plant in the seedling stage is still not mature, so the development of early sex identification of the herpetospermum pedunculosum has important significance in the production.

Aiming at the phenomena that the male and female values of the pedunculate herpetospermum pedunculosum are differentiated greatly and the forms during nutrition are extremely similar, the selection of the sex identification method is particularly important in order to provide practical basis and technical guidance for artificial cultivation as soon as possible. At present, methods for identifying plant sex are mostly based on differences of external morphology, physiological and biochemical differences, chromosome group types, isozyme maps, specific protein content and nucleotide differences of male and female plants, but most of the methods are used for carrying out sex difference research on mature individuals, and before flowering, the sex is difficult to identify by adopting a morphological or cytological method. With the development of molecular biology, based on the advantage that a molecular marker method is not affected by external environmental factors, more and more molecular markers (such as RAPD, SSR, ISSR, SRAP, AFLP and the like) are applied to early sex determination research of plants, in particular to a Sequence Characterized Amplified Regions (SCAR) marker technology which is derived from the RAPD molecular marker and has the advantages of insensitivity to reaction conditions, high specificity, good repeatability, stable and reliable identification result and the like.

Therefore, in the artificial cultivation process, if the molecular marker (such as SCAR marker) closely linked with the sex determination gene of the pedunculate herpetospermum is used for auxiliary selection, the male and female sex can be rapidly and accurately distinguished in the seedling stage, which has very important application value for production practice.

Disclosure of Invention

The invention aims to provide a specific SCAR molecular marker, a primer group, a kit, an identification method and application thereof for sex identification of herpetospermum pedunculosum.

In order to achieve the purpose, the invention adopts the technical scheme that:

a specific SCAR molecular marker for sex identification of the herpetospermum pedunculosum is a DNA molecule with a nucleotide sequence shown in SEQ.ID.NO1.

A primer group for identifying sex of the herpetospermum pedunculosum for amplifying molecular markers comprises a forward primer with a nucleotide sequence shown as SEQ ID.NO2 and a reverse primer with a nucleotide sequence shown as SEQ ID.NO3.

A kit for sex determination of the herpetospermum pedunculosum comprises the primer group.

A method for identifying a specific SCAR molecular marker for sex identification of pedunculate herpetospermum comprises the following steps:

1) extracting genome DNA of a leaf of the pedunculate herpetospermum fruit to be detected;

2) taking the genomic DNA of the leaf of the cantaloupe as a template, and carrying out RAPD-SCAR-PCR amplification reaction by using a primer group with a nucleotide sequence shown as SEQ.ID.NO2 and SEQ.ID.NO3 to obtain an amplification product;

3) carrying out gel electrophoresis detection on the amplification product;

4) observing an electrophoresis result, and if a target band of 423bp exists, determining the detected pedunculate herpetospermum fruit to be a female plant; if the target band of 423bp does not exist, the detected herpetospermum pedunculosum is a male plant.

Further, the reaction system of RAPD-SCAR-PCR amplification reaction comprises every 20 mu L: 10 x buffer2 μ L, forward primer 0.75 μ L with concentration of 10 μmol/L, reverse primer 0.75 μ L with concentration of 10 μmol/L, template DNA2 μ L with concentration of 1.25-20 ng/μ L, Taq DNA polymerase 4 μ L with concentration of 5U/μ L, dNTPs 1.6 μ L with concentration of 2.5mmol/L, and ddH2O for the rest.

Further, the RAPD-SCAR-PCR amplification program is as follows: pre-denaturation at 95 ℃ for 3 min; the following cycle was then repeated 35 times: denaturation at 95 deg.C for 30s, renaturation at 35 deg.C for 30s, and extension at 72 deg.C for 1 min; finally, extension is carried out for 5min at 72 ℃ to obtain an amplification product.

Furthermore, the tested pedunculate herpetospermum pedunculosum leaves are selected from fresh and non-insect-and-disease-spot pedunculate herpetospermum pedunculosum young leaves in national Tibetan medicine planting demonstration base of Tibetan glossy ganoderma.

Further, step 3) comprises the following steps: separating the amplification product obtained in the step 2) by using 8% polyacrylamide gel electrophoresis, staining the amplification product by ethidium bromide, observing the amplification product by using a gel imaging analyzer, and photographing and retaining the amplification product.

The specific SCAR molecular marker or primer group or kit or identification method for sex identification of the herpetospermum pedunculosum is applied to sex identification or breeding of the herpetospermum pedunculosum.

Based on the advantage of easy operation of RAPD technology, the molecular marker technology has become a commonly used molecular identification technology for hermaphrodite plants. However, RAPD is sensitive to reaction conditions, and the repeatability and stability are poor, so that establishment of a stable and optimal RAPD reaction system is the basis of utilizing RAPD molecular markers to carry out male and female identification research.

The SCAR marker is insensitive to reaction conditions, has good repeatability and is not influenced by external environmental factors, so that more information sites are provided compared with RAPD. The RAPD marker is converted into the SCAR marker to carry out early sex determination on plants, so that a more stable, reliable and accurate determination result can be obtained. Therefore, the invention firstly defines the optimal reaction system of the herpetospermum pedunculosum RAPD-SCAR-PCR reaction, and successfully transforms a female-related SCAR molecular marker which can be stably used for female and male identification of the herpetospermum pedunculosum on the basis, and the marker amplification band only stably appears in a female individual plant. In addition, the SCAR molecular marker obtained by the invention can be widely used for identifying the plant with unknown sex in the seedling stage at the later stage, and can be used for auxiliary identification by combining the shape and the photosynthetic physiological test (a male plant has higher photosynthetic efficiency than a female plant), so that the accuracy and the reliability of the developed molecular marker are ensured.

The invention has the beneficial effects that:

the molecular marker, the primers and the reaction system can be used for stably and quickly identifying the sex of the herpetospermum pedunculosum, can distinguish male and female before flowering of the herpetospermum pedunculosum, have important significance for early sex identification, crossbreeding and the like of the herpetospermum pedunculosum, and have higher guiding value for practical production of the herpetospermum pedunculosum.

Drawings

FIG. 1 shows the effect of Taq enzyme dosage (A), dNTPs (B), template DNA (C), primer concentration (D), cycle number (E) and annealing temperature (F) on PCR (M: AL2000DNA ladder);

FIG. 2 shows the result of amplification of male and female pools of Goya DNA by RAPD primers (M: AL2000 DNAlladder);

FIG. 3 shows the results of amplification between male and female individuals of the RAPD primers S15(A) and S94(B) (M: AL2000 DNAlladder);

FIG. 4 shows the results of the amplification of SCAR primer HP-94 between male and female of the Goya (M: AL2000 DNAladder; 1, female pool; 12, male pool; 2-11, female sample; 13-22, male sample).

Detailed Description

The following describes embodiments of the present invention in detail. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. All references mentioned herein are incorporated herein by reference. Unless defined to the contrary, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless indicated to the contrary, the techniques used or referred to herein are standard techniques well known to those of ordinary skill in the art. The materials, methods, and examples are illustrative only and not intended to be limiting. The 423bp female SCAR molecular marker sequence described in the following examples is SEQ ID. NO1.

SEQ.ID.NO1：

5'-ggatgagaccacgaattaaaaatgtctggaaaagctcgattgcgatttcgcgaggcaatcacatcgatgtaatgaaagtgagggacctacaacaatcacagaacgtcctgaataatcgacccgtttgccaagcagagtctcacgaaatcttccctctttgccttcaattacatcggaaaacgacttgtaaaccttattatggccatccctcattggttgtccgcggattccattatcaagaagtgtatccacggcttcttgtaccaatttctcctgacacattactaattctcctggcgtagatctactggttgttaatagatcaataagagtattgttccgatagataactcttctatagagttcattaatatccgagctcattagcctacccccatctatctgaatgatcggtctcatcca-3'

Example 1

1 materials and methods

1.1 test materials

Collecting fresh young leaf of fructus Herpetospermi Pueraria Bodinieri without insect spot and disease spot as test material in national Tibetan medicine planting demonstration base in Tibetan Ganoderma, noting sex, collecting, placing into self-sealing bag filled with silica gel, and taking back to laboratory, and storing at-20 deg.C in refrigerator for use.

1.2 reagents

The plant genome DNA extraction kit (DP305-02) and Taq DNApolymerase were purchased from Tiangen Biotechnology (Beijing) science and technology Co., Ltd, the polyacrylamide gel DNA recovery kit (D1250) was purchased from Solebao Biotechnology Co., Ltd, the pBLUE-T rapid cloning kit (ZC204) was purchased from Beijing Zhuang Union International Biogene technology Co., Ltd, and PCR primer synthesis and gene sequencing were completed by Beijing Optimalaceae Biotechnology Co., Ltd.

1.3 test methods

1.3.1 extraction of DNA and construction of Male and female DNA pools

Extracting genome DNA of leaf of the cantaloupe by using a plant genome DNA extraction kit of Tiangen corporation, detecting the integrity of the DNA by using 1% agarose gel electrophoresis, and determining the concentration by using a NanoVue ultramicro spectrophotometer. Based on the principle of a mixed segregation population analysis method (BSA), 10 parts of DNA of a single sample of the female/male germplasm material are respectively mixed in equal quantity to complete the construction of a female or male DNA sample pool.

1.3.2 optimization of RAPD reaction System

Taking adult herpetospermum pedunculosum DNA as a template, and sequentially carrying out PCR optimization on a herpetospermum pedunculosum RAPD system on Taq DNA polymerase, dNTPs, template DNA concentration, primer concentration, cycle times and annealing temperature, thereby ensuring the RAPD amplification stability. The RAPD primers (Table 1) were synthesized from Beijing Liu-He Hua Dageney Co., Ltd., and the RAPD system optimization parameters are shown in Table 2.

1.3.3 detection of polymorphism of male and female DNA of Herpetospermum undulatum and screening of molecular identification marker

And (3) taking the constructed male and female DNA reaction pools as templates, performing RAPD amplification by using 103 random primers one by one according to optimized RAPD optimal reaction conditions (table 3), carrying out electrophoresis separation on amplification products by using 8% polyacrylamide gel, dyeing by using ethidium bromide, observing by using a gel imaging analyzer, and taking pictures for storage.

1.3.4 Crabapple male and female SCAR molecular marker development and identification

The RAPD male and female specific fragment of 1.3.3 is recovered by utilizing a polyacrylamide gel DNA recovery kit (D1250) of Solebao Biotechnology limited, then the fragment is subjected to T cloning by utilizing a pBLUE-T rapid cloning kit, and a specific primer is designed after DNA sequencing of Beijing Optimalaceae Biotechnology limited. Specific primer PCR amplification is carried out by taking the male and female herpetospermum pedunculosum plant DNA with definite sex as a template (Table 4), and whether the specific strip amplification primer can be successfully converted into the SCAR molecular marker for male and female herpetospermum pedunculosum identification is verified.

TABLE 1 RAPD primer sequences

TABLE 2 RAPD reaction System optimization experiment influencing factors

TABLE 3 optimized RAPD reaction System

TABLE 4 RAPD-SCAR reaction System

2 results and analysis

2.1 optimization of RAPD reaction System

Preliminary experiment researches show that RAPD pattern bands with the genomic DNA of the cantaloupe as a template and the S15 as a primer are rich and clear, and the same primer and the same template cause great difference in amplification band types due to different reaction systems, so that RAPD optimization experiments are carried out by taking the reaction as a research object (figure 1). The concentration of Taq DNA polymerase directly affects the amplification result, small fragments are generated when the concentration is too high, few products are generated when the concentration is too low, the band is weak, 4 mu l, 6 mu l and 8 mu l of Taq enzyme (5U/. mu.l) amplification products are obvious as shown in figure 1A, and therefore, the enzyme concentration is 4 mu l from the cost-saving point of view. dNTPs serve as substrates in the PCR reaction, and the probability of mismatch with Taq DNA polymerase is increased due to excessively high final concentration, and as can be seen from FIG. 1B, the band is clear and reproducible when the volume is 1.6. mu.l, so that the concentration of 1.6. mu.l dNTPs (2.5mmol/L) is optimal. When the concentration of the template DNA is too low, the amplified band is unstable and even cannot be amplified. A10-fold dilution of template DNA (2. mu.l template DNA at a concentration of 1.25-20 ng/. mu.l) as shown in FIG. 1C initially amplified a stable band. The primer concentration is one of important factors influencing PCR reaction, and when the concentration is too low, a band cannot be amplified, and when the concentration is too high, a fuzzy small fragment and a primer dimer can be amplified in a non-specific manner. Since a stable and clear band was amplified with 1.5. mu.l of primer (10. mu. mol/L) (FIG. 1D), 1.5. mu.l was initially used as the optimal volume of the primers for the balconia RAPD reaction system. In the RAPD reaction, if the PCR amplification cycle number is too small, the reaction product is less and the band is not obvious. The results in FIG. 1E show that when only 25 cycles were performed, the reaction synthesis products were significantly lower than the other 4 conditions, and no significant difference in band brightness and number was observed at the beginning of 35 cycles, and 35 cycles were selected to improve machine utilization. The primers used for RAPD are typically 10bp in length, so the annealing temperature is no higher than 40 ℃ as shown in FIG. 1F at 32 ℃ and more clearly at 35 ℃ and the high temperature reduces non-specific binding between the primers and the template, so 35 ℃ is better than 32 ℃.

And (3) combining the analysis results, determining that each 20 mu L of the reaction system of the amplification reaction of the RAPD-SCAR-PCR of the herpetospermum pedunculosum comprises: 10 x buffer2 mul, forward primer 0.75 mul with concentration of 10 mul mol/L, reverse primer 0.75 mul with concentration of 10 mul mol/L, template DNA2 mul with concentration of 1.25-20 ng/mul, Taq DNA polymerase 4 mul with concentration of 5U/mul, dNTPs 1.6 mul with concentration of 2.5mmol/L, and ddH2O for the rest. The optimal RAPD amplification procedure is: pre-denaturation at 95 ℃ for 3 min; the following cycle was then repeated 35 times: denaturation at 95 deg.C for 30s, renaturation at 35 deg.C for 30s, and extension at 72 deg.C for 1 min; finally, extension is carried out for 5min at 72 ℃.

2.2 detection of polymorphism of male and female DNA of Herpetospermum undulatum and screening of molecular identification marker

And (3) RAPD amplification is carried out by using 103 random primers one by one under the optimized RAPD optimal reaction conditions by using the genomic DNA of the male and female herpetospermum pedunculosum mixed pool as a template, and then screening the molecular identification markers of male and female plants. 2 potential molecular markers were co-screened by RAPD amplification mapping (FIG. 2). From FIG. 2, it can be seen that the bands indicated by the RAPD result under the combination of the primers S15 and S94 are likely to be the sequence of the male and female molecular identification marker, and the molecular identification result is consistent with the PCR result of the male and female mixed pool (FIG. 3) through single-sample PCR verification of male and female, which indicates that the amplification stability of the primers S15 and S94 is high.

2.3 SCAR molecular marker development and identification of sex of herpetospermum pedunculosum

The male and female specific bands amplified by the primers S15 and S94 are recovered by a polyacrylamide gel DNA recovery kit, and related sequences are obtained and analyzed after T vector cloning and sequencing respectively (the S94 molecular marker is mainly analyzed because the subsequent SCAR marker of the S15 amplified band fails to be transformed). The S94 female specific band nucleic acid sequence was searched for the missing homologous sequence by the NCBI nucleic acid database using BLASTn. In order to convert the S94 label into a more stable SCAR molecular label, a Primer5 Primer design software is used for designing a specific amplification Primer of the specific band, and the sequence of a forward Primer is ggatgagaccgcgcatg; the reverse primer sequence was gatgagacctgttgcacgag. The DNA of the male and female plants of the pedunculate herpetospermum fruit is subjected to PCR amplification by using the pair of primers, a 423bp female SCAR molecular marker is obtained, and the corresponding band does not exist in all 10 male individuals (figure 4). Therefore, the S94 SCAR molecular marker (SEQ. ID. NO1) obtained by the invention can complete the identification of the male and female plants of the herpetospermum pedunculosum, and the developed SCAR marker has good male and female distinguishing capability.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and although the technical solutions of the present invention are described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the present invention, which should be covered by the protection scope of the present invention.

Sequence listing

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