title: Linkage mapping, molecular cloning and functional analysis of soybean gene Fg3 encoding flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase.
abstract: BACKGROUND
  Flavonol glycosides (FGs) are major components of soybean leaves and there are substantial differences in FG composition among genotypes. The first objective of this study was to identify genes responsible for FG biosynthesis and to locate them in the soybean genome. The second objective was to clone the candidate genes and to verify their function. Recombinant inbred lines (RILs) were developed from a cross between cultivars Nezumisaya and Harosoy.
  
  
  RESULTS
  HPLC comparison with authentic samples suggested that FGs having glucose at the 2″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Nezumisaya, whereas FGs of Harosoy were devoid of 2″-glucose. Conversely, FGs having glucose at the 6″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Harosoy, whereas these FGs were absent in Nezumisaya. Genetic analysis suggested that two genes control the pattern of attachment of these sugar moieties in FGs. One of the genes may be responsible for attachment of glucose to the 2″-position, probably encoding for a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase. Nezumisaya may have a dominant whereas Harosoy may have a recessive allele of the gene. Based on SSR analysis, linkage mapping and genome database survey, we cloned a candidate gene designated as GmF3G2″Gt in the molecular linkage group C2 (chromosome 6). The open reading frame of GmF3G2″Gt is 1380 bp long encoding 459 amino acids with four amino acid substitutions among the cultivars. The GmF3G2″Gt recombinant protein converted kaempferol 3-O-glucoside to kaempferol 3-O-sophoroside. GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].
  
  
  CONCLUSIONS
  GmF3G2″Gt encodes a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase and corresponds to the Fg3 gene. GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee. Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan. dishaokang@affrc.go.jp.
authors.forename: Shaokang
authors.initials: S
authors.lastname: Di
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan. yanfan@affrc.go.jp.
authors.forename: Fan
authors.initials: F
authors.lastname: Yan
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan. felipe-r@hotmail.es.|Current address: Universidad Católica de Oriente, Rionegro-Antioquia, Colombia. felipe-r@hotmail.es.
authors.forename: Felipe Rojas
authors.initials: FR
authors.lastname: Rodas
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan. rodtor@gmail.com.
authors.forename: Tito O
authors.initials: TO
authors.lastname: Rodriguez
authors.affiliation: Department of Botany, National Museum of Nature and Science, Tsukuba, Ibaraki, 305-0005, Japan. murai@kahaku.go.jp.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Murai
authors.affiliation: Department of Botany, National Museum of Nature and Science, Tsukuba, Ibaraki, 305-0005, Japan. iwashina@kahaku.go.jp.
authors.forename: Tsukasa
authors.initials: T
authors.lastname: Iwashina
authors.affiliation: RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, 230-0045, Japan. satoko.sugawra@riken.jp.
authors.forename: Satoko
authors.initials: S
authors.lastname: Sugawara
authors.affiliation: RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, 230-0045, Japan. tetsuya.mori@riken.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Mori
authors.affiliation: RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, 230-0045, Japan. ryo.nakabayashi@riken.jp.
authors.forename: Ryo
authors.initials: R
authors.lastname: Nakabayashi
authors.affiliation: RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, 230-0045, Japan. yskeiko@riken.jp.
authors.forename: Keiko
authors.initials: K
authors.lastname: Yonekura-Sakakibara
authors.affiliation: RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, 230-0045, Japan. kazuki.saito@riken.jp.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Saito
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan. masako@affrc.go.jp.|National Institute of Crop Science, Tsukuba, Ibaraki, 305-8518, Japan. masako@affrc.go.jp.
authors.forename: Ryoji
authors.initials: R
authors.lastname: Takahashi
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002874:Chromosome Mapping / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods*; D044948:Flavonols / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006027:Glycosides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D007178:Inbreeding; D040582:Inheritance Patterns / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011110:Polymorphism, Genetic; D011995:Recombination, Genetic / Q000235:genetics; D013025:Glycine max / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D044948:Flavonols; D006027:Glycosides; D005964:Glucosyltransferases