The glycosylation patterns of flavonol glycosides (FGs) in soybean leaves are associated with productivity and insect resistance. The structure of FGs is conditioned by four flavonoid glycoside glycosyltransferase (FGG) genes, Fg1 (6"-glucoside present), Fg2 (6"-rhamnoside present), Fg3 (2"-glucoside present), and Fg4 (2"-rhamnoside present). We previously cloned and characterized the Fg1 to Fg3 genes. This study was conducted to comprehensively identify FGG genes by isolating the Fg4 gene. Leaves of the cultivar, Clark (Fg4 allele), contained FGs with rhamnose at the 4"-position, whereas those of the cultivar A.K. (fg4 allele), were devoid of 4"-rhamnoside. Their F2 population segregated in three Clark-type: 1 A.K.-type ratios, suggesting that a single gene controlled the existence of 4"-rhamnoside. Based on genetic mapping, we cloned a candidate gene, Glyma.06G235000, with three amino acid substitutions between cultivars. Recombinant protein of Clark acted on kaempferol 3-O-rhamnosyl-(1→6)-galactoside and generated a product with a similar retention time to kaempferol 3-O-rhamnosyl-(1→4)-[rhamnosyl-(1→6)-glalactoside]. Furthermore, it acted on kaempferol 3-O-glucoside/galactoside and generated products with similar retention times to kaempferol 3-O-rhamnosyl-(1→2)-glucoside/galactoside. The A.K. protein did not function presumably due to G293R mutation. The cleaved amplified polymorphic sequence (CAPS) marker to detect the mutation co-segregated with FG composition. These results suggest that Glyma.06G235000 corresponds to the Fg4 gene and that the protein attaches rhamnose to the 2"- or 4"-position, depending on the substrates. Information of all FGG genes may be useful for developing cultivars with the desired FGG composition.

Pubescence color of soybean is controlled by two genes, T and Td. In the presence of a dominant T allele, dominant and recessive alleles of the Td locus generate tawny and light tawny (or near-gray) pubescence, respectively. Flavones, responsible for pubescence color, are synthesized via two copies of flavone synthase II genes (FNS II-1 and FNS II-2). This study was conducted to map and clone the Td gene. Genetic and linkage analysis using an F2 population and F3 families derived from a cross between a Clark near-isogenic line with light tawny pubescence (genotype: TT tdtd) and a Harosoy near-isogenic line with tawny pubescence (TT TdTd) revealed a single gene for pubescence color around the end of chromosome 3. Genome sequence alignment of plant introductions revealed an association between premature stop codons in Glyma.03G258700 (R2R3 MYB transcription factor) and recessive td allele. Cultivars and lines having near-gray or light tawny pubescence and a gray pubescence cultivar with td allele had premature stop codons in the gene. These results suggest that Glyma.03G258700 corresponds to the Td gene. It was predominantly expressed in pubescence. Compared to a tawny pubescence line, a near-isogenic line with td allele produced extremely small amounts of transcripts of Glyma.03G258700, FNS II-1, and FNS II-2 in pubescence. The promoter of FNS II-1 and FNS II-2 shared cis-acting regulatory elements for binding of MYB proteins. These results suggest that the wild type of Glyma.03G258700 protein may bind to the promoter of FNS II genes and upregulate their expression, resulting in increased flavone content and deeper pubescence color. In contrast, mutated Glyma.03G258700 protein may fail to upregulate the expression of FNS II genes, resulting in decreased flavone content and dilute pubescence color.

A new flavonol triglycoside, Quercetin 3- O-α-rhamnopyranosyl-(1→4)-[α-rhamnopyranosyl-(1→6)-β-galactopyranoside], was isolated from the leaves of soybean ( Glycine max) cultivar “Clark” and identified by UV spectra, LC-ESI-MS, acid hydrolysis, and1H and13C NMR. The compound was found together with 7 known flavonol glycosides, quercetin 3- O-robinobioside, quercetin 3- O-rutinoside, kaempferol 3- O-rhamnosyl-(1→4)-[rhamnosyl-(1→6)-galactoside], kaempferol 3- O-robinobioside, kaempferol 3- O-rutinoside, isorhamnetin 3- O-robinobioside, and isorhamnetin 3- O-rutinoside.

A soybean [Glycine max (L.) Merr.] mutant line producing deep purple flowers (E013‐C‐1) was developed from an ethylmethane sulfonate‐treated population of the cultivar ‘Bay’, which has purple flowers. Genetic analysis was performed on a cross between ‘E013‐C‐1’ and the cultivar ‘Clark’ that had purple flowers. F1 plants had purple flowers whereas F2 plants segregated into a 3:1 purple/deep purple ratio. The results suggest that a single gene controls flower color and that purple color is dominant to deep purple. F3 plants derived from F2 plants with deep purple flowers were fixed for deep purple flowers. F3 plants derived from F2 plants with purple flowers segregated into two families segregating for flower color and one family that was fixed for purple flower color. The results confirmed that a single gene controls flower color and its recessive allele is responsible for deep purple flower color. The gene was designated Wd. Linkage mapping with simple sequence repeat (SSR) markers suggested that the Wd gene was located between Satt612 and Sct_199 in chromosome 18. Deep purple petals contained 50% higher levels of anthocyanins than purple petals. The vacuolar pH of deep purple flowers was similar to that of purple ones. These results suggest that the Wd gene controls the amount of anthocyanins and it is responsible for the development of deep purple flowers.

Greatest potential, QTLs for hypoxia and waterlogging tolerance in soybean roots were detected using a new phenotypic evaluation method.

i) Graduate School ofScience and 71echnology, Chiba Uhiversity, 648 Matsudo, Matsudo, Chiba 271-851O, Japan 2} Ndtional Institute ofAgrobiotogical Resources, 21 -2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan 3) 77te institute of the Society for 71echno-innovation ofAgricutture, forestT:y and Fisheries (S7Aif), 446-1 Ipp Kamiyokoba, Tsukuba, Ibaraki 305-0854, Japan 4) IVationalAgriculturResearch Center, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan aizuka,

The T and W1 loci determine color and anthocyanin content in soybean hypocotyls. In purple hypocotyls, the anthocyanins were: A1, a delphinidin-type anthocyanin; A2 made of two unresolved peaks: peonidin 3,5-di-O-glucoside and a delphinidin-type anthocyanin; and A3, accounting for >90% of the total, was malvidin 3,5-di-O-glucoside. A single peak, peonidin 3,5-di-O-glucoside, was observed in the bronze hypocotyls.

A soybean line producing light purple flowers (E023‐H‐12) was developed from an ethyl methanesulfonate (EMS)‐treated population of cultivar Bay. The objective of this study was to investigate the genetic and molecular basis of flower color variation in E023‐H‐12. Genetic analysis suggested that the W1 gene encoding a flavonoid 3′5′‐hydroxylase (F3′5′H) controls light purple flower color. A single plant with purple flowers was generated in an F2 population derived from a cross between E023‐H‐12 and Clark‐w1 with white flowers, probably because of intragenic recombination of the F3′5′H gene. The allele for light purple flower was designated as w1‐lp2. The dominance relationships of the locus were W1 > w1‐lp2 > w1. Flower petals of E023‐H‐12 had similar expression levels of the F3′5′H gene but they had 42% less anthocyanins compared with Bay. The lower anthocyanin content may account for the light purple color of this mutant line. The nucleotide sequence of the F3′5′H gene of E023‐H‐12 had a single nucleotide polymorphism (SNP) resulting in alteration of an amino acid (H137L). A derived cleaved amplified polymorphic sequence (dCAPS) marker to discriminate the SNP co‐segregated with flower color. Thus, the amino acid substitution may be responsible for the lower anthocyanin content, and, consequently, light purple flower color.

Flavonoids are important secondary metabolites in plants. Sugar–sugar glycosyltransferases are involved in the final step of flavonoid biosynthesis and contribute to the structural diversity of flavonoids. This manuscript describes the first cloning of a sugar–sugar glucosyltransferase gene in the UGT family that attaches glucose to the 6″-position of sugar bound to a flavonol. The results provide a glimpse on the possible evolution of sugar–sugar glycosyltransferase genes and identify putative amino acids responsible for the recognition of the hydroxyl group of the sugar moiety and specification of sugar. A scheme for the genetic control of flavonol glycoside biosynthesis is proposed.

Time to flowering and maturity in soybean is controlled by loci E1 to E5, and E7 to E9. These loci were assigned to molecular linkage groups (MLGs) except for E5. This study was conducted to map the E5 locus using F2 populations expected to segregate for E5. F2 populations were subjected to quantitative trait locus (QTL) analysis for days to flowering (DF) and maturity (DM). In Harosoy-E5 × Clark-e2 population, QTLs for DF and DM were found at a similar position with E2. In Harosoy × Clark-e2E5 population, QTLs for DF and DM were found in MLG D1a and B1, respectively. In Harosoy-E5Dt2 × Clark-e2 population, a QTL for DF was found in MLG B1. Thus, results from these populations were not fully consistent, and no candidate QTL for E5 was found. In Harosoy × PI 80837 population, from which E5 was originally identified, QTLs corresponding to E1 and E3 were found, but none for E5 existed. Harosoy and PI 80837 had the e2-ns allele whereas Harosoy-E5 had the E2-dl allele. The E2-dl allele of Harosoy-E5 may have been generated by outcrossing and may be responsible for the lateness of Harosoy-E5. We conclude that a unique E5 gene may not exist.

Physical dormancy, a structural feature of the seed coat known as hard seededness, is an important characteristic for adaptation of plants against unstable and unpredictable environments. To dissect the molecular basis of qHS1, a quantitative trait locus for hard seededness in soybean (Glycine max (L) Merr.), we developed a near-isogenic line (NIL) of a permeable (soft-seeded) cultivar, Tachinagaha, containing a hard-seed allele from wild soybean (G. soja) introduced by successive backcrossings. The hard-seed allele made the seed coat of Tachinagaha more rigid by increasing the amount of β-1,4-glucans in the outer layer of palisade cells of the seed coat on the dorsal side of seeds, known to be a point of entrance of water. Fine-mapping and subsequent expression and sequencing analyses revealed that qHS1 encodes an endo-1,4-β-glucanase. A single-nucleotide polymorphism (SNP) introduced an amino acid substitution in a substrate-binding cleft of the enzyme, possibly reducing or eliminating its affinity for substrates in permeable cultivars. Introduction of the genomic region of qHS1 from the impermeable (hard-seeded) NIL into the permeable cultivar Kariyutaka resulted in accumulation of β-1,4-glucan in the outer layer of palisade cells and production of hard seeds. The SNP allele found in the NIL was further associated with the occurrence of hard seeds in soybean cultivars of various origins. The findings of this and previous studies may indicate that qHS1 is involved in the accumulation of β-1,4-glucan derivatives such as xyloglucan and/or β-(1,3)(1,4)-glucan that reinforce the impermeability of seed coats in soybean.

The R gene of soybean, presumably encoding a MYB transcription factor, controls seed coat color. The gene consists of multiple alleles, R (black), r-m (black spots and (or) concentric streaks on brown seed), and r (brown seed). This study was conducted to determine the structure of the MYB transcription factor gene in a near-isogenic line (NIL) having r-m allele. PCR amplification of a fragment of the candidate gene Glyma.09G235100 generated a fragment of about 1 kb in the soybean cultivar Clark, whereas a fragment of about 14 kb in addition to fragments of 1 and 1.4 kb were produced in L72-2040, a Clark 63 NIL with the r-m allele. Clark 63 is a NIL of Clark with the rxp and Rps1 alleles. A DNA fragment of 13 060 bp was inserted in the intron of Glyma.09G235100 in L72-2040. The fragment had the CACTA motif at both ends, imperfect terminal inverted repeats (TIR), inverse repetition of short sequence motifs close to the 5' and 3' ends, and a duplication of three nucleotides at the site of integration, indicating that it belongs to a CACTA-superfamily transposable element. We designated the element as Tgm11. Overall nucleotide sequence, motifs of TIR, and subterminal repeats were similar to those of Tgm1 and Tgs1, suggesting that these elements comprise a family.

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.

There are substantial genotypic differences in the levels of flavonol glycosides (FGs) in soybean leaves. The first objective of this study was to identify and locate genes responsible for FG biosynthesis in the soybean genome. The second objective was to clone and verify the function of these candidate genes. Recombinant inbred lines (RILs) were developed by crossing the Kitakomachi and Koganejiro cultivars. The FGs were separated by high performance liquid chromatography (HPLC) and identified. The FGs of Koganejiro had rhamnose at the 6″-position of the glucose or galactose bound to the 3-position of kaempferol, whereas FGs of Kitakomachi were devoid of rhamnose. Among the 94 RILs, 53 RILs had HPLC peaks classified as Koganejiro type, and 41 RILs had peaks classified as Kitakomachi type. The segregation fitted a 1:1 ratio, suggesting that a single gene controls FG composition. SSR analysis, linkage mapping and genome database survey revealed a candidate gene in the molecular linkage group O (chromosome 10). The coding region of the gene from Koganejiro, designated as GmF3G6″Rt-a, is 1,392 bp long and encodes 464 amino acids, whereas the gene of Kitakomachi, GmF3G6″Rt-b, has a two-base deletion resulting in a truncated polypeptide consisting of 314 amino acids. The recombinant GmF3G6″Rt-a protein converted kaempferol 3-O-glucoside to kaempferol 3-O-rutinoside and utilized 3-O-glucosylated/galactosylated flavonols and UDP-rhamnose as substrates. GmF3G6″Rt-b protein had no activity. These results indicate that GmF3G6″Rt encodes a flavonol 3-O-glucoside (1 → 6) rhamnosyltransferase and it probably corresponds to the Fg2 gene. GmF3G6″Rt was designated as UGT79A6 by the UGT Nomenclature Committee.