Genetic Relationship among 29 Yellow Camellia Germplasms in Nanning Golden Park by ISSR Analysis

Xiao Zheng¹  Li Ji-yuan^1*  Li Zhi-hui²  Huang Lian-dong² Fan Zheng-qi¹  Jiang Chang-jie²
1. Research Institute of Subtropical Forestry, CAF, Fuyang, 311400, Zhejiang, China
2. Nanning Golden Camellia Park, Nanning, Guangxi, 530023, China)

Introduction

Yellow camellias are one group of evergreen shrubs or small trees which belong to Genus Camellia of Sect. Chrysantha Chang of Theaceae, and are mainly distributed in the south of Guangxi Region, Yunnan, Guizhou and Sichuan of China, and Vientam. The colors of Camellia flowers are commonly red, pink, white and so on, but lack of yellow. Yellow camelliasare rare and endangered species in genus of Camellia, with unique yellow flowers, and have great values in scientific research, breeding, ornamentals as well as medical materials. Nanning Golden Camellia Park established a yellow camellia germplasm bank in 1995, probably largest one in the world. However, some yellow taxa may be confused due to transplanting, loss of labels, errors of records, and open hybridization etc. According to current morphological taxonomy systems by three authorizing experts, Prof. Chang, Liang and Min, there are certain differences in taxonomy among yellow camellia taxa. Molecular markers are one kind of new technology, manly including RAPD, AFLP, SSR and ISSR and so on, which provides an effective analysis method on germplasm resources. ISSR markers was used to examine genetic relationship and polymorphism of 29 yellow camellia taxain Nanning Golden Camellia Park.

1 Material and Methods

1.1 Experimental Material

Leaves of 29 yellow camellia samples were collected from the Nanning Golden Camellia Park and Research Institute of Subtropical Forestry. The details are given in table 1.

1.2 Genome DNA extraction

DNA of yellow camellias were extracted by CTAB (slightly improved), then tested the quality of DNA by way of 1 percent of agarose gel electrophoresis. DNA concentrations were determined by Nanodrop ND-2000.

Table 1  Taxa of yellow camellias used and their origins

1.3 Primers and PCR amplification

According to previous experiments, 14 primers shown in table 2 are selected from 50 ISSR primers. They were synthesized by Dingguo biological engineering and technology services limited company. The ISSR reaction used in this study was 20 ul. The suitable concentration of PCR system was: Mg²⁺ 1.5mmol/L, dNTP 0.2mmol/L, primers 0.5umol/L, Taq 2.5u, buffer 2ul and 12.7 ul sterilized water. Amplification reactions program: 94℃， degeneration, 5min; one cycles; 94℃，degeneration，40S，specific temperature annealing 45S (different primers with different annealing temperature); 72℃，extension, 1.5min; 40cycles; 72℃, extension, 7min; termination reaction, preservation at 4℃. Products of ISSR-PCR were electrophoresed in the 2% of agarose gel electrophoresis for 90min. Voltage was 5v/cm and taken photos by FR-200A gel imager.

1.4 Data analysis

14 primers were analyzed by way of ISSR，as well as DNA samples of each taxon. Difference of samples amplified belts can be observed, the bands were read by artificial methods. To the same primers in electrophoresis maps, each band which had bands in some samples recorded as “1”, no band recorded as “0”. The strong or weak bands were all recorded as “1”, original matrix was then established.

Y=(C,T), R=(A,G).

The data was analyzed with Popgen32. We can get Nei’s gene diversity, Shannon’s information index. We observed number of alleles and effective number of alleles. The UPGMA cluster analysis based on genetic similarity coefficient was carried out by NTSYSpc2.10e, the relationship tree can be obtained.

2 Results and Analysis

2.1 Results of DNA amplification

14 primers were selected from 50 primers. They were used in the PCR reaction of 29 yellow camellia taxa. Each primer has 29 amplification product map of ISSR. One of amplifications with primer UBC835 is shown in the following Fig.1. 133 bands were amplified with 14 primers. The size of product ranged from 100 to 2000 bp. The number of bands from each primer ranged from 8 to 12. On average, each primer can be amplified with 9.5 bands. There were only 7 bands are common, and 126 were polymorphism. The percentage of polymorphism bands was 94.74% (Table 2).

 Fig 1. Amplification of primer UBC835
M: marker; 1~29: Codes of lanes are the same as the accession number in Table 1

 2.2 The level of genetic diversity

Diversity was analyzed with the software of POPGEN. The results showed that the genetic similarity coefficient calculated from ISSR data ranging from 0.48 to 0.83. The highest genetic similarity was 0.83 between C. xiashiensis and C. micrantha was highest. And the lowest was 0.48 between C. pingguoensis var. terminalis and C.chuangtsoensis. The Nei’s genetic diversity was 0.3606 and the effective number of alleles was 1.6307.

2.3 Results of clustering analysis

According to the matrix of genetic distance, clustering analysis was carried out by the UPGMA method. Relationship tree of 29 tested samples was formed. The 29 samples can be divided into three major categories. Group I was only composed of C. achrysantha, Group II, the largest one, contained 26 taxa. Group III included two taxa, i.e. C. pingguoensis var. terminalis and C. longzhouensis. Furthermore, in Group II there are three sub-groups classified in Fig.2.

Fig 2  Dendrogram of 29 yellow camellia taxa resulted from UPGMA analysis based on Dice’s similarity coefficient from the ISSR data

3 Discussions and Conclusions

The technology of ISSR markers is a new kind of molecular markers widely used to amplify the region between micro-satellites. In this study, the percentage of polymorphic bands was 94.74%, Nei’s genetic diversity was 0.3606, Shannon’s information index was 0.5314. That was similar to the result 93.2% analyzed by Tang Shaoqing using AFLP. Compared to the level of polymorphism of 35 tea samples (84.5% ) and 20 native tea resources (57.8%) in Korea, ten tea varieties (73.3%) in India, 8 tea varieties in Japan (56.9%) respectively, indicating that yellow camellias conserved in the park have high genetic diversity and were collected from certain wide distribution.

The pair of C. xiashiensis and C. micrantha was classified into one group at very early stage, indicating two taxa are very similar. This great similarity is same as those in major morphological characteristics such as flower form, flower size, color, shape and size of leaves between them, and also agrees with results by AFPL (Tang et al, 2004c).Whereas, the results were different from those in taxonomic systems set by Liang, Zhang and Ming. These two taxa were treated as two separated species by Liang, and merged to C.parvipetala by Zhang and merged to C.chrysanthoides by Ming. C. micrantha was much similar to C. limonia except its ovary only puberulent. Liang suggested that they should be merged to C. limonia. The ISSR results suggested that C. xiashiensis and C. micrantha should be immerged to C. limonia.

C.xiashiensis	C. micrantha

The similarity coefficient between C.tunghinensis and C.leptopetala was 0.805, but there were differences in some important morphological characteristics, such as smaller leaves, long flower pedicels, light yellow flowers in C. tunghinensis, and large and narrow elliptic leaves, shorter pedicels and yellow flowers in C.leptopetala.

C.tunghinensis	C.leptopetala

The ISSR results showed that the two pairs of C. tianeensis and C. flavisa, C. wumingensis and C. parvipetala were similar, respectively. Whereas C. tianeensis was larger than C. flavisa, and C. parvipetala was smaller than C. wumingensis in flower size.

The one pair of C.wumingensisand C. parvipetala are very similar according to ISSR dengrogram, but very differences among important morphological characteristics in leaves and flower. The former has larger, and dense yellow flowers, and the latter has smaller, light yellow flowers.

C. parvipetala	C.wumingensis

 C.pingguoensis var. terminalis was treated as a distinct specie by Liang, and regarded as one variety of C. pingguoensis by Zhang and Ming. The result showed that C.pingguoensis was very similar to C. pingguoensis, the similarity coefficient was up to 0.647, which was the highest compared with other camellia species. Therefore, C. pingguoensis var. terminalis was not a distinct specie, should be merged to C. pingguoensis. C. longzhouensis was similar to C. chrysanthoides except bud and ovary pubescent. They were treated as the same taxon by Liang and Zhang suggested that they were different species. ISSR analysis showed that they were much different in relationship, should be two distinct species.

There were certain relationship between C.pinguoensis and the ‘unknown’ sample. They were very similar in flower color and flower size. The results showed that low relationship between C. nitidissima var. phaeopubisperma and C.chrysantha, which supported Liang’s treatment as two separated species, and disagreed with combination of these two taxa by Zhang and Ming.

C.chuangtsoensis is a separated species according to our ISSR and other morphological characteristics.  

Acknowledgments:

This work was supported by the National Key Twelfth-Five Science and Technology Program (2012BAD01B0703), International Cooperation Project of China (2011DFA30490), and Zhejiang Key Flower Breeding Program (2012C12909-6).

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