小麦PPO活性基因等位变异的区域分布研究

为了解中国不同生态区小麦种质资源籽粒多酚氧化酶(PPO)活性基因的等位变异的差异与分布,利用小麦PPO活性基因的功能标记PPO16、PPO29与PPO18,检测了来自中国7个不同生态麦区的379份小麦种质资源的等位变异和分布差异。结果表明:(1)在2AL染色体该基因位点有2种等位变异类型:Ppo-A1a(高PPO)和Ppo-A1b(低PPO),其频率分别为51.5%和48.5%。(2)在2DL染色体该基因位点有3种等位变异类型:Ppo-D1a(低PPO)、Ppo-D1b(高PPO)和Ppo-D1ab(中间型),其频率分别为57.8%、32.5%和9.8%。(3)该基因在2AL和2DL染色体上的位点变异有6种不同类型的组合:Ppo-A1a/D1a(中间型)、Ppo-A1a/D1b(高PPO)、Ppo-A1a/Ppo-D1ab(中间型)、Ppo-A1b/D1a(低PPO)、Ppo-A1b/D1b(中间型)和Ppo-A1b/Ppo-D1ab(中间型),其中,与低PPO活性相关的基因型组合Ppo-A1b/D1a的频率为25.6%。(4)小麦PPO活性基因不同变异类型在各生态区的分布存在明显差异,基因型Ppo-A1b在北部冬麦区和西南冬麦区的比例较大,基因型Ppo-D1a在黄淮冬麦区和北部冬麦区的比例较大,基因型组合Ppo-A1b/D1a在北部冬麦区的比例较大。研究认为,结合采用分子标记辅助选择(MAS),有利于小麦籽粒外观品质的遗传改良和新品种选育。     

In-depth genetic analysis reveals conditioning of polyphenol oxidase activity in wheat grains by cis regulation of TaPPO2A-1 expression level

Time-dependent darkening and discoloration of wheat product caused by high polyphenol oxidase enzymes (PPO) activity is the most undesirable character in wheat processing industry. We performed GWAS of PPO activity in wheat grains utilizing an association panel and identified 22 significant SNPs. The most significant GWAS peak on chromosome 2A was verified by QTL analysis of PPO activity. The candidate gene for this GWAS peak was identified as TaPPO2A-1, which was the highest expressed PPO gene in wheat grains. The expression level of TaPPO2A-1 was significantly correlated with PPO activity. The most significant association signal for GWAS of the expression values of TaPPO2A-1 pinpointed to the genomic region containing TaPPO2A-1. The results suggested that cis regulation of TaPPO2A-1 expression is the key factor in regulation of PPO activity in wheat grains. The conclusion was further enhanced by haplotype analysis of seven SNPs in the promoter of TaPPO2A-1.     

Co‐ordinated expression of the VEGF system components in granulosa cells to develop a proangiogenic autocrine milieu during ovarian follicle development

In the present study, we investigated the temporal relationship between angiogenic and antiangiogenic vascular endothelial growth factor isoforms (VEGFxxxa and VEGFxxxb, respectively), the receptors VEGFR1 and VEGFR2, their soluble forms, and the kinases and the splicing factors regulating the synthesis of VEGF isoforms in healthy and atretic antral follicles. The results show a higher (p < 0.05) messenger RNA (mRNA) expression of VEGF120a, VEGF164a, and VEGF120b in healthy than in atretic follicles, but the mRNA expression of VEGF164b was not detected. The mRNA of serine–arginine protein kinase 1 ( SRPK1) was higher ( p < 0.05) in large healthy follicles than in large atretic follicles. In contrast, atretic follicles had higher mRNA expression of a soluble form of the receptor 2 of VEGF ( sVEGFR2) than healthy follicles ( p < 0.05). Additionally, we observed a positive relationship ( p < 0.05) between SRPK1 and serine–arginine‐rich splicing factor 1 ( SRSF1) with the angiogenic isoforms VEGF120a and VEGF164a and between CDC‐like kinases‐1 ( CLK1) and SRSF6 with the antiangiogenic VEGF120b isoform. Principal components analysis (PCA) resulted in two PC explaining 71% of the variation, which was formed by the VEGF isoforms, the kinases and the splicing factor (PC1) and by the VEGF receptors (PC2). When PC analysis was carried out within follicular health status, there were no differences for PC1 between follicular status, whereas PC2 differed between healthy and atretic follicles. In conclusion, the higher mRNA expression for VEGF120a and VEGF164a, the low expression of sVEGFR2, and absent expression of mRNA for VEGF164b provide evidence of a proangiogenic autocrine milieu to support granulosa cells during follicle development.     

A Novel STS Marker for Polyphenol Oxidase Activity in Bread Wheat

The enzyme activity of polyphenol oxidase (PPO) in grain has been related to undersirable brown discoloration of bread wheat (Triticum aestivum L.) based end-products, particularly for Asian noodles. Breeding wheat cultivars with low PPO activity is the best approach to reduce the undesirable darkening. Molecular markers could greatly improve selection efficiency in breeding programs. Based on the sequences of PPO genes (GenBank Accession Numbers AY596268, AY596269 and AY596270) conditioning PPO activity during kernel development, 28 pairs of primers were designed using the software ‘DNAMAN’. One of the markers from AY596268, designated as PPO18, can amplify a 685-bp and an 876-bp fragment in the cultivars with high and low PPO activity, respectively. The difference of 191-bp size was detected in the intron region of the PPO gene. The STS marker PPO18 was mapped to chromosome 2AL using a DH population derived from a cross Zhongyou 9507× CA9632, a set of nulli-tetrasomic lines and ditelosomic line 2AS of Chinese Spring. QTL analysis indicated that the PPO gene co-segregated with the STS marker PPO18 and is closely linked to Xgwm312 and Xgwm294 on chromosome 2AL, explaining 28–43% of phenotypic variance for PPO activity across three environments. A total of 233 Chinese wheat cultivars and advanced lines were used to validate the correlation between the polymorphic fragments of PPO18 and grain PPO activity. The results showed that PPO18 is a co-dominant, efficient and reliable molecular marker for PPO activity and can be used in wheat breeding programs targeted for noodle quality improvement.     

Expression analysis of Fgf8a & Fgf8b in early stage of P19 cells during neural differentiation

Fgf8 is a member of the fibroblast growth factor (FGF) family that plays an important role in early neural development. Cellular aggregation and retinoic acid (RA) are needed for mouse embryonic carcinoma (EC) P19 cell neural differentiation. We have examined the Fgf8 gene in P19 cells during neural differentiation and identified 2 alternatively spliced Fgf8 isoforms, Fgf8a and Fgf8b, among the 8 known splicing isoforms in mammals. The expression of Fgf8a and Fgf8b mRNAs transiently and rapidly increased in the early stage of P19 cells during RA-induced neural differentiation, followed by a decline in expression. The relative amount of Fgf8b was clearly higher than that of Fgf8a at different time-points measured within 24 h after RA treatment. Increased Fgf8b mRNA expression was cellular-aggregation dependent. The results demonstrated that cellular-aggregation-induced Fgf8b, but not Fgf8a, may play a pivotal role in early neural differentiation of P19 cells.     

A novel molecular marker for the polyphenol oxidase gene located on chromosome 2B in common wheat

Polyphenol oxidase (PPO) is a major cause of time-dependent darkening and discoloration in Asian noodles and other wheat-based products. One of the best ways to reduce this undesirable darkening is to breed new wheat cultivars with low PPO activity using efficient and reliable markers. Based on the sequence of a PPO gene SSPPO-B1 (GenBank accession no. AB254804) located on chromosome 2B of common wheat, 26 pairs of primers were designed to detect polymorphisms between wheat cultivars with low and high PPO activity. F-8, one of these primer pairs, amplified double fragments (band ??a?? of approximately 400?bp and band ??b?? of approximately 600?bp) in the cultivars with low PPO activity, and a single fragment (only band a) in the cultivars with high PPO activity. The differences between the fragments a and b include five indels and several single nucleotide polymorphisms, which occurred in intron II of the PPO gene. F-8 can be used as a sequence-tagged site marker to discriminate between two alleles Ppo-B1a (GQ303713) and Ppo-B1b (AB254804). The screening of 284 accessions of the core collection of Chinese wheat germplasms using the marker F-8 showed that the double fragments were present in 188 accessions, and the single fragments were present in the remaining 96 accessions. Statistical analysis revealed that the cultivars with the double fragments had significantly lower mean PPO activity than those with the single fragments. We also screened the 284 accessions using two additional markers, PPO18 for Ppo-A1 on chromosome 2A and STS01 for Ppo-D1 on chromosome 2D. Results showed that the combination of markers F-8, PPO18, and STS01 could reliably predict PPO activity. These markers can be used in wheat breeding programs for low PPO activity selection to improve the quality of wheat-based products.     

Identification of an exonic splicing silencer in exon 6A of the human VEGF gene

Background  

The different isoforms of vascular endothelial growth factor (VEGF) play diverse roles in vascular growth, structure and function. Alternative splicing of the VEGF gene results in the expression of three abundant isoforms: VEGF121, VEGF165 and VEGF189. The mRNA for VEGF189 contains the alternatively spliced exon 6A whereas the mRNA for VEGF165 lacks this exon. The objective of this study was to identify the cis elements that control utilization of exon 6A. A reporter minigene was constructed (pGFP-E6A) containing the coding sequence for GFP whose translation was dependent on faithful splicing for removal of the VEGF exon 6A. To identify cis-acting splicing elements, sequential deletions were made across exon 6A in the pGFP-E6A plasmid.     

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.     

Induction of peroxidase and polyphenol oxidase in Arachis hypogaea in response to treatment with Pseudomonas fluorescens and inoculation with Alternaria alternata

Abstract

The application of talc-formulation through seed, seed treatment plus foliar spray and foliar spray alone significantly reduced the leaf blight incidence both under greenhouse and field conditions. The groundnut plants treated with biocontrol agent and challenge inoculated with Alternaria alternata recorded significantly increased activity of Peroxidase isozyme (PO), Polyphenol oxidase isozymes (PPO) activity. Expression of PO2, PPO1 and PPO2 isoforms were found in all the plants treated with Pf1 while additional PO1, PPO3, PPO4 and PPO5 were observed in Pf1-treated plants followed by challenge inoculation with the pathogen.     

Genetic mapping of new seed-expressed polyphenol oxidase genes in wheat (Triticum aestivum L.)

Polyphenol oxidase (PPO) enzymatic activity is a major cause in time-dependent discoloration in wheat dough products. The PPO-A1 and PPO-D1 genes have been shown to contribute to wheat kernel PPO activity. Recently a novel PPO gene family consisting of the PPO-A2, PPO-B2, and PPO-D2 genes has been identified and shown to be expressed in wheat kernels. In this study, the sequences of these five kernel PPO genes were determined for the spring wheat cultivars Louise and Penawawa. The two cultivars were found to be polymorphic at each of the PPO loci. Three novel alleles were isolated from Louise. The Louise X Penawawa mapping population was used to genetically map all five PPO genes. All map to the long arm of homeologous group 2 chromosomes. PPO-A2 was found to be located 8.9 cM proximal to PPO-A1 on the long arm of chromosome 2A. Similarly, PPO-D1 and PPO-D2 were separated by 10.7 cM on the long arm of chromosome 2D. PPO-B2 mapped to the long arm of chromosome 2B and was the site of a novel QTL for polyphenol oxidase activity. Five other PPO QTL were identified in this study. One QTL corresponds to the previously described PPO-D1 locus, one QTL corresponds to the PPO-D2 locus, whereas the remaining three are located on chromosome 2B.     

Allelic variation of polyphenol oxidase (PPO) genes located on chromosomes 2A and 2D and development of functional markers for the PPO genes in common wheat

Polyphenol oxidase (PPO) activity is highly related to the undesirable browning of wheat-based end products, especially Asian noodles. Characterization of PPO genes and the development of their functional markers are of great importance for marker-assisted selection in wheat breeding. In the present study, complete genomic DNA sequences of two PPO genes, one each located on chromosomes 2A and 2D and their allelic variants were characterized by means of in silico cloning and experimental validation. Sequences were aligned at both DNA and protein levels. Two haplotypes on chromosome 2D showed 95.2% sequence identity at the DNA level, indicating much more sequence diversity than those on chromosome 2A with 99.6% sequence identity. Both of the PPO genes on chromosomes 2A and 2D contain an open reading frame (ORF) of 1,731 bp, encoding a PPO precursor peptide of 577 amino acids with a predicted molecular mass of ∼64 kD. Two complementary dominant STS markers, PPO16 and PPO29, were developed based on the PPO gene haplotypes located on chromosome 2D; they amplify a 713-bp fragment in cultivars with low PPO activity and a 490-bp fragment in those with high PPO activity, respectively. The two markers were mapped on chromosome 2DL using a doubled haploid population derived from the cross Zhongyou 9507/CA9632, and a set of nullisomic–tetrasomic lines and ditelosomic line 2DS of Chinese Spring. QTL analysis indicated that the PPO gene co-segregated with the two STS markers and was closely linked to SSR marker Xwmc41 on chromosome 2DL, explaining from 9.6 to 24.4% of the phenotypic variance for PPO activity across three environments. In order to simultaneously detect PPO loci on chromosomes 2A and 2D, a multiplexed marker combination PPO33/PPO16 was developed and yielded distinguishable DNA patterns in a number of cultivars. The STS marker PPO33 for the PPO gene on chromosome 2A was developed from the same gene sequences as PPO18 that we reported previously, and can amplify a 481-bp and a 290-bp fragment from cultivars with low and high PPO activity, respectively. A total of 217 Chinese wheat cultivars and advanced lines were used to validate the association between the polymorphic fragments and grain PPO activity. The results showed that the marker combination PPO33/PPO16 is efficient and reliable for evaluating PPO activity and can be used in wheat breeding programs aimed for noodle and other end product quality improvement.     

Identification of alternatively spliced dab1 isoforms in zebrafish

We have investigated the genomic organization, the occurrence of alternative splicing and the differential expression of the zebrafish disabled1 (dab1) gene. Dab1 is a key effector of the Reelin pathway, which regulates neuronal migration during brain development in vertebrates. The coding region of the zebrafish dab1 gene spans over 600 kb of genomic DNA and is composed of 15 exons. Alternative splicing in a region enriched for tyrosine residues generates at least three different isoforms. These isoforms are developmentally regulated and show differential tissue expression. Comparison with mouse and human data shows an overall conservation of the genomic organization with different alternative splicing events generating species-specific isoforms. Because these alternative splicing events give rise to isoforms with different numbers of phosphorylateable tyrosines, we speculate that alternative splicing of the dab1 gene in zebrafish and in other vertebrates regulates the nature of the cellular response to the Reelin signal.Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.     

Genetic and biochemical analysis of common wheat cultivars lacking puroindoline a

Puroindoline a (Pin-a) and puroindoline b (Pin-b), two basic isoforms encoded by the Pina-D1 and Pinb-D1 loci respectively, involved in controlling grain texture in wheat, were isolated from starch granules of soft wheat cultivars using three different extraction procedures, and fractionated by acidic polyacrylamide gel electrophoresis (A-PAGE). Tris buffer containing 1% Triton X-114 extracted Pin-a and small amounts of Pin-b, whereas 1% SDS preferably extracted Pin-b. Large amounts of both puroindolines were isolated by a solution containing 50% propan-2-ol and 50 mM NaCl. This solution extracted reduced amounts of Pin-b and no traces of Pin-a from starch granules of 20 hard common wheats containing the null allele Pina-D1b. The absence of Pin-a was confirmed by immunostaining with an anti-Pin-a antiserum. With the exception of two cultivars, null Pin-a cultivars gave no PCR fragment with three primer pairs specific to either the coding region or the promoter region of Pina-D1a, suggesting that major changes had occurred at the Pina-D1 locus in these genotypes. Cultivars Fortuna and Glenman were unique in giving size-specific PCR fragments with all primer pairs for the allele Pina-D1a and showed a cytosine deletion at position 267 in the coding region of the Pin-a gene, which resulted in a TGA stop codon at position 361. However, there was no evidence of a mutated protein in the A-PAGE or SDS-PAGE patterns of Fortuna and Glenman. The novel gene, provisionally named Pina-D1c, is the first null allele due to a point mutation that has been identified at the Pina-D1 locus.     

Mitochondrial polymorphism. III. Heterosis,complementation, and spectral properties of purified cytochrome oxidase of wheat

Cytochrome oxidase was purified twentyfold from mitochondria of seedlings of wheat genotypes 28, 31 MS, and 31 MS/28. The enzyme of the hybrid exceeded in activity the parental enzymes. Mixtures of cytochrome oxidase of the parents exhibited complementation in that they approached the activity of the hybrid cytochrome oxidase. Hybrid mitochondria also exhibited heterosis in NADH: cytochrome c reductase activity. Complementation by parent mitochondria was observed for this enzyme also. The Michaelis constant of cytochrome oxidase and NADH: cytochrome reductase was markedly less in the hybrid and the mixture than in the parents. Difference spectra revealed the following: strain 28 had cytochromes a and b but was deficient in cytochrome c; strain 31 MS had cytochromes b and c but no a; the hybrid had all three cytochromes, as did the mixture. The relationship of cytochromes to heterosis and complementation is considered.This work was supported by DeKalb AgResearch, Inc.     

Functional gene markers for polyphenol oxidase locus in bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Higher polyphenol oxidase (PPO) activity in wheat kernels and flour has been implicated in the time dependent darkening of various end-products. Previous study conducted on a bread wheat (Triticum aestivum L.) doubled haploid (DH) mapping population derived from Chara (medium-high PPO) and WW2449 (low PPO) identified a major QTL for PPO activity located on the long arm of chromosome 2A. Physical mapping of SSR markers accounting for up to 84% of phenotypic variation for PPO activities suggests that the candidate PPO locus is localised in the deletion bin delimited by 2AL 0.77–0.85. In order to develop functional gene markers, nine wheat ESTs mapped to this deletion bin and partial PPO reference genes were explored for their sequence identities and linkage with PPO locus in a mapping population. In the present study, two markers: one SNP and one CAPS based upon BQ161439 sequence variation between the parents were identified which exhibited a tight linkage (0–0.6 cM) with the PPO loci designated as XTc1 and XPPO- _LDOPA. We also mapped the reference PPO gene (GenBank AY526268) characterised from developing kernels of wheat, on the long arm of chromosome 2A which exhibited a complete linkage with XPPO- _L DOPA locus. Results suggest that PPO variation displayed in the DH population from Chara/WW2449 is due to the same reference PPO gene. Allelic homoplasy of tightly linked markers, indicated that these markers are ‘diagnostic’ for the selection of low PPO gene in a range of germplasm being used in different Australian breeding programs. Identification and validation of ‘functional gene markers’ would facilitate in enhancing the selection efficiency for low PPO activity in wheat breeding programs.     

Genetic and in silico comparative mapping of the polyphenol oxidase gene in bread wheat (Triticum aestivum L.)

Polyphenol oxidases (PPOs) are involved in the time-dependent darkening and discolouration of Asian noodles and other wheat end products. In this study, a doubled haploid (DH) population derived from Chara (moderately high PPO activity)/WW2449 (low PPO activity) was screened for PPO activity based on l-DOPA and l-tyrosine assays using whole seeds. Both these assays were significantly genetically correlated (r=0.91) in measuring the PPO activity in this DH population. Quantitative trait loci (QTLs) analysis utilising a skeleton map enabled us to identify a major QTL controlling PPO activity based on l-DOPA and l-tyrosine on the long arm of chromosome 2A. The simple sequence repeat (SSR) marker GWM294b explained over 82% of the line mean phenotypic variation from samples collected in both 2000 and 2003. Four SSR markers were validated for PPO linkage in genetically diverse backgrounds and proven to correctly predict the PPO activity in more than 92% of wheat lines. Physical mapping using deletion lines of Chinese Spring has confirmed the location of the GWM294b, GWM312 and WMC170 on chromosome 2AL, between deletion breakpoints 2AL-C to 0.85. In order to identify functional gene markers, data searches for alignments between rice BAC/PAC clones assembled on chromosome 1 and 4, chromosome 7, and (1) the wheat expressed sequence tags mapped in deletion bin (2AL-C to 0.85) and (2) the coding sequence of a previously cloned wheat PPO gene were made and found significant sequence similarities with the PPO gene or common central domain of tyrosinase. Available PPO gene sequences in the National Centre for Biotechnology Information (NCBI) database have revealed that there is a significant molecular diversity at the nucleotide and amino acid level in the wheat PPO genes.Electronic Supplementary Material Supplementary material is available for this article at .     

Antioxidant status and physiological responses of wheat (Triticum aestivum L.) to cycocel application and bio fertilizers under water limitation condition

In order to study antioxidant status and physiological responses of wheat to cycocel (CCC) and bio fertilizers application under water limitation condition, a factorial experiment was conducted based on randomized complete block design with three replications in 2015. Treatments included water limitation in three levels [normal irrigation (I₁) as control; moderate water limitation (I₂) or irrigation withholding at 50% of heading stage; severe water limitation (I₃) or irrigation withholding at 50% of booting stage]; four bio fertilizer levels [(no bio fertilizer (F₀), seed inoculation by Azotobacter chrocoocum strain 5 (F₁), Pseudomonas putida strain 186 (F₂), Azotobacter?+?Pseudomonas (F₃))] and four CCC levels [(without CCC as control (C₀), application of 400 (C₁), 800 (C₂) and 1200 (C₃) mg/l)]. The results showed that water limitation decreased the chlorophyll-a, chlorophyll-b, total chlorophyll, carotenoid, stomata conductance, leaf area index (LAI) and relative water content of wheat, but activity of catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO) enzymes and proline content were increased. Similar results were observed in CAT, POD and PPO activities due to bio fertilizers and CCC application. Besides the water limitation effects, CCC-treated plants displayed a significant decrease in stomata conductance and LAI. Generally, it was concluded that the application of bio fertilizers and CCC can be a proper tool for increasing wheat yield under water limitation.     

Composition and phylogenetic analysis of wheat cryptochrome gene family

Cryptochrome (CRY) gene family encodes photoreceptors mediating developmental responses to blue light throughout the life of plants. We report here the characterization of CRY gene family in hexaploid wheat. Degenerate PCR amplification of the regions encoding the conserved flavin-binding domain of CRY proteins yielded seven bands, resulting from amplification of CRY1a, CRY1b and CRY2 homologous genes. Assignment of individual amplicons to subgenomes was accomplished by comparing their sequence compositions with those from the ancestor species of wheat. ESTs coding for CRY-DASH like proteins were identified in wheat EST database in GenBank. Southern blot showed that TaCRY1a, TaCRY1b and TaCRY2 are single copy genes. We mapped TaCRY1a and TaCRY2 to chromosomes of homoeologous group 6, TaCRY1b to group 2, and TaCRY-DASH to group 7. Phylogenetic analysis showed that CRY subfamily diversification occurred before the divergence of monocots and dicots. The regulatory and functional changes of CRY members within subfamily are discussed.