Studies On Genetic Diversity Of Broussonetia Papyrifera Based On AFLP Markers

Broussonetia papyrifera is mainly distributed in China's northwest, north, south China, southeast and southwest around. B. papyrifera is widely suitable, sprout strongly, easy for propagation, and it is an important tree species for roadsides afforestation and preventing dikes as well as protecting banks. The plant of B. papyrifera can be all used, such as the bark, wood, branch, leaf, flower, fruit, seed, root, latex and so on. Therefore, its economic benefits have enormous potentialities. The plants of Broussonetla had attracted scholars'eyes in recent years.AFLP (amplified fragment length polymorphism) was a new DNA molecular marker technique. Many studies of life scientific fields having been done rapidly using AFLP markers since its producing day, because of its many merits, such as the identification of a great number of polymorphisms, highly reproducible and stable. The genetic diversity, genetic structure and relationships of B. papyrifera were firstly analyzed by AFLP markers in this paper. Based on the habitats of its distribution, possible reasons for genetic differentiation and genetic diversity of B. papyrifera were discussed, and some strategies and suggestions for conservation and breeding were also put forward. The results were summarized as follows:1. 296 samples of B. papyrifera collected from Yunnan Province were used as tested materials in this study. The extraction method of genomic DNA was studied on young leaves of B. papyrifera is contrasted by some methods of standard SDS, standard CTAB, SDS-CTAB, improved CTAB and improved SDS. And results showed that improved SDS method was suitable for extracting DNA from young leaves of B. papyrifera, and the value of OD260/OD280 for DNA samples obtained with this method was between 1.8 and 2.0; the DNAs were completely digestible with EcoRI and MseI. AFLP fingerprinting figures were smoothly obtained. It is a rapid and high efficient method, and especially suitable for the situation that the study has a lot of samples, amount of works and a few time.2. The system suitable for B. papyrifera genomic DNA analysis was established according to systematical study of the main factors involved in AFLP technique system. The digestion of genomic DNA was implemented by using of two-step digestion with the two restriction enzymes. 4μL genomic DNA was digested with 5 units of EcoRI and MseI respectively in a reaction total volume of 30μL. Double strainded adaptor was ligated to the restriction fragments at 37℃overnight (8-12h). The preamplification products were diluted to 40 times with ddH2O and used in selective amplification. The PCR products added 4μL loading buffer were denatured at 95℃for 5min, which were isolated in 6% denatured polyacrylamide gel on stable power (95W) and then stained by silver nitrate.3. 13 primer combinations that produced clearly and highly polymorphic bands were screened from 64 EcoRI/ MseI primer combinations. The genetic diversity of 296 samples of B. papyrifera from Yunnan province was analyzed. And the clear polymorphic fingerprints were obtained which showed that it was feasible for using these primer combinations in B. papyrifera AFLP analysis. Genetic diversity on different river systems and different families of B. papyrifera was analyzed by AFLP with 7 primer combinations. 786 bands were obtained, 632 bands (80.4%) of them were polymorphic, 112.3 bands were obtained per primer pair on average. The amplification fragment ranged from 50bp to 450bp. The polymorphism amplified with E64/M54 was the highest, up to 86.4%, and which with E40/M34 was the lowest, 75.9%. Genetic diversity on different types of B. papyrifera was analyzed by AFLP with 6 primer combinations. 831 bands were obtained, 610 bands (73.4%) of them were polymorphic, and 138.5 bands were obtained per primer pair on average. The amplification fragment ranged from 80bp to 500bp. The polymorphism amplified with E34/M44 was the highest, up to 78.0%, and which with E44/M60 was the lowest, 67.1%. These results showed that the genetic diversity of B. papyrifera was abundant.4. The binary data matrix was constructed. The Dice similarity coefficient (SC) matrix was obtained by using NTSYS.pc2.11f software (Applied Biosystems, Setauket, NY. USA), and cluster analysis based on Nei's genetic distance was performed with unweighted pair-group methods of arithmetic averages (UPGMA).5. Genetic diversity among different levels ranged from 0.1328 to 0.1706, respectively for four River systems, ten families and for four types. The data of the relative magnitude of genetic differentiation among populations showed that the genetic variation within population was higher than that of among population, so the degree of the genetic variation among population was lower. And gene flows of different levels were over 2, which suggested that there existed sufficient genetic exchanges to prevent the genetic differentiation made by genetic drift among population.6. Among the four River systems, the greatest genetic distances is between Jinsha River and Yuanjiang River (0.0098), the lowest genetic distance is between Yuanjiang River and Nujiang River, Honghe River (0.0055). Four River systems can be divided into two groups according to the combine line 0.003 of the genetic distance: the first group contains Yuanjiang River, Nujiang River and Honghe River; the second group is only Jinsha River.7. Among the ten families, the greatest genetic distance is between Shiping family I and Jiangchuan family (0.0373), the lowest genetic distance is between Sichuan family I and Sichuan family II (0.0030). Four River system can be divided into two groups according to the combine line 0.008 of the genetic distance: the first group contains Nujiang family I, Nujiang family II, Nujiang family III, Shiping family I, Shiping family II, and Jianshui family I; the second group contains Jianshui family II, Sichuan family I, Sichuan family II, and Jiangchuan family.8. Among the four types, the greatest genetic distance is between Green-bark B. papyrifera (GB) and Red-bark with white-stripe B. papyrifera (RWB) (0.0296), the lowest genetic distance is between Red-bark B. papyrifera (RB) and White-bark with red-stripe B. papyrifera (WRB) (0.0087). Four types can be divided into two groups according to the combine line 0.006 of the genetic distance: the first group contains RWB and GB; the second group contains RB and WRB.