TAC1, a major quantitative trait locus controlling tiller angle in rice

A critical step during rice (Oryza sativa) cultivation is dense planting: a wider tiller angle will increase leaf shade and decrease photosynthesis efficiency, whereas a narrower tiller angle makes for more efficient plant architecture. The molecular basis of tiller angle remains unknown. This research demonstrates that tiller angle is controlled by a major quantitative trait locus, TAC1 (Tiller Angle Control 1). TAC1 was mapped to a 35-kb region on chromosome 9 using a large F(2) population from crosses between an indica rice, IR24, which displays a relatively spread-out plant architecture, and an introgressed line, IL55, derived from japonica rice Asominori, which displays a compact plant architecture with extremely erect tillers. Genetic complementation further identified the TAC1 gene, which harbors three introns in its coding region and a fourth 1.5-kb intron in the 3'-untranslated region. A mutation in the 3'-splicing site of this 1.5-kb intron from 'AGGA' to 'GGGA' decreases the level of tac1, resulting in a compact plant architecture with a tiller angle close to zero. Further sequence verification of the mutation in the 3'-splicing site of the 1.5-kb intron revealed that the tac1 mutation 'GGGA' was present in 88 compact japonica rice accessions and TAC1 with 'AGGA' was present in 21 wild rice accessions and 43 indica rice accessions, all with the spread-out form, indicating that tac1 had been extensively utilized in densely planted rice grown in high-latitude temperate areas and at high altitudes where japonica rice varieties are widely cultivated.     

Inheritance of an RNA splicing mutation (G+ 1 IVS20) in the type III procollagen gene (COL3A1) in a family having aortic aneurysms and easy bruisability: phenotypic overlap between familial arterial aneurysms and Ehlers-Danlos syndrome type IV.

Inheritance of a single base mutation in the type III procollagen gene (COL3A1) was studied in a family with aortic aneurysms and easy bruisability. The mutation was a substitution of A for G+ 1 of intron 20 of the gene and caused aberrant splicing of RNA transcribed from the mutated allele. The phenotype in the family included aortic aneurysms that ruptured and produced death. It also included easy bruisability, but it did not include other characteristic features of Ehlers-Danlos syndrome type IV, such as ecchymoses, abnormal scarring, or prominent subcutaneous blood vessels. The data from the family, together with a review of other probands with mutations in the type III procollagen gene, indicated that there is phenotypic overlap between Ehlers-Danlos syndrome type IV and familial arterial aneurysms not associated with any overlap between Ehlers-Danlos syndrome type IV and familial arterial aneurysms not associated with any of the striking changes in skin originally cited as a characteristic feature of Ehlers-Danlos syndrome type IV. In addition, the results suggested that DNA tests for mutations in the type III procollagen gene may be useful to identify individuals predisposed to developing arterial aneurysms.     

Identification of the barley accessions to serve as donors of thermostable β-amylase alleles

Molecular analysis was performed with 88 barley accessions, including varieties cultivated in Belarus and those obtained from the collection of the All-Russian Institute of Plant Industry (IPI). A marker directed to the single nucleotide substitution (SNP) C⁶⁹⁸ → T of the Bmy1 β-amylase gene showed that the Sd2H and Sd3 alleles were absent from the Belarussian varieties. Five accessions with the Bmy1 alleles determining thermostable isoforms of β-amylase I were identified among the accessions from the IPI collection.     

Structural characteristics of the chloroplast <Emphasis Type="Italic">rpS16</Emphasis> intron in <Emphasis Type="Italic">Allium sativum</Emphasis> and related <Emphasis Type="Italic">Allium</Emphasis> species

The intron sequence of chloroplast rpS16 and the secondary structure of its pre-mRNA were characterized for the first time in 26 Allium sativum accessions of different ecologo-geographical origins and seven related Allium species. The boundaries and main stem-loop consensus sequences were identified for all six domains of the intron. Polymorphism was estimated for the total intron and its regions. The structural regions of the rpS16 intron proved to be heterogeneous for mutation rate and spectrum. Mutations were most abundant in domains II and IV, and transition predominated in domains I, III, V, and VI. In addition to structural elements and motifs typical for group IIB introns, several Allium-specific micro- and macrostructural mutations were revealed. A 290-bp deletion involving domains III and IV and part of domain V was observed in A. altaicum, A. fistulosum, and A. schoenoprasum. Several indels and nucleotide substitutions were found to cause a deviation of the pre-mRNA secondary structure from the consensus model of group II introns.     

A single nucleotide polymorphism at the Vrn-D1 promoter region in common wheat is associated with vernalization response

Facultative wheat varieties adapt to a particular environment. But the molecular basis for the facultative growth habit is not clear relative to winter and spring growth habit. Two sets of wheat varieties were chosen for this study. Set 1 comprised ten spring accessions and Set 2 comprised ten facultative accessions. All accessions had been tested by the previously described allele-specific markers and shown having the same allelic composition of vrn-A1 vrn-B1 Vrn-D1 and vrn-B3. Here we examined whether differences in growth habit might be associated with as yet unidentified sequence variation at Vrn-D1 locus. A region including the intron 1 deletion, the entire reading frame from a cDNA template and a part of promoter region of the dominant Vrn-D1 gene in each of the accessions was sequenced, and a single nucleotide polymorphism was found between facultative accessions and spring accessions in the CArG-box at the promoter region. The novel allele in facultative accessions was designated as Vrn-D1b. The investigation of an F₂ population segregating for Vrn-D1b and Vrn-D1a (previously, Vrn-D1) in the greenhouse under long days without vernalization showed that the plants with Vrn-D1b homozygous allele headed 32?days later and had about three more leaves than the plants with Vrn-D1a homozygous allele. As Vrn-D1b has the same deletion in intron 1 as Vrn-D1a, and, in addition, a single nucleotide mutation at promoter region, and is associated with facultative growth habit, we suggest that the promoter mutation may modify the basal activity level of an allele of VRN1 that is already active (due to the loss of segments in intron 1). Our finding further supports that both the promoter and intron 1 regulatory affect vernalization response and work independently.     

Phylogenetic relationships of the sweetpotato in Ipomoea series Batatas (Convolvulaceae) based on nuclear beta-amylase gene sequences

Phylogenetic relationships of 13 accessions and a cultivar representing the sweetpotato, Ipomoea batatas (L.) Lam., and its wild progenitors, were investigated using the nucleotide sequence variation of a nuclear-encoded beta-amylase gene. A 1.1-1.3 kb fragment of the gene spanning two exons separated by a long intron was PCR-amplified, cloned, and sequenced. Exon sequences proved highly conservative, while intron sequences yielded large differences. Intron analyses grouped species in a phylogenetic context according to the presence of two genome types: A and B. These groups are consistent with results of previous analyses, save for the novel placement of I. tiliacea, among the A-genome species. Sequences specific to both A and B genome species have been identified. Exon sequences indicate that I. ramosissima and I. umbraticola are quite different from other A-genome species. Placement of I. littoralis is questionable; its intron is similar to other B-genome species, but its exons are quite different. Exon evolution indicates that the B-genome has evolved faster than the A-genome. Interspecific intron and exon variation indicates I. trifida, I. tabascana, and I. batatas form a monophyletic group.     

Mutations of barley β-amylase that improve substrate-binding affinity and thermostability

Three allelic forms of barley beta-amylase (Sd1, Sd2H and Sd2L) exhibit different thermostability and kinetic properties. These differences critically influence the malting quality of barley varieties. To understand the molecular basis for the different properties of these three allelic forms, Sd1 and Sd2L beta-amylase cDNAs were cloned, and the effects of the amino acid substitutions between them were evaluated by site-directed mutagenesis. The results showed that an R115C mutation is responsible for the difference in kinetic properties. This substitution resulted in an additional hydrogen bond which may create a more favourable environment for substrate-binding. The different thermostabilities of the beta-amylase forms are due to two amino acid substitutions (V233A and L347S), which increased the enzyme's thermostability index T50 by 1.9 degrees C and 2.1 degrees C, respectively. The increased thermostability associated with these two mutations may be due to relief of steric strain and the interaction of the protein surface with solvent water. Although both V233A and L347S mutations increased thermostability, they affected the thermostability in different ways. The replacement of L347 by serine seems to increase the thermostability by slowing thermal unfolding of the protein during heating, while the replacement of V233 by alanine appears to cause an acceleration of the refolding after heating. Because the different beta-amylase properties determined by the three mutations (R115C, V233A and L347S) are associated with malting quality of barley variety, a mutant with high thermostability and substrate-binding affinity was generated by combining the three preferred amino acid residues C115, A233 and S347 together. A possible approach to producing barley varieties with better malting quality by genetic engineering is discussed.     

A causal C–A mutation in the second exon of GS3 highly associated with rice grain length and validated as a functional marker

Comparative sequencing of GS3, the most important grain length (GL) QTL, has shown that differentiation of rice GL might be principally due to a single nucleotide polymorphism (SNP) between C and A in the second exon. A total of 180 varieties representing a wide range of rice germplasm were used for association analysis between C–A mutation and GL in order to confirm the potential causal mutation. A cleaved amplified polymorphic sequence (CAPS) marker, SF28, was developed based on the C–A polymorphism in the GS3 gene. A total of 142 varieties carried allele C with GL from 6.4 to 8.8 mm, while the remaining 38 varieties carried allele A with GL from 8.8 to 10.7 mm. Twenty-four unlinked SSR markers were selected to genotype 180 varieties for population structure analysis. Population structure was observed when the population was classified to three subpopulations. Average GL of either genotype A or genotype C within japonica among the three subpopulations had no significant difference from that in indica, respectively, although indica rice had longer grains on average than japonica in the 180 varieties. However, genotype C always had longer grain length on average than genotype A among three subpopulations. The mutation could explain 79.1, 66.4 and 34.7% of GL variation in the three subpopulations, respectively. These results clearly confirmed the mutation between C and A was highly associated with GL. The SF28 could be a functional marker for improvement of rice grain length.     

Beta-amylase gene variability in introgressive wheat lines

Variability of the beta-amylase gene in bread wheat, artificial amphidiploids, and derived introgression wheat lines was analyzed. Variation in homeologous beta-amylase sequences caused by the presence of MITE (Miniature Inverted-Repeat Transposable Element) and its footprint has been identified in bread wheat. The previously unknown location of MITE in Triticum urartu and T. aestivum L. beta-amylase gene has been found. These species have a MITE sequence in the third intron of beta-amylase, as opposed to Aegilops comosa and a number of other Triticeae species, which have it in the fourth intron. These two MITEs from Ae. comosa and T. aestivum were shown to have low identity scores. Miosa, an artificial amphidiploid, which has the M genome from Ae. comosa was shown to lose the MITE sequences. This loss might be caused by genomic shock due to allopolyploidization.     

Parental somatic and germ-line mosaicism for a multiexon deletion with unusual endpoints in a type III collagen (COL3A1) allele produces Ehlers-Danlos syndrome type IV in the heterozygous offspring.

Ehlers-Danlos syndrome (EDS) type IV is a dominantly inherited disorder that results from mutations in the type III collagen gene (COL3A1). We studied the structure of the COL3A1 gene of an individual with EDS type IV and that of her phenotypically normal parents. The proband was heterozygous for a 2-kb deletion in COL3A1, while her father was mosaic for the same deletion in somatic and germ cells. In fibroblasts from the father, approximately two-fifths of the COL3A1 alleles carried the deletion, but only 10% of the COL3A1 alleles in white blood cells were of the mutant species. The deletion in the mutant allele extended from intron 7 into intron 11. There was a 12-bp direct repeat in intron 7 and intron 11, the latter about 60 bp 5' to the junction. At the breakpoint there was a duplication of 10 bp from intron 11 separated by an insertion of 4 bp contained within the duplicated sequence. The father was mosaic for the deletion so that the gene rearrangement occurred during his early embryonic development prior to lineage allocation. These findings suggest that at least some of the deletions seen in human genes may occur during replication, rather than as a consequence of meiotic crossing-over, and that they thus have a risk for recurrence when observed de novo.     

Mutation characteristics in type I collagen genes in Chinese patients with osteogenesis imperfecta

Osteogenesis imperfecta is normally caused by an autosomal dominant mutation in the type I collagen genes COL1A1 and COL1A2. The severity of osteogenesis imperfecta varies, ranging from perinatal lethality to a very mild phenotype. Although there have been many reports of COL1A1 and COL1A2 mutations, few cases have been reported in Chinese people. We report on five unrelated families and three sporadic cases. The mutations were detected by PCR and direct sequencing. Four mutations in COL1A1 and one in COL1A2 were found, among which three mutations were previously unreported. The mutation rates of G>C at base 128 in intron 31 of the COL1A1 gene and G>A at base 162 in intron 30 of the COL1A2 gene were higher than normal. The patients' clinical characteristics with the same mutation were variable even in the same family. We conclude that mutations in COL1A1 and COL1A2 also have an important role in osteogenesis imperfecta in the Chinese population. As the Han Chinese people account for a quarter of the world's population, these new data contribute to the type I collagen mutation map.     

DNA fingerprinting of jute germplasm by RAPD

The genotype characteristic of cultivars was investigated, along with varieties of both of the jute species, Corchorus olitorius and Corchorus capsularis, in the germplasm collection at the Bangladesh Jute Research Institute (BJRI). DNA fingerprinting was generated for 9 different varieties and 12 accessions of jute cultivars by using random amplified polymorphic DNA (RAPD). A total of 29 arbitrary oligonucleotide primers were screened. Seven primers gave polymorphism within the varieties, and 6 primers detected polymorphism within the accessions that were tested. A dendrogram was engendered from these data, and this gave a distinct clustering of the cultivated species of jute. Therefore, we generated RAPD markers, which are species-specific. These primers can distinguish between C. olitorius and C. capsularis. From the dendrogram that we generated between the various members of these two species, we found the existing genetic classification that agrees with our molecular marking data. A different dendrogram showed that jute accessions could be clustered into three groups. These data will be invaluable in the conservation and utilization of the genetic pool in the germplasm collection.     

TP53 codon 72 and intron 3 polymorphisms and mutational status in gastric cancer: an association with tumor onset and prognosis

Although TP53 alterations have been studied in human tumors, data considering the role of two common TP53 polymorphisms (Pro72Arg in codon 72 and Ins16bp in intron 3) and their associations with TP53 mutations in gastric cancer are very limited. Thus, we analyzed these parameters taking into consideration the clinicopathological data. DNA from 106 gastric tumor samples was available for TP53 Pro72Arg and TP53 Ins16bp polymorphism genotyping by PCR-RFLP and PCR, respectively. The mutational status of the TP53 exons 5-7 was assessed by the single-strand conformational polymorphism test. The TP53 72ArgArg genotype was statistically associated with patients aged ≥65 years (p = 0.039), and the intron 3 A2A2 genotype was correlated with late-stage tumors (III and IV; p = 0.043). Considering both polymorphisms, a negative correlation between the TP53 Pro-A1 haplotype and age <65 years (r = -0.211; p = 0.030) was found. Taking into account the TP53 mutations, the Pro/Pro genotype was positively correlated with the presence of exon 7 mutations (p = 0.049), and a correlation between this genotype and the number of mutations in TP53 was observed (p = 0.019). This study corroborates the understanding of TP53 polymorphisms in gastric carcinogenesis, especially regarding the genetic features in tumor onset and prognosis.     

Identical G+1 to A mutations in three different introns of the type III procollagen gene (COL3A1) produce different patterns of RNA splicing in three variants of Ehlers-Danlos syndrome. IV. An explanation for exon skipping some mutations and not others

Identical G+1 mutations in three different introns of the gene for type III procollagen (COL3A1) that cause aberrant splicing of RNA were found in three probands with life-threatening variants of Ehlers-Danlos syndrome. Because the three mutations were in a gene with multiple and homologous exons, they provided an interesting test for factors that influence aberrant splicing. The G+1 to A mutation in intron 16 caused extensive exon skipping, the G+1 to A mutation in intron 20 caused both use of a cryptic splice site and retention of all the intron sequences, and the G+1 to A mutation in intron 42 caused efficient use of a single cryptic splice site. The different patterns of RNA splicing were not explained by evaluation of potential cryptic splice sites in the introns by either their homology with 5'-splice sites from other genes or by their delta G(0)37 values for binding to U1 RNA. Instead, the results suggested that the patterns of aberrant RNA splicing were primarily determined by the relative rates at which adjacent introns were normally spliced.     

Molecular genetic study of the factor VIII gene in families from Bashkir with hemophilia A

The distribution of hemophilia A was studied in Bashkortostan. The factor VIII gene of the blood coagulation system was analyzed in 34 patients with hemophilia A and 48 of their close relatives. Inversion of intron 22 of the factor VIII gene was revealed in nine cases, which comprised 30% of the total sample analyzed. The type II and type III of this mutation occurred at a relatively high frequency, which may be explained by the founder effect and genetic drift. The allelic frequencies of the polymorphic locus HindIII at intron 19 were similar; a substantial allelic heterogeneity of both microsatellite (CA)-repeats at intron 13 and the DXS52 locus were found on normal and mutant X chromosomes. The molecular genetic analysis of (CA)-repeats and the loci HindIII and DXS52 in families with hemophilia A makes it possible to reveal up to 89% of the informative families.     

Polymorphism of bone morphogenetic protein 4 gene and its relationship with litter size of Jining Grey goats

Two pairs of primers (P1 and P2) were designed to detect single nucleotide polymorphisms of exon 2 and intron 2 of bone morphogenetic protein 4 (BMP4) gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Boer, Angora and Inner Mongolia Cashmere goats) by single strand conformation polymorphism. Results showed that no polymorphism was detected for exon 2 (primer P1) of BMP4 gene in four goat breeds. For intron 2 (primer P2), three genotypes (AA, AB and BB) were detected in Jining Grey and Inner Mongolia Cashmere goats, two genotypes (AB and BB) in Angora goats, and only one genotype (AA) in Boer goats. Sequencing revealed one mutation (2203G>A) of BMP4 gene in the genotype BB in comparison to the genotype AA. The differences of litter size between AA, AB and BB genotypes were not significant (P > 0.05) in Jining Grey goats. A pair of primer (P3) was designed to detect polymorphism in the 3' flanking region of BMP4 gene that contained dinucleotide repeated sequence (CA) in the four goat breeds by microsatellite analysis. For primer P3, three genotypes (CC, CD and DD) were detected in four goat breeds. Sequencing revealed one more CA dinucleotide in genotype DD than in genotype CC. The Jining Grey does with genotype CC had 0.55 (P < 0.05) or 0.72 (P < 0.05) kids more than those with genotype CD or DD. These results preliminarily indicated that allele C of BMP4 gene is a potential DNA marker for improving litter size in goats.