Allelic variation and distribution independence of Puroindoline b-B2 variants and their association with grain texture in wheat

In this study, we identify the allelic variation of the Pinb-B2v3 variant, which could be divided into three different alleles, Pinb-B2v3a, Pinb-B2v3b and Pinb-B2v3c. The result of χ² tests showed that the distribution of Puroindoline b-2 variants has different frequencies in common and durum wheats. Analysis of the association of Pinb-B2v with grain hardness indicated that wheat cultivars with Pinb-B2v3b possessed relatively higher single kernel characterization system (SKCS) hardness indices in soft wheat in the 2006–2007 cropping season. Further analysis of SKCS hardness among different Puroindoline B-b2 variants by an F₈ recombinant inbred line (RIL) population containing 350 RILs indicated that lines with Pinb-2v3b were on average 5.4 SKCS hardness index units harder than those carrying the Pinb-2v2 haplotype. Derived cleaved amplified polymorphic sequence markers were developed for identification of Pinb-B2v3b and Pinb-B2v3c alleles and will be useful for screening early generation materials by marker-assisted selection during wheat breeding. The results of quantitative real-time PCR indicated that the relative expression level of Pinb-B2v3b was significantly higher than those of Pinb-B2v2, Pinb-B2v3a and Pinb-B2v3c, that four Pinb-B2 alleles showed the highest relative expression level on the 14th day after anthesis during grain development, and that relative expression levels of Pinb-B2v3b and Pinb-B2v2 in leaf were significantly higher than those in root, suggesting that PINB-2 are possibly not seed-specific proteins and that the expression level of Pinb-B2v3 was possibly positively correlated with grain hardness.     

Comparative analysis on the diversity of <Emphasis Type="Italic">Auricularia auricula</Emphasis>-<Emphasis Type="Italic">judae</Emphasis> by physiological characteristics,somatic incompatibility and TRAP fingerprinting

Phenotypic traits (physiological characteristics and somatic incompatibility) and genotypic traits (target region amplification polymorphism TRAP) were used to study the diversity of 32 main cultivars of Auricularia auricula-judae in China. Twenty seven important and stable physiological indexes were evaluated; somatic incompatibility test (SIT) reaction was described from three aspects: type, pigment, and intensity; 16 pairs of TRAP primer combinations produced 535 unambiguous and reproducible DNA fragments, of these 524 (97.9%) were polymorphic. Dendrograms were constructed by Unweighted Pair-group Method with Arithmetic Averages (UPGMA) method, and the principal coordinate analysis (PCO) of the three methods (physiological characteristics, SIT intensity and TRAP) exhibited similar clustered patterns, revealing that all the tested strains could be divided into three distinct groups, each of which was correlated with different geographical regions. Most strains originated from the same area were with a narrow genetic basis and could possibly be domesticated from the local wild-type strains, some strains were suspected to be synonymous. The grouping information obtained in the present work provides significant information for further genetic improvement of A. auricula-judae.     

A new multiplex PCR test for the determination of Vrn-B1 alleles in bread wheat (Triticum aestivum L.)

Winter wheat requires vernalization, a long exposure to low but non-freezing temperatures, to promote reproductive development. The vernalization requirement in bread wheat (Triticum aestivum L.) is mainly controlled by the Vrn-1 genes that are located on chromosomes 5A, 5B and 5D. Dominant alleles confer spring habit and are epistatic to the recessive winter alleles which means that spring varieties carry at least one dominant allele. To date, two dominant and one recessive Vrn-B1 alleles have been described. Vrn-B1a (formerly designated as Vrn-B1) differs from the winter vrn-B1 allele by a large deletion in intron 1. Vrn-B1b has an additional small deletion and is probably derived from Vrn-B1a. The novel allele described here and designated as Vrn-B1c also has a large deletion within intron 1 but with different breakpoints from Vrn-B1a or b, and sequence duplication, showing that this is an independently derived spring allele. By combining an exon 1 primer with previously published PCR primers it was possible to develop a multiplex PCR that distinguished all four alleles simultaneously. The multiplex PCR was validated by testing 320 winter wheat and 137 spring wheat varieties. This demonstrated that the novel Vrn-B1c allele was present in 25 spring varieties of diverse origin, showing this allele to be widely distributed.     

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.     

Identification of SNPs in the<Emphasis Type="Italic"> waxy</Emphasis> gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice

Common non-waxy (Wx) rice cultivars contain two different alleles at the waxy locus, designated Wx ^a and Wx ^b, which encode different levels of granule-bound starch synthases and are hence involved in the control of endosperm amylose content. The Wx ^a allele was predominant in non-waxy indica cultivars, whereas the Wx ^b allele was common to the non-waxy japonica variety. Recently, some of the molecular mechanisms underlying the differentiation of Wx ^a from Wx ^b have been characterized. One structural difference between these two alleles was shown to be due to alternative splicing caused by a single-base substitution (AGGT to AGTT) at a donor site of the first intron within the Wx gene. In the case of waxy (wx) rice, it was not possible to distinguish whether the each wx allele was derived from Wx ^a or Wx ^b alleles by phenotypic analysis. However, we succeeded in developing a derived cleaved amplified polymorphic sequence (dCAPS) marker for the detection of the one-base splicing mutation without the need for sequencing. A mismatch primer was used to generate a restriction site in the Wx ^a allele (AGGT) but not in the Wx ^b allele (AGTT). Three hundred fifty-three waxy rice strains that are widely found in Asia were then employed for analysis using this dCAPS marker. Our findings suggested that waxy rice strains have both Wx ^a- and Wx ^b-derived alleles, but that the Wx ^b-derived allele was predominant, and its distribution was independent of indica-japonica differentiation. The wild relatives of cultivated rice all possessed the AGGT allele. It was concluded that the waxy mutations, and the corresponding rice cultivation, originated from japonica during the evolution and domestication process of rice and was preferentially selected by most Asian peoples.Communicated by J. Heslop-Harrison     

Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector

Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethroid resistance in the major African malaria vector Anopheles funestus and detected key mutations controlling this resistance. An Africa-wide polymorphism analysis of the duplicated genes CYP6P9a and CYP6P9b revealed that both genes are directionally selected with alleles segregating according to resistance phenotypes. Modelling and docking simulations predicted that resistant alleles were better metabolizers of pyrethroids than susceptible alleles. Metabolism assays performed with recombinant enzymes of various alleles confirmed that alleles from resistant mosquitoes had significantly higher activities toward pyrethroids. Additionally, transgenic expression in Drosophila showed that flies expressing resistant alleles of both genes were significantly more resistant to pyrethroids compared with those expressing the susceptible alleles, indicating that allelic variation is the key resistance mechanism. Furthermore, site-directed mutagenesis and functional analyses demonstrated that three amino acid changes (Val¹⁰⁹Ile, Asp³³⁵Glu and Asn³⁸⁴Ser) from the resistant allele of CYP6P9b were key pyrethroid resistance mutations inducing high metabolic efficiency. The detection of these first DNA markers of metabolic resistance to pyrethroids allows the design of DNA-based diagnostic tools to detect and track resistance associated with bednets scale up, which will improve the design of evidence-based resistance management strategies.     

Development and validation of a new Ppo-A1 marker useful for marker-assisted selection in tetraploid wheats

Polyphenol oxidase (PPO) activity is a major cause of undesirable brown color of semolina. In tetraploid wheat, the Ppo-A1 gene is significantly involved in the phenotypic expression of PPO activity. The main goal of this study was to develop and validate a more efficient marker for Ppo-A1 to facilitate marker-assisted selection for low PPO activity in tetraploid wheat breeding programs. A large tetraploid wheat collection, including durum cultivars, domesticated and wild accessions, was used to evaluate the PPO activity. The heritability values indicated that the phenotypic expression of PPO activity was mainly due to genotypic effect. PPO18, and a new marker named MG18, were used to study the Ppo-A1 allelic variation in a tetraploid wheat collection. PPO18 analysis detected four alleles (Ppo-A1b, Ppo-A1e, Ppo-A1f and Ppo-A1g). The high frequency of Ppo-A1g (no PCR product) detected in the tetraploid wheat collection, led to the development of a new genome-specific Ppo-A1 marker (MG18). MG18 analysis identified the same alleles as PPO18 which were associated with low or high PPO activity. The new MG18 marker was more efficient than PPO18 in detecting the four different alleles of Ppo-A1 in the tetraploid wheat collection. Indeed, the accessions assigned to the Ppo-A1g group, according to PPO18, when tested with MG18, were better classified in the four alleles of the Ppo-A1 gene. The MG18 analysis proved that the PPO18 marker overestimated the number of accessions with Ppo-A1g. Therefore, MG18 can be applied to large-scale marker-assisted selection for PPO activity in durum breeding programs.     

Cloning and expression of <Emphasis Type="Italic">Perilla frutescens FAD2</Emphasis> gene and polymorphism analysis among cultivars

?12 fatty acid desaturase (FAD2) is a key enzyme for linoleic acid and linolenic acid biosynthesis. Perilla frutescens is a special oil plant species with highest linolenic acid content. In this study, based on RACE, two alleles for one FAD2 gene were isolated from P. frutescens cultivar C2: the 3956 bp PfFAD2a and the 3959 bp PfFAD2b, both with a full-length cDNA of 1526 bp, and both encoding a 382aa basic protein. The alleles have identities of over 98%, and their encoded proteins differ only by substitution of a strongly similar residue. Saccharomyces cerevisiae heterologous expression suggested that PfFAD2a/b both encode a bio-functional FAD2 enzyme. Phylogenetic analyses indicated that PfFAD2 shows the highest homologies to FAD2 genes from dicots such as Boraginaceae and Burseraceae. PfFAD2a/b expressions are mainly restricted to developing seeds. PfFAD2a/b expression in the seedling leaf is upregulated by cold (4 °C) and repressed by heat (42 °C). Each of the eight cultivars contains two alleles for one PfFAD2 and 40 SNP sites are found. One allelic gene in cultivars C1 and P1 is pseudogene because of premature stop codon mutation in 5′ coding region. All other normal PfFAD2 genes/allelic genes encode identical or very similar proteins. PfFAD2a/b expression level in developing seeds also varies among the eight cultivars. This study provides systemic molecular and functional features of PfFAD2 and enables its application in the study of plant fatty acids traits.     

Responses of antioxidant defenses and membrane damage to drought stress in fruit bodies of Auricularia auricula-judae

Fruit bodies of Auricularia auricula-judae are often subjected to drought stress and became dormant. The responses of antioxidant defenses and membrane damage to drought stress were investigated in this study. Picked fruit bodies were exposed to sunlight and dehydrated naturally and samples were collected at different levels of water loss (0, 10, 30, 50, and 70 %) for determination of electrolyte leakage (EL); contents of malondialdehyde (MDA), ascorbic acid (AsA) and reduced glutathione (GSH); and activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD). Results showed that membrane permeability (assessed by EL) and membrane lipid peroxidation (MDA content) remained unchanged at all levels of water loss studied. Contents of AsA and GSH showed no change at 0, 10 and 30 % of water loss, however, both of them increased significantly at 50 and 70 % of water loss. SOD activity significantly increased with the rising of water loss from 0 to 30 %, reached the peak at 30 and 50 % of water loss, and then significantly decreased at 70 % of water loss. A gradual increase in POD and CAT activities was observed when water loss rose from 0 to 50 %. As water loss went up to 70 %, POD activity remained the same as that at 50 %, but CAT activity decreased. The results indicate that the increased activities of enzymatic antioxidants (SOD, CAT and POD) and contents of non-enzymatic antioxidants (AsA and GSH) in fruit bodies of A. auricula-judae can effectively scavenge reactive oxygen species, cause no damage to cell membranes as demonstrated by the unchanged EL and MDA content, and contribute to dormancy under drought stress.     

Allelic variation of a soluble acid invertase gene (SAI-1) and development of a functional marker in sweet sorghum [Sorghum bicolor (L.) Moench]

Sweet sorghum [Sorghum bicolor (L.) Moench] accumulates large amounts of sugars in the stem parenchyma and can be used to manufacture sucrose and alcohol. Soluble acid invertase (SAI) plays an important role in sucrose accumulation in the sweet sorghum stem. Characterization of SAI genes and development of functional markers are important for marker-assisted selection in breeding programs. Here, the full-length genomic DNA sequence of a SAI gene (SAI-1) was cloned by in silico cloning and experimentally validated. The SAI-1 gene comprises three exons and two introns, totaling 4,966 bp, with an open reading frame of 2,025 bp encoding a polypeptide of 674 amino acids. The protein encoded by SAI-1 (molecular weight 73.4 kDa) contains a glycosyl hydrolase family 32 conserved domain, which catalyzes the hydrolysis of sucrose. Four allelic variants at the SAI-1 locus, SAI-1a, SAI-1b, SAI-1c and SAI-1d (GenBank accession nos. JX535515, JX535516, JX535517 and JX535518), were identified in 165 sweet sorghum cultivars. A co-dominant marker, SBX1, was developed to discriminate these variants. The mean Brix value from cultivars with a 141-bp (SAI-1c) PCR product was higher than in cultivars with a 136-bp (SAI-1b) product, and the mean Brix value from cultivars with a 136-bp (SAI-1b) product was significantly higher than those with a 132-bp (SAI-1a) product. The results showed that a domain of ATTGA repeat sequences in the second intron might be an important regulatory site, and that the functional marker SBX1 was closely related to the Brix value, and could therefore be used in sweet sorghum breeding programs.     

A gene locus affecting tolerance to BGG in mice.

A genetic analysis was made of the ease of tolerance induction to bovine γ-globulin (BGG) in DBA/2, BALB/c, F₁ and backcross generation mice. Like parental DBA/2 mice, the F₁ generation of BALB/c × DBA/2 becomes tolerant when treated with 2 mg BGG. A backcross of this F₁ to DBA/2 parents produced mice that all became tolerant to this dose of BGG. A backcross of F₁ mice to BALB/c parents produced 50% offspring tolerized by the same dose of BGG and 50% resistant to tolerance induction.The data suggest a single autosomal locus affecting tolerance induction. Data presented elsewhere suggest that the locus affects macrophage function. We propose that this locus be called tolerance (symbol Tol-l) and the two alleles be (Tol-l^a (DBA/2 type) and Tol-l^b (BALB/c type) with Tol-l^a being dominant.     

Allelic Variability in the Intronic Region of the Fibroin Heavy-Chain Gene in Silkworm Bombyx mori L. Strains of Brazilian Germplasm Bank

The domesticated silkworm Bombyx mori L. is currently found only in germplasm banks. Therefore, characterization and conservation of this genetic resource is crucial. Based on previous studies that revealed nucleotide differences in silkworm strains, the intron of the fibroin heavy chain gene (H-fib) can be used for molecular silkworm characterization. The H-fib gene has two exons and a unique intron, and encodes the principal component of the silk fiber, the fibroin heavy chain. Therefore, this study aimed to identify the genetic variability of the unique intron of H-fib gene of 20 silkworm strains maintained at the Universidade Estadual de Maringá Brazilian Germplasm Bank (UBGB) by conformation-sensitive gel electrophoresis (CSGE) and nucleotide sequencing. Genomic DNA extracted from silkworm moths was PCR amplified. CSGE revealed that most of the analyzed silkworm strains had only homoduplex molecules. However, DNA from the Japanese strains B106, B82, and M12-2 had two extra DNA fragments produced by heteroduplex molecules, revealing variation between alleles. Sequencing of the H-fib intron was used to confirm the variation previously detected by CSGE and detected a significant polymorphism characterized by a 17-base pair (bp) deletion, a 2-bp insertion, and eight nucleotide substitutions. Although genetic and allelic variability was detected in some silkworm strains, the intron of the H-fib gene revealed not to be the best molecular marker for the characterization of B. mori strains from UBGB.     

Genetic control of two esterases of rat plasma (Rattus norvegicus)

Three electrophoretic variants of plasma esterase in the albumin zone, presumably carboxylesterase, have been demonstrated in 250 rats representing a laboratory population of Wistar rats. Electrophoretic variants of the enzyme are believed to be controlled by two codominant alleles at the autosomal locus referred to as Es-2. The variant of carboxylesterase represented by a fast-migrating single band on starch gel electrophoresis is determined by the gene named Es-2 ^a, whereas the slow-migrating variant, represented by two bands, is under control of the allelic gene Es-2 ^b. Animals with Es-2 ^a/Es-2 ^b genotype have three bands of carboxylesterase in the albumin zone. Genetically determined polymorphism of plasma esterase, presumably carboxylesterase, in the prealbumin zone was shown in both laboratory and wild populations of rats. Breeding tests suggest that the gene referred to as Es-1 ^a, responsible for the presence of carboxylesterase in the prealbumin zone, is inherited dominantly, whereas animals homozygous for the allele Es-1 ^b locked this esterase fraction.     

Functional differentiation of bmp2a and bmp2b genes in zebrafish

Bone morphogenetic protein 2 plays an important role in the regulation of osteoblast proliferation and differentiation. Phylogenetic analysis showed that the bmp2 ortholog evolved from the same ancestral gene family in vertebrates and was duplicated in teleost, which were named bmp2a and bmp2b. The results of whole-mount in situ hybridization showed that the expression locations of bmp2a and bmp2b in zebrafish were different in different periods (24 hpf, 48 hpf, 72 hpf), which revealed potential functional differentiation between bmp2a and bmp2b. Phenotypic analysis showed that bmp2a mutations caused partial rib and vertebral deformities in zebrafish, while bmp2b^−/− embryos died massively after 12 hpf due to abnormal somite formation. We further explored the expression pattern changes of genes (bmp2a, bmp2b, smad1, fgf4, runx2b, alp) related to skeletal development at different developmental stages (20 dpf, 60 dpf, 90 dpf) in wild-type and bmp2a^−/− zebrafish. The results showed that the expression of runx2b in bmp2a^−/− was significantly downregulated at three stages and the expression of other genes were significantly downregulated at 90 dpf compared with wild-type zebrafish. The study revealed functional differentiation of bmp2a and bmp2b in zebrafish embryonic and skeletal development.     

L' α-Glycérophosphate-déshydrogénase du moustique Culex pipiens: Génétique formelle,linkage et étude de populations

THE -CLYCEROPHOSPHATE-DEHYDROGENASE IN THE MOSOUITO CULEX PIPIENS: HEREDITY, LINKAGE AND POPULATION STUDIES. Culex pipiens -glycerophosphate-dehydrogenase is coded by an autosomal locus which is shown to have a demonstrable activity in nymphs just prior to imago emergence and in adults of any age and sex. The -Gpd locus has two codominant alleles, -Gpd ^1.00 and -Gpd ^1.30, and heterozygotes present three isozymes. The -Gpd locus is localized on the same chromosomes as the two linked esterase loci Est-2 and Est-1, at 37.05 and 44.88 cross-over units, respectively, of these loci. Population studies seem to indicate that the -Gpd locus has a low variability in Culex pipiens as in most other organisms.     

Allelic differentiation at the <Emphasis Type="Italic">Sg-1</Emphasis> locus for the terminal sugar of the C-22 position of group A saponin in Chinese wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. &amp; Zucc.)

Group A saponins are thought to be the cause of bitter and astringent tastes in processed foods of soybean (Glycine max), and the elimination of group A saponins is an important breeding objective. The group A saponins include two main Aa and Ab types, controlled by codominant alleles at the Sg-1 locus that is one of several key loci responsible for saponin biosynthesis in the subgenus Glycine soja. However, A0 mutant lacking group A saponin is a useful gene resource for soybean quality breeding. Here, eight Chinese wild soybean A0 accessions were sequenced to reveal the mutational mechanisms, and the results showed that these mutants were caused by at least three kinds of mechanisms involving four allelic variants (sg-1^0-b2, sg-1^0-b3, Sg-1^b-0, and Sg-1^b-01). The sg-1^0-b2 had two nucleotide deletions at positions +?72 and +?73 involving in the 24th and 25th amino acids. The sg-1^0-b3 contained a stop codon (TGA) at the 254th residue. The Sg-1^b-0 and Sg-1^b-01 were two novel A0-type mutants, which likely carried normal structural alleles, and nevertheless did not encode group A saponin due to unknown mutations beyond the normal coding regions. In addition, to reveal the structural features, allelic polymorphism, and mechanisms of the abiogenetic absence of group A (i.e., A0 phenotype), nucleotide sequence analysis was performed for the Sg-1 locus in wild soybean (Glycine soja). The results showed that Sg-1 alleles had a lower conservatism in the coding region; as high as 18 sequences were found in Chinese wild soybeans in addition to the Sg-1^a (Aa) and Sg-1^b (Ab) alleles. Sg-1^a and Sg-1^b alleles were characterized by eight synonymous codons and nine amino acid substitutions. Two evolutionarily transitional allelic sequences (Sg-1^a7 and Sg-1^b2) from Sg-1^a toward Sg-1^b were detected.     

Genetic study of pancreatic proteinase and α-amylase in mice (Mus musculus)

Genetic variants were found at two loci for pancreatic proteinase in mice. The Prt-1 locus contains a pair of allelic genes, Prt-1 ^a and Prt-1 ^b , ad the Prt-2 locus contains two codominant allelic genes, Prt-2 ^a and Prt-2 ^b .Expression of the two genetic variants of proteinase allowed mice strains used in this study to be classified into three phenotypic classes. Prt-1 ^b andPrt-2 ^a were found in most of the Japanese inbred strains, Prt-1 ^b andPrt-2 ^a were found in most of the inbred strains imported from the United States, and, furthermore, Prt-1 ^b and Prt-2 ^b were present in Japanese feral-origin mice strains. Prt-1, Prt-2, and Amy-2 loci did not belong to the same linkage group.