SSR (Simple Sequence Repeat) marker primer group for identifying filial generation of hibiscus varieties and detection method

Abstract

The invention provides an SSR (Simple Sequence Repeat) marker primer group and a detection method for identifying filial generations of hibiscus varieties, and belongs to the technical field of molecular markers, and the SSR marker HBG18 is used for carrying out PCR (Polymerase Chain Reaction) detection on a male parent'hibiscus rosa 'and female parent'chiffon' hybrid combination and a male parent'blue bird 'and female parent'Han-plus' hybrid combination offspring; the SSR marker HBG7 is used for carrying out PCR detection on the hybridized combination of the male parent pink heart double petals and the female parent Chinese chiffon; the SSR marker HBG21 is used for carrying out PCR detection on a hybridized combination of a male parent'common powder 'and a female parent'red heart' and a hybridized combination of a male parent'blue chiffon 'and a female parent'long bract double petal', agarose gel electrophoresis detection shows that the three SSR markers can accurately distinguish hibiscus parents and hybrid filial generations of the hibiscus parents, and the purpose of rapidly identifying the hybrid F1 generation is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular markers, and particularly relates to an SSR marker primer set and a detection method for identifying hybrid offspring of hibiscus varieties. Background Art

[0002] Hibiscus syriacus Linn is a deciduous shrub of the genus Hibiscus in the family Malvaceae. It is also known as the morning glory, vitex, and hedge flower. It is native to central my country and is distributed throughout China except for parts of North China, Northwest China, and Northeast China. There are about 200 species of Hibiscus plants worldwide, 25 of which are distributed in China, including 13 woody plants. Hibiscus blooms in summer and autumn, with a long flowering period. The flowers are large and brightly colored. It is often used as an ornamental tree species for garden flowers and also has medicinal value. Hibiscus has a strong dust-retaining function and is highly resistant to harmful gases such as sulfur dioxide and chlorides. It is often used as a greening tree species for polluted factories.

[0003] At present, the research on hibiscus mainly focuses on breeding technology, genetic diversity and genetic structure. In terms of molecular marker application, it mainly involves the following aspects: 1. Application of SRAP markers to identify kinship, 2. Genetic diversity analysis of hibiscus plants using ISSR molecular markers, 3. Analysis of genetic relationships of hibiscus varieties based on RAPD method, 4. Application of EST-SSR to construct DNA fingerprint of hibiscus varieties. In general, the research on hibiscus molecular markers is relatively lagging, and the existing molecular markers cannot meet the research needs of hibiscus genetic breeding. Hybrid breeding is one of the main ways to create new hibiscus germplasm and cultivate new varieties. The hybrid offspring of hibiscus has rich variation and good ornamental value. The leaves and other nutritional organs of some hybrid offspring individuals have the characteristics of both parents; some individuals have similar morphological characteristics to the mother parent, with no obvious differences, and cannot be identified by morphological characteristics. In addition, the cycle is too long and is affected by growth and development and the environment, which to a certain extent limits the accurate and rapid identification of hybrid offspring. Therefore, there is an urgent need to screen new molecular markers for different parental combinations and establish a fast, accurate and efficient molecular identification technology system for hibiscus hybrid progeny, in order to shorten the breeding cycle, speed up the breeding process, and promote the selection and promotion of hibiscus varieties and high-quality development of the seed industry in my country. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a combination of SSR marker primers and a detection method for identifying the hybrid offspring of hibiscus varieties. The molecular marker technology is not affected by the plant development period and environment, and only requires a trace amount of fresh tissue or silica gel rapid drying tissue samples to accurately and efficiently detect plant varieties or the hybrid offspring (F1) of two parents. Based on transcriptome sequencing technology, simple sequence repeats (SSR) length polymorphic sites suitable for the identification of hibiscus hybrid F1 generation are mined, SSR markers that can be used to identify hybrid offspring are screened, and then the genomic DNA of hibiscus parents and their hybrid F1 generation is used to verify the candidate markers, and markers with good specificity and high resolution are obtained, so as to achieve the purpose of rapid and accurate identification of F1 generation.

[0005] To achieve the above object, the present invention first provides an SSR marker primer set for identifying hybrid offspring of hibiscus varieties, wherein the SSR marker primer set includes HBG7, HBG18, and HBG21;

[0006] The primer set sequence of HBG7 is shown in SEQ ID NO.1 to SEQ ID NO.2;

[0007] The primer set sequence of HBG18 is shown in SEQ ID NO.3 to SEQ ID NO.4;

[0008] The primer set sequence of HBG21 is shown in SEQ ID NO.5 to SEQ ID NO.6.

[0009] Based on a general inventive concept, the present invention also provides a method for detecting an SSR marker primer set for identifying hybrid offspring of hibiscus varieties, comprising the following steps:

[0010] S1. Fresh tissues or silica gel quick-dried tissue samples of the hybrid F1 generation of the male parent of hibiscus, the female parent of hibiscus and the hibiscus variety to be tested are used as the samples to be tested, and the total DNA of the samples is extracted by the modified CTAB method;

[0011] S2, using the total DNA obtained in step S1 as a template and HBG18, HBG21 or HBG7 SSR marker primer set as amplification primers for PCR amplification;

[0012] S3, performing sequence analysis on the amplified product of step S2, and judging whether the F1 generation to be tested is a hybrid F1 generation by whether the amplified product has a specific band. Preferably, when the male parent and the female parent of hibiscus are 'Rose Hibiscus' and 'China Chiffon', respectively, HBG18 is used as the marker primer set, and if the PCR amplified product has the specific bands of the male parent and the female parent, the F1 generation to be tested is a hybrid offspring of 'Rose Hibiscus' and 'China Chiffon'.

[0013] Preferably, when the male and female parents of Hibiscus syriacus are 'Bluebird' and 'Hanbo' respectively, HBG18 is used as the marker primer set. If the PCR amplification product has a specific band of the male parent, the F1 generation to be tested is the hybrid offspring of 'Bluebird' and 'Hanbo'.

[0014] Preferably, when the male and female parents of Hibiscus syriacus are 'Pink Heart Double Petal' and 'China Chiffon' respectively, HBG7 is used as the marker primer set. If the PCR amplification product has specific bands of both the male and female parents, the F1 generation to be tested is the hybrid offspring of 'Pink Heart Double Petal' and 'China Chiffon'.

[0015] Preferably, when the male parent and the female parent of Hibiscus syriacus are 'Plain Pink' and 'Red Heart' respectively, HBG21 is used as the marker primer set. If the PCR amplification product has a specific band of the male parent, the F1 generation to be tested is the hybrid offspring of 'Plain Pink' and 'Red Heart'.

[0016] Preferably, when the male and female parents of Hibiscus syriacus are 'Blue Chiffon' and 'Long-bracted Double Petals' respectively, HBG21 is used as the marker primer set. If the PCR amplification product has a specific band of the male parent, the F1 generation to be tested is the hybrid offspring of 'Blue Chiffon' and 'Long-bracted Double Petals'.

[0017] Preferably, the system for PCR amplification in step S2 is: 1 μL (about 20 ng) of DNA template of hibiscus parent or material to be tested, 1 μL each of 10 μmol / L upstream and downstream primers, 10 μL of 2×Taq Master Mix, 7 μL of ddH2O, and the total volume of the system is 20 μL.

[0018] Preferably, the procedure of the PCR amplification is:

[0019] (1) HBG7-labeled PCR reaction procedure: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 57°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min, 4°C storage;

[0020] (2) HBG18-labeled PCR reaction procedure: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 57.5°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min, 4°C storage;

[0021] (3) HBG21-labeled PCR reaction procedure: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 61.5°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min, 4°C storage

[0022] The identification method of this scheme is as follows:

[0023] The SSR marker HBG18 in this scheme was used to perform PCR detection on the offspring of the hybrid combination of the male parent 'Rose Hibiscus' and the female parent 'China Chiffon', and the hybrid combination of the male parent 'Blue Bird' and the female parent 'Hanbo'. The SSR marker HBG7 was used to perform PCR detection on the hybrid combination of the male parent 'Pink Heart Double Petal' and the female parent 'China Chiffon'. The SSR marker HBG21 was used to perform PCR detection on the hybrid combination of the male parent 'Pufen' and the female parent 'Red Heart', and the hybrid combination of the male parent 'Blue Chiffon' and the female parent 'Long-bract Double Petal'. Detection by agarose gel electrophoresis showed that the three SSR markers could accurately distinguish the hibiscus parents and their hybrid offspring, achieving the purpose of rapid identification of the hybrid F1 generation.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The existing detection technology is morphological identification, which mainly distinguishes through the characteristics of tissues and organs such as leaves, branches, flowers and fruits. It has a long cycle and is limited by time and the growth and development stage of the plant. It cannot meet the needs of rapid and accurate identification of the hybrid F1 generation. This program uses SSR markers to identify the hybrid F1 generation. It is not affected by the plant development period, growth stage and environment. Only a small amount of fresh tissue or silica gel quick-dried tissue samples are needed to accurately and quickly identify the samples to be tested. Under the premise of complete instruments and equipment, the test results can be obtained in only about 4 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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 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.

[0027] Figure 1 The PCR product bands of the paternal parent, maternal parent and progeny amplified by primers HBG18, HBG7 and HBG21 in Experimental Example 1;

[0028] Figure 2 The PCR product bands of the paternal, maternal and progeny amplified by primer HBG21 in Experimental Example 1 are shown. DETAILED DESCRIPTION

[0029] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of the present invention.

[0031] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the examples are commercially available.

[0032] Example 1

[0033] Development of SSR markers

[0034] 1. Experimental Materials

[0035] Young leaves of hibiscus parents and their hybrid F1 generations were collected, totaling 24 materials, for identification, screening and specificity verification of SSR markers;

[0036] 2. Development of SSR markers

[0037] 2.1 RNA extraction

[0038] The total RNA of Hibiscus truncatum genome was extracted using Trizol kit (Invotrigen, USA). The specific method was referred to the kit manual, and 1% agarose gel was used for electrophoresis detection.

[0039] 2.2 Library construction and sequencing

[0040] use The RNA Sample Preparation Kit constructs the library. After enriching the eukaryotic mRNA with polyA tails by magnetic beads with Oligo (dT), the mRNA is cut by ultrasound. The fragmented mRNA is used as a template and random oligonucleotides are used as primers to synthesize the first chain of cDNA in the M-MuLV reverse transcriptase system. The RNA chain is then degraded with RNaseH, and the second chain of cDNA is synthesized with dNTPs as raw materials in the DNA polymerase I system. The purified double-stranded cDNA is end-repaired, A-tailed, and connected to the sequencing adapter. The cDNA of about 200bp is screened with AMPure XPbeads, PCR amplification is performed, and the PCR product is purified again using AMPure XPbeads to finally obtain the library.

[0041] The library quality detection standards are as follows: (1) agarose gel electrophoresis: analyze the integrity of sample RNA and the presence of DNA contamination; (2) NanoPhotometer spectrophotometer: detect RNA purity (OD260 / 280 and OD260 / 230 ratio); (3) Qubit2.0 Fluorometer: accurately quantify RNA concentration; (4) Agilent 2100 bioanalyzer: accurately detect RNA integrity.

[0042] After the library inspection was qualified, the Illumina platform was used for transcriptome sequencing to obtain double-end sequencing data with a data volume of 6Gb.

[0043] 2.3 SSR site mining

[0044] The MISA (http: / / pgrc.ipk-gatersleben.de / misa / ) online tool was used to identify simple repeat sequences, with the parameters set to 10 or more mononucleotide repeats, 6 or more dinucleotide repeats, and 5 or more other types of repeats. A total of 17,098 SSR loci were mined.

[0045] 2.4. SSR locus primer design

[0046] The detected SSR loci were preliminarily screened, and the loci with the number of repeated bases greater than 30 bp were randomly selected. The Primer-BLAST software (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC=BlastHome) was used to design specific primers. The design conditions were as follows: primer length 20-25 bp, Tm value difference between forward and reverse primers not exceeding 3°C, no palindromic structure at the end of the primer, low self-pairing probability, GC content 40-60%, and PCR product length 100-300 bp. Finally, 137 pairs of primers were screened and commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0048] After screening the 137 pairs of primers one by one, three pairs of polymorphic primers were obtained, namely HBG7, HBG18 and HBG21. The primer sequences are shown in Table 1 below:

[0049] Table 1 Specific marker primer information

[0050]

[0051] Experimental Example 1

[0052] Molecular Identification of F1 Generation of Hybrids in Hibiscus truncatum

[0053] The DNA of the hibiscus parent and its hybrid F1 generation extracted in Example 1 was used as an amplification template, and PCR amplification was performed using specific primers of three SSR markers.

[0054] 1. PCR reaction system

[0055] The PCR reaction system was 20 μL, including 10 μL of 2×Taq Master Mix, 1 μL of upstream and downstream primers (10 μmol / L), 7 μL of ddH2O, and 1 μL of DNA of the hibiscus parent or the material to be tested (about 20 ng).

[0056] 2. PCR reaction procedure

[0057] The PCR reaction program for HBG7 labeling was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0058] The HBG18-labeled PCR reaction program was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 57.5°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0059] The PCR reaction program for HBG21 labeling was as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 61.5°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

[0060] 3. Electrophoresis detection of PCR amplification products

[0061] The PCR amplification products were electrophoresed on a 2% agarose gel (5 μL of Gel Red 10000× stock solution was added to every 50 mL of agarose solution) and electrophoresed for 80 min at a voltage of 105 V in 1×TAE buffer. The electrophoresis results were photographed and recorded in a gel imaging system. If the size of the PCR product was consistent with the expectation and there were no non-specific bands, the target nucleic acid fragment was considered to be successfully amplified.

[0062] 4. Identification results

[0063] The results are as follows Figure 1 As shown, 1 is the male parent 'Rose Hibiscus', 2 is the female parent 'China Chiffon', 3, 4 and 5 are three seedlings of their hybrid offspring, amplified with primer HBG18, the PCR product has the specific bands of the male and female parents, and is a true hybrid F1 generation.

[0064] 6 is the male parent 'Blue Bird', 7 is the female parent 'Hanbo', 8, 9 and 10 are three seedlings of their hybrid offspring. The PCR product was amplified with primer HBG18 and had the specific band of the male parent, which is a true hybrid F1 generation.

[0065] 11 is the male parent 'Pink Heart Double Petal', 12 is the female parent 'China Chiffon', 13, 14 and 15 are three seedlings of their hybrid offspring. The PCR product was amplified with primer HBG7 and had specific bands of the male and female parents, indicating that it was a true hybrid F1 generation.

[0066] No. 16 is the male parent 'Pufen', No. 17 is the female parent 'Hongxin', and No. 18, 19 and 20 are three seedlings of their hybrid offspring. The PCR product was amplified with primer HBG21 and had a male-specific band, indicating that it was a true hybrid F1 generation.

[0067] Figure 2 As shown, 1 is the male parent 'Blue Chiffon', 2 is the female parent 'Long-bracted Double Petal', 3, 4 and 5 are three seedlings of their hybrid offspring. The PCR product was amplified with primer HBG21 and had a male-specific band, indicating that it was a true hybrid F1 generation.

[0068] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. For those skilled in the art, improvements and changes obtained without departing from the technical concept of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A SSR marker primer set for identifying hybrid offspring of hibiscus varieties, characterized in that: The SSR marker primer set includes HBG7, HBG18, and HBG21; The primer set sequence of HBG7 is shown in SEQ ID NO.1 to SEQ ID NO.2; The primer set sequence of HBG18 is shown in SEQ ID NO.3 to SEQ ID NO.4; The primer set sequence of HBG21 is shown in SEQ ID NO.5 to SEQ ID NO.

6.

2. A detection method for the SSR marker primer set for identifying the hybrid offspring of hibiscus varieties as claimed in claim 1, characterized in that: The following steps are involved: S1. Fresh tissues or silica gel quick-dried tissue samples of the hybrid F1 generation of the male parent of hibiscus, the female parent of hibiscus and the hibiscus variety to be tested are used as the samples to be tested, and the total DNA of the samples is extracted by the modified CTAB method; S2, using the total DNA obtained in step S1 as a template and HBG18 or HBG21 or HBG7 SSR marker primer set as amplification primers for PCR amplification; S3. Perform sequence analysis on the amplified product of step S2, and determine whether the F1 generation to be tested is a hybrid F1 generation based on whether the amplified product has a specific band.

3. The detection method according to claim 2, characterized in that: When the male parent and the female parent of hibiscus are 'Rose Hibiscus' and 'China Chiffon' respectively, HBG18 is used as the marker primer set. If the PCR amplification product has the specific bands of the male and female parents, the F1 generation to be tested is the hybrid offspring of 'Rose Hibiscus' and 'China Chiffon'.

4. The detection method according to claim 2, characterized in that: When the male and female parents of hibiscus are 'Blue Bird' and 'Hanbo' respectively, HBG18 is used as the marker primer set. If the PCR amplification product has a specific band of the male parent, the F1 generation to be tested is the hybrid offspring of 'Blue Bird' and 'Hanbo'.

5. The detection method according to claim 2, characterized in that: When the male parent and the female parent of Hibiscus syriacus are 'Pink Heart Double Petal' and 'China Chiffon' respectively, HBG7 is used as the marker primer set. If the PCR amplification product has specific bands of both the male and female parents, the F1 generation to be tested is the hybrid offspring of 'Pink Heart Double Petal' and 'China Chiffon'.

6. The detection method according to claim 2, characterized in that: When the male parent and the female parent of hibiscus are 'Pufen' and 'Hongxin' respectively, HBG21 is used as the marker primer set. If the PCR amplification product has a specific band of the male parent, the F1 generation to be tested is the hybrid offspring of 'Pufen' and 'Hongxin'.

7. The detection method according to claim 2, characterized in that: When the male parent and the female parent of hibiscus are 'Blue Chiffon' and 'Long-bracted Double Petal' respectively, HBG21 is used as the marker primer set. If the PCR amplification product has a specific band of the male parent, the F1 generation to be tested is the hybrid offspring of 'Blue Chiffon' and 'Long-bracted Double Petal'.

8. The detection method according to claim 2, characterized in that: The PCR amplification system in step S2 is: 1 μL of DNA template of the hibiscus parent or the material to be tested, 1 μL of each of the upstream and downstream primers of 10 μmol / L, 10 μL of 2×Taq Master Mix, 7 μL of ddH2O, and the total volume of the system is 20 μL.

9. The detection method according to claim 2, characterized in that: The procedure of the PCR amplification is: (1) HBG7-labeled PCR reaction procedure: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 57°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min, 4°C storage; (2) HBG18-labeled PCR reaction procedure: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 57.5°C annealing for 30 s, 72°C extension for 30 s, 35 cycles; 72°C extension for 5 min, 4°C storage; (3) HBG21-labeled PCR reaction procedure: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 61.5°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 5 min, and storage at 4°C.

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