A single-base deletion in soybean flavonoid 3'-hydroxylase gene is associated with gray pubescence color.
ABSTRACT The T locus of soybean (Glycine max (L.) Merr.) controls pubescence and seed coat color and is presumed to encode flavonoid 3'-hydroxylase (F3'H). The dominant T and the recessive t allele of the locus produce brown and gray pubescence, respectively. PCR primers were constructed based on the sequence of a soybean EST clone homologous to the F3'H gene. A putative full-length cDNA, sf3'h1 was isolated by 3' and 5' RACE. Sequence analysis revealed that sf3'h1 consists of 1690 nucleotides encoding 513 amino acids. It had 68% and 66% homology with corresponding F3'H protein sequences of petunia and Arabidopsis, respectively. A conserved amino acid sequence of F3'H proteins, GGEK, was found in the deduced polypeptide. Sequence analysis of the gene from a pair of near-isogenic lines for T, To7B (TT, brown) and To7G (tt, gray) revealed that they differed by a single C deletion in the coding region of To7G. The deletion changed the subsequent reading frame resulting in a truncated polypeptide lacking the GGEK consensus sequence and the heme-binding domain. Genomic Southern analysis probed by sf3'h1 revealed restriction fragment length polymorphisms between cultivars with different pubescence color. Further, sf3'h1 was mapped at the same position with T locus on LG3(c2). PCR-RFLP analysis was performed to detect the single-base deletion. To7B and three cultivars with brown pubescence exhibited shorter fragments, while To7G and three cultivars with gray pubescence had longer fragments due to the single-base deletion. The PCR-RFLP marker co-segregated with genotypes at the T locus in a F2 population segregating for the T locus. The above results strongly suggest that sJ3'h1 represents the T gene of soybean responsible for pubescence color and that the single-base deletion may be responsible for gray pubescence color.
Article: Three minimum tile paths from bacterial artificial chromosome libraries of the soybean (Glycine max cv. 'Forrest'): tools for structural and functional genomics.[show abstract] [hide abstract]
ABSTRACT: The creation of minimally redundant tile paths (hereafter MTP) from contiguous sets of overlapping clones (hereafter contigs) in physical maps is a critical step for structural and functional genomics. Build 4 of the physical map of soybean (Glycine max L. Merr. cv. 'Forrest') showed the 1 Gbp haploid genome was composed of 0.7 Gbp diploid, 0.1 Gbp tetraploid and 0.2 Gbp octoploid regions. Therefore, the size of the unique genome was about 0.8 Gbp. The aim here was to create MTP sub-libraries from the soybean cv. Forrest physical map builds 2 to 4. The first MTP, named MTP2, was 14,208 clones (of mean insert size 140 kbp) picked from the 5,597 contigs of build 2. MTP2 was constructed from three BAC libraries (BamHI (B), HindIII (H) and EcoRI (E) inserts). MTP2 encompassed the contigs of build 3 that derived from build 2 by a series of contig merges. MTP2 encompassed 2 Gbp compared to the soybean haploid genome of 1 Gbp and does not distinguish regions by ploidy. The second and third MTPs, called MTP4BH and MTP4E, were each based on build 4. Each was semi-automatically selected from 2,854 contigs. MTP4BH was 4,608 B and H insert clones of mean size 173 kbp in the large (27.6 kbp) T-DNA vector pCLD04541. MTP4BH was suitable for plant transformation and functional genomics. MTP4E was 4,608 BAC clones with large inserts (mean 175 kbp) in the small (7.5 kbp) pECBAC1 vector. MTP4E was suitable for DNA sequencing. MTP4BH and MTP4E clones each encompassed about 0.8 Gbp, the 0.7 Gbp diploid regions and 0.05 Gbp each from the tetraploid and octoploid regions. MTP2 and MTP4BH were used for BAC-end sequencing, EST integration, micro-satellite integration into the physical map and high information content fingerprinting. MTP4E will be used for genome sequence by pooled genomic clone index. Each MTP and associated BES will be useful to deconvolute and ultimately finish the whole genome shotgun sequence of soybean.Plant Methods 02/2006; 2:9. · 2.83 Impact Factor
Article: Expression of a putative flavonoid 3'-hydroxylase in sorghum mesocotyls synthesizing 3-deoxyanthocyanidin phytoalexins.[show abstract] [hide abstract]
ABSTRACT: In sorghum, ingress of Cochliobolus heterostrophus stimulates the synthesis of 3-deoxyanthocyanidins that act as phytoalexins. Apigeninidin and luteolinidin are two major phytoalexins induced in the first 24 h after infection. In an attempt to understand genetic regulation of the biosynthesis of sorghum phytoalexins, we isolated a differentially expressed partial cDNA. Characterization and comparison showed that this cDNA sequence corresponds to a putative flavonoid 3'-hydroxylase. Full length sequence characterization allowed us to establish that the sorghum putative f3'h cDNA encodes a peptide of 517 amino acids that has domains conserved among cytochrome P450 proteins functioning in the flavonoid biosynthetic pathway. Heterologous expression of the putative f3'h cDNA in Escherichia coli yielded a membrane preparation that catalyzed the hydroxylation of naringenin. We show here that transcription of the flavonoid 3'-hydroxylase was coordinately regulated with that of chalcone synthase and dihydroflavonol reductase, and expression of these genes was induced within the first 24 h of fungal challenge. Synthesis of apigeninidin and luteolinidin followed the induced expression of the f3'h gene, implicating its role in fungal induced expression of sorghum phytolaexins.
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ABSTRACT: In soybean (Glycine max [L.] Merr.), varieties with seed-flooding tolerance at the geminating stage are desirable for breeding in countries with much rainfall at sowing time. Our study revealed great intervarietal variation in seed-flooding tolerance as evaluated by germination rate (GR) and normal seedling rate (NS). Pigmented seed coat and small seed weight tended to give a positive effect on seed-flooding tolerance. Subsequently, QTL analysis of GR and NS were performed and a total of four QTLs were detected. Among them, Sft1 on the linkage group H (LG_H) exhibited a large effect on GR after a 24-h treatment; however, Sft2 near the I locus on LG_A2 involved in seed coat pigmentation exhibited the largest effect on seed-flooding tolerance. Sft1, Sft3 and Sft4 were independent of seed coat color and seed weight. Based on the results, we discussed the physiological effects of genetic factors responsible for seed-flooding tolerance in soybean.Plant Science.