Genetic Diversity Of Areca Catechu L. From Baoting Of Hainan Province Detected With ISSR Markers

Areca catechu L. is a species of perennial evergreen arbor, cultivated originally in Malaysia and now planted mostly across the tropical regions of Asia and Americas. The cultivation history of Areca catechu in China is about 2000 years, which planted dominatingly in Hainan and Taiwan province. In Hainan province, Areca catechu is the second-important economic crop followed the rubber plant. Besides Hainan and Taiwan, Areca catechu is also cultivated in some areas of Guangdong, Guangxi, Fujian and Yunnan. The seed of Areca catechu contains tannin and various alkaloids, that make it becomes the most important of the four famous medicines in south China and has great values in traditional Chinese medicine. The variety of cultivated Areca catechu in China was introduced from foreign countries. During the long cultivation history, many kinds of types had appeared under various growth conditions. In China, Areca catechu is the most common species of the Areca family. However there are few reports on the genetic diversity of this important crop for the moment. In this research,the genetic diversity of Areca catechu materials collected from three different regions of Baoting county of Hainan province using ISSR markers with Areca triandra Roxb. was studied, Archontophoenix alexandrac (F. Muell.) H. Wendl. et Drude and Cocos nucifera L. were used as out-group checks. The aim of this research is to acquire the knowledge about the genetic variation of ISSR molecular markers of the Areca catechu in Hainan province and provide scientific basis for the collection and preservation of Areca catechu germplasm resources. After the research, following results were obtained:Firstly, the effective and detailed procedure of DNA extraction from Areca catechu was established. The traditional CTAB method to extract DNA from the leaves of Areca catechu with high amylose content was improved. This improved method is easily handle and can get the Areca catechu DNA with high purity and good quality.Secondly, the ISSR reaction system for Areca catechu was established. After probing into the various affecting factors of ISSR reaction, a suited and effective reaction system for Areca catechu was established, which consists of 60 ng template DNA,1.0 U Taq DNA polymerase, 2.5 mmol/L Mg2+,0.4 mmol/L dNTPs and 0.8μmol/L primers with total 20μL reaction solution..Thirdly,30 polymorphic primers were selected and used to amplify the total DNA of Areca catechu. The size of the amplified products were from 250bp to 2000bp. Three hundred and forty seven bands were obtained in all 99 accessions with 30 primers.302 bands were polymorphic with the percentage of polymorphism 87%.4-16 polymorphic bands were amplified using one primer with a mean of 10.. The rate of polymorphic bands produced with one primer was 0.160-1.000, with an average of 0.665. The genetic diversity level of Areca catechu materials used in this study was low as a whole. The genetic diversity within-population was about 70% of the total genetic diversity detected (68.2% and 66.7% for Nei's and Shannon's index respectively). These indicated that the population genetic structure of Areca catechu was in the rank of cross-pollinatinged plant.Fourthly, the level of genetic diversity of Areca catechu materials from Baoting was from modest to low with a total diversity index (I) 0.4398. And the variation among populations was great (the variation range from 0.1928 to 0.3946). The populations from Qixianling and Bacun village had higher genetic diversity than those from Ganshiling. It was detected that about 70% genetic diversity existed within Areca catechu populations. This means Areca catechu had a characteristic genetic structure of cross-pollinating plant.Fifthly, Areca catechu materials of 12 populations studied could be classified into 5 clusters or types:Ganshiling type (I), Ganshiling-Qixianling transition type (II), Bacun village type (III), Qixianling type (IV) and Baoting-Qixianling transition type (V). This would provide basis for genetic modificationof Areca catechu.Sixthly, there was no clear correlation between the fruittypes and the ISSR markers of Areca catechu. However, some fruittypes could be classified into together although this was likely due to the result of geographical consistency.Seventhly, there was clear geographically phylogenetic relationship among the populations of Areca catechu from Baoting county of Hainan. The populations from Qixianling were phylogenetically close to those from Bacun village and distant to those of Ganshiling. In addition, there were some intercrossing populations between different areas.Based on these results, it was necessary to extend the sampling regions in order to enrich the germplasm bank. When collecting and preserving the germplasm resources of Areca catechu more individual plants shoud be collected from the populations with higher genetic diversity. The genetic variations of parents used in cross-breeding should be high for Areca catechu in order to gain stronger hybrid vigor. B11, B22, B23 from Bacun village and Q19 and Q112 from Qixianling farm should be considered as one of the parents due to the presence of higher genetic variations than the other material used in this study.