Abstract

In this study, 49 samples of 43 Rhododendron sp. were studied by using RAPD to analyze their genetic diversity, relative ship and phylogeny. Four hundred and seven DNA fragments were amplified by 24 primers, the polymorphic rate was up to 98.03%. Their coefficient of Genetic Similarity (GS) was ranging from 0.2623~0.9059, which indicated rich genetic diversity in Rhododendron. The results showed that: 43 Rhododendron sp. were divided into 3 groups by RAPD, which was consistent with the division based on morphological characters, subgenus Hymenanthes had a closer relationship with subgenus Rhododendron than subgenus Pseudorhodorastrum and subgenus Hymenanthes was more primitive in phylogeny, subgenus Pseudorhodorastrum was the evolutive group in morphological characters, while subgenus Rhododendron was the transition one.

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INTRODUCTION

Rhododendron distributes mountain region widely in Southeastern China and formed many large populations. Studies on its relationship and classification system are good for resource conservation. Rhododendron is the largest genus in Ericaceae family, 1000 species have been found up to now (Yang et al., 1999). After genus of Rhododendron was posted in 1753, a large number of studies have been done on its classification and many kinds of classification system were proposed, including 8 subgenuses by Sleumer (1949, 1980), 5 subgenuses by Cullen (1980), 8 subgenuses by Chamberlain and Rae (1990) and Chamberlain et al. (1996), 8 subgenuses by Philipson and Philipson (1982) and 9 subgenuses. Which, one is more authoritative is still under arguing. Whether, lepidote Pseudorhodorastrum whose anthotaxy is axillary, belonged to subgenus Pseudorhodorastrum or should be regarded as a subgroup of lepidote subgenus Rhododendro is the controversial focus.

Random Amplified Polymorphic DNA (RAPD) technique is convenient to operate, with good polymorphism can be used in analyzing genetic diversity and the relation between species has been used in analyzing the relationships between strains belonging to the same genera and genetic diversity on many plants, such as Nothopanax Miq., Caragana Fabr., Prunus Persica and Lespedeza. Up to now, there had been few reports on Rhododendron in molecule level. In abroad, Kron (1997) and Kurashige et al. (1998, 2001) analyzed two genes trnK and matK of Rhododendron. At home, Gao et al. (2002, 2003) studied the relationship between subg. Tsutsusi and Phylogeny and development of section Azaleastrum. RAPD analysis only was reported on 11 Rhododendron (Zhao et al., 1996) in home.

Southwest of China is the largest distribution center of Rhododendron, where has a large distribution of numerous and valuable subgenus Hymenanthes and subgenus Rhododendron. In this study, genetic diversity, relationship and phylogeny of 43 species distributed in Southeast in China were analyzed by RAPD, which provided some proofs for the classification of Rhododendron.

MATERIALS AND METHODS

Forty nine samples belong to 43 species were studied. Twenty nine species were collected from southwestern mountain region of Sichuan in China, where, the altitude is 2020~3435 m; while, the other 14 species were collected from West China Subalpine Botanical Garden, Institute of Botany, the Chinese Academy of Sciences, Dujiangyan in Sichuan. Selected four individuals of each sample and mixed, then put into silica gel to dry and conserved at -80°C.

Eight subgenuses system proposed by Sleumer (1949, 1980) was adopted. Forty three species (Table 1) belong to 3 subgenuses, including 23 subgenus Hymenanthes, 18 subgenus Rhododendron and 2 subgenus Pseudorhodorastrum.

Table 1:	Materials of experiment

DNA extraction: DNA extraction method suitable for RAPD analysis has been established by means of the nucleus deposition method (Zou et al., 2001). The concentration and quality of each sample of DNA were calculated from the Optical Density (OD) values at 230, 260 and 280 nm and DNA was regarded as being of good quality when, the ratio of OD_260/280 to OD_260/230 was near 1.8. DNA samples were diluted to standardized DNA (20 ng μL^-1).

PCR amplification: The reaction was carried out in a volume of 20 μL and was prepared as follows: 40 ng of genomic DNA, 1.5 U TaqE, 1.5 μL MgCl₂ (25 mmol L^-1), 2.0 μL 10x reaction buffer, 5 mmol L^-1 dNTPs and 0.36 μmol L^-1 primer. Each reaction solution was overlaid with one drop of mineral oil to prevent evaporation. Amplification reactions was performed in a 96-well thermocycler (Eppendorf Authorized Thermal Cycler PCR) programmed as follows: an initial denaturizing at 94°C for 3 min followed by 40 cycles of 1 min at 94°C, 39 sec at 36°C, 1 min at 72°C and finally extended at 72°C for 10 min. The amplified products were analyzed for band presence and absence after electrophoretic separation on 1.5% agarose gels and staining with ethidium bromide. Each amplified reaction was carried out 3 times to ensure results were consistent.

Statistics and analysis of data: Four hundred and twenty RAPD primers screened, 24 primers produced distinct, reproducible, polymorphic profiles among the samples tested (Table 2). Evaluated 1 for band and 0 for bandless to form a binary matrix. Analyzed the (0, 1) binary matrix by using NTSYSpc software and calculated GS by Nei and Li (1979) method with equation:

GS = 2 Nij/(Ni + Nj)

 

Where,
Nij	=	The number of bands sample i and j amplified
Ni	=	The number of bands amplified by sample i and
Nj	=	Amplified by sample j. The samples tested were classified by UPGMA based on GS.

 

RESULTS AND DISCUSSION

RAPD amplification results: Four hundred and seven bands were amplified by 24 primers among 49 samples, 399 were polymorphic bands, up to 98.03%. Average bands amplified by a primer were 16.96. The results (Table 2) showed that different primers could amplify different bands on the same sample and different samples could amplify different bands by the same primer, which reflects complexity in genetic background and genetic diversity.

Table 2:	Sequences of 24 primers and the number of amplified strips of Rhododendron

 

Fig. 1:	RAPD figure amplified by primer SBS-J9 and SBS-X3. M is DNA Marker; 1~49: Sample number

 

Only 8 bands were shared by all samples, which indicated their homology to a certain extent. Among the three subgenuses, some bands shared by one subgenus, 6 in subgenus Hymenanthes; 5 in subgenus Rhododendron; and 4 in subgenus Pseudorhodorastrum, which showed the characteristics of subgenus. Figure 1 was the result amplified by primer J₉ and X₃.

The similarity coefficient was calculated by software. Average GS of 49 samples was 0.4631, which reflected the difference between samples was small. GS 0.2623 between R. rubiginosum and R. hemitrichotum was the least, while GS 0.9059 between 2 R. gonggashanenses was the largest and followed the GS 0.8736 between 2 R. fortunei 3 R. racemosum and 3 R. hemitrichotum had a higher GS within their species, which were 0.7547 and 0.7342, respectively. Although, some Rhododendron belongs to the same species, their GS was higher, which indicated their high homology in genetic background.

Fig. 2:	Dendrogram obtained from RAPD data of 43 species (49 specimens) of Rhododendron by UPGMA

The GS of 49 samples between 3 subgenuses were different, GS between subgenus Hymenanthes and subgenus Rhododendron was 0.3982, which was higher than the GS 0.3713 between subgenus Rhododendron and subgenus Pseudorhodorastrum. GS 0.3519 between subgenus Hymenanthes and subgenus Pseudorhodorastrum was the least, which reflected that subgenus Rhododendron and subgenus Hymenanthes had a closer relationship than subgen Pseudorhodorastrum. The relationship between subgenus Pseudorhodorastrum and subgenus Rhododendron was far, while subgenus Pseudorhodorastrum had a much farther relationship with subgenus Hymenanthes. The low GS between 3 subgenuses showed their great differences in background. GS of 25 samples in subgenus Hymenanthes was 0.4457~0.8714, 18 samples in subgenus Rhododendron was 0.4457~0.8488, 2 samples in subgenus Pseudorhodorastrum was 0.5291~0.7059, their averages were 0.5660, 0.5983 and 0.6527, respectively. The results showed that the difference in subgenus less than between them.

Forty nine samples were divided into 3 groups by UPGMA based on GS (Fig. 2). The first group included 25 samples belonged to subgenus Hymenanthes; the second group contained 18 samples belonged to subgenus Rhododendron and the other 6 samples of subgenus Pseudorhodorastrum were divided into the third group. The result was accorded with the classification based on morphology. The first two groups clustered at GS 0.40 then gathered with the third group at GS 0.37.

Only section Ponticum was gathered into the first group, 25 samples of 23 species were divided into two subgroups at GS 0.54, 17 samples of 15 species were divided into the first subgroup including subsection Fortunea, subsection Falconera and subsection Maculifera, the other 8 species were divided into the second subgroup. Eighteen samples in the second group were 18 species, which could be divided into 3 subsections by morphology. While, they were divided into two subgroups in GS 0.59, 12 species of, which were in the first subgroup containing subsection Heliolepids and most of subsection Triflora. The other 6 species in the subsecond group contained 5 species of subsection Triflora and a species R. rubiginosum of subsection Heliolepids. The results showed that in subgenus Rhododendron, there was a closest relativeship among R. triflorum, R. ambiguum and R. lutescens and they were gathered together at GS 0.83. Followed by R. intricatum, R. thymifolium and R. nitidulum in subsection Lapponica, they were clustered together at GS 0.80. In the third group, 6 samples of subgenus Pseudorhodorastrum were divided into two subgroups, 3 samples of R. racemosum were in the first small subgroup, the other 3 samples in the second subgroup were R. hemitrichotum.

RAPD genetic diversity: Four hundred and seven bands were amplified by 24 primers among 49 samples, 399 of, which were polymorphic bands, up to 98.03% (Table 2 and Fig. 1), which reflected a high genetic diversity among 49 Rhododendron samples. The morphology diversity was caused by genetic diversity and the complex environment. High genetic diversity was good for the breeding of excellent cultivars. High diversity of Rhododendron reflected its strong adaptability to environment, which is benefit to its propagation, resource conservation, domestication and screen.

Clustering result is consistent with the classification based on morphology, which showed that morphological traits can reflect the genetic characters. Figure 2, subgenus Hymenanthes and subgenus Rhododendron gathered together first and then gathered with subgenus Pseudorhodorastrum, which indicated that subgenus Hymenanthes and subgenus Rhododendron had a closer relationship than with subgenus Pseudorhodorastrum. In many classification system, Hymenanthes were divided as one subgenus, so it was improper to divide subgenus Pseudorhodorastrum as a subsection in subgenus Rhododendron. In this study, the results did not support Cullen and Chaimberlain’s view, but we can agree Sleumer’s standpoint to divide subgenus Pseudorhodorastrum as a subgenus, which could show the difference between subgenus Pseudorhodorastrum and subgenus Rhododendron.

The genetic difference was obvious between species in a subgenus. R. gonggashanense, R. davidii and R. fortunei in subgenus Hymenanthes, their GS can be up to 0.814, which was approached to the value calculated by morphology. R. davidii and R. wiltonii had a close relationship, their GS was 0.4508. R. triflorum, R. ambiguum and R. lutescens in section Ponticum were sole semi-deciduous species and with yellow flower, RAPD markers showed these characters, their GS was the largest (0.8280), we can show from the clustering dendrograph that their genetic difference was the least. Although, R. intricatum, R. thymifolium and R. nitidulum in subsection Lapponica were distributed far away, they clustered together in GS 0.80, which reflected their high genetic homology. GS between R. rubiginosum in subsection Heliolepids and 14 species in subsection Triflora was 0.5000~0.6867, the average was 0.6039, while the GS in subsection Triflora was 0.4700~0.8488, their average was 0.5981, which showed that subsection Heliolepids had a closer relationship with subsection Triflora. It can be reflected by morphology. So, we could consider to merge subsection Heliolepids and subsection Triflora as one subsection. R. racemosum and R. hemitrichotum in subgenus Pseudorhodorastrum were divided into section Rhodobotrys and section Trachyrhodion, respectively based on morphology, their GS was 0.5915 close to the GS 0.5983 within section Rhododendron and higher than the GS 0.5660 within section Ponticum. It was obviously that section Rhodobotrys and section Trachyrhodion had a closer relationship; we should reconsider that whether they could be divided into different sections.

CONCLUSION

Forty three Rhododendron sp. tested can be divided into 3 groups by RAPD, which was consistent with the division based on morphological characters. The 407 bands were amplified by 24 primers among 49 samples, the number of polymorphic bands was 399 up to 98.03%, which reflected high genetic diversity in Rhododendron.

How to cite this article:

Zhou Lanying, Wan Yongqing and Zhang Li. Genetic Diversity and Relationship of 43 Rhododendron sp. Based on RAPD Analysis.
DOI: https://doi.org/10.36478/brj.2009.1.6
URL: https://www.makhillpublications.co/view-article/1995-4751/brj.2009.1.6