| Rattan belongs to the subfamily Calamoideae of the family Palmae and is an important non-timber forest product in the tropics and sub-tropics. However, historical over-exploitation of rattan has led to a decrease of this natural resource. To meet the domestic demands for rattan materials, plantations have been established widely in Guangdong, Guangxi and Fujian provinces in China. Under most circumstances, seeds for these plantations were highly mixed ones, traditionally collected from natural forests or other plantations, and failed to produce adequate canes of high quality. Therefore, it is needed to develop rattan breeding for improved seed quality. Moreover, the knowledge of genetic structure and genetic diversity of rattan is critical for genetic resources conservation and breeding strategy deployment, especially for Daemonorops margaritae (Hance) Beccari and Calamus simplificifolius C. F. Wei., which are both domestic to China. In addition, D. margaritae and C. simplificifolius are dioecious plants, which represents an impediment to seed orchard establishment because only a few male plants are needed to ensure the fertilization of female plants, and the isolation of sex-linked DNA sequence will promote breeding programs as well as practical cultivation. (1) Genomic DNA was successfully extracted from leaf tissues of D. margaritae and C. simplificifolius using a modified CTAB method. This method could remove phenolic compounds, polysaccharides and proteins, and the quality and quantity of DNA extracted were reliably characterized. In addition, the reaction composition and amplification program for RAPD analysis were developed. This study provides a technological basis for future molecular studies in rattan and the related species. (2) Analysis of genetic diversity was based on the investigation of RAPD data of four natural populations of D. margaritae and five natural populations of C. simplicifolius. The results about D. margaritae were as follows: a total of 154 fragments were detected with 15 primers, of which 117 fragments (75.97%) were polymorphic across the populations. The Nei′s index and Shannon information index were 0.2584 and 0.3888, respectively. The total genetic diversity was 0.2578. Genetic diversity of D. margaritae mainly distributed within population, which the coefficient of gene differentiation was 0.1412. When genetic diversity was estimated by Shannon indes of phenotypic diversity, it was the highest in Jianfengling population and was the lowest in Bawangling population. The average genetic diversity of within populations was 2.7650, and average total genetic diversity was 3.2541. Genetic diversity of D. margaritae mainly distributed within population, which the coefficient of gene differentiation was 0.1503.Genetic identity between Maoganxiang and Jianfengling populations was the highest while that between Diaoluoshan and Bawangling populations was the lowest. The results of AMOVA indicated that most of the variation (97.38%) resided within populations. The results about C. simplicifolius were as follows: a total of 124 fragments were detected with 11 primers, of which 96 fragments (77.42%) were polymorphic across the proportions. The Nei′s index and Shannon information index were 0.2237 and 0.3442, and total genetic diversity was 0.2231. Genetic diversity of C. simplicifolius mainly distributed within population, which the coefficient of gene differentiation was 0.1555. When genetic diversity was estimated by Shannon indes of phenotypic diversity, it was the highest in Qiongzhong population and was the lowest in Bawangling population. The average genetic diversity of within populations was 2.7278, and average total genetic diversity was 3.5468. Genetic diversity of C. simplicifolius mainly distributed within population, which the coefficient of gene differentiation was 0.2284.Genetic identity between Wuzhishan and Bawangling populations was the highest while that between Jianfengling and Qiongzhong populations was the lowest. The results of AMOVA indicated that most of the variation (86.91%) resided within populations. (3) Seed traits and seedling traits were measured for four natural populations of D. margaritae and five natural populations of C. simplicifolius, which were used to analyze phenotypic diversity. Variation patterns were similar in both species with the results using RAPD markers. The ANOVA analysis revealed significant differences at 0.01 level among and within populations in all traits in C. simplicifolius and, except for sprout length, in D. margaritae. The nested variance analysis indicated that the variance within population was the main part of the phenotypic variation in the two rattan species studied, similar with the result of RAPD analysis, which were 37.034% and 37.660% in average over the traits investigated for D. margaritae and C. simplicifolius, respectively. The phenotypic differentiations among populations were 1.594% and 15.560% in average over the traits investigated for D. margaritae and C. simplicifolius, respectively. (4) The variation of seed and seedling traits were investigated based on the field data ofprogenies of D. margaritae and C. simplicifolius families. The results revealed that differences in all seed and seedling traits of two rattan were significant at 0.01 level among families and those of seedling traits were significant at 0.05 level within families. The heritabilities of spout traits, leaf number and largest leaf length of two rattan were higher than 80% according to genetic variation. In analysis of the growth trend, air temperature had correlations with leaf number and largest leaf length for D. margaritae, but only with leaf number for C. simplicifolius. The growth trends of largest leaf length and leaf number were same as that of D. margaritae. With regression analysis of seed traits and leaf number with largest leaf length, leaf number, germinate ratio, seed length and 1000-seed weight had linear correlations with largest leaf length for D. margaritae, while seed length and germinate ratio had linear correlations with largest leaf length for C. simplicifolius. D. margaritae and C. simplicifolius gained 4 and 5 main factors with factor analysis, respectively. In clustering according to reference oblique factor value, families 102019, 102020 and 402012 of D. margaritae and families 101007, 101008 and 101009 of C. simplicifolius were identified among the best ones. (5) RAPD molecular marker technique was used to detect sex-specific marker in C. simplicifolius. DNA samples were extracted individually from ten male and ten female plants. After a total of 1040 decamer primers had been tested, an approximate 500 bp male-specific DNA fragment was generated with the primer S1443. The RAPD marker was converted into SCAR marker, which was suitable for a precise, repetitive and rapid identification of male plants in C. simplicifolius, and should be of benefit for breeding programs of this dioecious species.
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