113species in the genus Camellia, with unique yellow flowers, and have great values in scientific research, breeding, for 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, Professors Chang, Liang and Min, there are certain differences in taxonomy among yellow camellia taxa. Molecular markers are one kind of new technology, mainly including RAPD, AFLP, SSR and ISSR and so on, which provide an effective analysis method for germplasm resources. ISSR markers were used to examine genetic relationship and polymorphism of 29 yellow camellia taxa in 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 for the quality of DNA by way of 1 percent of agarose gel electrophoresis. DNA concentrations were determined by Nanodrop ND-2000.

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

Genetic Relationship among 29 Yellow Camellia Germplasms in Nanning

Golden Park by ISSR AnalysisXIAO Zheng1 LI Ji-yuan1* 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

Abstract: 29 Samples of yellow camellias were analyzed by inter-simple sequence repeat (ISSR) markers to detect their genetic diversity and relationship. 133 discernible loci were obtained from all the samples by using 14 primers and 126 bands were polymorphic. The average percentage of polymorphic bands was 94.74%, Nei’s gene diversity and Shannon’s information index was 0.3606 and 0.5314 respectively, indicating that the genetic diversity of yellow camellias was relatively high. According to the dendrogram, 29 materials could be divided into 3 groups. Camellia chrysantha formed the first group; C. pingguoensis var. terminalis and C. lungzhouensis were classified into the third group, and the others into the second group. The pair of C. xiashiensis and C. micrantha was classified into one group at very early stage, indicating two taxa are very similar. The pair of C. tunghinensis and C. leptopetala was much similar. The two pairs of C. tianeensis and C. flavida, C. wumingensis and C. parvipetala were similar, respectively. There are certain relationship between C. pingguoensis and the hybrid sample, but low relationship between C. nitidissima and C. chrysantha. C. chuongtsoensis is a separated species.Key words: Yellow camellias; Genetic relationship; Inter-simple sequence repeat (ISSR)

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 Vietnam. The colors of camellia flowers are commonly red, pink, white and so on, but not yellow. Yellow camellias are rare and endangered

114reaction, 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. Differences in the 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 was recorded as “1”, no band was recorded as “0”. The strong or weak bands were all recorded as “1” The original matrix was then established.

Y=(C,T), R=(A,G).The data was analyzed with Popgen32. We could now get Nei’s gene diversity and Shannon’s information index. We observed the number of alleles and the effective number of alleles. The UPGMA cluster analysis based on genetic similarity coefficient was carried out by NTSYSpc2.10e, to obtain the relationship tree.

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

Table1 Taxa of yellow camellias used and their origins

No. Taxa Origin No. Taxa Origin1 C. achrysantha Guangxi 16 C. chrysanthoides Guangxi2 C.pingguoensis var. terminalis Guangxi 17 C. microcarpa Guangxi3 C. tianeensis Guangxi 18 An Unknown taxon Guangxi4 C. flavida Guangxi 19 C. multipetala Guangxi5 C. euphlebia Guangxi 20 C. ptilosperm Guangxi6 C. longzhouensis Guangxi 21 C. tunghinensis Guangxi7 C. longruiensis Guangxi 22 C. leptopetala Guangxi8 A natural hybrid Guangxi 23 C. chuangtsoensis Guangxi9 C. limonia Guangxi 24 C. long gangensis Guangxi10 C. pingguoensis Guangxi 25 C. fascicularis Yunnan11 C. xiashiensis Guangxi 26 C. parvipetala Guangxi12 C. micrantha Guangxi 27 C. wumingensis Guangxi13 C. chrysantha Guangxi 28 C. nitidissima var. phaeopubisperma Guangxi14 C. impressinervis Guangxi 29 C. tienii Vietnam15 C. pubipetala Guangxi

Table 2 Primers used for ISSR amplification

Primer Sequence (5’-3’) Annealing temperature (℃)

No. of amplified bands

No. of polymorphic bands

Percentage of polymorphic bands (%)

UBC810 (GA)8T 55 8 8 100UBC811 (GA)8C 55 9 9 100UBC818 (CA)8G 51 8 7 87.5UBC824 (TC)8G 55 10 10 100UBC827 (AC)8G 55 8 7 87.5UBC834 (AG)8YT 54 11 9 81.82UBC835 (AG)8YC 56 12 12 100UBC841 (GA)8YC 56 12 11 91.67UBC843 (CT)8RA 54 9 9 100UBC848 (CA)8RG 55 8 8 100UBC853 (TC)8RT 52 10 10 100UBC873 (GACA)4 52 10 9 90IR43 (GA)8CT 53 9 8 88.89IR53 (CAA)8G 57 9 9 100Total 　 　 133 126 94.74

115product 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).

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

Fig1. Amplification of primer UBC835

M: marker; 1~29: Codes of lanes are the same as the accession number in Table 1

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

116was 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.

3 Discussions and ConclusionsThe 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 very similar to C. limonia except its ovary was only

C.xiashiensis

C. micrantha

C.xiashiensis

C. micrantha

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.

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.

117

C.tunghinensis

C.leptopetala

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

The one pair of C. wumingensis and 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 distinct species 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 species and should be merged with C. pingguoensis. C. longzhouensis was similar to C. chrysanthoides except both bud and ovary are pubescent. They were treated as the same taxon by Liang and Zhang suggesting that they were different species. ISSR analysis showed that they were very different in relationship, and should be two distinct species.

There were certain relationship between C. pingguoensis and the unknown sample. They were very similar in flower color and flower size. The results showed a 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).

118Literature cited

[1] Liang Sheng Ye. Yellow Camellias. Beijing: China Forestry Press, 1993.

[2] Chang Hungta, Ren Shanxiang. Flora Reipublicae Popularis Sinicae, Tomus 49(3). Beijing: Science Press, 1998.

[3] Ming Tien-lu. Monograph of the genus Camellia. Yunnan: Yunnan Science and Technology Press, 2000.

[4] Gao Jiyin, Clifford R. Parks, Du Yueqiang. Collected Species of the genus Camellia: an illustrated outline. Zhejiang: Zhejiang Science and Technology Press,2005.

[5] Xiao Tiaojiang, Clifford R. Parks. Molecular analysis of the Genus Camellia. International Camellia Journal, 2003, 57-65.

[6] Lin Li, Li Jiyuan*, Ni Sui*, Chen Yue, Fan Zhengqi, Li Xinlei, Ying Zhen.The influence of Insular Geographical Isolation on Population Gentic Structure of Camellia japonica. Forest Research, 2012, 25(3):378~384.

[7] Lin Li, Ni Sui, Li Jiyuan*, Chen Yue, Ying Zhen.Study on population genetic diversity of Camellia japonica in 5 islands between China and Japan. Guihaia, 2012, 32(3): 298~303.

[8] Lin Li, Hu Zhongyi, Li Jiyuan, Zhu Zhiyong, Ni Sui. Analysis on Genetic Diversity of Ten Insular Populations of Camellia japonica. Acta Horticulturae Sinica, 2012, 39(8):3531-1538.

[9] TANG Shao-qing, SHI Su-hua, ZHONG Yang, WANG Yan, 2004a. Phylogenetic relationships of golden camellias (sect.Chrysantha, Camellia)in China: evidence from ITS sequences of nuclear ribosomal DNA. Guihaia, 24(6): 488~492.

[10] Tang Shaoqing, Shi Suhua, Chen Yueqin, Qu Lianghu, Zhang Hongda, 1998.Phylogenetic Relationship of Camellia nitidissima Chi and Its Allied Species Based on Random Amplified Polymorphic DNA. ACTA SCIENTIARUM NATURALIU MUNIVERSIT AT IS SUNYAT SENI, 37(4): 28-32.

[11] Tang Shaoqing, Du LinFang, WANG Yan. AFLP Analysis of Sect. Chrysantha Chang ( Camellia Sect. Chrysantha), 2004C. Journal of Wuhan Botanial Research, 22(1):44~48.

[12] Park Y G, Shiv SK. Genetic variation on wild tea populations in Korea[M]. Proceedings of the International Symposium on Molecular Biology and Tea Breeding . Hangzhou : Zhejiang University , 2000: 1-18

[13] Mirshra R K, Sen-M and iS. Genome analysis and isozyme studies for developing molecular markers associated with drought tolerance in tea plant[M].Proceedings of 2001 International Conference on O-Cha(Tea) Culture and Science(Session II). Japan :Shizuoka, 2001

[14] Kaundun S S , Zhyvoloup A, Park Y G. Evaluation of the genetic diversity among elite tea (Camellia sinensis var. sinensis) accessions using RAPD markers[J] . Euphytica, 2000, l15 : 7-l6