CN112725426B - Specific SCAR molecular marker for sex identification of cantaloupe, primer group, kit, identification method and application of specific SCAR molecular marker - Google Patents
Specific SCAR molecular marker for sex identification of cantaloupe, primer group, kit, identification method and application of specific SCAR molecular marker Download PDFInfo
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Abstract
The invention discloses a specific SCAR molecular marker, a primer group, a kit, an identification method and application thereof for sex identification of the cantaloupe, which belong to the technical field of plant molecular genetics, wherein the cantaloupe is a hermaphrodite plant taking seeds as medicinal materials, the utilization value of the cantaloupe female plant is higher, sex is difficult to distinguish by adopting morphological and physiological methods before flowering, and the invention utilizes RAPD and SCAR molecular marker technology to research molecular markers related to the sex research of the cantaloupe; on the basis of establishing an optimal reaction system for RAPD amplification of the cantaloupe, a female-related molecular identification marker is found, and the SCAR molecular marker stably amplifies a specific band with the size of 423bp only in a female single plant; the obtained 1 SCAR molecular marker related to female gender realizes the rapid and accurate identification of the cantaloupe with different sexes, and is used for identifying plants with unknown seedling stage sexes and detecting large-scale germplasm materials.
Description
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 cantaloupe.
Background
The cantaloupe (Herpetospermum pedunculosum (Ser.) C.B.Clarke), tibetan language called Golgi Duoduo, belongs to the family Cucurbitaceae, belongs to annual climbing herbaceous plants, mainly produces the high altitude areas with the altitude of 2300-3500m in Sichuan, yunnan, tibet and the like at the east of Qinghai-Tibet plateau, and the dried and mature seeds contain rich fatty acid, lignan, polysaccharide, amino acid and microelements, have the functions of clearing heat, detoxicating and softening liver, and particularly contain lignan compounds which can obviously reduce immune liver injury and are mainly applied to the treatment of anti-hepatitis B in modern medicine. In view of the unique curative effect of the borygmus dorsalis in liver disease treatment, the research on various aspects of application of the borygmus dorsalis is increasingly carried out at home and abroad, and the previous research on the borygmus dorsalis is mainly focused on aspects of tissue culture, cultivation technology, extraction, separation and purification of specific active ingredients, medicinal function research and the like. The bordeaux is a hermaphrodite plant taking seeds as medicinal materials, in production practice, in order to improve the resource utilization rate of the bordeaux, save resources such as manpower, material resources, land and the like, ensure the seed yield, the economic value of female plants is higher than that of male plants, but the sex of the female plants is difficult to distinguish before flowering, and the identification system for the sex of the bordeaux in the seedling stage is still immature, so that the development of early identification of the sex of the bordeaux is of great significance in production practice.
Aiming at the phenomenon that the male and female values of the cantaloupe are large in differentiation and the morphology is very similar in nutrition period, practical basis and technical guidance are provided for artificial cultivation as soon as possible, and the selection of the sex identification method is very important. The existing plant sex identification methods are mostly based on the external morphology, physiological and biochemical differences, chromosome sets, isozymes maps, specific protein content and nucleotide differences of male and female plants, and the methods are mostly used for sex difference research on mature individuals, and are difficult to identify the sex by adopting morphological or cytological methods before flowering. With the development of molecular biology, based on the advantage that a molecular marking method is not influenced by external environmental factors, more and more molecular markers (such as RAPD, SSR, ISSR, SRAP, AFLP and the like) are applied to early sex identification research of plants, in particular to a characteristic sequence amplification region (sequence characterized amplified regions, SCAR) marking technology, which is derived from RAPD molecular markers 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 molecular markers (such as SCAR markers) closely linked with the sex-determining genes of the cantaloupe are used for auxiliary selection, the sex can be rapidly and accurately distinguished in the seedling stage, and the method 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 the cantaloupe.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a specific SCAR molecular marker for sex identification of Herpetum erinaceus is a DNA molecule with a nucleotide sequence shown as SEQ ID NO1.
Based on the advantage of easy operation of RAPD technology, the molecular marker technology has become a common hermaphrodite plant molecular identification technology. However, RAPD is sensitive to reaction conditions, and has poor repeatability and stability, so that the establishment of a stable and optimal RAPD reaction system is the basis of carrying out male and female identification research by utilizing RAPD molecular markers.
Because SCAR marks are insensitive to reaction conditions, the repeatability is good, the SCAR marks are not influenced by external environment factors, and more information sites are provided compared with RAPD. The RAPD marker is converted into the SCAR marker to carry out early sex identification of plants, so that more stable, reliable and accurate identification results can be obtained. Therefore, the invention firstly defines the optimal reaction system of the RAPD-SCAR-PCR reaction of the borduring melon, and successfully converts a SCAR molecular marker which can be used for stabilizing female-related male identification of the borduring melon on the basis of the optimal reaction system, and the marker amplified band only appears stably in female single plants. In addition, the SCAR molecular marker obtained by the invention can be widely used for identifying plants with unknown sex in seedling stage in later period, and assisted identification is carried out by combining morphology and photo-biological test (male plants have higher photosynthetic efficiency than female plants), so that the accuracy and reliability of the developed molecular marker are ensured.
The invention has the beneficial effects that:
the molecular marker, the primer and the reaction system can stably and rapidly identify the sex of the cantaloupe, can distinguish the sex before the cantaloupe flowers, have important significance for early sex identification, hybridization breeding and the like of the cantaloupe, and have higher guiding value for practical production of the cantaloupe.
Drawings
FIG. 1 shows the effect of Taq enzyme amount (A), dNTPs (B), template DNA (C), primer concentration (D), cycle number (E) and annealing temperature (F) on PCR, respectively (M: AL2000 DNAlader);
FIG. 2 shows the amplification results of RAPD primer amplification of the male and female DNA pools of Pimpinella Brachycarpa (M: AL2000DNA ladder);
FIG. 3 shows the result of amplification between male and female individuals of the RAPD primers S15 (A) and S94 (B) (M: AL2000DNA ladder);
FIG. 4 shows the amplification results of SCAR primer HP-94 between the male and female wells and the male and female single samples of the Herpetum poinsettii (M: AL2000DNA ladder;1, female well; 12, male well; 2-11, female single sample; 13-22, male single sample).
Detailed Description
Embodiments of the present invention are described in detail below. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not 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 sequence of the 423bp female SCAR molecular marker 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
Fresh, pest-free and disease-spot-free young leaves of the Begonia microphylla are collected at a national Tibetan medicine material planting demonstration base in a Tibetan forest-glossy ganoderma area and used as test materials, sex is noted, the fresh, pest-spot-free and disease-spot-free young leaves are collected and put into a self-sealing bag filled with silica gel, and the self-sealing bag is brought back to a laboratory and put into a refrigerator for preservation at the temperature of minus 20 ℃ for standby.
1.2 reagents
Plant genomic DNA extraction kits (DP 305-02) and Taq DNA Polymerase were purchased from Tiangen Biochemical (Beijing) technologies, inc., polyacrylamide gel DNA recovery kit (D1250) was purchased from Soy Bao Biotechnology, inc., pBLUE-T rapid cloning kit (ZC 204) was purchased from Beijing village allied biosystems, inc., and PCR primer synthesis and gene sequencing were performed by Beijing Optimago, inc.
1.3 test methods
1.3.1DNA extraction and construction of Male and female DNA pool
Extracting the genomic DNA of the leaf blade of the cantaloupe by using a plant genomic DNA extraction kit of the Tiangen company, 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 mixed segregation population analysis (BSA), 10 parts of DNA of each single sample of female/male germplasm materials are mixed in equal quantity respectively, and the construction of a female or male DNA sample pool is completed.
Optimization of 1.3.2RAPD reaction System
PCR optimization of the rapD system of the rapD is sequentially performed on Taq DNA polymerase, dNTPs, template DNA concentration, primer concentration, cycle times and annealing temperature by taking adult rapD with known gender as a template, so that the stability of the amplification of the rapD is ensured. RAPD primers (Table 1) were synthesized from Beijing Liuhua big Gene Inc. and the RAPD system optimization parameters are shown in Table 2.
1.3.3 screening of Male and female DNA polymorphism detection and molecular identification markers of Herpetum Willemottle
And (3) taking the male and female DNA reaction tanks constructed as above as templates, carrying out RAPD amplification by using 103 random primers one by one according to the optimized RAPD optimal reaction conditions (table 3), separating amplified products by 8% polyacrylamide gel electrophoresis, and observing and photographing for preservation by a gel imaging analyzer after ethidium bromide staining.
1.3.4 development and identification of SCAR molecular markers of male and female plants of Herpetum recited in the genus Herpetum
1.3.3 RAPD male and female specific fragments were recovered using a polyacrylamide gel DNA recovery kit (D1250) from Sorption Biotechnology Co., ltd., followed by T cloning of the fragments using a pBLUE-T rapid cloning kit, and specific primers were designed after DNA sequencing from Beijing Trignoto Biotechnology Co., ltd. And (3) carrying out PCR amplification of a specific primer by taking DNA of male and female plants of the Herpetum erinaceus with definite gender as a template (Table 4), and verifying whether the specific band amplification primer can be successfully converted into SCAR molecular markers for male and female identification of the Herpetum erinaceus.
TABLE 1RAPD primer sequences
TABLE 2 influence factors of RAPD reaction System optimization experiments
TABLE 3 RAPD reaction System after optimization
TABLE 4 RAPD-SCAR reaction system
2 results and analysis
2.1 optimization of RAPD reaction System
The pre-experimental study shows that RAPD pattern strips with the genome 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 can cause great differences in amplified patterns due to different reaction systems, so that RAPD optimization experiments are carried out by taking the reaction as a study object (figure 1). The concentration of Taq DNA polymerase directly affects the result of amplification, and too high will result in small fragments, too low will result in few products and weak bands, 4. Mu.l, 6. Mu.l, 8. Mu.l of Taq enzyme (5U/. Mu.l) amplified product shown in FIG. 1A is evident, so the enzyme concentration of 4. Mu.l is chosen from the viewpoint of cost saving. As can be seen from FIG. 1B, the bands are clear and reproducible at a volume of 1.6. Mu.l, and thus the concentration of 1.6. Mu.l dNTPs (2.5 mmol/L) is optimal. When the concentration of the template DNA is too low, the amplified band is unstable and even no band is amplified. The template DNA (2. Mu.l of template DNA at a concentration of 1.25 to 20 ng/. Mu.l) diluted 10-fold as shown in FIG. 1C was initially amplified to give a stable band. Primer concentration is one of the important factors affecting PCR reaction, and too low a concentration will not amplify the band, and too high a concentration will nonspecifically amplify the small fragment and primer dimer. When the primer (10. Mu. Mol/L) was 1.5. Mu.l, a stable and clear band was amplified (FIG. 1D), so that 1.5. Mu.l was initially used as the optimal volume of the primer for the RAPD reaction system of the Piano-cantaloupe. In RAPD reaction, if the PCR amplification cycle times are too small, the reaction products are few, and the bands are not obvious. The results in FIG. 1E show that when only 25 cycles were performed, the reaction product was significantly lower than the other 4 conditions, while 35 cycles were started with no significant difference in strip brightness and number, and 35 cycles were selected to increase machine utilization. The primers used for RAPD are typically 10bp in length, so the annealing temperature is no higher than 40℃and in FIG. 1F 32℃35℃is more clear, whereas high temperatures reduce non-specific binding between the primers and the template, and 35℃is therefore better than 32 ℃.
And (3) combining the analysis results to determine the reaction system of the RAPD-SCAR-PCR amplification reaction of the cantaloupe, wherein each 20 mu L of the reaction system comprises: 10 XBuffer 2. Mu.L, forward primer 0.75. Mu.L, reverse primer 0.75. Mu.L, template DNA 2. Mu.L, taq DNA polymerase 4. Mu.L, dNTPs 1.6. Mu.L, ddH2O, the remainder, the concentration of 10. Mu.mol/L, the concentration of 1.25-20 ng/. Mu.L. The optimal RAPD amplification procedure is: pre-denaturation at 95℃for 3min; the following cycle was then repeated 35 times: denaturation at 95℃for 30s, renaturation at 35℃for 30s, extension at 72℃for 1min; finally, the extension is carried out for 5min at 72 ℃.
2.2 detection of male and female DNA polymorphism of Herpetum Willemotschia, and screening of molecular identification markers
And (3) taking genome DNA of the hybrid male and female cantaloupe pools as a template, carrying out RAPD amplification by using 103 random primers one by one under the optimized RAPD optimal reaction condition, and screening male and female plant molecular identification markers. Through RAPD amplification profiling, a total of 2 potential molecular identification markers were screened (fig. 2). As can be seen from FIG. 2, the band indicated by the RAPD result combined by the primers S15 and S94 is likely to be the sequence of the male and female molecular identification mark, and the male and female single PCR verification shows that the molecular mark identification result is consistent with the male and female mixed pool PCR result (FIG. 3), which shows that the amplification stability of the primers S15 and S94 is high.
2.3 development and identification of SCAR molecular markers of the sex of the Pelargonium
And (3) recovering the male and female specific bands amplified by the primers S15 and S94 by using a polyacrylamide gel DNA recovery kit, cloning and sequencing by using a T vector to obtain related sequences respectively and analyzing (mainly aiming at S94 molecular markers because the subsequent SCAR marker conversion of the S15 amplified bands fails). The S94 female specific band nucleic acid sequence was searched for homologous sequences by BLASTN using NCBI nucleic acid database. 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 strip. PCR amplification of the DNA of male and female plants of the Herpetum Willemottle with the pair of primers gave a 423bp female SCAR molecular marker, whereas in all 10 male individuals, the corresponding band was absent (FIG. 4). Therefore, the S94 SCAR molecular marker (SEQ. ID. NO1) obtained by the invention can finish the identification of the male and female strains of the cantaloupe, and shows that the developed SCAR marker has good male and female distinguishing capability.
Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention, but not for limiting the same, and although the technical solution of the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the present invention, and all such modifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of adult
<120> specific SCAR molecular marker, primer set, kit, identification method and application thereof for sex identification of Pelargonium
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 423
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ggatgagacc acgaattaaa aatgtctgga aaagctcgat tgcgatttcg cgaggcaatc 60
acatcgatgt aatgaaagtg agggacctac aacaatcaca gaacgtcctg aataatcgac 120
ccgtttgcca agcagagtct cacgaaatct tccctctttg ccttcaatta catcggaaaa 180
cgacttgtaa accttattat ggccatccct cattggttgt ccgcggattc cattatcaag 240
aagtgtatcc acggcttctt gtaccaattt ctcctgacac attactaatt ctcctggcgt 300
agatctactg gttgttaata gatcaataag agtattgttc cgatagataa ctcttctata 360
gagttcatta atatccgagc tcattagcct acccccatct atctgaatga tcggtctcat 420
cca 423
<210> 2
<211> 17
<212> DNA
<213> Artificial sequence (Artificial Sequence)
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
Claims (1)
1. A specific SCAR molecular marker for sex identification of the cantaloupe is characterized by being a DNA molecule with a nucleotide sequence shown as SEQ ID NO. 1.
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