What is the impact of SRAP molecular marker technology on the genetic diversity of Rhododendron sinensis?
Foreword: Rhododendron huading is born in the mixed coniferous and broad-leaved forest at an altitude of 700~1200m, the plant height is about 1~4m, the bark is patchy longitudinal fissure, the flowering period is long, the flower color is gorgeous, and it has good ornamental value, but the interspecific relationship between Rhododendron huading is loose, the overall correlation is poor, and the population has different degrees of decline, and the species needs to be protected urgently.
Correlation Sequence Amplification Polymorphism (SRAP) is a novel PCR-based molecular marker technique that uses specific primers to amplify the open reading frame (ORF) with refolding as the core.
Compared with molecular markers such as simple sequence repeat interval (ISSR), random amplification DNA polymorphism (RAPD), and simple repeat sequence (SSR), SRAP has the advantages of high co-dominance, good reproducibility, and easy operation, and is widely used in the identification and evaluation of germplasm resources, gene localization cloning, and genetic diversity analysis.
In recent years, research on R. sinensis has mainly focused on community structure, seed germination and seedling raising, but there are few studies at the molecular level.
The leaves of different plants were collected, rinsed, dried and grinded, and the genomic DNA of the leaves of R. huading was extracted by sodium dodecyl sulfonate method (SDS method) for electrophoresis detection. A PCR reaction was used to select combinations with good reproducibility and high polymorphism from 140 pairs of primers for SRAP-PCR amplification.
After the cycle, "0" and "1" were used to indicate the absence and existence of bands, and the statistical results of SRAP-PCR were used to calculate the genetic parameters at the population level and the species level, and then UPGMA cluster analysis was performed.
A total of 8 primer combinations were screened, and the bands amplified by these primers were characterized by high definition, good stability and rich polymorphism, which could be used for subsequent band reading and statistical analysis.
A total of 263 loci were amplified by 8 pairs of primers, including 261 polymorphic sites, with a percentage of 99.24% polymorphic sites, and an average of 32.87 loci were amplified from each pair of primers.
At the species level, the H values of R. sinensis were 0.2215 and I were 0.3579, while the mean PPB values at the population level were 36.96%, and the mean values of I and H were 0.1817 and 0.1199, respectively, which were lower than those at the species level.
There were also great differences in the genetic parameters of each population, and the genetic parameters (Na, Ne, H, I) of KCS were larger than those of other populations, indicating high genetic diversity.
The genetic distance between populations ranged from 0.1167~0.2173, and the average genetic distance was 0.1567.
Among them, the genetic distance between KCS and CZ was the largest, which was 0.2173, and the genetic agreement was the smallest, which was 0.8047, indicating that there was a large genetic difference between populations, and their relationship was farther.
The genetic distance between DCJ and CZ was the smallest (0.1167), and the genetic similarity coefficient was the largest (0.8899), indicating that the genetic difference between the two populations was the largest.
The calculated results of genetic differentiation coefficient (Gst) and gene flow (Nm) showed that the Gst of the five populations was 0.4596, that is, 45.96% of the genetic variation came from the population, 54.04% existed within the population, and the Nm was 0.5878, which was less than 1, indicating that the genetic exchange between the populations was less.
The results of cluster analysis showed that when the genetic similarity coefficient was 0.31, the five populations were clustered into two different branches, the first one included the population TGJ, KCS and TTS, and the other included the population DCJ and CZ, indicating that the relationship between DCJ and CZ and the other three populations was relatively distant.
With the increase of the similarity coefficient, the genetic relationship between the individuals in the population is more clear, and the samples of the same geographical origin are clustered into a group, and there is no crossover between the populations.
When the genetic similarity coefficient was about 0.56, the 10 samples of the TTS population were divided into two branches, and TTS3 was clustered in one group, indicating that there were some differences between TTS3 and other individuals in the population.
At the species level, the PPB value of R. sinensis was as high as 99.24%, the I value was 0.3579, and the H value was 0.2215, which was higher than that of many endangered shrub species, while the genetic parameters at the population level were much smaller than those at the species level, indicating that the level of genetic variation in the population was relatively low.
The genetic variation of plant populations is mainly affected by factors such as population size, reproductive system, and gene flow, among which the migration of gene flow can prevent population differentiation and prevent the reduction of genetic variation within populations.
The gene exchange of plants needs to rely on pollen, spores and other mediums carrying genetic material, and the breeding system of Rhododendron sinensis is heterogeneous, and the gene exchange between and within populations can be carried out through the diffusion of seeds or pollen.
Conclusion:
In summary, the genetic differentiation coefficient among the populations of C. sinensis was 0.4596, indicating that most of the genetic variation occurred within the population, and the rest occurred among the populations.
When Nm<1, genetic drift would lead to obvious genetic differentiation between populations, and the Nm value of R. huading was 0.5878, indicating that genetic drift occurred between populations, which was one of the important reasons for genetic differentiation between populations.
SRAP molecular markers clarified the genetic diversity of R. sinensis, which provided a basis for the subsequent conservation and rational utilization of R. sinensis.
