The phylogenetic relationship among 30 accessions belonging to nine species of the genus Echinochloa Beauv. was studied on the basis of the sequence of three non-coding regions ( trn T-L, trn L-F intergenic spacers, and trn L intron) of chloroplast DNA (cpDNA). A strict consensus parsimonious tree of the three most parsimonious trees derived from 25 polymorphic sites (six indels and 19 substitutions) in the total sequences, ranging from 1715-1760 bp, represented five groups: (i) Echinochloa oryzicola Vasing. and Echinochloa stagnina Beauv. from Thailand; (ii) Echinochloa crus-galli Beauv. complex; (iii) Echinochloa crus-pavonis Schult; (iv) Echinochloa colonum Link. and Echinochloa frumentacea Link.; and (v) the African species, Echinochloa obtusiflora Stapf and Echinochloa stagnina . Japanese barnyard millet ( Echinochloa esculenta H. Scholz) and various weedy varieties of E. crus-galli and Echinochloa oryzoides Fritsch had quite similar sequences and formed the E. crus-galli complex, which was characterized by six substitutions. A cultivated form of E. oryzicola (Mosuo barnyard millet) and various morphological and agronomical forms of E. oryzicola were characterized by two indels. Indian barnyard millet ( E. frumentacea ) and its wild counterpart ( E. colonum ) were characterized by five substitutions. Domestication as millets and adaptation to paddy environments as mimic weeds might occur after the divergence of species in the Asian Echinochloa .

BackgroundGlycine soja is a wild relative of soybean that has purple flowers. No flower color variant of Glycine soja has been found in the natural habitat.ResultsB09121, an accession with light purple flowers, was discovered in southern Japan. Genetic analysis revealed that the gene responsible for the light purple flowers was allelic to the W1 locus encoding flavonoid 3'5'-hydroxylase (F3'5'H). The new allele was designated as w1-lp. The dominance relationship of the locus was W1 >w1-lp >w1. One F2 plant and four F3 plants with purple flowers were generated in the cross between B09121 and a Clark near-isogenic line with w1 allele. Flower petals of B09121 contained lower amounts of four major anthocyanins (malvidin 3,5-di-O-glucoside, petunidin 3,5-di-O-glucoside, delphinidin 3,5-di-O-glucoside and delphinidin 3-O-glucoside) common in purple flowers and contained small amounts of the 5'-unsubstituted versions of the above anthocyanins, peonidin 3,5-di-O-glucoside, cyanidin 3,5-di-O-glucoside and cyanidin 3-O-glucoside, suggesting that F3'5'H activity was reduced and flavonoid 3'-hydroxylase activity was increased. F3'5'H cDNAs were cloned from Clark and B09121 by RT-PCR. The cDNA of B09121 had a unique base substitution resulting in the substitution of valine with methionine at amino acid position 210. The base substitution was ascertained by dCAPS analysis. The polymorphism associated with the dCAPS markers co-segregated with flower color in the F2 population. F3 progeny test, and dCAPS and indel analyses suggested that the plants with purple flowers might be due to intragenic recombination and that the 65 bp insertion responsible for gene dysfunction might have been eliminated in such plants.ConclusionsB09121 may be the first example of a flower color variant found in nature. The light purple flower was controlled by a new allele of the W1 locus encoding F3'5'H. The flower petals contained unique anthocyanins not found in soybean and G. soja. B09121 may be a useful tool for studies of the structural and functional properties of F3'5'H genes as well as investigations on the role of flower color in relation to adaptation of G. soja to natural habitats.

Polymerase chain reaction (PCR) and polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) techniques were applied for establishing the reliable practice in identification of Echinochloa oryzicola Vasing. and E. crus‐galli (L.) Beauv. (barnyardgrass). Total DNA was extracted from 18 accessions and 86 individuals of E. oryzicola, 33 accessions and 140 individuals of E. crus‐galli var. crus‐galli, 23 individuals of E. crus‐galli var. praticola, and six individuals of E. crus‐galli var. formosensis that were collected from Japan. A partial region of intergenetic spacer between trnT and trnL, and an intron of trnL were amplified separately using a trn‐a and trn‐b1 primer set, and a trn‐c and trn‐d primer set, respectively. All individuals of E. oryzicola showed the same fragment amplified by the trn‐a and trn‐b1 primer set. The fragment was 481 bp in length, and was undigested by EcoR I, whereas all individuals of E. crus‐galli, including three botanical varieties, showed the same fragment with a 449‐bp length. The fragment was digested by EcoR I into two fragments (178 and 271 bp). The fragment amplified by the trn‐c and trn‐d primer set in all individuals of E. oryzicola was digested by Alu I into two fragments (174 and 452 bp), but undigested by Dra I. In contrast, the fragment amplified by the trn‐c and trn‐d primer set in all individuals of E. crus‐galli was digested by Dra I into two fragments (134 and 487 bp), but undigested by Alu I. There was no intraspecific variation in these regions; thus, these two species are easily identifiable by using our method.

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