[Analysis of transicriptomes and exploring flavonoid biosynthetic pathway genes in Lithocarpus polystachyus].

The sweet taste and health effect of Lithocarpus polystachyus are mainly related flavonoid. To obtain Lithocarpus transcriptome database and flavonoid biosynthesis-related genes, the RNA-Seq techology (Illumina HiSeq 4000) was used to sequence its transcriptome. Six Gb database was assembled after assembly steps, and 41 043 of L. polystachyus unigenes were obtained. With blasting them with 7 data banks, all unigenes were involved in 51 GO-terms and 237 metabolic pathways. And furthermore 28 genes of the flavonoid biosynthesis-related were found. After using the MicroSatallite, 18 161 SSR were obtained, the single-nucleotide-repeated was the richest at 7 346. These data represent abundant messages about transcripts and provide valuable genome data sources in molecular biology of L. polystachyus.

Variation in pollen-donor composition among pollinators in an entomophilous tree species, Castanea crenata, revealed by single-pollen genotyping.

BACKGROUND: In plants, reproductive success is largely determined by the composition of pollen (i.e., self-pollen and outcross-pollen from near and distant pollen-donors) transported as a result of pollinator foraging behavior (e.g., pollen carryover). However, little evidence is available on how and to what extent the pollen carryover affects the pollen-donor composition and on which insect taxa are effective outcross-pollen transporters under field conditions. In this study, we explored roles of foraging behavior of insect pollinators on pollen-donor composition and subsequent reproductive success in a woody plant. METHODS: We performed paternity analyses based on microsatellite genotyping of individual pollen grains found on diurnal pollinators (i.e., bumblebee, small bee, fly, small beetle, and honeybee) visiting Castanea crenata trees. RESULTS: The outcross-pollen rate was highest in bumblebees (66%), followed by small bees (35%), flies (31%), and small beetles (18%). The effective number of pollen donors, representing pollen carryover, was greater in bumblebees (9.71) than in flies (3.40), small bees (3.32), and small beetles (3.06). The high percentages of pollen from outside the plot on bumblebees (65.4%) and flies (71.2%) compared to small bees (35.3%) and small beetles (13.5%) demonstrated their longer pollen dispersal distances. CONCLUSIONS: All of the diurnal insects carried outcross-pollen grains for long distances via pollen carryover. This fact suggests that a wide range of insect taxa are potential outcross-pollen transporters for the self-incompatible C. crenata.

Genetic differentiation and genetic diversity of Castanopsis (Fagaceae), the dominant tree species in Japanese broadleaved evergreen forests, revealed by analysis of EST-associated microsatellites.

The broadleaved evergreen forests of the East Asian warm temperate zone are characterised by their high biodiversity and endemism, and there is therefore a need to extend our understanding of its genetic diversity and phylogeographic patterns. Castanopsis (Fagaceae) is one of the dominant tree species in the broadleaved evergreen forests of Japan. In this study we investigate the genetic diversity, genetic structure and leaf epidermal morphology of 63 natural populations of C. sieboldii and C. cuspidata, using 32 Expressed Sequence Tag associated microsatellites. The overall genetic differentiation between populations was low (GST = 0.069 in C. sieboldii and GST = 0.057 in C. cuspidata). Neighbor-joining tree and Bayesian clustering analyses revealed that the populations of C. sieboldii and C. cuspidata were genetically clearly differentiated, a result which is consistent with the morphology of their epidermal cell layers. This suggests that C. sieboldii and C. cuspidata should be treated as independent species, although intermediate morphologies are often observed, especially at sites where the two species coexist. The higher level of genetic diversity observed in the Kyushu region (for both species) and the Ryukyu Islands (for C. sieboldii) is consistent with the available fossil pollen data for Castanopsis-type broadleaved evergreen trees during the Last Glacial Maximum and suggests the existence of refugia for Castanopsis forests in southern Japan. Within the C. sieboldii populations, Bayesian clustering analyses detected three clusters, in the western and eastern parts of the main islands and in the Ryukyu Islands. The west-east genetic differentiation observed for this species in the main islands, a pattern which is also found in several plant and animal species inhabiting Castanopsis forests in Japan, suggests that they have been isolated from each other in the western and eastern populations for an extended period of time, and may imply the existence of eastern refugia.

Habitat loss other than fragmentation per se decreased nuclear and chloroplast genetic diversity in a monoecious tree.

Generally, effect of fragmentation per se on biodiversity has not been separated from the effect of habitat loss. In this paper, using nDNA and cpDNA SSRs, we studied genetic diversity of Castanopsis sclerophylla (Lindl. & Paxton) Schotty populations and decoupled the effects of habitat loss and fragmentation per se. We selected seven nuclear and six cpDNA microsatellite loci and genotyped 460 individuals from mainland and island populations, which were located in the impoundment created in 1959. Number of alleles per locus of populations in larger habitats was significantly higher than that in smaller habitats. There was a significant relationship between the number of alleles per locus and habitat size. Based on this relationship, the predicted genetic diversity of an imaginary population of size equaling the total area of the islands was lower than that of the global population on the islands. Re-sampling demonstrated that low genetic diversity of populations in small habitats was caused by unevenness in sample size. Fisher's α index was similar among habitat types. These results indicate that the decreased nuclear and chloroplast genetic diversity of populations in smaller habitats was mainly caused by habitat loss. For nuclear and chloroplast microsatellite loci, values of F(ST) were 0.066 and 0.893, respectively, and the calculated pollen/seed dispersal ratio was 162.2. When separated into pre-and post-fragmentation cohorts, pollen/seed ratios were 121.2 and 189.5, respectively. Our results suggest that habitat loss explains the early decrease in genetic diversity, while fragmentation per se may play a major role in inbreeding and differentiation among fragmented populations and later loss of genetic diversity.

Patterns of pollen flow in a dense population of the insect-pollinated canopy tree species Castanopsis sieboldii.

Insect pollinations of tree species with high-density populations have rarely been studied. Since the density of adults can affect effective pollen dispersal, short-distance pollination, even by insects, may frequently occur in high-density populations. To test this prediction, we investigated pollination patterns in a high-density population of the insect-pollinated canopy tree species Castanopsis sieboldii by paternity analysis using genotypes at 8 microsatellite loci of 145 adult trees and 439 seeds from 11 seed parents in a 4-ha plot. We then explored their genetic effects on the population by calculating other population genetics parameters. Although C. sieboldii has high potential for long-distance dispersal of pollen (as indicated by a fat-tailed dispersal kernel), the cumulative pollination at the local scale was spatially limited and strongly dependent on the distance between parents due to the high density of adults. Genetic diversity estimates for pollen pools accepted by each seed parent converged on a maximum as the effective number of pollen parents increased. The genetic diversity of pollen pool bulked over all the seed parents from inside the plot did not differ from that of the total pollen pools. Therefore, although pollen flow from distant pollen parents may help to maintain the genetic diversity of offspring, pollen parents neighboring seed parents may be the main contributors to the genetic diversity of the offspring at the seed stage.

Pollen donor composition during the early phases of reproduction revealed by DNA genotyping of pollen grains and seeds of Castanea crenata.

In plants, pollen donor composition can differ during the early phases of reproduction through various selection mechanisms favouring self, related or nonrelated pollen donors, but such differences have not been examined under natural conditions because paternity is difficult to analyse in a natural setting. Here, we performed paternity analyses based on microsatellite genotyping of individual pollen grains deposited on female flowers (n = 773) and seeds (n = 304) to evaluate pollen donor composition from three individuals of the insect-pollinated monoecious tree Castanea crenata in a natural forest. Spatial genetic structure was also investigated. A mean self-pollen rate of 90.2% was observed at the pollination stage, but a low selfing rate of 0.3% was observed at the seed stage. In outcross events, however, pairwise distance and relatedness between maternal and paternal parents were not different between pollination and seed stages. We also observed significant positive relatedness, based on clear fine-scale genetic structure of individual trees within 80 m of one another, and 71% of seeds were derived using pollen grains of related trees within 80 m. The results suggest that the mechanism of self-incompatibility strongly avoids self-pollen before seed production. However, the avoidance of biparental inbreeding was not obvious between pollination and seed stages.

In vitro germination and transient GFP expression of American chestnut (Castanea dentata) pollen.

The development of the male reproductive structures of American chestnut (Castanea dentata) is described to advance our understanding of its reproductive behavior. This information has been vital in the development of a strategy to collect pollen grains from male catkins suitable for in vitro germination and transformation experiments. Cutting male catkins into small segments and rolling them over a culture plate resulted in evenly dispersed and large amounts of pollen with minimal unwanted accessory floral parts. To optimize pollen viability, the effect of various storage conditions on in vitro germination was examined. Our results showed that initial storage at 4 degrees C for 2 weeks significantly increased percent germination as compared to freshly collected pollen and those stored directly at -20 degrees C or -80 degrees C. This also means that for long-term storage of American chestnut pollen, the catkins should first be kept at 4 degrees C for a couple of weeks and then at -80 degrees C. The use of pollen grains with high viability is necessary for the transformation of American chestnut pollen. To optimize pollen transformation via particle bombardment, the effects of target distance, target pressure, and pollen developmental stage were examined. Statistical analysis showed that bombardment of ungerminated pollen at 1,100 psi resulted in the highest percent transient GFP expression (4.1%).