Polymorphic primer of Rhododendron pulchrum whole genome SSR molecular marker and application of polymorphic primer

Abstract

The invention belongs to the technical field of biology, and particularly discloses a Rhododendron pulchrum whole genome SSR molecular marker polymorphic primer and application thereof.g-SSR molecular marker primers are massively developed on the basis of Rhododendron pulchrum whole genome data, 30 pairs of molecular marker primers with good polymorphism are screened, 96 Rhododendron pulchrum germplasm resources are amplified, and the Rhododendron pulchrum whole genome SSR molecular marker polymorphic primer is obtained. Group structure analysis is carried out, and a plurality of SSR molecular markers linked with flowering traits of rhododendron are identified through genotype and phenotype correlation analysis; the developed g-SSR has the advantages of large quantity, strong polymorphism, high degree of association with phenotypic characters and accurate and reliable experimental results, and provides a new tool for researches of rhododendron crossbreeding, genetic relationship identification, molecular marker-assisted selective breeding and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a polymorphic primer for a whole-genome SSR molecular marker of Rhododendron splendidum and an application thereof. Background Art

[0002] Rhododendron is one of the most species-rich genera in the Ericaceae family, with over 1,000 species worldwide, widely distributed in temperate and alpine regions of Asia, Europe, and North America. Plants in this genus have attracted considerable attention for their exceptional ornamental value, ecological adaptability, and medicinal properties. China, as the distribution center of Rhododendron, is home to approximately 600 species, accounting for over 60% of the global total. However, due to the high morphological plasticity and frequent interspecific hybridization of Rhododendron species, their classification and phylogenetic relationships have long been subject to considerable controversy. Traditional classification methods rely primarily on phenotypic traits such as floral morphology and leaf shape, but these traits are susceptible to environmental influences and fail to accurately reflect genetic differences between species. Therefore, the introduction of molecular marker technology to analyze genetic diversity, identify species, and conduct phylogenetic studies of Rhododendron is of great significance.

[0003] In the study of Rhododendron plants, the application of molecular marker technology has made certain progress. Many studies have used different SSR marker combinations to conduct systematic analysis of Rhododendron germplasm resources, including the study of genetic diversity and population genetic structure of Rhododendron species (including varieties), identification of kinship, etc. These studies have laid a molecular biological foundation for the protection of germplasm resources, variety improvement and systematic evolutionary analysis of Rhododendron plants. Although researchers have focused on the development of SSR molecular markers for Rhododendron plants for many years. However, compared with other economically or ecologically important plant groups, molecular marker research on Rhododendron is still insufficient. So far, there are few SSR markers that have been made public, and the only ones are derived from plants of the same genus, such as Rhododendron grandiflorum and Rhododendron dahurica. So far, there have been no reports on the large-scale development and application of g-SSR markers based on the whole genome of Rhododendron splendidum. Summary of the Invention

[0004] The purpose of the present invention is to provide a polymorphic primer for SSR molecular marker of whole genome of Rhododendron splendidum and application thereof in view of the existing problems.

[0005] The present invention is achieved through the following technical solutions:

[0006] A set of polymorphic primers for whole-genome SSR molecular markers of Rhododendron splendidum consists of the following 30 pairs of polymorphic primers, the nucleotide sequences of which are as follows:

[0007] The forward primer of RsgSSR26 is shown in SEQ ID NO. 1, and the reverse primer is shown in SEQ ID NO. 2;

[0008] The forward primer of RsgSSR62 is shown in SEQ ID NO. 3, and the reverse primer is shown in SEQ ID NO. 4;

[0009] The forward primer of RsgSSR94 is shown in SEQ ID NO. 5, and the reverse primer is shown in SEQ ID NO. 6;

[0010] The forward primer of RsgSSR122 is shown in SEQ ID NO. 7, and the reverse primer is shown in SEQ ID NO. 8;

[0011] The forward primer of RsgSSR123 is shown in SEQ ID NO. 9, and the reverse primer is shown in SEQ ID NO. 10;

[0012] The forward primer of RsgSSR126 is shown in SEQ ID NO. 11, and the reverse primer is shown in SEQ ID NO. 12;

[0013] The forward primer of RsgSSR128 is shown in SEQ ID NO. 13, and the reverse primer is shown in SEQ ID NO. 14;

[0014] The forward primer of RsgSSR143 is shown in SEQ ID NO. 15, and the reverse primer is shown in SEQ ID NO. 16;

[0015] The forward primer of RsgSSR148 is shown in SEQ ID NO. 17, and the reverse primer is shown in SEQ ID NO. 18;

[0016] The forward primer of RsgSSR154 is shown in SEQ ID NO. 19, and the reverse primer is shown in SEQ ID NO. 20;

[0017] The forward primer of RsgSSR185 is shown in SEQ ID NO. 21, and the reverse primer is shown in SEQ ID NO. 22;

[0018] The forward primer of RsgSSR188 is shown in SEQ ID NO. 23, and the reverse primer is shown in SEQ ID NO. 24;

[0019] The forward primer of RsgSSR198 is shown in SEQ ID NO. 25, and the reverse primer is shown in SEQ ID NO. 26;

[0020] The forward primer of RsgSSR200 is shown in SEQ ID NO. 27, and the reverse primer is shown in SEQ ID NO. 28;

[0021] The forward primer of RsgSSR207 is shown in SEQ ID NO. 29, and the reverse primer is shown in SEQ ID NO. 30;

[0022] The forward primer of RsgSSR212 is shown in SEQ ID NO. 31, and the reverse primer is shown in SEQ ID NO. 32;

[0023] The forward primer of RsgSSR220 is shown in SEQ ID NO. 33, and the reverse primer is shown in SEQ ID NO. 34;

[0024] The forward primer of RsgSSR222 is shown in SEQ ID NO. 35, and the reverse primer is shown in SEQ ID NO. 36;

[0025] The forward primer of RsgSSR227 is shown in SEQ ID NO. 37, and the reverse primer is shown in SEQ ID NO. 38;

[0026] The forward primer of RsgSSR229 is shown in SEQ ID NO. 39, and the reverse primer is shown in SEQ ID NO. 40;

[0027] The forward primer of RsgSSR232 is shown in SEQ ID NO. 41, and the reverse primer is shown in SEQ ID NO. 42;

[0028] The forward primer of RsgSSR235 is shown in SEQ ID NO. 43, and the reverse primer is shown in SEQ ID NO. 44;

[0029] The forward primer of RsgSSR236 is shown in SEQ ID NO. 45, and the reverse primer is shown in SEQ ID NO. 46;

[0030] The forward primer of RsgSSR249 is shown in SEQ ID NO. 47, and the reverse primer is shown in SEQ ID NO. 48;

[0031] The forward primer of RsgSSR253 is shown in SEQ ID NO. 49, and the reverse primer is shown in SEQ ID NO. 50;

[0032] The forward primer of RsgSSR259 is shown in SEQ ID NO. 51, and the reverse primer is shown in SEQ ID NO. 52;

[0033] The forward primer of RsgSSR260 is shown in SEQ ID NO. 53, and the reverse primer is shown in SEQ ID NO. 54;

[0034] The forward primer of RsgSSR261 is shown in SEQ ID NO. 55, and the reverse primer is shown in SEQ ID NO. 56;

[0035] The forward primer of RsgSSR262 is shown in SEQ ID NO. 57, and the reverse primer is shown in SEQ ID NO. 58;

[0036] The forward primer of RsgSSR268 is shown as SEQ ID NO.59, and the reverse primer is shown as SEQ ID NO.60.

[0037] Application of polymorphic primers for genome-wide SSR molecular markers of Rhododendron splendidum in rhododendron hybrid breeding, kinship identification, and molecular marker-assisted selection breeding.

[0038] Compared with the prior art, the present invention has the following advantages:

[0039] The present invention develops a large number of g-SSR molecular marker primers based on the whole genome data of Rhododendron splendidum and Hedera zingiberensis, screens 30 pairs of molecular marker primers with good polymorphism, amplifies 96 Rhododendron germplasm resources, conducts population structure analysis, and identifies multiple SSR molecular markers linked to the flowering traits of Rhododendron through genotype and phenotype association analysis; the g-SSRs developed by the present invention are numerous, highly polymorphic, and highly correlated with phenotypic traits, and the experimental results are accurate and reliable, providing a new tool for research on Rhododendron hybrid breeding, kinship identification, molecular marker-assisted selection breeding, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Statistical results for 12 phenotypic traits;

[0041] Figure 2 is the density distribution of genomic SSRs;

[0042] Figure 3 Statistics of the number of different types of SSRs;

[0043] Figure 4 For genetic structure analysis. DETAILED DESCRIPTION

[0044] In order to further explain the present invention, it is described below with reference to the following specific embodiments.

[0045] Test materials

[0046] The Rhododendron plant materials used in this study were all obtained from the nursery of Zhejiang Shengyan Agricultural Development Co., Ltd. (see Table 1 for details). A total of 96 Rhododendron germplasm resources were collected, primarily representing current mainstream commercial cultivars. Their geographic origins were as follows: 6 (6.25%) originated in China, 21 (21.88%) originated in the United States, and 69 (71.88%) originated in Japan. This sample composition fully reflects the distribution characteristics of East Asia (particularly Japan) and North America as the main sources of modern commercial rhododendron varieties, and is representative of commercial breeding.

[0047] Table 1

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] Example 1

[0054] 1.1 DNA extraction

[0055] Disease-free young leaves from each accession were collected, quickly frozen in liquid nitrogen, and stored at -80°C until use. DNA was extracted using a new plant genomic DNA extraction kit (Tiangen, Beijing). DNA quality was tested using a NanoDrop spectrophotometer and 1.2% agarose gel electrophoresis. Qualified DNA was stored at -20°C until use.

[0056] 1.2 Phenotypic trait determination and statistical analysis

[0057] According to the requirements of the Forestry Industry Standard of the People's Republic of China, "Guidelines for the Conduct of Tests for Distinctness, Uniformity, and Stability - Rhododendron (Rhododendron subgenus Hymenanthes (Blume) K. Koch and subgenus Rhododendron L.)" (LY / T 1852-2009), 12 phenotypic traits (blade length, blade shape, petiole length, inflorescence type, pedicel length, flower diameter, flower shape, petal type, stamen number, corolla lobe shape, initial flowering period, and remontant) were observed. Each trait was uniquely coded for each resource according to the Guidelines, and each trait was observed with at least three replicates. Basic descriptive statistics and general linear regression analyses were performed using built-in functions in the R language.

[0058] 1.3 Comprehensive evaluation of flowering traits of azalea germplasm resources

[0059] The 12 phenotypic traits can be divided into 2 to 5 levels, and the Shannon-Werner index ranges from 0.234 to 1.360 ( Figure 1Among them, blade length, petiole length, petal type, stamen number, and remontant can each be divided into two levels, inflorescence type, pedicel length, corolla lobe shape, and initial flowering period can be divided into three levels, and flower shape and blade shape can be divided into four and five levels, respectively. Among them, two traits with H greater than 1, including blade shape and flower diameter, indicate rich diversity. In terms of blade length, 87.50% (84) of the materials were “very short”; in terms of blade shape, 92.71% of the materials were “oblong-elliptical” (48) and “oval” (41); in terms of petiole length, 93.75% (90) of the materials were “extremely short”; in terms of inflorescence type, 85.42% (82) of the materials were “umbel”; in terms of pedicel length, 82.29% (79) of the materials were “short”; in terms of flower diameter, 57.29% (55) of the materials were “large”; in terms of flower shape, 73.96% (71) of the materials were “broadly funnel-shaped”; in terms of petal type, 60.42% (58) of the materials were “single”; in terms of Stamen In terms of number, 89.58% (86) of the materials showed "few"; in terms of corollalobe shape, 92.71% (89) of the materials showed "broadly elliptical"; in terms of initial flowering period, 65.63% (63) of the materials began to bloom in April; in terms of remontant, 82.29% (79) of the materials showed "absent" (non-multi-season flowering).

[0060] Example 2

[0061] 2.1 Identification of SSRs in the Rhododendron genome

[0062] Based on the whole-genome data of Rhododendron spp. (GenBank database: PRJNA869534), the MISA program (http: / / www.pgrc.ipkgatersleben.de / misa) was used to identify SSR (simple sequence repeat) sites. The MISA program identified 1-6 bp motifs, with a minimum threshold of 10 or more repeats for mononucleotide repeats, 6 or more for dinucleotide repeats, and 5 or more for trinucleotide to hexanucleotide repeats. The SSR density distribution along chromosomes was plotted and heat maps were created using TBtools (v2.048). Descriptive statistical analysis was performed using R (v4.2.3), and a general linear regression model was constructed. The correlation between chromosome length and SSR number was tested (Pearson's r and significance level P < 0.01). Specific primers were designed using Primer Premier 5.0 software, with the parameters set as primer length 18-25 bp, amplified fragment size 100-300 bp, annealing temperature 55-65°C, and GC content 40%-60%.

[0063] 2.2 Screening of polymorphic SSR markers

[0064] 190 primer pairs were synthesized by Shanghai Sangon Biotechnology Co., Ltd. First, PCR amplification was performed using 154 representative accessions, RS1, RS2, RS97, and RS11, as templates. Primers that consistently amplified bands were screened by electrophoresis on 1.5% agarose gel. Then, polymorphism amplification was performed on these four accessions using these same templates. A primer with the sequence TGTAAAACGACGGCCAGT was inserted at the 3' end of the forward primer, leaving the reverse primer sequence unchanged. The TP-M13 reaction system and protocol were based on the study by Ye et al. (https: / / www.mdpi.com / 2077-0472 / 13 / 1 / 200). Amplified products were analyzed on an ABI 3730XL DNA analyzer. Finally, markers with strong polymorphism and a size consistent with the expected product were selected for PCR amplification of 96 rhododendron accessions.

[0065] 2.3 Identification and characterization of SSRs in the Rhododendron genome

[0066] 2.3.1 Identification and distribution of SSRs in the Rhododendron genome

[0067] A total of 324,674 SSR sites were identified in the 509,494.34 Kb sequence of the azalea genome, with an average of 1.57 SSR sites per Kb. Among them, the number of mononucleotides and dinucleotides accounted for the vast majority, with 186,861 and 114,321 identified, respectively, accounting for 57.55% and 35.21% of the total SSR content, respectively. Trinucleotides, Tetranucleotides, Pentanucleotides, and Hexanucleotides accounted for only 7.24% in total. The number of different types of SSRs gradually decreased with the increase in the number of repetitions (Table 2). The density distribution of these SSR sites on the 13 chromosomes is shown in the figure below. Figure 2 As shown in Table 2, the 13 chromosomes contained 21,146 to 29,100 SSR markers, with an additional 4,234 markers distributed across other short scaffold sequences. Chromosome RsChr2 contained the largest number of SSRs. Linear regression revealed a correlation between chromosome length and the number of SSR loci in the Rhododendron genome, with a correlation coefficient of 0.87, indicating a highly significant correlation between chromosome length and the number of SSR loci (P < 0.01).

[0068] Table 2

[0069] Chromosome number Length (Mb) SSR quantity RsChr1 37.81445 24382 RsChr2 46.9999 29100 RsChr3 37.25137 25229 RsChr4 30.0301 21146 RsChr5 35.24487 24841 RsChr6 35.22913 22770 RsChr7 36.34218 23843 RsChr8 35.09778 24668 RsChr9 41.74717 26966 RsChr10 38.47903 25528 RsChr11 37.96297 21939 RsChr12 36.17999 23157 RsChr13 41.71885 26871

[0070] 2.3.2 Quantitative characteristics of main motifs of different types of SSRs

[0071] The 324,674 SSR sites contain 265 motif types. In Mononucleotide, there are two motifs, A / T and C / G, of which A / T accounts for an absolute majority (93.6%). Figure 3 A). There are four types of dinucleotides: AG / CT, AT / AT, AC / GT, and CG / CG, among which AG / CT accounts for the largest proportion (67.2%) ( Figure 3 B). Among trinucleotides, there are 10 motifs including ACC / GGT, AAG / CTT, AAT / ATT, AGG / CCT, AAC / GTT, CCG / CGG, ATC / ATG, AGC / CTG, ACT / AGT, and ACG / CGT. ACC / GGT (38.5%) accounts for the largest proportion, followed by AAG / CTT (19.7%) and AAT / ATT (13.6%). The proportions of the other seven motifs are all less than 10% ( Figure 3C). Tetranucleotides include AAAT / ATTT, AAAG / CTTT, ACAT / ATGT, AGGG / CCCT, AGAT / ATCT AAAC / GTTT, ACCC / GGGT, AATT / AATT, AAGG / CCTT, and 23 other motifs. Among them, AAAT / ATTT, AAAG / CTTT, ACAT / ATGT, and AGGG / CCCT account for the largest proportion, while the proportions of the other 28 motifs are all less than 10% ( Figure 3 D). In Pentanucleotide, there are 73 motif types, of which AAAAG / CTTTT (15.9%), AAACC / GGTTT (10.6%), AAAAT / ATTTT (8.8%), AAACT / AGTTT (6.4%), AAAAC / GTTTT (5.6%), ACCCC / GGGGT (4.8%), AGAGG / CCTCT (3.7%), AGCCG / CGGCT (3.5%), and AGGGG / CCCT (3.1%) are the top nine motif types with the largest proportion ( Figure 3 E). In hexanucleotides, there are 144 motif types, of which 15 have a number ≥ 10. Among them, AGAGGG / CCCTCT and AAAAAG / CTTTTT account for the highest proportions, 13.5% and 12.3% respectively ( Figure 3 F) Among all motif types, there are 24 motif types with a number greater than 100, and the top 10 motif types are A / T, AG / CT, AT / AT, AC / GT, C / G, ACC / GGT, AAG / CTT, AAT / ATT, AGG / CCT, and AAC / GTT.

[0072] Example 3

[0073] 3.1 Genetic diversity analysis of 96 rhododendron resources based on 30 SSR markers

[0074] Sequencing data from SSR amplification products were collected and analyzed simultaneously using GeneMarker Version 1.51 and GeneMapper Version 4.0 (Applied Biosystems). Multiple indicators reflecting genetic diversity were analyzed, including the polymorphism information index (PIC), expected heterozygosity (He), observed heterozygosity (Ho), average number of alleles (Na), and Shannon diversity index (I). Data were calculated using POPGENE Version 1.31. The polymorphism information index (PIC) was calculated using The Excel Microsatellite Toolkit 3.1 and GenAlEx software, and population structure analysis was calculated using STRUCTRE 2.3.1 software.

[0075] 3.2 Polymorphism analysis of SSR markers

[0076] After PCR amplification of the four templates, 124 (65.3%) g-SSR markers were able to stably amplify the target bands. Although 25 markers were able to amplify bands, the product length was obviously not in line with expectations. Such markers were not required for further research. The remaining markers could not amplify bands and were all discarded. For the 124 pairs of markers that could be stably amplified, after capillary fluorescence detection, it was found that 32.3%) markers were polymorphic in the four samples. 30 pairs of primers with strong polymorphism were selected from them to perform PCR amplification on 96 rhododendron resources. The primer information is shown in Table 3. The results showed that a total of 597 polymorphic bands were amplified by the 30 pairs of g-SSR markers. The number of amplified bands for each marker ranged from 4 to 56. The most was RsgSSR212, and the least was RsgSSR185. On average, each marker could amplify 12.467 polymorphic fragments. The average Ne, Ho, He, and I values ​​for all markers were 6.406, 0.630, 0.768, and 2.055, respectively. The PIC value, an important indicator for evaluating the genetic information index (GII) of a marker, ranged from 0.240 to 0.949, with an average of 0.748 among the 30 markers. The highest PIC value was for RsgSSR212, while the lowest was for RsgSSR185 (Table 4). In this study, 27 markers, representing 90% of all markers, had PIC values ​​greater than 0.5, indicating that the EST-SSR markers developed in this study are highly polymorphic and suitable for subsequent genetic diversity analysis.

[0077] Table 3

[0078]

[0079]

[0080] Table 4

[0081]

[0082]

[0083] 3. Results of population structure analysis of 3,396 rhododendron resources

[0084] The results showed that the samples included 4 original species of Rhododendron (sample numbers: 92, 97, 101, 114), 2 cultivated species of Rhododendron (sample numbers: 98, 115) and 90 commercial varieties of Rhododendron ( Figure 4 ). The presence of three genetic components, C1, C2 and C3, was detected in all samples, which is consistent with the characteristics that commercial azalea varieties are mainly obtained through hybrid breeding. Based on the analysis of the proportion of genetic components, the samples can be divided into three main groups: C1-dominant group (55.21%, a total of 53 samples): This group is mainly composed of azalea varieties from Japan (n=52), and only sample 106 is the American Anku azalea. Genetic analysis shows that these samples are mostly hybrid offspring between azalea varieties. C2-dominant group (20.83%, a total of 20 samples): This group shows a more complex genetic background, and the sources of samples include 12 samples from Japan, 5 samples from the United States and 3 samples from China. C3-dominant group (23.96%, a total of 23 samples): This group is mainly composed of American Anku azalea. Among them, Rhododendron fortunei and Rhododendron × pulchrum are also included in this category, indicating that the genes of these varieties may have been introduced into the gene pool of commercial rhododendrons.

[0085] Example 4

[0086] 4.1 Genotype-phenotype association analysis

[0087] TASSEL 4.0 software was used to perform genome-wide association analysis on SSR genotype and phenotypic data. First, a mixed linear model (MLM) was used to correct for the population structure effect. The Q value of the population genetic structure analysis (calculated by STRUCTURE software) and the kinship matrix (calculated by TASSEL software) were included in the model as covariates to control the false positive rate. Subsequently, a threshold of P < 0.05 was used to determine the significant association between SSR loci and target traits.

[0088] 4.2 Results of association analysis of SSR markers

[0089] Association analysis of 12 phenotypic traits and SSR data using the MLM model revealed that 17 markers were significantly associated with 10 phenotypic traits (P < 0.05), explaining 5.68% to 13.35% of the phenotypic variation (Table 5). Blade length was significantly associated with RsgSSR261 and had the highest phenotypic explanation rate. In addition, multiple traits were significantly associated with more than one marker, for example, Remontant was significantly associated with three markers, RsgSSR227, RsgSSR62, and RsgSSR126; Corollalobe shape was significantly associated with two markers, RsgSSR198 and RsgSSR261; Initial flowering period was significantly associated with three markers, RsgSSR259, RsgSSR207, and RsgSSR26; Flower diameter was significantly associated with three markers, RsgSSR185, RsgSSR188, and RsgSSR198; Petal type was significantly associated with two markers, RsgSSR126 and RsgSSR268; Petiole length was significantly associated with two markers, RsgSSR222 and RsgSSR188; Stamen number was significantly associated with three markers, RsgSSR235, RsgSSR128, and RsgSSR154. In addition, there are 5 markers that can be significantly associated with multiple traits, including RsgSSR198, RsgSSR261, RsgSSR188, RsgSSR126, and RsgSSR207, which are significantly associated with 3, 2, 2, 2, and 2 traits, respectively.

[0090] Table 5

[0091]

[0092]

[0093] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A polymorphic primer for SSR molecular markers of the whole genome of Rhododendron splendidum, characterized by: It consists of the following 30 pairs of polymorphic primers, the nucleotide sequences of which are as follows: The forward primer of RsgSSR26 is shown in SEQ ID NO. 1, and the reverse primer is shown in SEQ ID NO. 2; The forward primer of RsgSSR62 is shown in SEQ ID NO. 3, and the reverse primer is shown in SEQ ID NO. 4; The forward primer of RsgSSR94 is shown in SEQ ID NO. 5, and the reverse primer is shown in SEQ ID NO. 6; The forward primer of RsgSSR122 is shown in SEQ ID NO. 7, and the reverse primer is shown in SEQ ID NO. 8; The forward primer of RsgSSR123 is shown in SEQ ID NO. 9, and the reverse primer is shown in SEQ ID NO. 10; The forward primer of RsgSSR126 is shown in SEQ ID NO. 11, and the reverse primer is shown in SEQ ID NO. 12; The forward primer of RsgSSR128 is shown in SEQ ID NO. 13, and the reverse primer is shown in SEQ ID NO. 14; The forward primer of RsgSSR143 is shown in SEQ ID NO. 15, and the reverse primer is shown in SEQ ID NO. 16; The forward primer of RsgSSR148 is shown in SEQ ID NO. 17, and the reverse primer is shown in SEQ ID NO. 18; The forward primer of RsgSSR154 is shown in SEQ ID NO. 19, and the reverse primer is shown in SEQ ID NO. 20; The forward primer of RsgSSR185 is shown in SEQ ID NO. 21, and the reverse primer is shown in SEQ ID NO. 22; The forward primer of RsgSSR188 is shown in SEQ ID NO. 23, and the reverse primer is shown in SEQ ID NO. 24; The forward primer of RsgSSR198 is shown in SEQ ID NO. 25, and the reverse primer is shown in SEQ ID NO. 26; The forward primer of RsgSSR200 is shown in SEQ ID NO. 27, and the reverse primer is shown in SEQ ID NO. 28; The forward primer of RsgSSR207 is shown in SEQ ID NO. 29, and the reverse primer is shown in SEQ ID NO. 30; The forward primer of RsgSSR212 is shown in SEQ ID NO. 31, and the reverse primer is shown in SEQ ID NO. 32; The forward primer of RsgSSR220 is shown in SEQ ID NO. 33, and the reverse primer is shown in SEQ ID NO. 34; The forward primer of RsgSSR222 is shown in SEQ ID NO. 35, and the reverse primer is shown in SEQ ID NO. 36; The forward primer of RsgSSR227 is shown in SEQ ID NO. 37, and the reverse primer is shown in SEQ ID NO. 38; The forward primer of RsgSSR229 is shown in SEQ ID NO. 39, and the reverse primer is shown in SEQ ID NO. 40; The forward primer of RsgSSR232 is shown in SEQ ID NO. 41, and the reverse primer is shown in SEQ ID NO. 42; The forward primer of RsgSSR235 is shown in SEQ ID NO. 43, and the reverse primer is shown in SEQ ID NO. 44; The forward primer of RsgSSR236 is shown in SEQ ID NO. 45, and the reverse primer is shown in SEQ ID NO. 46; The forward primer of RsgSSR249 is shown in SEQ ID NO. 47, and the reverse primer is shown in SEQ ID NO. 48; The forward primer of RsgSSR253 is shown in SEQ ID NO. 49, and the reverse primer is shown in SEQ ID NO. 50; The forward primer of RsgSSR259 is shown in SEQ ID NO. 51, and the reverse primer is shown in SEQ ID NO. 52; The forward primer of RsgSSR260 is shown in SEQ ID NO. 53, and the reverse primer is shown in SEQ ID NO. 54; The forward primer of RsgSSR261 is shown in SEQ ID NO. 55, and the reverse primer is shown in SEQ ID NO. 56; The forward primer of RsgSSR262 is shown in SEQ ID NO. 57, and the reverse primer is shown in SEQ ID NO. 58; The forward primer of RsgSSR268 is shown as SEQ ID NO.59, and the reverse primer is shown as SEQ ID NO.

60.

2. Application of the polymorphic primers for the whole genome SSR molecular markers of Rhododendron splendidum according to claim 1 in azalea hybrid breeding, kinship identification, and molecular marker-assisted selection breeding.

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