Allele-specific marker development and selection efficiencies for both flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes in soybean subgenus soja

Color is one of the phenotypic markers mostly used to study soybean (Glycine max L. Merr.) genetic, molecular and biochemical processes. Two P450-dependent mono-oxygenases, flavonoid 3′-hydroxylase (F3′H; EC1.14.3.21) and flavonoid 3′,5′-hydroxylase (F3′5′H, EC1.14.13.88), both catalyzing the hydroxylation of the B-ring in flavonoids, play an important role in coloration. Previous studies showed that the T locus was a gene encoding F3′H and the W1 locus co-segregated with a gene encoding F3′5′H in soybean. These two genetic loci have identified to control seed coat, flower and pubescence colors. However, the allelic distributions of both F3′H and F3′5′H genes in soybean were unknown. In this study, three novel alleles were identified (two of four alleles for GmF3′H and one of three alleles for GmF3′5′H). A set of gene-tagged markers was developed and verified based on the sequence diversity of all seven alleles. Furthermore, the markers were used to analyze soybean accessions including 170 cultivated soybeans (G. max) from a mini core collection and 102 wild soybeans (G. soja). For both F3′H and F3′5′H, the marker selection efficiencies for pubescence color and flower color were determined. The results showed that one GmF3′H allele explained 92.2 % of the variation in tawny and two gmf3′h alleles explained 63.8 % of the variation in gray pubescence colors. In addition, two GmF3′5′H alleles and one gmF3′5′h allele explained 94.0 % of the variation in purple and 75.3 % in white flowers, respectively. By the combination of the two loci, seed coat color was determined. In total, 90.9 % of accessions possessing both the gmf3′h-b and gmf3′5′h alleles had yellow seed coats. Therefore, seed coat colors are controlled by more than two loci.     

A deletion mutation in the βA1/A3 crystallin gene (CRYBA1/A3) is associated with autosomal dominant congenital nuclear cataract in a Chinese family

Congenital cataracts are an important cause of blindness worldwide. In a family of Chinese descent, a dominant congenital nuclear cataract locus was mapped to chromosome 17q11.1-12. The maximum LOD score, 2.49, at recombination fraction 0, was obtained for marker D17S1294. The results of both linkage and haplotype analyses defined a disease-gene to an 11.78-cM region harboring the gene coding for A1/A3 crystallin (CRYBA1/A3). Mutation analysis of the CRYBA1/A3 gene identified a 3-bp deletion in exon 4, which cosegregated with the disease risk in this family and was not observed in 100 normal chromosomes. This mutation resulted in the deletion of a highly conserved glycine at codon 91 (G91) and could be associated with an incorrect folding of A1/A3 crystallin. It highlights the physiological importance of crystallin and supports the role of CRYBA1/A3 in human cataracts formation.Y. Qi and H. Jia contributed equally to this work     

Isolation and functional characterization of a novel gene coding for flavonoid 3′-hydroxylase from globe artichoke

Globe artichoke (Cynara cardunculus L. var. scolymus) is rich in flavonoids which contribute to its health-promoting properties. With the aim of understanding the genetic control of flavonoid accumulation in artichoke, we isolated an artichoke full-length cDNA sequence encoding flavonoid 3′-hydroxylase (F3′H), a major enzyme of the flavonoid hydroxylation pattern. In silico studies confirmed that the deduced amino acid sequence of CcF3′H is highly similar to F3′Hs isolated from other Asteraceae. The Northern blot analysis demonstrated that CcF3′H was highly expressed in leaves and in specific parts of the heads. Its expression differed slightly among artichoke cultivars. The overexpression of CcF3′H in tobacco plants led to the accumulation of flavonoids and to an increase of flower colour intensity, thus identifying CcF3′H as promising candidate for genetic engineering. CcF3′H represents the first structural gene of the flavonoid biosynthesis isolated from C. cardunculus, and its characterization sheds light on the accumulation of flavonoids.     

Gene-dependent flavonoid 3′-hydroxylation in maize

The influence of the gene Pr on flavonoid 3-hydroxylase activity in maize is described. Specific activities are presented for the hydroxylase in seedlings and aleurone tissue homozygous dominant and recessive and heterozygous for Pr. Specific activity levels in both tissues increased in a nearly direct proportion with the increase in Pr dosage, which is consistent with Pr being the structural gene for the hydroxylase. Regression analysis of the gene dosage:enzyme activity comparison yielded correlation coefficients of 0.979 and 0.959 for the seedlings and aleurone, respectively. Quantitative identification of the cyanidin and pelargonidin in the aleurone indicated that cyanidin increased with an increase in dominant Pr, while pelargonidin decreased, although the increases and decreases observed were not directly proportional to the gene dosage. Comparison of the cyanidin/pelargonidin ratio to the gene dosage ratio in the different tissues showed a strong correlation (0.998), which demonstrates that the dosage of Pr controls the ratio of cyanidin to pelargonidin. Cyanidin was found at a low concentration in aleurone homozygous for pr. Hydroxylase activity in maturing field plants reaches its peak concentration near anthesis and is present at an appreciable concentration in mature plant tissue homozygous for pr, as well as in seedlings homozygous for pr. Suggestion is made that pr could be a hypomorphic allele or that a duplicate gene for Pr could exist to account for the hydroxylase activity in homozygous pr tissue. Evidence for the hydroxylase in the aleurone and the seedlings and the pigment ratio data from the aleurone suggest that Pr is indeed a structural gene for NADPH:flavonoid 3-hydroxylase.Cooperative Investigations, Agricultural Research Service, United States Department of Agriculture, and Missouri Agricultural Experiment Station, Columbia, Missouri 65211. Journal Series No. 9958.     

The potato P locus codes for flavonoid 3′,5′-hydroxylase

The potato P locus is required for the production of blue/purple anthocyanin pigments in any tissue of the potato plant such as tubers, flowers, or stems. We have previously reported, based on RFLP mapping in tomato, that the gene coding for the anthocyanin biosynthetic enzyme flavonoid 3,5-hydroxylase (f35h) maps to the same region of the tomato genome as P maps in potato. To further evaluate this association a Petunia f35h gene was used to screen a potato cDNA library prepared from purple-colored flowers and stems. Six positively hybridizing cDNA clones were sequenced and all appeared to be derived from a single gene that shares 85% sequence identity at the amino acid level with Petunia f35h. The potato gene cosegregated with purple tuber color in a diploid F₁ sub-population of 37 purple and 25 red individuals and was found to be expressed in tuber skin only in the presence of the anthocyanin regulatory locus I. A potato f35h cDNA clone was placed under the control of a doubled CaMV 35S promoter and introduced into the red-skinned cultivar Désirée. Tuber and stem tissues that are colored red in Désirée were purple in nine of 17 independently transformed lines.An erratum to this article can be found at     

An active hAT transposable element causing bud mutation of carnation by insertion into the flavonoid 3′-hydroxylase gene

The molecular mechanisms underlying spontaneous bud mutations, which provide an important breeding tool in carnation, are poorly understood. Here we describe a new active hAT type transposable element, designated Tdic101, the movement of which caused a bud mutation in carnation that led to a change of flower color from purple to deep pink. The color change was attributed to Tdic101 insertion into the second intron of F3′H, the gene for flavonoid 3′-hydroxylase responsible for purple pigment production. Regions on the deep pink flowers of the mutant can revert to purple, a visible phenotype of, as we show, excision of the transposable element. Sequence analysis revealed that Tdic101 has the characteristics of an autonomous element encoding a transposase. A related, but non-autonomous element dTdic102 was found to move in the genome of the bud mutant as well. Its mobilization might be the result of transposase activities provided by other elements such as Tdic101. In carnation, therefore, the movement of transposable elements plays an important role in the emergence of a bud mutation.     

Molecular cloning and characterization of a flavonoid 3′-hydroxylase gene from purple-fleshed sweet potato (Ipomoea batatas)

Flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21) is an important enzyme which determines the hydroxylation pattern of anthocyanins. In this study, the full-length cDNA and genomic DNA of F3′H were isolated and characterized from the purple-fleshed sweet potato (Ipomoea batatas). IbF3’′H was 1,789 bp containing a 1,554 bp open reading frame (ORF) encoding 518 amino acids. Comparative and bioinformatic analysis revealed that IbF3′H was highly homologous with F3′Hs from other plant species. Conserved domain search revealed that IbF3′H was a cytochrome P450 dependent enzyme. Three F3′H-specific motifs (V₇₅VVAAS₈₀, G₄₂₇GEK₄₃₀ and V₄₃₃DVKG₄₃₇) were conserved in IbF3′H. Phylogenetic analysis revealed that IbF3′H was clustered into the same subgroup with the homologues from I. purpurea, I. tricolor and I. nil. There were multiple copies of the IbF3′H gene in the genome of I. batatas. IbF3′H was constitutively expressed in all tested tissues including fibrous roots, thick roots, storage roots, stems and leaves. During storage root formation, IbF3′H was expressed most abundantly in the storage roots, suggesting that the anthocyanin biosynthesis is also active in the under-ground organs. IbF3′H expression was associated with anthocyanin accumulation in five different sweet potato cultivars tested. Complementative analysis implied that the full-length cDNA of IbF3′H could encode a functional protein and had a special catalytic activity of flavonoid 3′-hydroxylase.     

A ∼ 4.1 kb deletion in IRX1 gene upstream is completely associated with rumplessness in Piao chicken

Piao chicken, a Chinese indigenous rumpless chicken breed, lacks pygostyle, caudal vertebra, uropygial gland and tail feathers. The rumplessness in Piao chicken presents an autosomal dominant inheritance pattern. However, the molecular genetic mechanisms underlying the rumplessness in Piao chicken remains unclear. In this study, whole-genome resequencing was performed for 146 individuals from 10 chicken breeds, including 9 tailed chicken breeds and Piao rumpless breed. Tailbone CT scan for Piao chickens and WL chickens, revealed that some Piao chicken tails were normal in number, and for a few Piao chickens tail length and tail bone numbers were between the rumpless and the normal tailed chickens. The results showed that the rumpless phenotype has not been completely fixed in Piao chicken breed. Using selection signature analysis and structural variation detection, we found a 4174 bp deletion located in the upstream region of IRX1 gene on chromosome 2 related to rumpless phenotype. Structural variation genotyping showed that the deletion was present in all 32 rumpless Piao chickens (del/del, wild/del) and absent from all 112 tailed chickens included in the dataset for the other 9 breeds and 2 tailed Piao chickens (wild/wild). In summary, all rumpless Piao chickens tested here carry this deletion mutation, to show a complete linkage association with rumplessness trait. We suggested that the 4174 bp deletion could be causative for rumpless phenotype in Piao chicken since this is the only mutation to show the complete linkage disequilibrium with rumplessness on whole genome level across all of 146 chickens from the 10 breeds. This study could facilitate a better understanding of the genetic characteristics of Piao chicken.     

Agrobacterium tumefaciens–mediated genetic transformation and overexpression of the flavonoid 3′5′-hydroxylase gene increases the flavonoid content of the transgenic Aconitum carmichaelii Debx. plant

In Vitro Cellular & Developmental Biology - Plant - Aconitum carmichaelii Debx. is a medicinal plant that contains a variety of valuable medicinal substances, including flavonoids, alkaloids,...     

FSHβsubunit gene is associated with major gene controlling litter size in commercial pig breeds

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Identification of a novel splicing mutation and 1-bp deletion in the 17α-hydroxylase gene of Japanese patients with 17α-hydroxylase deficiency

We report studies of two unrelated Japanese patients with 17α-hydroxylase deficiency caused by mutations of the 17α-hydroxylase (CYP17) gene. We amplified all eight exons of the CYP17 gene, including the exon-intron boundaries, by the polymerase chain reaction and determined their nucleotide sequences. Patient 1 had novel, compound heterozygous mutations of the CYP17 gene. One mutant allele had a guanine to thymine transversion at position +5 in the splice donor site of intron 2. This splice-site mutation caused exon 2 skipping, as shown by in vitro minigene expression analysis of an allelic construct, resulting in a frameshift and introducing a premature stop codon (TAG) 60 bp downstream from the exon 1-3 boundary. The other allele had a missense mutation of His (CAC) to Leu (CTC) at codon 373 in exon 6. These two mutations abolished the 17α-hydroxylase and 17,20-lyase activities. Restriction fragment length polymorphism (RFLP) analysis with a mismatch oligonucleotide showed that the patient’s mother and brother carried the splice-site mutation, but not the missense mutation. Patient 2 was homozygous for a novel 1-bp deletion (cytosine) at codon 131 in exon 2. This 1-bp deletion produces a frameshift in translation and introduces a premature stop codon (TAG) proximal to the highly conserved heme iron-binding cysteine at codon 442 in microsomal cytochrome P450 steroid 17α-hydroxylase (P450c17). RFLP analysis showed that the mother was heterozygous for the mutation. Received: 15 November 1997 / Accepted: 15 March 1998     

A SNP in the 5′ flanking region of the myostatin-1b gene is associated with harvest traits in Atlantic salmon (Salmo salar)

Background

Myostatin (MSTN) belongs to the transforming growth factor-β superfamily and is a potent negative regulator of skeletal muscle development and growth in mammals. Most teleost fish possess two MSTN paralogues. However, as a consequence of a recent whole genome-duplication event, salmonids have four: MSTN-1 (?1a and -1b) and MSTN-2 (?2a and -2b). Evidence suggests that teleost MSTN plays a role in the regulation of muscle growth. In the current study, the MSTN-1b gene was re-sequenced and screened for SNP markers in a commercial population of Atlantic salmon. After genotyping 4,800 progeny for the discovered SNPs, we investigated their association with eight harvest traits - four body-weight traits, two ratios of weight traits, flesh colour and fat percentage - using a mixed model association analysis.

Results

Three novel SNPs were discovered in the MSTN-1b gene of Atlantic salmon. One of the SNPs, located within the 5′ flanking region (g.1086C?>?T), had a significant association with harvest traits (p?<?0.05), specifically for: Harvest Weight (kg), Gutted Weight (kg), Deheaded Weight (kg) and Fillet Weight (kg). The haplotype-based association analysis was consistent with this result because the two haplotypes that showed a significant association with body-weight traits, hap4 and hap5 (p?<?0.05 and p?<?0.01, respectively), differ by a single substitution at the g.1086C?>?T locus. The alleles at g.1086C?>?T act in an additive manner and explain a small percentage of the genetic variation of these phenotypes.

Conclusions

The association analysis revealed that g.1086C?>?T had a significant association with all body-weight traits under study. Although the SNP explains a small percentage of the variance, our results indicate that a variation in the 5′ flanking region of the myostatin gene is associated with the genetic regulation of growth in Atlantic salmon.

     

A 5-base insertion in the γC-crystallin gene is associated with autosomal dominant variable zonular pulverulent cataract

A seven-generation family with 30 members affected by highly variable autosomal dominant zonular pulverulent cataracts has been previously described. We have localized the cataracts to a 19-cM interval on chromosome 2q33-q35 including the gamma-crystallin gene cluster. Maximum lod scores are 4.56 (theta=0.02) with D2S157, 3.66 (theta=0.12) with D2S72, and 3.57 (theta=0.052) with CRYG. Sequencing and allele-specific oligonucleotide analysis of the pseudo gammaE-crystallin promoter region from individuals in the pedigree suggest that activation of the gammaE-crystallin pseudo gene is unlikely to cause the cataracts in the family. In addition, base changes in the TATA box but not the Sp1-binding site have been found in unaffected controls and can be excluded as a sole cause of cataracts. In order to investigate the underlying genetic mechanism of cataracts in this family further, exons of the highly expressed gammaC- and gammaD-crystallin genes have been sequenced. The gammaD-crystallin gene shows no abnormalities, but a 5-bp duplication within exon 2 of the gammaC-crystallin gene has been found in one allele of each affected family member and is absent from both unaffected family members and unaffected controls. This mutation disrupts the reading frame of the gammaC-crystallin coding sequence and is predicted to result in the synthesis of an unstable gammaC-crystallin with 38 amino acids of the first "Greek key" motif followed by 52 random amino acids. This finding suggests that the appropriate association of mutant betagamma-crystallins into oligomers is not necessary to cause cataracts and may give us new insights into the genetic mechanism of cataract formation.     

Functional analysis of <Emphasis Type="Italic">Antirrhinum kelloggii</Emphasis> flavonoid 3′-hydroxylase and flavonoid 3′,5′-hydroxylase genes; critical role in flower color and evolution in the genus <Emphasis Type="Italic">Antirrhinum</Emphasis>

The enzymes flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) play an important role in flower color by determining the B-ring hydroxylation pattern of anthocyanins, the major floral pigments. F3′5′H is necessary for biosynthesis of the delphinidin-based anthocyanins that confer a violet or blue color to most plants. Antirrhinum majus does not produce delphinidin and lacks violet flower colour while A. kelloggii produces violet flowers containing delphinidin. To understand the cause of this inter-specific difference in the Antirrhinum genus, we isolated one F3′H and two F3′5′H homologues from the A. kelloggii petal cDNA library. Their amino acid sequences showed high identities to F3′Hs and F3′5′Hs of closely related species. Transgenic petunia expressing these genes had elevated amounts of cyanidin and delphinidin respectively, and flower color changes in the transgenics reflected the type of accumulated anthocyanidins. The results indicate that the homologs encode F3′H and F3′5′H, respectively, and that the ancestor of A. majus lost F3′5′H activity after its speciation from the ancestor of A. kelloggii.