Genome sequencing reveals the genetic architecture of heterostyly and domestication history of common buckwheat.

Common buckwheat, Fagopyrum esculentum, is an orphan crop domesticated in southwest China that exhibits heterostylous self-incompatibility. Here we present chromosome-scale assemblies of a self-compatible F. esculentum accession and a self-compatible wild relative, Fagopyrum homotropicum, together with the resequencing of 104 wild and cultivated F. esculentum accessions. Using these genomic data, we report the roles of transposable elements and whole-genome duplications in the evolution of Fagopyrum. In addition, we show that (1) the breakdown of heterostyly occurs through the disruption of a hemizygous gene jointly regulating the style length and female compatibility and (2) southeast Tibet was involved in common buckwheat domestication. Moreover, we obtained mutants conferring the waxy phenotype for the first time in buckwheat. These findings demonstrate the utility of our F. esculentum assembly as a reference genome and promise to accelerate buckwheat research and breeding.

Recombinant inbred lines and next-generation sequencing enable rapid identification of candidate genes involved in morphological and agronomic traits in foxtail millet.

We constructed recombinant inbred lines (RILs) between a Japanese and a Taiwanese landrace of foxtail millet and employed next-generation sequencing, such as flexible ddRAD-seq and Nanopore sequencing to identify the candidate genes involved in the crop evolution of foxtail millet. We successfully constructed a linkage map using flexible ddRAD-seq with parents and RILs and detected major QTLs for each of three traits: leaf sheath colors, spikelet-tipped bristles (stb), and days to heading (DTH). (1) For leaf sheath colors, we identified the C gene on chromosome IV. (2) We identified a homeobox (HOX14) gene for stb on chromosome II, which shows homology with HvVrs1 in barley. (3) Finally, we identified a QTL with a large effect on DTH on chromosome II. A parent of the RILs from Taiwan and Yugu1 had a Harbinger-like TE in intron 3 of this gene. We also investigated the geographical distribution of the TE insertion type of this gene and found that the insertion type is distributed in the northern part of East Asia and intensively in South and Southeast Asia, suggesting that loss/reduction of function of this gene plays an important role in spreading into the northern part of East Asia and subtropical and tropical zones.

Classification and systematics of the Fagopyrum species.

Fagopyrum (Polygonaceae) is a small genus including less than 30 species, mostly endemic to southern China. The genus includes two cultivated species, common buckwheat F. esculentum and Tartary buckwheat F. tataricum. Fagopyrum is clearly distinguished from other genus in Polygonaceae by the central position of embryo in achene. The genus is divided into two major groups, namely the cymosum group and the urophyllum group, based on morphological characters and molecular systematics. In the last three decades the number of species in the genus has doubled by the discovery of new species by Japanese and Chinese groups. Most of them are revealed to be included in the urophyllum group based on morphological and molecular genetic analyses. Molecular systematic surveys have also detected inappropriate treatment of some non-Fagopyrum species as new species or combination in the genus.

Analysis of Fruit Lignin Content, Composition, and Linkage Types in Pear Cultivars and Related Species.

Lignin content, composition, and linkage types were investigated in pear fruit cultivars and related species. Lignin content increased during early stages and then decreased toward ripening in the core and flesh of "Gold Nijisseiki" and "Alexandrine Douillard". The lignin content was highest at harvest in Chinese quince. Only trace amounts of lignin were detected in apple flesh. The lignin content was low in Japanese pears "Ohshu", "Hosui", and "Kosui", and the noncondensed lignin index was high in flesh. The lignin type was guaiacyl-syringyl (GS) in these pears and related species. The S/G ratio at harvest varied widely (0.75-2.64) and increased during early stages and remained constant toward harvest in "Gold Nijisseiki" and "Alexandrine Douillard". "Gold Nijisseiki" and "Alexandrine Douillard" were determined to be G- and S-lignin-rich types, respectively. ß-Aryl ether, phenylcoumaran, and resinol interunit linkage types were detected among monolignol bonds, and ß-Aryl ether units were the main linkages in the pear.

Life Cycle and Genetic Diversity of Symplocarpus nipponicus (Araceae), an Endangered Species in Japan.

Symplocarpus nipponicus, a member of the Araceae family, is an endangered plant in several prefectures in Japan. For the conservation of this wild species, we investigated the morphology, life cycle, and genetic diversity of three wild populations. By fixed-point observation over several years, we found that it takes at least four years for the plant to set the inflorescences consisting of spadices and spathes, and another two years for it to set mature seeds. To examine the genetic diversity in the wild population, we developed 11 novel microsatellite markers and investigated the genetic variation in three populations in Kyoto Prefecture: Ayabe, Hanase, and Momoi. The Ayabe population carried less genetic variation than the other two areas, suggesting the isolation of the habitat and thus a higher risk of extinction. Our results provide basic knowledge of the ecological aspects of S. nipponicus, as well as molecular techniques for the assessment of its genetic diversity, and thus are useful for the conservation of this endangered species.

Isolation and characterization of microsatellite loci in Fimbristylis sericea (Cyperaceae)(1.).

PREMISE OF THE STUDY: We developed microsatellite markers for Fimbristylis sericea, a coastal herb found in sand dunes in Asia and Australia. • METHODS AND RESULTS: Twelve microsatellite loci were isolated, and the genetic variability within and among local populations was characterized. The number of alleles per locus was two to five with a mean of 3.5, total expected heterozygosity per locus was 0.069-0.645 with a mean of 0.336, and average expected heterozygosity within a population per locus was 0.051-0.230. Most of the loci deviated significantly from Hardy-Weinberg equilibrium. • CONCLUSIONS: All 12 microsatellite loci were polymorphic within and among populations. These loci could be useful genetic markers for population genetic studies of F. sericea populations.

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.

Isolation and characterization of microsatellite loci in Schoenoplectus juncoides (Cyperaceae)1.

PREMISE OF THE STUDY: We developed microsatellite markers for Schoenoplectus juncoides, a weed found in rice paddy fields in eastern Asia. METHODS AND RESULTS: Ten dinucleotide microsatellite loci were isolated, and genetic variability within and among local populations were characterized. The number of alleles per locus ranged from 2 to 6 with a mean of 3.6. Expected heterozygosity per locus ranged from 0.067 to 0.471, whereas observed heterozygosity showed much lower values ranging from 0.000 to 0.083. Most of the loci showed significant deviation from Hardy-Weinberg equilibrium and high inbreeding coefficient values (0.525-1.000). CONCLUSIONS: All the 10 microsatellite loci displayed polymorphism within and among populations. These loci are useful genetic markers for microevolution and ecological studies of S. juncoides populations.

Clonal and spatial genetic structure within populations of a coastal plant, Carex kobomugi (Cyperaceae).

Clarification of clonal growth pattern is critical for understanding the population dynamics and reproductive system evolution of clonal plant species. The contribution of clonality to the spatial genetic structure (SGS) within populations is also an important issue. I examined the spatial distribution of genetic variability within two populations of the coastal plant Carex kobomugi using seven microsatellite loci. Genotyping of 226 and 140 ramets within 14 × 40 m and 14 × 34 m plots on two populations revealed 36 and 33 multilocus genotypes, respectively. To quantify the extent of intermingling among clones, for each genet, I calculated the dominance of ramets belonging to a particular genet within a spatial range of the genet. Furthermore, I analyzed spatial distribution of genotypes within 2 × 2 m and 1 × 2 m quadrats using second-order spatial statistics. These analyses indicated that clones are highly intermingled, suggesting a low level of spatial interaction among clones. Spatial autocorrelation analysis of kinship coefficient including all pairs of ramets showed significantly stronger SGS than analysis considering only pairs between different genets. I conclude that clonal propagation largely contributes to SGS at a fine scale.

Nucleotide sequence variability of the Adh gene of the coastal plant Calystegia soldanella (Convolvulaceae) in Japan.

Calystegia soldanella (Convolvulaceae) is a self-incompatible perennial herb distributed on sandy seashores throughout the temperate zone of the world. In Japan, the species occasionally grows on the sandy shores of Lake Biwa. To clarify the genetic differentiation among local populations, we investigated the nucleotide sequence variability of the Adh gene. In a 1625-bp sequence between exon 2 and the 3' noncoding region of the Adh gene, a total of 44 polymorphic sites were found among 91 individuals from 19 populations. The nucleotide diversity for the entire sample was 0.00212. Similar values were determined for geographical groups of populations. No genetic differentiation among the groups of populations was found. The complete lack of genetic differentiation between the sea coastal populations and the inland populations could not be attributed to gene flow. Although the inland populations are geographically isolated from the sea coastal populations, the time since separation might be insufficient to establish significant genetic differentiation.

Nucleotide substitutions in the acetolactate synthase genes of sulfonylurea-resistant biotypes of Monochoria vaginalis (Pontederiaceae).

Some point mutations in acetolactate synthase (ALS) confer resistance to ALS-inhibiting herbicides in weeds. To clarify the evolution of the herbicide resistance of Monochoria vaginalis, a weed in rice fields in Japan, the nucleotide sequences of four genes encoding ALS were surveyed in five sulfonylurea-resistant (SU-R) and five sulfonylurea-susceptible (SU-S) biotypes. In the ALS1 gene, two SU-R biotypes showed nucleotide substitutions changing Pro197 to Ser and Leu, respectively. In a different gene, ALS3, three other SU-R biotypes showed either of the two nonsynonymous nucleotide substitutions seen in ALS1. Only two biotypes geographically located distantly from each other shared the same mutation conferring SU resistance in the same gene. These patterns of nucleotide substitutions indicate that the SU-R phenotype was acquired independently by different biotypes. Nucleotide diversity values of the genes showing SU-R mutations were higher than those of ALS2 lacking any SU-R mutation and of a putative pseudogene, ALS4. This result suggests that the maintenance of nucleotide variability within target genes provides an opportunity for the evolution of SU-R phenotypes by herbicide-driven selection for mutations conferring resistance.

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.