Ohmi Ohnishi

Intra- and interspecific phylogeny of wild Fagopyrum (Polygonaceae) species based on nucleotide sequences of noncoding regions in chloroplast DNA.

The intra- and interspecific phylogeny of Fagopyrum (Polygonaceae) species was studied using nucleotide sequence data from two noncoding regions in chloroplast DNA, the trnK (UUU) intron and the trnC (GCA)-rpoB spacer. Thirty-seven accessions of ten species and two unidentified samples in the urophyllum group of Fagopyrum were analyzed. Both of the studied regions showed high variability, including nucleotide substitutions, insertion/deletions, and inversions. Separate parsimony analyses of the two regions generated phylogenies that were largely consistent with each other. A single most parsimonious tree derived from the combined data of the two regions suggested that (1) either F. statice or F. leptopodum was derived from the ancestor more than once, (2) F. gracilipes, a tetraploid species, has recently been derived from diploid ancestor and rapidly spread out to its present distribution areas, and (3) F. pleioramosum, F. macrocarpum, and F. callianthum, three newly discovered species endemic to the upper Min River valley, differentiated from their common ancestral species in the present distribution area.

Search for the wild ancestor of buckwheat III. The wild ancestor of cultivated common buckwheat, and of tatary buckwheat

By surveying wild Fagopyrum species and their distribution in southern China and the Himalayan hills, I arrived at the conclusion that the newly discovered subspecies F. esculentum ssp. ancestralis Ohnishi is the wild ancestor of cultivated common buckwheat, while previously known wild tatary buckwheat,F. tataricum ssp. potanini Batalin is the wild ancestor of tatary buckwheat. Their original birthplace is revealed to be northwestern corner of Yunnan province for common buckwheat judging from the distribution of wild ancestor, and to be the northwest part of Sichuan province for tatary buckwheat judging from allozyme variability in wild tatary buckwheat. F. cymosum is not the ancestor of cultivated buckwheat; it is only distantly related to cultivated buckwheat, in morphology, isozymes and cpDNA. Several genetic, ecological and taxonomic categories which should be taken into consideration in examining the origin of buckwheat were discussed. Key Words: Fagopyrum esculentum ssp. ancestralis; Fagopyrum tataricum ssp. potanini; southern China theory of origin of buckwheat.

Asian PERILLA Crops and Their Weedy Forms: Their Cultivation, Utilization and Genetic Relationships

The cultivation and utilization of two Perilla crops were surveyed in Asia. Perilla frutescens var. frutescens is essentially an oil crop and is now widely cultivated in China and Korea. Its seeds are also used as a flavor for traditional foods in Japan, Korea, China and Nepal. In Korea, leaves of var. frutescens are used as a fresh vegetable and for making pickles. Whereas P. frutescens var. crispa is a Chinese medicine and afresh vegetable in the Far East, it has almost disappeared in many parts of Asia. Cultivation of var. crispa is still continued in Japan and Vietnam. In particular, it is cultivated in a large scale for coloring pickles in the areas where a large amount of plum pickles are produced in Japan. In China and Korea, it remains only as a relict form. Weedy plants ofPerilla are found in Japan, Korea and China. We can classify them into two forms; one, which is closely related to var. frutescens, and the other, which is similar to var. crispa We foundP citriodora andR hirtella in Guandong and Jiangxi provinces of China, respectively. It is clear that they are not endemic to Japan. A phylogenetic tree of samples of two Perilla crops and their weedy forms based on RAPD markers revealed that the weedy forms similar to var. crispa and var. frutescens are genetically closely related to var. crispa and var. frutescens, respectively. Var. crispa and its closely related weedy form seem to be more primitive.

Phylogenetic relationships among cultivated types of Brassica rapa L. em. Metzg. as revealed by AFLP analysis

The cultivated types of Brassica rapa L. em. Metzg. consist of morphologically distinct subspecies such as turnip, turnip rape, Chinese cabbage, pak choi and pot herb mustard which are classified as ssp. rapa, ssp. oleifera, ssp. pekinensis, ssp. chinensis and ssp. nipposinica (syn. ssp. japonica), respectively. We attempted to elucidate the phylogenetic relationships among the cultivated types of B. rapa. Thirty-two accessions from the Eurasian Continent were analyzed using AFLP markers with a cultivar of B. oleracea as an outgroup. In total, 455 bands were detected in the ingroup and 392 (86.6%) were polymorphic. The Neighbor-Joining tree based on the AFLP markers indicated that the accessions of B. rapa were congregated into two groups according to geographic origin. One group consisted of ssp. rapa and ssp. oleifera of Europe and Central Asia and the other included all the subspecies of East Asia. Our results suggest that cultivars from East Asia were probably derived from a primitive cultivated type, which originated in Europe or in Central Asia and migrated to East Asia. This primitive cultivated type was probably a common ancestor of ssp. rapa and ssp. oleifera. The Neighbor-Joining tree also shows that leafy vegetables in East Asia such as ssp. pekinensis, ssp. chinensis and ssp. nipposinica were differentiated several times from the distinct cultivars of ssp. oleifera in East Asia.

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.

Genetic relationships among cultivated types of Perilla frutescens and their weedy types in East Asia revealed by AFLP markers

AFLP markers were employed to detect genetic diversity in two cultivated Perilla frutescens (i.e. var. frutescens and var. crispa) and their weedy types and to assess their genetic relationships. Analysis of 60 Perilla accessions from China, Korea and Japan by seven AFLP primer combinations identified a total of 125 fragments, of which 80 (64%) were polymorphic at the species level. The phenotypic diversity meassured by Shannons index of information for the cultivated type of var. frutescens, the weedy type of var. frutescens, the cultivated type of var. crispa and the weedy type of var. crispa were on average 1.07, 2.23, 1.24 and 1.75, respectively. The weedy types exhibited high within-type variation in spite of a small number of samples. In the neighbor joining tree, two major clusters were recognized: (1) cultivated type of var. frutescens, (2) weedy type of var. frutescens, and cultivated and weedy types of var. crispa. The cultivated types of var. frutescens and of var. crispa were sharply separated by AFLPs. However, there remained ambiguities in regard to the intraspecific relaltionships, due to the clustering of the weedy types which occured in each of the clusters of the cultivated types. Two cultivated types of P. frutescens and their weedy types should be taxonomically considered as a P. frutescens complex. The present AFLP data are consistent with the hypothesis that China is the original place of cultivation of var. frutescens and Korea is a secondary center of diffusion of var. frutescens.

Origin of cultivated Tatary buckwheat (Fagopyrum tataricum Gaertn.) revealed by RAPD analyses

The phylogenetic relationships among cultivated landraces and natural populations of wild subspecies of Tatary buckwheat were investigated at the individual level by constructing a phylogenetic tree based on RAPD markers. As the PCR templates, DNA of individuals rather than bulked samples, was used. Ten individuals from 10 cultivated landraces, 71 individuals from 21 natural populations of wild subspecies, and 7 individuals from 3 weedy Tatary buckwheat were provided for RAPD analyses. Three groups were recognized: (1) all cultivated landraces and wild subspecies from central Tibet and northern Pakistan; (2) 10 individuals of wild subspecies from northwestern Yunnan; (3) the remaining individuals of wild subspecies from northwestern Yunnan and all individuals of wild subspecies from Sichuan. Group (2) was phylogenetically closely related to group (1). The origin of cultivated Tatary buckwheat, the hybrid origin of weedy Tatary buckwheat and of the wild populations from central Tibet and northern Pakistan are discussed. We arrive at the conclusion that cultivated Tatary buckwheat probably originated in northwestern Yunnan in China.

The Distribution of Perilla Species

Perilla (Lamiaceae) contains one tetraploid species, P. frutescens (L.) Britt. and three diploid species, P. citriodora (Makino) Nakai, P. hirtella Nakai and P. setoyensis G. Honda. Tetraploid species have been traditionally cultivated in Asia for their seed oil and for their fragrant leaves that are used as medicine or as a garnish for fish. The center of diversity is still obscure. To conserve the genetic resources, it is important to know the diversity of the tetraploid species. The three diploid species, which are possible parents of the tetraploid species, are all believed to be indigenous to Japan. Their distribution in China and Korea was clarified on the basis of herbarium and field surveys. The tetraploid species is assumed to have originated somewhere around the mid-to downstream area of the Changjiang River. Though Perilla is not cultivated as often in these areas as in northern China, Korea, the Himalayan region, or Myanmar, these areas should also be important for the conservation of genetic resources of tetraploid Perilla crops because of the expected high genetic diversity.

Phylogenetic position of east Tibetan natural populations in Tartary buckwheat (Fagopyrum tataricum Gaert.) revealed by RAPD analyses

Phylogenetic relationships among cultivated landraces and natural populations of wild subspecies of Tartary buckwheat were investigated by constructing an NJ tree based on RAPD markers focussing on east Tibetan natural populations. Ten plants from three cultivated landraces and 29 plants from five natural populations of wild subspecies in eastern Tibet were used for RAPD analyses. The wild subspecies from eastern Tibet was classified into three types; (1) same type as cultivated landraces; (2) closely related to natural populations of northwestern Yunnan; and (3) an exceptional population, Zhuka, which was closely related to Sichuan populations. Since the type (2) is considered as the wild ancestral type of cultivated Tartary buckwheat, we conclude that eastern Tibet too may be one of the center of origin of this crop.

Phylogenetic relationships among natural populations of perennial buckwheat, Fagopyrum cymosum Meisn., revealed by allozyme variation

Twenty natural populations of F. cymosum, an insect-pollinated allogamous wild species of Fagopyrum, were investigated for their chromosome number and allozyme variation at 10 Ioci encoding 8 enzymes. Diploid populations were obtained in Sichuan, Yunnan and Tibet in China, whereas tetraploid populations were obtained in Tibet, the Himalayan hills, Thailand and southern China. Both diploid and tetraploid populations maintained a large amount of allozyme variation. The average heterozygosity, He, ranged from 0.045 to 0.389 (0.213 on average). Tetraploid populations showed higher He values than diploid populations. Natural populations of F. cymosum were locally well differentiated (GST = 0.322), probably due to reproductive isolation between the two ploidy levels and to distribution over wide areas from southern China to the Himalayan hills. The phylogenetic tree constructed by the neighbor-joining method based on allozyme variation clarified two distinct groups of diploid populations, the Sichuan and Yunnan groups. As for the tetraploid populations, polyploidization occurred twice independently, once in Yunnan and once in eastern Tibet. Tetraploid F. cymosum diffused to warmer areas such as southern Yunnan and Thailand as well as to cooler areas of Tibet and the high Himalayan hills and became the most widely distributed of the Fagopyrum species.