Phylogenetic analysis of AGAMOUS sequences reveals the origin of the diploid and tetraploid forms of self-pollinating wild buckwheat, Fagopyrum homotropicum Ohnishi.

Fagopyrum homotropicum Ohnishi is a self-pollinating wild buckwheat species indigenous to eastern Tibet and the Yunnan and Sichuan Provinces of China. It is useful breeding material for shifting cultivated buckwheat (F. esculentum ssp. esculentum Moench) from out-crossing to self-pollinating. Despite its importance as a genetic resource in buckwheat breeding, the genetic variation of F. homotropicum is poorly understood. In this study, we investigated the genetic variation and phylogenetic relationships of the diploid and tetraploid forms of F. homotropicum based on the nucleotide sequences of a nuclear gene, AGAMOUS (AG). Neighbor-joining analysis revealed that representative individuals clustered into three large groups (Group I, II and III). Each group contained diploid and tetraploid forms of F. homotropicum. We identified tetraploid plants that had two diverged AG sequences; one belonging to Group I and the other belonging to Group II, or one belonging to Group II and the other belonging to Group III. These results suggest that the tetraploid form originated from at least two hybridization events between deeply differentiated diploids. The results also imply that the genetic diversity contributed by tetraploidization of differentiated diploids may have allowed the distribution range of F. homotropicum to expand to the northern areas of China.

S-LOCUS EARLY FLOWERING 3 is exclusively present in the genomes of short-styled buckwheat plants that exhibit heteromorphic self-incompatibility.

The different forms of flowers in a species have attracted the attention of many evolutionary biologists, including Charles Darwin. In Fagopyrum esculentum (common buckwheat), the occurrence of dimorphic flowers, namely short-styled and long-styled flowers, is associated with a type of self-incompatibility (SI) called heteromorphic SI. The floral morphology and intra-morph incompatibility are both determined by a single genetic locus named the S-locus. Plants with short-styled flowers are heterozygous (S/s) and plants with long-styled flowers are homozygous recessive (s/s) at the S-locus. Despite recent progress in our understanding of the molecular basis of flower development and plant SI systems, the molecular mechanisms underlying heteromorphic SI remain unresolved. By examining differentially expressed genes from the styles of the two floral morphs, we identified a gene that is expressed only in short-styled plants. The novel gene identified was completely linked to the S-locus in a linkage analysis of 1,373 plants and had homology to EARLY FLOWERING 3. We named this gene S-LOCUS EARLY FLOWERING 3 (S-ELF3). In an ion-beam-induced mutant that harbored a deletion in the genomic region spanning S-ELF3, a phenotype shift from short-styled flowers to long-styled flowers was observed. Furthermore, S-ELF3 was present in the genome of short-styled plants and absent from that of long-styled plants both in world-wide landraces of buckwheat and in two distantly related Fagopyrum species that exhibit heteromorphic SI. Moreover, independent disruptions of S-ELF3 were detected in a recently emerged self-compatible Fagopyrum species and a self-compatible line of buckwheat. The nonessential role of S-ELF3 in the survival of individuals and the prolonged evolutionary presence only in the genomes of short-styled plants exhibiting heteromorphic SI suggests that S-ELF3 is a suitable candidate gene for the control of the short-styled phenotype of buckwheat plants.

Construction of a BAC library for buckwheat genome research - an application to positional cloning of agriculturally valuable traits.

We have constructed a BAC library for common buckwheat Fagopyrum esculentum Moench. The library includes 142,005 clones with an average insert size of approximately 76 kb, equivalent to approximately a 7 to approximately 8-fold coverage of the genome. Polymerase chain reaction based screening of the library with AGAMOUS and FLORICAULA/LEAFY primers, has identified 7 and 9 BACs, respectively, which are consistent with the genome coverage. This library represents the first large insert genomic library for F. esculentum and it can be served as a genetic resource facilitating agricultural, pharmacological, physiological, and evolutionary studies of the species. To demonstrate the utilization of the library for characterizing agriculturally valuable traits, we developed a sequence tagged site marker tightly linked to the dwarf E locus as well as to the self-incompatibility complex locus and screened the library to initiate positional cloning of the causative genes.

Close genetic relationship between cultivated and natural populations of common buckwheat in the Sanjiang area is not due to recent gene flow between them-An analysis using microsatellite markers.

Natural populations of wild common buckwheat have been found growing adjacent to cultivated populations of common buckwheat. Gene flow between the cultivated and natural populations would be expected in such cases. To evaluate the amount of gene flow, two sets composed of a cultivated buckwheat population and an adjacent natural population of wild common buckwheat were chosen, one from Yanjing village in the Sanjiang area, which is presumed to be the original birthplace of common buckwheat, and one from Jinhe village, Yanyuan district of Sichuan province in China. The genotypes of 45 individuals from each population were examined at eight microsatellite marker loci to estimate the magnitude of gene flow between the cultivated and wild common buckwheat populations. The Bayesian method with a Markov chain Monte Carlo approach estimated that the magnitude of gene flow between the populations in the Sanjiang area at 0.002-0.008 was not significantly different from that found in Yanyuan district at 0.002-0.008. The gene flow between cultivated populations was higher, usually at 0.002-0.044 (exceptionally high at 0.255 between cultivated populations of Yanjing and Jinhe), than that found between a cultivated population and a natural population (0.002-0.008) or between two natural populations (0.002-0.003). Therefore, the genetic similarity found between the cultivated populations and natural populations observed in the Sanjiang area (Konishi et al., 2005) was not due to recent gene flow between them. This in turn suggests that the close genetic relationship found in the Sanjiang area may be due to the common ancestry of the natural populations and cultivated common buckwheat.

Two distinct groups of natural populations of Fagopyrum urophyllum (Bur. et Franch.) Gross revealed by the nucleotide sequence of a noncoding region in chloroplast DNA.

Fagopyrum urophyllum is a cross-pollinating perennial woody shrub species belonging to the urophyllum group of Fagopyrum. Natural populations of F. urophyllum were morphologically classified into two distinct groups, the Dali group and the Kunming group without exception. This grouping was verified by molecular phylogeny based on the nucleotide sequence of chloroplast DNA. The reproductive isolation found between the two groups was almost perfect as the distribution of two groups did not overlap each other. The net nucleotide substitution rate (Da) between the two groups was at the same level as between two distinct species. These data suggests that the two groups should be classified into distinct species if future studies on F. urophyllum confirm complete reproductive isolation and no ambiguously classified populations in the border area of the central Yunnan province of China.

Original birthplace of cultivated common buckwheat inferred from genetic relationships among cultivated populations and natural populations of wild common buckwheat revealed by AFLP analysis.

The genetic relationships among seven cultivated populations and eight natural populations of wild common buckwheat were analyzed using amplified fragment length polymorphism (AFLP). The genetic distance was estimated for each pair of the 15 populations based on the AFLP data, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method based on the genetic distance. All the cultivated populations were grouped in a cluster. The natural populations were grouped into two clusters composed of (1) the Sanjiang group (three populations from eastern Tibet and one population from Adong village of Yunnan province) and (2) two populations from Yunnan province and two populations from Sichuan province. The Sanjiang group is more closely related to cultivated populations. These results indicate that the direct ancestor of common buckwheat was natural populations of wild common buckwheat from the Sanjiang area.

Amplified fragment length polymorphism linkage analysis of common buckwheat (Fagopyrum esculentum) and its wild self-pollinated relative Fagopyrum homotropicum.

Common buckwheat (Fagopyrum esculentum) (2n = 2x = 16) and Fagopyrum homotropicum (2n = 2x = 16) were mated in an interspecific cross and amplified fragment length polymorphism (AFLP) linkage maps were constructed by analyzing segregation in the F2 population. Six hundred and sixty-nine bands were identified using 20 AFLP primer combinations, of which 462 (69%) segregated in the F2 population. The map of F. esculentum has eight linkage groups with 223 markers covering a total of 508.3 cM. The map of F. homotropicum has eight linkage groups with 211 markers covering 548.9 cM. There was one to one correspondence of the esculentum and homotropicum linkage groups. Three morphological markers, distylous self-incompatibility, shattering habit, and winged seed, were located on the AFLP map. Distylous self-incompatibility and shattering habit are tightly linked to each other (1.3 cM) and are located near the center of linkage group 1. Winged seed is located on linkage group 4.

Topological incongruence between nuclear and chloroplast DNA trees suggesting hybridization in the urophyllum group of the genus Fagopyrum (Polygonaceae).

We performed phylogenetic analyses of Fagopyrum species in the urophyllum group based on nucleotide sequences of two nuclear genes, FLORICAULA/LEAFY (FLO/LFY) and AGAMOUS (AG), and three segments of chloroplast DNA (cpDNA), rbcL-accD, trnK intron, and trnC-rpoB spacer. The FLO/LFY and AG sequences turned out to be phylogenetically more informative at the intrageneric level than the cpDNA sequences. Congruence among these gene trees, inferred by a maximum-likelihood (ML) method, demonstrated that topologies were partially incongruent between the nuclear and chloroplast DNA phylogenies. The nuclear DNA sequence data supported a monophyletic relation of F. statice, F. gilesii, and F. jinshaense, whereas the former two species formed another monophyletic relation with the F. capillatum-F. gracilipes-F. gracilipedoides-F. rubifolium clade excluding F. jinshaense in the synthetic cpDNA phylogeny. In addition, two divergent sequences of FLO/LFY were found in F. rubifolium (tetraploid). One of these was sister to F. gracilipedoides and another was sister to F. statice, and a monophyletic relation of these two genes was rejected by a bootstrap analysis. These results suggest that hybridization may have occurred during diversification of Fagopyrum species in the urophyllum group, and that F. rubifolium is possibly allotetraploid species.

Intraspecific cpDNA variations of diploid and tetraploid perennial buckwheat, Fagopyrum cymosum (Polygonaceae).

We investigated the phylogenetic and biogeographic relationships of natural populations of diploid and tetraploid Fagopyrum cymosum (Polygonaceae). Intraspecific variation of chloroplast DNA sequences was detected in three regions approximately 5 kb long in total: the 3' end of rbcL, accD and associated intergenic spacer region, the trnC (GCA)-rpoB spacer region, the trnK (UUU) intron, and the matK region. The accessions of F. cymosum were divided into two major groups, a Tibet-Himalayan clade and a Yunnan-Sichuan clade, with a high bootstrap probability. It was estimated that these two clades diverged about 0.7 million years ago. The geographical and climatic interruption by the Hengduanshan mountains might have caused the genetic divergence in F. cymosum. Autotetraploid populations of F. cymosum have arisen allopatrically from a diploid progenitor at least twice, once in the Tibet-Himalayan area and once in the Yunnan-Sichuan area. This conclusion reinforces a previous study based on allozyme variation. We also found that F. tataricum, a close relative of F. cymosum, was completely included within the Tibet-Himalayan clade in the phylogenetic tree. This suggests that F. tataricum speciated from F. cymosum in the Tibet-Himalayan area.

Two new Fagopyrum (Polygonaceae) species, F. gracilipedoides and F. jinshaense from Yunnan, China.

Two new Fagopyrum (Polygonaceae) species, F. gracilipedoides and F. jinshaense, are described. Fagopyrum gracilipedoides was only found at Baoshan village, Lijiang district, Yunnan province, China. This species resembles the weedy species, F. gracilipes in morphology but they differ in both mating system and ploidy level; F. gracilipedoides is a heterostylous self-incompatible diploid species (2n = 16), whereas F. gracilipes is a self-compatible tetraploid species (2n = 32). Fagopyrum jinshaense was found along the Jinsha River valley near the border of Sichuan, Yunnan, and Tibet in China. Fagopyrum jinshaense is morphologically similar to F. gilesii, but they differ in the morphology of their inflorescences. Fagopyrum jinshaense has long spike-like inflorescences, whereas F. gilesii has a compact head-like one. Both F. jinshaense and F. gilesii are self-incompatible with heterostylous flowers and are diploid. Interspecific crosses between the two new species and already known species in the urophyllum group of Fagopyrum resulted in the production of self- and cross-sterile hybrids in the species combinations attempted. These results indicated that both new species are reproductively isolated from other Fagopyrum species. Molecular phylogenetic analysis based on the nucleotide sequence of the trnK gene intron in chloroplast DNA revealed the following points: 1) F. gracilipedoides, F. rubifolium and F. gracilipes / F. capillatum showed a trifurcating relationship and 2) F. jinshaense formed a clade with F. pleioramosum, F. callanthum, and F. macrocarpum, and was only distantly related to F. gilesii. In the clade of F. capillatum, F. gracilipes, F. gracilipedoides and F. rubifolium, the breakdown of self-incompatibility accompanying genome duplication seems to have occurred twice independently.