Clonal Structure And Genetic Diversity Of Bashania Fargesii

Bashania fargesii is the main food source of Qinling giant panda in winter and spring,mainly distribute in the south slope of Qinling Mountains and Daba Mountain. It plays an important role in the stability of ecosystem, soil and water conservation and animal diversity protection in this area. Previous studies focused on the studies of biological characteristic, clonal population ecology, bamboo forest thinning and regeneration, nutrition composition and development and untilization of B. fargesii, but the clonal strucuture and genetic structure are still unknown, therefore, the protection of this bamboo forest exist some blindness.In this study, simple sequence repeat fingerprints(SSR) were used to reveal the clonal structure and genetic diversity of B. fargersii populations. 11 natural reserves of the Qinling giant panda habitat and Zhenba and Wanyuan in Daba Moutain were selected as study areas.A total of 1647 DNA samples from 13 populations and 5 plots were collected in these study areas. Based on analysis of experiment and data, the development of microsatellite primer pairs, ploidy determination, genotype frequency determination, clonal structure of B. fargersii populations at different age, Clonal structure of B. fargesii and B. aristata, clonal structure of flowering population and genetics structure among 13 populations were examined comprehensively.Our study was trying to explore the clone number, size, spatial pattern, genetic variation and genetic structure of B. fargesii populations in populations’ establishment, development,recession and flowering stages, so as to provide some scientific references and theoretical basis for bamboo forest thinning and regeneration, artificial planting and giant panda protection strategy.The results are as follows:( 1) The bioinformatics method was used to develop microsatellite primers for B.fargesii and B. aristata. A total of 77 primer pairs were synthesized, among which 50 primers were designed by Oligo software and 27 primers were downloaded from references. After preparatory and repeated screening, fluorescence labeled 15 primers pairs were furtherscreened in B. fargesii and B. aristata(43 individuals per species). 15 microsatellite primers were shown to be polymorphic in B. fargesii and B. aristata. The number of alleles per locus ranged 4–20 and 5–18, respectively. For B. fargesii, the Ho and He ranged from 0.310 to 0.943 and 0.323 to 0.905, and for B. aristata, these ranged from 0.625 to 0.971 and 0.554 to 0.905. The PIC ranged from 0.322 to 0.901 and 0.537 to 0.898, respectively.(2)8 high polymorphism primers pairs were selected from 15 primers pairs using 52 samples from 13 populations of Qinling giant panda nature reserve and Daba Mountain.These 8 primers were used to study population clonal structure and genetic diversity of B.fargesii and B. aristata. The SSR data of 286 samples of 13 populations were analyzed. The average allele’s numbers of every primer pair per population were greater than 2, every primer pair data exhibited the maximum number of four alleles and the minimum number of one allele. Polysat software was used to estimate the ploidy of plant microsatellite data. B. fargesii is tetraploid plant by estimating the ploidy of 13 populations.(3)The MAC-PR(Microsatellite DNA Allele Counting—Peak Ratios) method was used to estimate tetraploid allelic configurations. Using this method, we were able to assign tetraploid allelic configurations in 8 loci for almost all of samples investigated. Also, it is further confirmed that B. fargesii is tetraploid plant. Based on the analyzing the data of 8primers allele amplification, the MAC-PR method could be a suitable means to get the quantitative information from SSR data of B. fargesii. This method could effectively estimate tetraploid allelic frequency and identify invalid alleles.(4)The clonal structure of B. fargersii populations at three different genet ages showed that the size of clones in the three plots increased and the number of clones decreased with population aging. The spatial distribution pattern of clones in 7 years and > 30 years plots exhibited a clumped distribution, while > 60 years plot showed two different patterns with simultaneously clumped and discrete distributions. The results showed that the genet generally formed a clumped distribution pattern during the seeding stage. However, with increasing clone size and genet age, compound axis mixed B. fargesii may expand into either the more monopodial type of ramets or long-distance clones representing a discrete distribution pattern when the genet are pressured by other strong clones. Both the Mantel test and spatial autocorrelation analysis supported the significant presence of positive spatial clonal structures in three plots at the small-scale level. The analysis of clonal diversity revealed that the distinguishable genotypes at the seeding stage(7 years) were much greater than that at the adult stage(30 years and 60 years). Although the genotypic diversity of clonal populations reduced with genet aging, because of initial seedling recruitment, the value remained high.(5)The flowering B. fargesii population was composed of two clones. Each clone had flowering, non-flowering and mixed flowering patches during the flowering period. The flowering time of this population is lasted at least four years. The first two flowering patch respectively appear in different locations of two clones. The mixed flowering patches of previous year became over time flowering patches, and non-flowering patches became both mixed flowering and flowering patches. Two clones had same peak blooming years in 2015,while a large number of seeds were produced. Finally, all patches of two clones flowered.( 6) The clonal structure of two staggered distribution bamboos B. fargesii and B.aristata demonstrated that the size and number of clones in plots were uneven. The clones showed two different patterns with simultaneously clumped and discrete distributions in both species. Both Mantel tests and spatial autocorrelation analysis revealed significant positive clonal structures in both species. The information about spatial genetic structure derived from the comparison of B. fargesii and B. aristata can be used to explain the propagation capacity of B. fargesii was stronger than B. aristata. In light of this clonal structure(clone number, size and spatial patterns), the results implied that competition plays an important role in changing the clonal structure of mixed-species stands.(7)The analysis of SSR data from 13 populations showed high genetic diversity within populations. A clear population structure was detected by the unweighted pair group method,principle coordinate analysis and Bayesian clustering. All clustering approaches supported that a division of the 13 populations into 3 major groups, among which 9 populations(Huangbaiyuan, Niuweihe, Changqing, Sangyuan, Panlong, Wuliangshan, Foping,Niangniangshan and Tianhuanshan in Qinling Mountains) belonged to group 1, Pingheliang and Zhenan populations belonged to group 2 and Zhenba and Wanyuan populatioans belonged to group 3. Moderate gene flow existed in between group 1 and group 2 and also in between group 1 and group 3. A certain level of genetic differentiation between group 3 and group 2 were detected. This genetic differentiation may be related to the serious fragmentation of population habitats and geographical isolation.