Rhododendron SSR molecular markers, primer combinations and their applications

Abstract

The invention discloses azalea SSR molecular marker, a primer composition and an application thereof, and belongs to the technical field of azalea molecular marker. The invention provides a total of 30 SSR molecular markers, of which 6 have dibase repeating units, 24 have tribase repeating units, 30 pairs of primers related to molecular markers, and the provided SSR and primer composition have clear amplified bands, high polymorphism, and good repeatability in various varieties of azalea, and can be used for germplasm identification, genetic diversity and kinship analysis among various subgenera of azalea, with stable and reliable results, laying a foundation for the management of germplasm resources of azalea, molecular marker-assisted breeding, genetic protection, and genetic diversity analysis among and within azalea populations; and also has the advantages of being simple to use and highly efficient.

Description

Technical Field

[0001] The invention relates to the technical field of Rhododendron molecular markers, in particular to Rhododendron SSR molecular markers, primer compositions and applications thereof. Background Art

[0002] Azalea is a general term for plants of the genus Rhododendron in the family Ericaceae. It is an evergreen or deciduous shrub with strong appreciation value and is often used as a greening plant in gardens. Azalea has high edible and medicinal value, and plays an important role in water conservation and soil consolidation, maintaining ecological balance, etc. It has a long history of cultivation in my country. The genus Rhododendron is the largest genus in the family Ericaceae, with more than 1,200 species in the world. There are more than 30,000 azalea varieties in the world, which are widely distributed in Asia, North America, and Europe. Among them, more than 90% of the species are concentrated in eastern Asia. China is the country with the richest wild azalea resources. Azaleas are mostly developed through hybrid breeding. However, due to the imperfect classification system and unclear kinship, breeding work is hindered by many obstacles.

[0003] Simple sequence repeat (SSR), also known as microsatellite, is a DNA sequence composed of multiple repetitions of a basic unit consisting of approximately 1-6 nucleotides in the genome. SSR molecular markers have the advantages of co-dominant inheritance, small amount of DNA required, simple operation, abundant quantity, strong versatility and polymorphism between species, and good experimental repeatability. TPM13-SSR (simple sequence repeat with tailed primer M13) technology combines SSR molecular marker technology with fluorescent sequencing technology, and has the advantages of high repeatability and accurate results. It has largely solved a series of problems in traditional gel electrophoresis analysis, such as low throughput, cumbersome amplification product detection process, and large workload of data recording.

[0004] In recent years, the application of SSR molecular markers in the study of Rhododendron has increased significantly. However, due to the incomplete mining of the genome sequence of Rhododendron, the publicly published genome data of Rhododendron that can be searched from the NCBI database is extremely limited. Only 10 species (including Lingbao Rhododendron R.henanense, White Rhododendron R.mucronatum, Rhododendron R.simsii, Red Rhododendron R.griersonianum, Red Horse Silver Flower R.vialii, Sheep Rhododendron R.molle, Horse Silver Flower R.ovatum, European Rhododendron R.ferrugineum, R.kiyosumense, Round-leaved Rhododendron R.williamsianum) have their genome data published, accounting for 0.8% of the total number of Rhododendron species. The data available for research is very scarce. In this case, it is meaningful to use SSR for research. SSR markers can make up for the limitation of insufficient genome data to a certain extent, and have high application value in genetic diversity, gene positioning, molecular marker-assisted breeding, genetic map construction, etc. Summary of the invention

[0005] The purpose of the present invention is to provide Rhododendron SSR molecular markers, primer compositions and applications thereof, so as to provide molecular markers with clear amplification bands, high polymorphism and good repeatability for Rhododendron germplasm resource identification, kinship analysis, genetic diversity research, gene positioning, molecular marker-assisted breeding and genetic map construction, in the current situation where Rhododendron genome data is limited and gene mining is incomplete.

[0006] To achieve the above purpose, the present invention provides 30 SSR molecular markers of Rhododendron, of which 6 repeating units are dibases and 24 are tribases, namely A-001, A-016, A-018, A-022, A-049, A-052, A-254, A-260, A-264, A-267, A-272, A-290, A-300, A-313, A-321, AA-334, A-335, A-336, A-341, A-345, A-346, A-349, A-350, A-353, A-354, A-356, A-362, A-363, A-368, and A-394.

[0007] Preferably, the repeating unit of A-001 is TA, with a repetition number of 8; the repeating unit of A-016 is TC, with a repetition number of 8; the repeating unit of A-018 is CA, with a repetition number of 9; the repeating unit of A-022 is AG, with a repetition number of 9; the repeating unit of A-049 is AT, with a repetition number of 8; the repeating unit of A-052 is CT, with a repetition number of 8; the repeating unit of A-254 is CCG, with a repetition number of 5; the repeating unit of A-260 is A The repeating unit of A-264 is GGT, which is repeated 5 times; the repeating unit of A-267 is GGA, which is repeated 6 times; the repeating unit of A-272 is AGA, which is repeated 5 times; the repeating unit of A-290 is GAA, which is repeated 5 times; the repeating unit of A-300 is CGA, which is repeated 7 times; the repeating unit of A-313 is GAC, which is repeated 5 times; the repeating unit of A-321 is CTT, which is repeated 7 times; The repeating unit of A-334 is ATG, with 5 repeats; the repeating unit of A-335 is GCT, with 5 repeats; the repeating unit of A-336 is TCT, with 5 repeats; the repeating unit of A-341 is GCG, with 5 repeats; the repeating unit of A-345 is CCT, with 7 repeats; the repeating unit of A-346 is GAA, with 5 repeats; the repeating unit of A-349 is GAA, with 5 repeats; the repeating unit of A-350 is The repeating unit of A-353 is CAC, with 5 repeating times; the repeating unit of A-354 is TCT, with 5 repeating times; the repeating unit of A-356 is GCG, with 5 repeating times; the repeating unit of A-362 is TCC, with 5 repeating times; the repeating unit of A-363 is TTC, with 5 repeating times; the repeating unit of A-368 is GTG, with 5 repeating times; the repeating unit of A-394 is CAG, with 5 repeating times.

[0008] Preferably, the forward primer sequence of A-001 is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO.2; the forward primer sequence of A-016 is shown as SEQ ID NO.3, and the sequence of the reverse primer is shown as SEQ ID NO.4; the forward primer sequence of A-018 is shown as SEQ ID NO.5, and the sequence of the reverse primer is shown as SEQ ID NO.6; the forward primer sequence of A-022 is shown as SEQ ID NO.7, and the sequence of the reverse primer is shown as SEQ ID NO.8; the forward primer sequence of A-049 is shown as SEQ ID NO.9, and the sequence of the reverse primer is shown as SEQ ID NO.10; the forward primer sequence of A-052 is shown as SEQ ID NO.11, and the sequence of the reverse primer is shown as SEQ ID NO.12; the forward primer sequence of A-254 is shown as SEQ ID NO.13, and the sequence of the reverse primer is shown as SEQ ID NO.14; the forward primer sequence of A-260 is shown as SEQ ID NO. The forward primer sequence of A-264 is shown in SEQ ID NO.17, and the sequence of the reverse primer is shown in SEQ ID NO.18; the forward primer sequence of A-267 is shown in SEQ ID NO.19, and the sequence of the reverse primer is shown in SEQ ID NO.20; the forward primer sequence of A-272 is shown in SEQ ID NO.21, and the sequence of the reverse primer is shown in SEQ ID NO.22; the forward primer sequence of A-290 is shown in SEQ ID NO.23, and the sequence of the reverse primer is shown in SEQ ID NO.24; the forward primer sequence of A-300 is shown in SEQ ID NO.25, and the sequence of the reverse primer is shown in SEQ ID NO.26; the forward primer sequence of A-313 is shown in SEQ ID NO.27, and the sequence of the reverse primer is shown in SEQ ID NO.28; the forward primer sequence of A-321 is shown in SEQ ID NO.29, and the sequence of the reverse primer is shown in SEQ ID NO. The forward primer sequence of AA-334 is shown in SEQ ID NO.31, and the sequence of the reverse primer is shown in SEQ ID NO.32; the forward primer sequence of A-335 is shown in SEQ ID NO.33, and the sequence of the reverse primer is shown in SEQ ID NO.34; the forward primer sequence of A-336 is shown in SEQ ID NO.35, and the sequence of the reverse primer is shown in SEQ ID NO.36; the forward primer sequence of A-341 is shown in SEQ ID NO.37, and the sequence of the reverse primer is shown in SEQ ID NO.38; the forward primer sequence of A-345 is shown in SEQ ID NO.The forward primer sequence of A-346 is shown in SEQ ID NO.41, and the sequence of the reverse primer is shown in SEQ ID NO.42; the forward primer sequence of A-349 is shown in SEQ ID NO.43, and the sequence of the reverse primer is shown in SEQ ID NO.44; the forward primer sequence of A-350 is shown in SEQ ID NO.45, and the sequence of the reverse primer is shown in SEQ ID NO.46; the forward primer sequence of A-353 is shown in SEQ ID NO.47, and the sequence of the reverse primer is shown in SEQ ID NO.48; the forward primer sequence of A-354 is shown in SEQ ID NO.49, and the sequence of the reverse primer is shown in SEQ ID NO.50; the forward primer sequence of A-356 is shown in SEQ ID NO.51, and the sequence of the reverse primer is shown in SEQ ID NO.52; the forward primer sequence of A-362 is shown in SEQ ID NO.53, and the sequence of the reverse primer is shown in SEQ ID NO. The forward primer sequence of A-363 is shown in SEQ ID NO.55, and the reverse primer sequence is shown in SEQ ID NO.56; the forward primer sequence of A-368 is shown in SEQ ID NO.57, and the reverse primer sequence is shown in SEQ ID NO.58; the forward primer sequence of A-394 is shown in SEQ ID NO.59, and the reverse primer sequence is shown in SEQ ID NO.60.

[0009] A product for the identification of Rhododendron germplasm, genetic diversity or kinship analysis, the product is a reagent or a kit, and the product contains the above-mentioned SSR molecular marker.

[0010] An application of the product as described above in the identification of Rhododendron germplasm, genetic diversity and kinship analysis.

[0011] The application of Rhododendron SSR molecular markers as described above in Rhododendron germplasm identification, genetic diversity and phylogenetic relationship analysis.

[0012] The method for using the Rhododendron SSR molecular markers in the identification of Rhododendron germplasm, genetic diversity and kinship analysis as described above is to add an M13 linker without a fluorescent label to the 5′ end of the forward primer, and the reverse primer remains unchanged. Then, an M13 primer with a fluorescent group is used, and a total of three primers are used to perform PCR amplification on the sample to be analyzed. After the amplification product is qualified by agarose gel electrophoresis, it is detected by fluorescence capillary electrophoresis. According to the obtained peak diagram and parameters, the fragment data is converted into a "0" and "1" matrix, and the Dice similarity coefficient of the SSR site is calculated using NTSYS-pc software, and a non-weighted group average cluster analysis is performed to determine the kinship and germplasm.

[0013] Preferably, the sequence of the M13 linker / primer is as shown in SEQ ID NO.61, and the fluorescent group is one of Fam, Hex, Tamrad and Rox.

[0014] Preferably, the PCR amplification program is 94°C, 3 min; 94°C, 94°C, 30 s; 56°C, 30 s, 72°C, 1 min, 35 cycles; 72°C, 10 min.

[0015] Therefore, the specific technical effects of the Rhododendron SSR molecular marker, primer composition and application thereof provided by the present invention are as follows:

[0016] (1) The SSR and primer combination provided by the present invention can amplify clear bands in various varieties of azalea, with high polymorphism and good repeatability, and can accurately, efficiently and stably identify azalea germplasm resources, and is easy to operate;

[0017] (2) The SSR and primer combination provided by the present invention can be used for germplasm identification, genetic diversity and kinship analysis among various subgenera of Rhododendron, and the results are stable and reliable, laying a foundation for the management of Rhododendron germplasm resources, breeding, genetic protection, and genetic diversity analysis among and within Rhododendron populations;

[0018] (3) The SSR and primer combination provided by the present invention can well distinguish various wild species and can be used for genetic analysis and classification research of different subgenera.

[0019] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0021] Figure 1 is a fragment length distribution diagram of Unigene in Example 1 of the present invention;

[0022] Figure 2is an electrophoresis diagram of PCR products of 40 azalea samples subjected to PCR using primer A321 in Example 2 of the present invention, wherein M is maker, 1 is twelve ethyl weight, 2 is ornamental, 3 is double flower red, 4 is flower peony, 5 is red coral, 6 is big mandarin duck brocade, 7 is cloud dress, 8 is red dress and white wrap, 9 is Edinburgh, 10 is white butterfly, 11 is magpie on plum, 12 is pink pearl, 13 is pink sky benefit, 14 is green fan, 15 is Shouguang reputation, 16 is sunlight, 17 is star brilliance, 18 is shanghai jiang jiang, 19 is guang jiang ... 8 is Wanlihong, 19 is Qiqihong, 20 is Taoyuan, 21 is Xueliqing, 22 is Yuling, 23 is Huabao, 24 is Dahongpao, 25 is Zijuan, 26 is Kangnaixin, 27 is Elsieli, 28 is Fenxia, ​​29 is Dajin, 30 is Yudaizhirong, 31 is Fenfendai, 32 is Hongyu, 33 is Xing'an Dujuan, 34 is Yodogawa Dujuan, 35 is Zhaoshanbai, 36 is Yingshanhong (red), 37 is Yunjin Dujuan, 38 is Yingshanhong, 39 is Hou'er Dujuan, 40 is Hainan Dujuan;

[0023] Figure 3 is a peak graph obtained by capillary electrophoresis detection of A321 in Example 2 of the present invention, wherein A is sample 7 (Yunshang), B is sample 11 (Xi Que Deng Mei), C is sample 28 (Fenxia), D is sample 34 (Yodogawa Rhododendron), E is sample 35 (Zhaoshanbai), and F is sample 40 (Hainan Rhododendron);

[0024] Figure 4 is the clustering tree of 40 azalea samples in Example 2 of the present invention;

[0025] Figure 5 is a peak diagram obtained by capillary electrophoresis detection of A267 in Example 3 of the present invention, wherein A is azalea (red), B is hericium erinaceus, C is pink rosa centifolia, and D is iris alba;

[0026] Figure 6 This is the clustering tree of 19 azalea samples in Example 3 of the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0028] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0029] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0030] Embodiment 1

[0031] The development of SSR primer combinations, the specific steps are as follows:

[0032] S11. Acquisition of experimental materials: Roots, stems, young leaves, flower buds and fruits of Rhododendron splendidum were collected. Roots and young leaves were collected in March, flower buds in April, stems in May, and fruits in September. The collected materials were quickly frozen in liquid nitrogen and transported to the laboratory, where they were stored in a -80°C refrigerator for use.

[0033] S12. Extraction of RNA from Rhododendron and construction of a normalized library:

[0034] ① RNA extraction: EASYspinPlus Plant RNA Rapid Extraction Kit (Aidlab, Beijing) was used to extract RNA from the roots, stems, young leaves, flower buds and fruits of Rhododendron splendidum according to the instructions.

[0035] ② RNA quality detection: The absorbance value was measured using Nanodrop 1000 to detect RNA purity, and the quality of the obtained RNA was detected by RNA agarose gel electrophoresis.

[0036] ④ Sequencing and data assembly: RNA samples with good test quality were sent to Beijing Biomark Biotechnology Co., Ltd. to complete the construction of cDNA library. Based on the sequencing by synthesis (SBS) technology, the cDNA library was sequenced using the Illumina HiSeq2500 high-throughput sequencing platform. After removing the redundancy, there were 8,442,884,533 high-quality reads (clean data), accounting for 97.84% of the total, of which the GC content was 46.52%, and the bases with a clean data quality value greater than or equal to Q30 reached 92.11%, and the transcriptome data was reliable.

[0037] 84,809 unigenes were assembled and sequenced using Trinity software. The N50 of the unigene was 1110, and the assembly integrity was high. The fragment length distribution of the unigene is as follows Figure 1 As shown, there are many short Unigene fragments, among which the fragments with a length of 200 to 300 bp are the most numerous.

[0038] S13. Detection and analysis of SSR molecular markers: MISA software was used to perform sequence analysis on 14,356 Unigenes above 1 kb obtained by screening, and 6,555 SSR sequences were identified, with a total of 6 types of SSRs. Among them, the number of double-base repeat and three-base repeat SSRs was relatively large, and the total proportion of the other types was only 15.88%.

[0039] S14. Transcriptome SSR types and distribution characteristics

[0040] By counting the repeat types of the obtained SSRs, it was found that the double-base AG / CT had the largest number of repeats, totaling 4,084, while the triple-base repeat was AAG / CTT.

[0041] S15. Primer design

[0042] According to the transcriptome of roots, stems, young leaves, flower buds and fruits of Rhododendron splendidum, the 300 bp sequence before and after the searched SSR loci was selected, and primers were designed using Primer 3 online software (www.simgene.com / Primer 3).

[0043] The primer design parameters are as follows: primer length 18-27 bp (20 bp is preferred), primer sequence GC 40%-70% (optimal 50%), annealing temperature 50-65°C (optimal 56°C), PCR amplification product length 100-300 bp, and other parameters are default settings. In addition, avoid the occurrence of mismatches, primer dimers, and hairpin structures. A total of 396 pairs of primers were synthesized by Shanghai Shenggong Bioengineering Co., Ltd.

[0044] Embodiment 2

[0045] Screen out SSR primers with clear amplification bands, rich polymorphism, and good repeatability. The specific steps are as follows:

[0046] S21. A total of 40 azalea varieties, including some wild species of the genus Rhododendron and domestic representative azaleas such as Eastern Rhododendron, Western Rhododendron, Hairy Rhododendron and Summer Rhododendron, were selected as test materials. The 40 samples were numbered in sequence. The sample information is shown in Table 1.

[0047] Table 1 Test material information

[0048]

[0049]

[0050] S22. The genomic DNA of each sample was extracted using the CTAB method, and the quality and concentration of the DNA were determined using 1% agarose gel electrophoresis and NanoDrop UV spectrophotometer. The DNA solution was then diluted to 20 ng / μL and stored in a -20°C refrigerator for later use.

[0051] S23, Screening and Detection of SSR Molecular Markers in Rhododendron

[0052] PCR amplification was performed on 40 azalea samples using the 396 pairs of primers synthesized in Example 1. 30 pairs of SSR primers with clear amplification bands, rich polymorphism and good repeatability were screened out, as shown in Table 2. The repeating unit and quantity information of each SSR are shown in Table 3.

[0053] Table 2 Information of 30 pairs of SSR primers obtained by screening

[0054]

[0055]

[0056] Table 3 Repeating units and quantity information of 30 SSRs

[0057]

[0058]

[0059] Note: This number of replicates only represents the results obtained in the transcriptome data of Rhododendron splendidum

[0060] An M13 adapter without a fluorescent marker was added to the 5′ end of the forward primer of the 30 pairs of primers, and the sequence was shown in SEQ ID NO.61. The reverse primer remained unchanged. Then, an M13 primer with a fluorescent group was used, and the sequence was shown in SEQ ID NO.61. The fluorescent group was Tamrad. PCR amplification was performed on all samples. The PCR reaction system was shown in Table 4, and the amplification conditions were shown in Table 5. The PCR products were electrophoresed on a 1% agarose gel. After 15 minutes, the results were observed on a UV gel imager and photographed and recorded. The results are shown in Figure 2 As shown, all SSR marker amplification bands are clear and reproducible, and the size of the amplified products is 200-300 bp. SEQ ID NO.61: TGTAAAACGACGGCCAGT

[0061] Table 4 PCR reaction system (20 μL)

[0062] Components Volume (μL) Upstream primer F (10mM) 0.4 Downstream primer R (10mM) 1 2×RapidTaqMasterMix 10 M13 primer with fluorescent group (10mM) 0.8 DNA template (20ng / μL) 2.5 <![CDATA[ddH2O]]> 5.3

[0063] Table 5 PCR program

[0064]

[0065] The fluorescently labeled amplified products that passed the agarose gel electrophoresis test were packaged and commissioned to Sangon Biotech (Shanghai) to perform capillary electrophoresis detection and fragment analysis. The fragment peak diagram and various parameters were obtained. Figure 3 .

[0066] ① The number of alleles (N) at each SSR locus was calculated using the software POPGENE 1.32. a ), effective number of alleles (N e ), observed heterozygosity (H o ), expected heterozygosity (He ), the specific parameters are set to the number of group is 40, the number of populations is 1, the number of loci is 30, and the rest of the parameters use the software default values. The polymorphic information content (PIC) is calculated using the software PIC_CALC, using the default parameters. The results are shown in Table 6, and it can be seen that all 30 sites show obvious genetic variation, the number of alleles (Na) of each site varies from 3 to 17, the average value is 7.63, the expected heterozygosity (He) is 0.1671-0.9073, the observed heterozygosity (Ho) is 0.0750-0.7500, the PIC value is between 0.1611-0.8853, and the vast majority of PIC values ​​are greater than 0.5, with an average value of 0.5886, indicating that the polymorphism of these 30 primers is good.

[0067] Table 6 Polymorphism analysis results of 40 rhododendron samples

[0068]

[0069]

[0070] ② Perform variety cluster analysis on the capillary electrophoresis results of PCR products of the 40 azalea varieties shown in Table 6

[0071] The fragment data were converted into "0" and "1" matrices, and the Dice similarity coefficient of the SSR loci was calculated using NTSYS-pc software, and unweighted pair-group method with arithmetic means (UPGMA) cluster analysis was performed.

[0072] A cluster tree of 40 azalea samples was constructed based on the Dice coefficient and UPGMA. The results are as follows Figure 4 As shown, all materials are divided into 5 groups at a relative distance of 0.47:

[0073] The first group (Ⅰ) has 10 samples, including multiple series of rhododendron, mainly western rhododendron and hairy rhododendron. It can be further divided into two branches, western rhododendron varieties are mainly in the first branch, and hairy rhododendron varieties are mainly in the second branch.

[0074] The second group (Ⅱ) has 10 samples, mainly eastern azalea and summer azalea.

[0075] The third group (III) has 9 samples, mainly including summer azalea samples and wild species samples. The summer azalea sample is a single branch, the wild azalea species is a single small branch, and the three wild species of Rhododendron cloudbrocade, Rhododendron, and Hericium erinaceus are gathered into a small branch.

[0076] The fourth group (IV) has 8 samples, most of which are species of the subgenus Azalea.

[0077] The fifth group (V) consists of only three samples clustered together. Rhododendron xinganense and Rhododendron sphenanthera both belong to the subgenus Rhododendron, while Rhododendron rosaceae belongs to a separate branch of the subgenus Azalea.

[0078] The results of cluster analysis showed that the screened SSR primer set could effectively distinguish the above sample materials, clearly distinguish varieties from wild species, and at the same time demonstrated the genetic similarities and differences between the samples, especially the high similarity between samples of the same line, which indicated that the SSR primer set could be used for the identification of azalea varieties.

[0079] Embodiment 3

[0080] The SSR primers screened in Example 2 were used to identify the common types and wild species in each subgenus. The specific steps were as follows:

[0081] S31. Species of the same genus as Rhododendron splendidum were selected, including 12 species of the subgenus Azalea, 3 species of the subgenus Rhododendron, 3 species of the subgenus Azalea, and 1 species of the subgenus Azalea. A total of 19 samples from the four subgenera (information on the subgenera is shown in Table 7) were selected to evaluate the inter-subgeneric universality and wild species identification ability of the newly developed 30 pairs of SSR markers.

[0082] Table 7 Subgenus information

[0083]

[0084]

[0085] S32. Extraction of genomic DNA: The CTAB method was used to extract genomic DNA. The reaction system shown in Table 4 (wherein the fluorescent group was Fam) and the PCR program shown in Table 5 were used for PCR amplification. The PCR products were subjected to agarose gel electrophoresis. The fluorescently labeled amplified products that passed the agarose gel electrophoresis test were packaged. Sangon Biotech (Shanghai) was commissioned to perform capillary electrophoresis detection and fragment analysis. The obtained fragment peak diagrams and various parameters are shown in Figure 5 .

[0086] Subgeneric generality display: The software POPGENE 1.32 was used to calculate the number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), and expected heterozygosity (He) of each SSR locus. The specific parameters were set as the number of groups to 19, number of populations to 1, number of loci to 30, and the rest of the parameters used the software default values. The software PIC_CALC was used to calculate the polymorphic information content (PIC) using default parameters.

[0087] The results are shown in Table 8. All 30 loci showed significant genetic variation. The number of alleles (Na) at each locus ranged from 2 to 12, with an average of 6, the expected heterozygosity (He) from 0.0526 to 0.9180, the observed heterozygosity (Ho) from 0.0526 to 0.6154, and the PIC value from 0.0499 to 0.8809. The 30 pairs of SSR primers obtained had good universality among the four subgenera and were suitable for genetic analysis and classification studies of different subgenera.

[0088] Table 8 Generality inspection results

[0089]

[0090]

[0091] S34. Identification of wild species: The amplification results of 30 pairs of SSR primers were converted into "0" and "1" matrices. The Dice similarity coefficient of the SSR loci was calculated using NTSYS-pc software and cluster analysis was performed using the unweighted pair-group method with arithmetic means (UPGMA).

[0092] The results are as follows Figure 6 As shown, in the clustering results, horticultural varieties are mostly clustered together, and the branches are consistent with the closeness of subgenera. Wild species have obvious branching structure. The SSR primers developed in the present invention can well distinguish various wild species and can be applied to genetic analysis and classification research of different subgenera.

[0093] Therefore, the present invention provides a total of 30 SSR molecular markers, of which 6 have dibase repeating units, 24 have tribase repeating units, and 30 pairs of primers related to molecular markers. The provided SSR and primer compositions have clear amplified bands in various azalea varieties, high polymorphism, and good repeatability, and can be used for germplasm identification, genetic diversity, and kinship analysis among various subgenera of azalea, with stable and reliable results, laying a foundation for the management of azalea germplasm resources, molecular marker-assisted breeding, genetic protection, and genetic diversity analysis among and within azalea populations; and also has the advantages of simple operation and high efficiency.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of a primer combination for identifying SSR molecular markers of Rhododendron in the identification of Rhododendron germplasm, genetic diversity and kinship analysis, characterized in that: The SSR molecular markers are A-001, A-016, A-018, A-022, A-049, A-052, A-254, A-260, A-264, A-267, A-272, A-290, A-300, A-313, A-321, AA-334, A-335, A-336, A-341, A-345, A-346, A-349, A-350, A-353, A-354, A-356, A-362, A-363, A-368, A-394; Among them, 6 repeating units are dibases, and 24 are tribases. The repeating unit of A-001 is TA, and the number of repetitions is 8; the repeating unit of A-016 is TC, and the number of repetitions is 8; the repeating unit of A-018 is CA, and the number of repetitions is 9; the repeating unit of A-022 is AG, and the number of repetitions is 9; the repeating unit of A-049 is AT, and the number of repetitions is 8; the repeating unit of A-052 is CT, and the number of repetitions is 8; the repeating unit of A-254 is CCG, and the number of repetitions is 5 ; The repeating unit of A-260 is ACC, with 5 repeats; the repeating unit of A-264 is GGT, with 5 repeats; the repeating unit of A-267 is GGA, with 6 repeats; the repeating unit of A-272 is AGA, with 5 repeats; the repeating unit of A-290 is GAA, with 5 repeats; the repeating unit of A-300 is CGA, with 7 repeats; the repeating unit of A-313 is GAC, with 5 repeats; the repeating unit of A-321 is CTT The repeating unit of A-334 is ATG, with 5 repeating times; the repeating unit of A-335 is GCT, with 5 repeating times; the repeating unit of A-336 is TCT, with 5 repeating times; the repeating unit of A-341 is GCG, with 5 repeating times; the repeating unit of A-345 is CCT, with 7 repeating times; the repeating unit of A-346 is GAA, with 5 repeating times; the repeating unit of A-349 is GAA, with 5 repeating times; the repeating unit of A-350 is The repeating unit of A-353 is CAC, which is repeated 5 times; the repeating unit of A-354 is TCT, which is repeated 5 times; the repeating unit of A-356 is GCG, which is repeated 5 times; the repeating unit of A-362 is TCC, which is repeated 5 times; the repeating unit of A-363 is TTC, which is repeated 5 times; the repeating unit of A-368 is GTG, which is repeated 5 times; the repeating unit of A-394 is CAG, which is repeated 5 times; The forward primer sequence of A-001 is shown in SEQ ID NO.1, and the sequence of the reverse primer is shown in SEQ ID NO.2; the forward primer sequence of A-016 is shown in SEQ ID NO.3, and the sequence of the reverse primer is shown in SEQ ID NO.4; the forward primer sequence of A-018 is shown in SEQ ID NO.5, and the sequence of the reverse primer is shown in SEQ ID NO.6; the forward primer sequence of A-022 is shown in SEQ ID NO.7, and the sequence of the reverse primer is shown in SEQ ID NO.8; the forward primer sequence of A-049 is shown in SEQ ID NO.9, and the sequence of the reverse primer is shown in SEQ ID NO.10; the forward primer sequence of A-052 is shown in SEQ ID NO.11, and the sequence of the reverse primer is shown in SEQ ID NO.12; the forward primer sequence of A-254 is shown in SEQ ID NO.13, and the sequence of the reverse primer is shown in SEQ ID NO.14; the forward primer sequence of A-260 is shown in SEQ ID NO. The forward primer sequence of A-264 is shown in SEQ ID NO.17, and the sequence of the reverse primer is shown in SEQ ID NO.18; the forward primer sequence of A-267 is shown in SEQ ID NO.19, and the sequence of the reverse primer is shown in SEQ ID NO.20; the forward primer sequence of A-272 is shown in SEQ ID NO.21, and the sequence of the reverse primer is shown in SEQ ID NO.22; the forward primer sequence of A-290 is shown in SEQ ID NO.23, and the sequence of the reverse primer is shown in SEQ ID NO.24; the forward primer sequence of A-300 is shown in SEQ ID NO.25, and the sequence of the reverse primer is shown in SEQ ID NO.26; the forward primer sequence of A-313 is shown in SEQ ID NO.27, and the sequence of the reverse primer is shown in SEQ ID NO.28; the forward primer sequence of A-321 is shown in SEQ ID NO.29, and the sequence of the reverse primer is shown in SEQ ID NO. The forward primer sequence of AA-334 is shown in SEQ ID NO.31, and the sequence of the reverse primer is shown in SEQ ID NO.32; the forward primer sequence of A-335 is shown in SEQ ID NO.33, and the sequence of the reverse primer is shown in SEQ ID NO.34; the forward primer sequence of A-336 is shown in SEQ ID NO.35, and the sequence of the reverse primer is shown in SEQ ID NO.36; the forward primer sequence of A-341 is shown in SEQ ID NO.37, and the sequence of the reverse primer is shown in SEQ ID NO.38; the forward primer sequence of A-345 is shown in SEQ ID NO.The forward primer sequence of A-346 is shown in SEQ ID NO.41, and the sequence of the reverse primer is shown in SEQ ID NO.42; the forward primer sequence of A-349 is shown in SEQ ID NO.43, and the sequence of the reverse primer is shown in SEQ ID NO.44; the forward primer sequence of A-350 is shown in SEQ ID NO.45, and the sequence of the reverse primer is shown in SEQ ID NO.46; the forward primer sequence of A-353 is shown in SEQ ID NO.47, and the sequence of the reverse primer is shown in SEQ ID NO.48; the forward primer sequence of A-354 is shown in SEQ ID NO.49, and the sequence of the reverse primer is shown in SEQ ID NO.50; the forward primer sequence of A-356 is shown in SEQ ID NO.51, and the sequence of the reverse primer is shown in SEQ ID NO.52; the forward primer sequence of A-362 is shown in SEQ ID NO.53, and the sequence of the reverse primer is shown in SEQ ID NO. The forward primer sequence of A-363 is shown in SEQ ID NO.55, and the reverse primer sequence is shown in SEQ ID NO.56; the forward primer sequence of A-368 is shown in SEQ ID NO.57, and the reverse primer sequence is shown in SEQ ID NO.58; the forward primer sequence of A-394 is shown in SEQ ID NO.59, and the reverse primer sequence is shown in SEQ ID NO.

60.

2. A product for the identification of Rhododendron germplasm, genetic diversity or kinship analysis, characterized in that: The product is a reagent or a kit, and the product comprises the primer composition for identifying the Rhododendron SSR molecular marker as described in claim 1.

3. Use of the product as claimed in claim 2 in the identification of Rhododendron germplasm, genetic diversity and kinship analysis.

4. The method for using the primer combination for identifying Rhododendron SSR molecular markers as claimed in claim 1 in the identification of Rhododendron germplasm, genetic diversity and kinship analysis, characterized in that: An M13 adapter without a fluorescent marker was added to the 5′ end of the forward primer, and the reverse primer remained unchanged. A total of three primers, one M13 primer with a fluorescent group, were used to perform PCR amplification on the sample to be analyzed. After the amplified product was qualified by agarose gel electrophoresis, it was detected by fluorescence capillary electrophoresis. According to the obtained peak diagram and parameters, the fragment data was converted into a "0" and "1" matrix. The Dice similarity coefficient of the SSR locus was calculated using NTSYS-pc software, and a non-weighted group average method cluster analysis was performed to determine the genetic relationship and germplasm.

5. The method for using the primer combination for identifying Rhododendron SSR molecular markers according to claim 4 in the identification of Rhododendron germplasm, genetic diversity and kinship analysis, characterized in that: The sequence of the M13 linker / primer is shown in SEQ ID NO.61, and the fluorescent group is one of Fam, Hex, Tamrad and Rox.

6. The method for using the primer combination for identifying Rhododendron SSR molecular markers according to claim 4 in the identification of Rhododendron germplasm, genetic diversity and kinship analysis, characterized in that: The PCR amplification program was 94°C, 3 min; 94°C, 94°C, 30 s; 56°C, 30 s, 72°C, 1 min, 35 cycles; 72°C, 10 min.

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