A set of ssr marker primers for identifying offsprings of a cross between varieties of hibiscus and a detection method

Abstract

The scheme provides a kind of identification of hybridization of hibiscus varieties SSR marker primer group and detection method, belong to molecular marker technical field, application SSR marker HBG18 is to the hybridization combination of male parent 'rose hibiscus' and female parent 'China chiffon', the offspring of hybridization combination of male parent 'blue bird' and female parent 'Han cloth' is carried out PCR detection;Application SSR marker HBG7 is to the hybridization combination of male parent 'pink heart double' and female parent 'China chiffon' is carried out PCR detection;Application SSR marker HBG21 is to the hybridization combination of male parent 'Pu powder' and female parent'red heart', the hybridization combination of male parent 'blue chiffon' and female parent 'long calyx double' is carried out PCR detection, through agarose gel electrophoresis detection, it shows that three SSR markers can accurately distinguish hibiscus parent and its hybrid offspring, reaches the purpose of rapid identification of hybrid F1 generation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular markers, and particularly relates to a SSR marker primer set for identifying hybrid offspring of Hibiscus varieties and a detection method. BACKGROUND

[0002] Hibiscus syriacus Linn is a deciduous shrub of the Malvaceae family and Hibiscus genus, also known as Zhongtianmulaohua, Jingzhu, and Huzhanghua, etc. It is native to the central part of China and is distributed in the whole country except for some parts of North China, Northwest China and Northeast China. Globally, there are about 200 species of the Hibiscus genus, of which 25 are distributed in China, including 13 woody plants. Hibiscus syriacus Linn blooms in summer and autumn, has a long flowering period, large and colorful flowers, and is often used as an ornamental tree species in gardens. It also has medicinal value. Hibiscus syriacus Linn has a strong dust-retaining function and strong resistance to harmful gases such as sulfur dioxide and chlorides, and is often used as a green tree species for factories with pollution.

[0003] At present, the research on Hibiscus syriacus Linn mainly focuses on propagation technology, genetic diversity and genetic structure. In terms of application of molecular markers, the following aspects are mainly involved: 1. application of SRAP markers to identify genetic relationship, 2. genetic diversity analysis of the Hibiscus genus using ISSR molecular markers, 3. genetic relationship analysis of Hibiscus varieties based on RAPD method, and 4. application of EST-SSR to construct the DNA fingerprint of Hibiscus varieties. Overall, the research on molecular markers of Hibiscus syriacus Linn is relatively lagging behind, and the existing molecular markers cannot meet the needs of genetic breeding research of Hibiscus syriacus Linn. Hybrid breeding is one of the main ways to create new germplasm and cultivate new varieties of Hibiscus syriacus Linn. The hybrid offspring of Hibiscus syriacus Linn is rich in variation and has good ornamental properties. Some individuals have the characteristics of both parents in their leaf blades and other vegetative organs. Some individuals have morphological characteristics similar to the mother plant and have no obvious differences, which cannot be identified by morphological characteristics. In addition, the long cycle and the influence of growth and development and environment to some extent limit the accurate and rapid identification of hybrid offspring. Therefore, it is urgent to screen new molecular markers for different parent combinations and establish a rapid, accurate and efficient molecular identification technology system for the hybrid offspring of Hibiscus syriacus Linn, so as to shorten the breeding cycle, speed up the breeding process, and promote the selection, popularization and high-quality development of Hibiscus syriacus Linn varieties in China. SUMMARY

[0004] In order to solve the above technical problems, the present application provides a kind of identification of hybrid offspring of SSR marker primer combination and detection method of Chinese hibiscus variety, molecular marker technology is not influenced by plant development period and environment, only needs trace fresh tissue or silica gel quick drying tissue sample can accurately and efficiently detect plant variety or two parent hybrid offspring (F1). Based on transcriptome sequencing technology, Simple Sequence Repeats (SSR) length polymorphism site suitable for identifying hybrid F1 generation of Chinese hibiscus is excavated, and the candidate marker is verified using the genomic DNA of the parent and its hybrid F1 generation, and the specific and high-resolution marker is obtained, to achieve the purpose of rapid and accurate identification of F1 generation.

[0005] To achieve the above purpose, the present application first provides a kind of identification of hybrid offspring of SSR marker primer combination of Chinese hibiscus variety, and the SSR marker primer combination includes HBG7, HBG18, HBG21;

[0006] The primer group sequence of HBG7 is as shown in SEQ ID NO.1-SEQ ID NO.2;

[0007] The primer group sequence of HBG18 is as shown in SEQ ID NO.3-SEQ ID NO.4;

[0008] The primer group sequence of HBG21 is as shown in SEQ ID NO.5-SEQ ID NO.6.

[0009] Based on a general inventive concept, the present application also provides a kind of detection method of identification of hybrid offspring of SSR marker primer combination of Chinese hibiscus variety, comprising the following steps:

[0010] S1, with fresh tissue or silica gel quick drying tissue sample of hybrid F1 generation of Chinese hibiscus father, Chinese hibiscus mother and the wood variety to be measured as the sample to be measured, the total DNA of the sample is extracted by improved CTAB method;

[0011] S2, with the total DNA obtained in S1 step as template, with HBG18, HBG21 or HBG7 SSR marker primer group as amplification primer for PCR amplification;

[0012] S3, sequence analysis is carried out to the amplification product of S2 step, and whether the amplification product has specific band determines whether the F1 generation to be measured is hybrid F1 generation. As preferred, when the Chinese hibiscus father and Chinese hibiscus mother are 'Rose Chinese hibiscus' and 'China chiffon' respectively, HBG18 is used as marker primer group, and if the PCR amplification product has the specificity band of the father and the mother, then the F1 generation to be measured is the hybrid offspring of 'Rose Chinese hibiscus' and 'China chiffon'.

[0013] As preferred, when the Hibiscus rosa-sinensis male parent and female parent are 'Lan Niao' and 'Han Bo' respectively, HBG18 is used as the marker primer set, if the PCR amplification product has the specific band of the male parent, the F1 generation to be tested is the hybrid offspring of 'Lan Niao' and 'Han Bo'.

[0014] As preferred, when the Hibiscus rosa-sinensis male parent and female parent are 'Fen Hong Xin Chong Pian' and 'Zhong Guo Xue Fang' respectively, HBG7 is used as the marker primer set, if the PCR amplification product has the specific band of the male parent and the female parent, the F1 generation to be tested is the hybrid offspring of 'Fen Hong Xin Chong Pian' and 'Zhong Guo Xue Fang'.

[0015] As preferred, when the Hibiscus rosa-sinensis male parent and female parent are 'Pu Fen' and 'Hong Xin' respectively, HBG21 is used as the marker primer set, if the PCR amplification product has the specific band of the male parent, the F1 generation to be tested is the hybrid offspring of 'Pu Fen' and 'Hong Xin'.

[0016] As preferred, when the Hibiscus rosa-sinensis male parent and female parent are 'Lan Se Xue Fang' and 'Chang Bao Chong Pian' respectively, HBG21 is used as the marker primer set, if the PCR amplification product has the specific band of the male parent, the F1 generation to be tested is the hybrid offspring of 'Lan Se Xue Fang' and 'Chang Bao Chong Pian'.

[0017] As preferred, the PCR amplification system in the S2 step is as follows: 1 μL (about 20 ng) of DNA template of the Hibiscus rosa-sinensis parent or the material to be tested, 1 μL of 10 μmol / L of the upper and lower primers respectively, 10 μL of 2 × Taq Master Mix, 7 μL of ddH2O, and the total volume of the system is 20 μL.

[0018] As preferred, the PCR amplification procedure is as follows:

[0019] (1) The PCR reaction procedure of HBG7 marker is as follows: 94 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 57 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 35 cycles; 72 ℃ extension for 5 min, 4 ℃ preservation;

[0020] (2) The PCR reaction procedure of HBG18 marker is as follows: 94 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 57.5 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 35 cycles; 72 ℃ extension for 5 min, 4 ℃ preservation;

[0021] (3) The PCR reaction procedure of HBG21 marker is as follows: 94 ℃ pre-denaturation for 3 min; 94 ℃ denaturation for 30 s, 61.5 ℃ annealing for 30 s, 72 ℃ extension for 30 s, 35 cycles; 72 ℃ extension for 5 min, 4 ℃ preservation

[0022] The method identified in the present application is as follows:

[0023] The SSR marker HBG18 in the application is applied to the PCR detection of the offspring of the cross combination of the male parent 'Rose Hibiscus' and the female parent 'Chinese Chiffon', the cross combination of the male parent 'Blue Bird' and the female parent 'Han Cloth', the cross combination of the male parent 'Pink Heart Double' and the female parent 'Chinese Chiffon' is detected by the SSR marker HBG7, and the cross combination of the male parent 'Pu Powder' and the female parent 'Red Heart' and the cross combination of the male parent 'Blue Chiffon' and the female parent 'Long-bract Double' are detected by the SSR marker HBG21. Through agarose gel electrophoresis detection, it is shown that the three SSR markers can accurately distinguish the hibiscus parent and its hybrid offspring, and achieve the purpose of rapidly identifying the hybrid F1 generation.

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

[0025] The existing detection technology is morphological identification, which is mainly distinguished by the characteristics of leaf, branch, flower and fruit and other tissue organs, and has a long cycle and is limited by time and plant growth and development stage, and cannot meet the needs of rapid and accurate identification of hybrid F1 generation. The application adopts the SSR marker to identify the hybrid F1 generation, which is not affected by the plant development period, growth stage and environment, and only needs a small amount of fresh tissue or silica gel rapid drying tissue sample to accurately and rapidly identify the sample. Under the premise of complete instrument equipment, the detection result can be obtained in about 4 hours. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0027] Figure 1 PCR product bands of the male parent, the female parent and the offspring amplified by the primer HBG21 in the experimental example 1.

[0028] Figure 2 PCR product bands of the male parent, the female parent and the offspring amplified by the primer HBG21 in the experimental example 1. DETAILED DESCRIPTION

[0029] In order to make the technical problems, technical solutions and advantages of the application more clear, the following will be described in detail in combination with the drawings and specific embodiments.

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

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

[0032] Example 1

[0033] Development of SSR Markers

[0034] 1. Experimental materials

[0035] Tender leaves were collected from the parent hibiscus and its F1 hybrids, totaling 24 materials, for the identification, screening, and specificity verification of SSR markers.

[0036] 2. Development of SSR tags

[0037] 2.1 RNA Extraction

[0038] Total RNA was extracted from the hibiscus genome using the Trizol kit (Invotrigen, USA), following the kit instructions. Electrophoresis was performed using a 1% agarose gel.

[0039] 2.2 Library Construction and Sequencing

[0040] use The RNA Sample Preparation Kit was used to construct a library. Eukaryotic mRNA with polyA tails was enriched using Oligo(dT) magnetic beads, and then fragmented using sonication. Using the fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA was synthesized in an M-MuLV reverse transcriptase system. The RNA strand was then degraded with RNase H, and the second strand of cDNA was synthesized using dNTPs in a DNA polymerase I system. The purified double-stranded cDNA underwent end repair, A-tailing, and ligation with sequencing adapters. CDNA samples of approximately 200 bp were screened using AMPure XPbeads, amplified by PCR, and the PCR products were purified again using AMPure XPbeads to obtain the final library.

[0041] The library quality detection criteria are as follows: (1) agarose gel electrophoresis: analyze sample RNA integrity and whether there is 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 passes the detection, the Illumina platform is used for transcriptome sequencing, and double-end sequencing data is obtained, with a data volume of 6Gb.

[0043] 2.3, SSR site mining

[0044] Simple sequence repeats are identified using the MISA (http: / / pgrc.ipk-gatersleben.de / misa / ) online tool, with the following parameters: single nucleotide repeats of 10 or more, di-nucleotide repeats of 6 or more, and other types of repeats of 5 or more. A total of 17098 SSR sites were mined.

[0045] 2.4, SSR site primer design

[0046] The detected SSR sites are preliminarily screened, and sites with repeat base number greater than 30bp are randomly selected. Primer-BLAST software (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi?LINK_LOC

[0047] =BlastHome) is used to design specific primers with the following design conditions: primer length 20-25bp, Tm value difference between forward and reverse primers no more than 3℃, primer ends without palindrome structure, small self-pairing probability, GC content 40-60%, and PCR product length 100-300bp. Finally, 137 pairs of primers are screened, and are synthesized by Shengong Biotechnology (Shanghai) Co., Ltd.

[0048] After screening the 137 pairs of primers one by one, 3 pairs of polymorphic primers are obtained, namely HBG7, HBG18 and HBG21, and the primer sequences are shown in Table 1 as follows:

[0049] Table 1 Specific marker primer information

[0050]

[0051] Experimental Example 1

[0052] Molecular identification of F1 generation of hybrid hibiscus

[0053] The DNA of the parent extracted in Example 1 and the hybrid F1 generation of the parent were used as the amplification template, and the specific primers of three SSR markers were used for PCR amplification, respectively.

[0054] 1. PCR reaction system

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

[0056] 2. PCR reaction procedure

[0057] The PCR reaction procedure of HBG7 marker: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min, 4℃ preservation.

[0058] The PCR reaction procedure of HBG18 marker: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 57.5℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min, 4℃ preservation.

[0059] The PCR reaction procedure of HBG21 marker: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 61.5℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; 72℃ extension for 5 min, 4℃ preservation.

[0060] 3. Electrophoretic detection of PCR amplification products

[0061] The PCR amplification products were subjected to electrophoresis on an agarose gel with a concentration of 2% (5 μL of Gel Red 10000x stock solution was added to 50 mL of agarose solution), and electrophoresis was performed in 1x TAE buffer at a voltage of 105 V for 80 min. The electrophoretic results were photographed and recorded in a gel imaging system. If the size of the PCR product was consistent with the expected size, and there was no non-specific band, it was considered that the amplification of the target nucleic acid fragment was successful.

[0062] 4. Identification results

[0063] The results are shown in Table 1. Figure 1 As shown in Table 1, 1 is the male parent 'Rose Hibiscus', 2 is the female parent 'China Chiffon', 3, 4 and 5 are three seedlings of the hybrid offspring, and the PCR products amplified by primer HBG18 have specific bands of the male parent and the female parent, which are the true hybrid F1 generation.

[0064] 6 is the male parent 'Blue Bird', 7 is the female parent 'Hanba', 8, 9 and 10 are three seedlings of the hybrid offspring, amplified by primer HBG18, the PCR product has the specificity band of the male parent, and is the true hybrid F1 generation.

[0065] 11 is the male parent 'Pink Heart Double', 12 is the female parent 'China Snow', 13, 14 and 15 are three seedlings of the hybrid offspring, amplified by primer HBG7, the PCR product has the specificity band of the male parent and the female parent, and is the true hybrid F1 generation.

[0066] 16 is the male parent 'Pu Powder', 17 is the female parent 'Red Heart', 18, 19 and 20 are three seedlings of the hybrid offspring, amplified by primer HBG21, the PCR product has the specificity band of the male parent, and is the true hybrid F1 generation.

[0067] Figure 2 As shown, 1 is the male parent 'Blue Snow', 2 is the female parent 'Long-bract Double', 3, 4 and 5 are three seedlings of the hybrid offspring, amplified by primer HBG21, the PCR product has the specificity band of the male parent, and is the true hybrid F1 generation.

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

Claims

1. A detection method for identifying a cross progeny of a variety of Hibiscus syriacus using a primer set of a SSR marker, characterized by, The method comprises the following steps: S1, taking fresh tissue or silica gel rapidly dried tissue sample of a hybrid F1 generation of a to-be-tested variety of Hibiscus as a to-be-tested sample, and extracting total DNA of the sample by using a modified CTAB method; S2, taking the total DNA obtained in the step S1 as a template, and performing PCR amplification by using an HBG18 SSR marker primer group as amplification primers; S3, performing sequence analysis on the amplification product of the step S2, and judging whether the to-be-tested F1 generation is a hybrid F1 generation according to whether the amplification product has a specific band; The primer group sequence of the HBG18 is shown as SEQ ID NO. 3-SEQ ID NO.

4.

2. A detection method of a primer set of an SSR marker for identifying a cross progeny of a variety of Hibiscus hamabo, characterized by, The method comprises the following steps: S1, taking fresh tissue or silica gel rapidly dried tissue sample of a hybrid F1 generation of a to-be-tested variety of Hibiscus as a to-be-tested sample, and extracting total DNA of the sample by using a modified CTAB method; S2, taking the total DNA obtained in the step S1 as a template, and performing PCR amplification by using an HBG18 SSR marker primer group as amplification primers; S3, performing sequence analysis on the amplification product of the step S2, and judging whether the to-be-tested F1 generation is a hybrid F1 generation according to whether the amplification product has a specific band; The primer group sequence of the HBG18 is shown as SEQ ID NO. 3-SEQ ID NO. 4.

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