Full-length Genomic DNA Sequences Research Articles

Identification of a candidate gene for the I locus determining the dominant white bulb color in onion (Allium cepa L.).

Through a map-based cloning approach, a gene coding for an R2R3-MYB transcription factor was identified as a causal gene for the I locus controlling the dominant white bulb color in onion. White bulb colors in onion (Allium cepa L.) are determined by either the C or I loci. The causal gene for the C locus was previously isolated, but the gene responsible for the I locus has not been identified yet. To identify candidate genes for the I locus, an approximately 7-Mb genomic DNA region harboring the I locus was obtained from onion and bunching onion (A. fistulosum) whole genome sequences using two tightly linked molecular markers. Within this interval, the AcMYB1 gene, known as a positive regulator of anthocyanin production, was identified. No polymorphic sequences were found between white and red AcMYB1 alleles in the 4,860-bp full-length genomic DNA sequences. However, a 4,838-bp LTR-retrotransposon was identified in the white allele, in the 79-bp upstream coding region from the stop codon. The insertion of this LTR-retrotransposon created a premature stop codon, resulting in the replacement of 26 amino acids with seven different residues. A molecular marker was developed based on the insertion of this LTR-retrotransposon to genotype the I locus. A perfect linkage between bulb color phenotypes and marker genotypes was observed among 5,303 individuals of segregating populations. The transcription of AcMYB1 appeared to be normal in both red and white onions, but the transcription of CHS-A, which encodes chalcone synthase and is involved in the first step of the anthocyanin biosynthesis pathway, was inactivated in the white onions. Taken together, an aberrant AcMYB1 protein produced from the mutant allele might be responsible for the dominant white bulb color in onions.

Cloning of Metalloproteinase 17 Genes from Oriental Giant Jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae).

We previously demonstrated that Nemopilema nomurai jellyfish venom metalloproteinases (JVMPs) play a key role in the toxicities induced by N. nomurai venom (NnV), including dermotoxicity, cytotoxicity, and lethality. In this study, we identified two full-length JVMP cDNA and genomic DNA sequences: JVMP17-1 and JVMP17-2. The full-length cDNA of JVMP17-1 and 17-2 contains 1614 and 1578 nucleotides (nt) that encode 536 and 525 amino acids, respectively. Putative peptidoglycan (PG) binding, zinc-dependent metalloproteinase, and hemopexin domains were identified. BLAST analysis of JVMP17-1 showed 42, 41, 37, and 37% identity with Hydra vulgaris, Acropora digitifera, Megachile rotundata, and Apis mellifera venom metalloproteinases, respectively. JVMP17-2 shared 38 and 36% identity with H. vulgaris and A. digitifera, respectively. Alignment results of JVMP17-1 and 17-2 with other metalloproteinases suggest that the PG domain, the tissue inhibitor of metalloproteinase (TIMP)-binding surfaces, active sites, and metal (ion)-binding sites are highly conserved. The present study reports the gene cloning of metalloproteinase enzymes from jellyfish species for the first time. We hope these results can expand our knowledge of metalloproteinase components and their roles in the pathogenesis of jellyfish envenomation.

A SNP variation in an expansin (EgExp4) gene affects height in oil palm.

Oil palm (Elaeis guineensis Jacq.), an Aracaceae family plant, is utilized for both consumable and non-consumable products, including cooking oil, cosmetics and biodiesel production. Oil palm is a perennial tree with 25 years of optimal harvesting time and a height of up to 18 m. However, harvesting of oil palm fruit bunches with heights of more than 2–3 meters is challenging for oil palm farmers. Thus, understanding the genetic control of height would be beneficial for using gene-based markers to speed up oil palm breeding programs to select semi-dwarf oil palm varieties. This study aims to identify Insertion/Deletions (InDels) and single nucleotide polymorphisms (SNPs) of five height-related genes, including EgDELLA1, EgGRF1, EgGA20ox1, EgAPG1 and EgExp4, in short and tall oil palm groups by PacBio SMRT sequencing technology. Then, the SNP variation’s association with height was validated in the Golden Tenera (GT) population. All targeted genes were successfully amplified by two rounds of PCR amplification with expected sizes that ranged from 2,516 to 3,015 base pair (bp), covering 5′ UTR, gene sequences and 3′ UTR from 20 short and 20 tall oil palm trees. As a result, 1,166, 909, 1,494, 387 and 5,384 full-length genomic DNA sequences were revealed by PacBio SMRT sequencing technology, from EgDELLA1, EgGRF1, EgGA20ox1, EgAPG1 and EgExp4 genes, respectively. Twelve variations, including eight InDels and four SNPs, were identified from EgDELLA1, EgGRF1, EgGA20ox1 and EgExp4. No variation was found for EgAPG1. After SNP through-put genotyping of 4 targeted SNP markers was done by PACE™ SNP genotyping, the association with height was determined in the GT population. Only the mEgExp4_SNP118 marker, designed from EgExp4 gene, was found to associate with height in 2 of 4 height-recordings, with p values of 0.0383 for height (HT)-1 and 0.0263 for HT-4. In conclusion, this marker is a potential gene-based marker that may be used in oil palm breeding programs for selecting semi-dwarf oil palm varieties in the near future.

BrcuHAC1 is a histone acetyltransferase that affects bolting development in Chinese flowering cabbage

Histone acetylation is an important posttranslational modification associated with gene activation. In Arabidopsis, histone acetyltransferase 1 (HAC1) can promote flowering by regulating the transcription of FLOWERING LOCUS C (FLC), a major floral repressor. The size of the full-length cDNA and genomic DNA sequences of the histone acetyltransferase 1 gene (BrcuHAC1) in Chinese flowering cabbage (Brassica rapa syn. campestris ssp. chinensis var. utilis) were 5846 bp and 7376 bp, with an open reading frame (ORF) coding for a peptide with 1689 amino acids. The expression levels of BrcuHAC1 in different tissues and different developmental stages were as follows: flower>leaf>stem>root, and completed bolting and flowering stage>5th true leaf-stage>4th true leaf-stage>3rd true leaf-stage>2nd true leaf-stage>1st true leaf-stage. Silencing of BrcuHAC1 resulted in slow growth, and delayed bolting and flowering time in Chinese flowering cabbage. Molecular analysis showed that the mRNA level of FLC was increased, indicating that the delayed flowering phenomenon was mediated by FLC in the silenced group. In contrast, the expression levels of the autonomous-pathway genes were not significantly affected in the silenced group. In addition, the histone modification of FLC chromatin was also not affected in the silenced group. FLC is not the direct target gene of BrcuHAC1. However, BrcuHAC1 may affect the bolting and flowering time of Chinese flowering cabbage through the epigenetic modification of upstream factors of FLC.

Identification of a novel haplotype of the Ms locus controlling restoration of male-fertility and its implication in origination of cytoplasmic male-sterility in onion (Allium cepa L.)

ABSTRACT Seeds of F1 hybrid cultivars of onions have been exclusively produced by utilising cytoplasmic male-sterility (CMS). A single locus, Ms, controls restoration of male-fertility of CMS onions. When a molecular marker tagging the AcPMS1 gene, a candidate gene for the Ms locus, was used to genotype diverse breeding lines, an unusual marker genotype showing an intermediate size of PCR product was identified in one breeding line (ORI854). The male-fertility phenotype of ORI854 was all male-sterile. Full-length genomic DNA sequence (16,464 bp) of this novel AcPMS1 allele from ORI854 was more closely related to a recessive AcPMS1 allele than to a dominant allele. However, there were more than 200 polymorphic sequences between this novel allele and the recessive allele, indicating that this novel AcPMS1 allele might not have recently diverged from the recessive allele. Phylogenetic analysis implied that onion male-sterility might have first appeared in a common ancestor before divergence of this novel allele and recessive allele of AcPMS1. Analysis of full-length cDNA sequences of additional 12 genes showing almost perfect linkage disequilibrium with the Ms locus indicated that no recombination might have happened among 13 genes including AcPMS1 in the novel Ms haplotype after divergence from the recessive Ms haplotype.

Cloning, expression analysis and molecular marker development of cinnamyl alcohol dehydrogenase gene in common wheat.

In common wheat, stem strength is one of the key factors for lodging resistance, which is influenced by lignin content. Cinnamyl alcohol dehydrogenase (CAD) is a vital enzyme in the pathway of lignin biosynthesis. Cloning and marker development of the CAD gene could be helpful for lodging resistance breeding. In this study, the full-length genomic DNA sequence of CAD gene in wheat was cloned by using homologous strategy. A marker 5-f2r2 was developed based on CAD sequence and used to genotype 258 wheat lines. Four haplotype combinations of CAD genes were identified in 258 wheat lines. Correction analyses among the CAD gene expression, CAD activity, and stem strength indicated significant positive correlation between CAD gene expression and CAD activity, between wheat CAD activity and wheat stem strength. The haplotype combination B is significantly associated with the lower enzyme activity and weak stem strength, which was supported by the level of CAD gene expression. The CAD activity and stem strength of wheat could be distinguished to some extent using this pair of specific primer 5-f2r2 designed in this study, indicating that the sequence targeted site (STS) marker 5-f2r2 could be used in marker assistant selection (MAS) breeding.

Molecular identification, characterization, and expression analysis of a novel trypsin inhibitor-like cysteine-rich peptide from the cotton bollworm, Helicoverpa armigera (H\xfcbner) (Lepidoptera: Noctuidae)

Trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors have been reported to inhibit proteases such as trypsin, cathepsin, elastase, and chymotrypsin, and thus play a critical role in several physiological processes. However, the information about TIL peptides in insects is limited. In the present study, a novel cysteine-rich trypsin inhibitor-like protease, designated as HaTIL2, was isolated from the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). The cDNA sequence of HaTIL2 is 470 nucleotides long, with 240 nucleotides open reading frame encoding 80 amino acid residues. The analysis of genomic DNA revealed that the full-length genomic DNA sequence of HaTIL2 was 574 bp with two exons and one intron. The predicted molecular weight of HaTIL2 is 8.632 kDa, with an isoelectric point of 4.41. The results of neighbor-joining tree demonstrated that HaTIL2 was closely related to H. armigera TIL3, DmCEI, and DsCtAPI followed by TcIMI and MdCEI. The mRNA of HaTIL2 was constitutively expressed at different levels in different stages of H. armigera. The HaTIL2 showed a high expression on different days of the pupal stage, which revealed that HaTIL2 might play a vital role during the pupal stage. Although the detailed function of HaTIL2 needs to be elucidated, the obtained results are of particular importance to open up new avenues of research into the functional studies of insect peptidase inhibitors.

Identification of a variant of CMS-T cytoplasm and development of high resolution melting markers for distinguishing cytoplasm types and genotyping a restorer-of-fertility locus in onion (Allium cepa L.)

Cytoplasmic male-sterility (CMS) has been used in F1 hybrid seed production in onion (Allium cepa L.). Two types of CMS (CMS-S and CMS-T) have been reported in onions. Almost complete mitochondrial genome sequences of normal, CMS-T, and CMS-S cytoplasm types have been obtained in previous studies. Unlike highly divergent CMS-S mitochondrial genome, there were only three single nucleotide polymorphisms (SNPs) between normal and CMS-T mitochondrial genomes except for orf725, the causal gene for CMS induction. Cleaved amplified polymorphic sequence (CAPS) and high resolution melting (HRM) markers were developed based on one of these SNPs. When these markers were tested for 243 diverse breeding lines, four of them containing CMS-T cytoplasm showed SNP genotypes of the normal cytoplasm. Although mitochondrial genome organizations of these four accessions were assumed to be similar to those of normal and CMS-T, the copy number of orf725 was less than half of that of general CMS-T, suggesting that male-sterility of this variant of CMS-T might be induced by an independent event of substoichiometric shifting of orf725. To distinguish normal and all types of CMS cytoplasms, an HRM marker was developed on the basis of cox1 and orf725. In addition, for genotyping the Ms locus, a nuclear restorer-of-fertility (Rf) gene, approximately 15 kb full-length genomic DNA sequences of AcPMS1, a candidate Rf gene, were obtained. Two reproducible HRM markers were developed among ten candidate HRM markers designed on the basis of more than 800 SNPs or InDels between AcPMS1 alleles.

Characterization of TaCOMT genes associated with stem lignin content in common wheat and development of a gene-specific marker

Stem lignin content (SLC) in common wheat (Triticum aestivum L.) contributes to lodging resistance. Caffeic acid 3-O-methyltransferase (COMT) is a key enzyme involved in lignin biosynthesis. Characterization of TaCOMT genes and development of gene-specific markers could enable marker-assisted selection in wheat breeding. In the present study, the full-length genomic DNA (gDNA) sequences of TaCOMT genes located on chromosomes 3A, 3B, and 3D were cloned by homologous cloning. Two allelic variants, TaCOMT-3Ba and TaCOMT-3Bb, were identified and differed by a 222-bp insertion/deletion (InDel) in the 3′-untranslated region (3′-UTR). A co-dominant gene-specific marker based on this InDel was developed and designated as TaCOMT-3BM. A total of 157 wheat cultivars and advanced lines grown in four environments were used to validate the associations between allelic patterns and SLC. The SLC of cultivars with TaCOMT-3Ba was significantly (P<0.01) higher than that of those with TaCOMT-3Bb, and the marker TaCOMT-3BM could be effectively used in wheat breeding.

Molecular characterization and function analysis of three RIG-I-like receptor signaling pathway genes (MDA5, LGP2 and MAVS) in Oreochromis niloticus

The recognition of microbial pathogens, which is mediated by pattern recognition receptors (PRRs), is critical to the initiation of innate immune responses. In the present study, we isolated the full-length cDNA and genomic DNA sequences of the MDA5, LGP2 and MAVS genes in Nile tilapia, termed OnMDA5, OnLGP2 and OnMAVS. The OnMDA5 gene encodes 974 amino acids and contains two caspase-associated recruitment domains (CARDs), a DExDc domain (DExD/H box-containing domain), a HELICc (helicase superfamily C-terminal) domain and a C-terminal regulatory domain (RD). The OnLGP2 gene encodes 679 amino acids and contains a DExDc, a HELICc and an RD. The OnMAVS gene encodes 556 amino acids and contains a CARD, a proline-rich domain, a transmembrane helix domain and a putative TRAF2-binding motif (269PVQDT273). Phylogenetic analyses showed that all three genes from Nile tilapia were clustered together with their counterparts from other teleost fishes. Real-time PCR analyses showed that all three genes were constitutively expressed in all examined tissues in Nile tilapia. OnMDA5 presented the highest expression level in the blood and the lowest expression level in the liver, while OnMAVS presented the highest expression level in the kidney. The highest expression level of OnLGP2 was detected in the liver. An examination of the expression patterns of these RIG-I-like receptors (RLRs) during embryonic development showed that the highest expression levels of OnMDA5 occurred at 2 days postfertilization (dpf), and the expression significantly decreased from 3 to 8 dpf. The expression levels of OnLGP2 significantly increased from 4 to 8 dpf. The expression levels of OnMAVS mRNA were stable from 2 to 8 dpf. Upon stimulation by intraperitoneal injection of Streptococcus agalactiae, the expression levels of OnMDA5 were first downregulated and then upregulated in the blood, gill and spleen. In the intestine and kidney, the expression of OnMDA5 was first upregulated, then downregulated, and then upregulated again. The expression of OnLGP2 was upregulated in the kidney and intestine, and the expression of OnMAVS was upregulated in the spleen. Overexpression of OnMAVS increased NF-κB activation in 293 T cells (p < 0.05), and after cotransfection with OnMDA5, the OnMAVS-dependent NF-κB activation was slightly increased (p > 0.05), after cotransfection with OnLGP2, the OnMAVS-dependent NF-κB activation was significantly decreased (p < 0.05). These findings suggest that, although the deduced protein structure of OnMDA5 is evolutionarily conserved with the structures of other RLR members, its signal transduction function is markedly different. The results also suggest that OnLGP2 has a negative regulatory effect on the OnMAVS gene. OnMDA5 and OnMAVS were uniformly distributed throughout the cytoplasm in 293 T cells, whereas OnLGP2 was distributed throughout the cytoplasm and nucleus. These results are helpful for clarifying the innate immune response against bacterial infection in Nile tilapia.

The evolution and functional characterization of lined seahorse (Hippocampus erectus) CCKs involved in fasting and thermal stress response

The peptide cholecystokinin (CCK) plays an important role in the regulation of vertebrate appetite and feeding behaviour. In the present study, the full-length cDNA and genomic DNA sequences of two CCK precursors were cloned and analysed in the Syngnathidae fish, the lined seahorse (Hippocampus erectus). Both CCK1 and CCK2 in the seahorse consist of four exons. The sequence of the octapeptide of seahorse CCK1 (DYMGWMDF) was the same as that of the chicken and human, while the octapeptide of seahorse CCK2 (DYEGWMDF) was unique among vertebrates. According to the phylogenetic analysis, two types of CCKs were produced by teleost-specific genome duplication (TGD). Both CCK1 and CCK2 were highly expressed in the brain, while detectable amounts of CCK1 mRNA in the brood pouch and CCK2 mRNA in the intestine were also found. Both CCK1 and CCK2 mRNA levels significantly increased during the transition from endogenous to exogenous nutrition. Additionally, fasting induced a significant increase in the CCK1 mRNA expression in the brain of juvenile seahorses but had no effect on CCK2 transcript levels. In addition, the CCK1 and CCK2 mRNA levels in the seahorse brain significantly increased after a high-temperature treatment. Thus, the mRNA expression of CCK had obvious tissue specificities and this preliminary study opens new avenues for further functional studies on the endocrine regulations of CCK in the transition from endogenous to exogenous nutrition, food intake regulation and metabolism in the seahorse.

Activity and expression of ADP-glucose pyrophosphorylase during rhizome formation in lotus (Nelumbo nucifera Gaertn.).

BackgroundLotus root is a traditional and popular aquatic vegetable in China. Starch is an important component of the rhizome and directly affects the quality of processed products. ADP -glucose pyrophosphorylase (AGPase) is a rate-limiting enzyme associated with starch biosynthesis in plants. Therefore, in the present study, AGPase activity and NnAGP expression during rhizome development of lotus were analyzed.ResultsAmong 15 cultivars analyzed, the contents of amylose and total starch in the rhizome were highest in ‘Mei Ren Hong’. ‘Su Zhou’ and ‘Zhen Zhu’ showed the lowest amylose, amylopectin and total starch contents. In the rhizome, activity of AGPase was highest at the middle swelling stage of development, and higher activity was observed in the ‘Hou ba’ leaf and terminational leaf at the same stage. Three AGPase genes, comprising two large subunit genes (NnAGPL1 and NnAGPL2) and one small subunit gene (NnAGPS), were isolated and identified. The deduced amino acid sequences showed 40.5 % similarity among the three genes. Full-length genomic DNA sequences of NnAGPL1, NnAGPL2, and NnAGPS were 4841, 11,346 and 4169 bp, respectively. Analysis of the temporal and spatial expression patterns revealed that the transcription levels of NnAGPL1 and NnAGPS were higher in the rhizome, followed by the ‘Hou ba’ leaf, whereas NnAGPL2 was significantly detected in the ‘Hou ba’ leaf and terminational leaf. The initial swelling stage of rhizome development was accompanied by the highest accumulation of mRNAs of NnAGPL1, whereas expression of NnAGPL2 was not detected during rhizome development. The transcript level of NnAGPS was highest at the initial swelling stage compared with the other rhizome developmental stages. Transcription of NnAGPL1, NnAGPL2, and NnAGPS was induced within 24 h after treatment with exogenous sucrose. The mRNA level of NnAGPL1 and NnAGPS was increased by exogenous ABA, whereas transcription of NnAGPL2 was not affected by ABA.ConclusionsThe three AGPase genes display marked differences in spatial and temporal expression patterns. Regulation of AGPase in relation to starch synthesis in lotus is indicated to be complex.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-016-0140-z) contains supplementary material, which is available to authorized users.

Molecular cloning and functional characterization of spexin in orange-spotted grouper (Epinephelus coioides).

Spexin is a newly discovered neuropeptide in vertebrates. Comprehensive comparative studies are required to unveil its biological functions. In order to ascertain the neuroendocrine function of spexin in orange-spotted grouper, its full-length cDNA and genomic DNA sequences were cloned and analyzed. Sequence analyses showed that the spexin gene structure is composed of six exons and five introns, and the amino acids of mature peptide (spexin-14) in grouper are identical to that of other fish. Tissue expression analysis found that grouper spexin is highly expressed in the brain, liver and ovary. Real time-PCR analysis demonstrated that the hypothalamic expression of spexin declined gradually during the ovarian development, and was up-regulated by food deprivation. Intraperitoneal administration of spexin-14 peptides to grouper significantly elevated the mRNA levels of proopiomelanocortin (pomc) and suppressed the orexin expression in the hypothalamus, but could not change the hypothalamic expression of gonadotropin releasing hormone 1 (gnrh1). Both in vivo and in vitro administration of spexin could not significantly influence the expression of follicle-stimulating hormone β (fshβ) and luteinizing hormone β (lhβ) in the pituitary with the exception of an inhibition of gh expression. Our data suggested that the spexin has a significant role in the regulation of energy metabolism and food intake in orange-spotted grouper.

Mechanistic insights into induction of vitellogenin gene expression by estrogens in Sydney rock oysters, Saccostrea glomerata

Marine molluscs, such as oysters, respond to estrogenic compounds with the induction of the egg yolk protein precursor, vitellogenin (Vtg), availing a biomarker for estrogenic pollution. Despite this application, the precise molecular mechanism through which estrogens exert their action to induce molluscan vitellogenesis is unknown. As a first step to address this question, we cloned a gene encoding Vtg from the Sydney rock oyster Saccostrea glomerata (sgVtg). Using primers designed from a partial sgVtg cDNA sequence available in Genbank, a full-length sgVtg cDNA of 8498bp was obtained by 5′- and 3′-RACE. The open reading frame (ORF) of sgVtg was determined to be 7980bp, which is substantially longer than the orthologs of other oyster species. Its deduced protein sequence shares the highest homology at the N- and C-terminal regions with other molluscan Vtgs. The full-length genomic DNA sequence of sgVtg was obtained by genomic PCR and genome walking targeting the gene body and flanking regions, respectively. The genomic sequence spans 20kb and consists of 30 exons and 29 introns. Computer analysis identified three closely spaced half-estrogen responsive elements (EREs) in the promoter region and a 210-bp CpG island 62bp downstream of the transcription start site. Upregulation of sgVtg mRNA expression was observed in the ovaries following in vitro (explants) and in vivo (tank) exposure to 17β-estradiol (E2). Notably, treatment with an estrogen receptor (ER) antagonist in vitro abolished the upregulation, suggesting a requirement for an estrogen-dependent receptor for transcriptional activation. DNA methylation of the 5′ CpG island was analysed using bisulfite genomic sequencing of the in vivo exposed ovaries. The CpG island was found to be hypomethylated (with 0–3% methylcytosines) in both control and E2-exposed oysters. However, no significant differential methylation or any correlation between methylation and sgVtg expression levels was observed. Overall, the results support the possible involvement of an ERE-containing promoter and an estrogen-activated receptor in estrogen signalling in marine molluscs.

A new antimicrobial peptide SCY2 identified in Scylla Paramamosain exerting a potential role of reproductive immunity.

A new antimicrobial peptide named SCY2 with 65.08% identity in amino acid sequence to the known scygonadin (SCY1) was first characterized in Scylla paramamosain based on its cloned full-length cDNA and genomic DNA sequences. The SCY2 gene was dominantly expressed in the ejaculatory duct of male crabs and its mRNA transcripts were discerned mainly in the glandular epithelium of the inner wall and the secretion inside the ejaculatory duct. Although the SCY2 gene could not be induced with the challenge of the bacteria and fungi tested, its induction reached the highest level at the peak period of mating in mature male crabs either in June or November, suggesting its induction was likely related to seasonal reproduction changes. Moreover, it was interesting to note that, from analysis of its transcripts and protein, SCY2 was significantly expressed only in the ejaculatory duct of pre-copulatory males before mating, however it was clearly detected in the spermatheca of post-copulatory females after mating accompanied by the decreased level of SCY2 expression in the ejaculatory duct. These results suggested that the SCY2 was probably transferred from the male during mating action with the female for the purpose of protecting fertilization. The recombinant SCY2 was more active against the Gram-positive than the Gram-negative bacteria tested. It was further observed that the SCY2 transcripts were significantly increased with addition of exogenous progesterone in tissue cultures whereas the several other hormones tested had no any effect on SCY2 expression, indicating that there might be a relationship between the SCY2 expression and the induction of hormones invivo. In summary, this study demonstrated that one role of SCY2 was likely to be involved in crab reproduction and it exerted its reproductive immune function through the mating action and the maintenance of inner sterility in the spermatheca of the female, thus leading to successful fertilization of S.paramamosain.

Mapping quantitative trait loci for peroxidase activity and developing gene-specific markers for TaPod-A1 on wheat chromosome 3AL.

Three novel QTL for peroxidase activity were mapped, and gene-specific markers for TaPod-A1 were developed and validated using RILs derived from the Doumai/Shi 4185 cross and 281 wheat cultivars. TaPod-A1 is within one of the three QTL. Peroxidase (POD) activity in grain is an important factor determining the color of flour and end-use products of wheat, such as noodles and steamed bread. Mapping QTL for POD activity, characterization of POD genes and development of gene-specific markers are important for molecular marker-assisted selection in wheat breeding. Quantitative trait loci (QTL) for POD activity in common wheat were mapped using a recombinant inbred line (RIL) population derived from a Doumai/Shi 4185 cross grown in four environments and genotyped using the wheat 90 K iSelect assay. Three novel QTL for POD activity, QPod.caas-3AL, QPod.caas-4BS and QPod.caas-5AS, were identified on chromosomes 3AL, 4BS and 5AS, explaining 5.3-21.2% of phenotypic variance across environments. The full-length genomic DNA (gDNA) sequence of a POD gene, designated TaPod-A1, on chromosome 3A was characterized by homolog cloning and PCR verification. Two complementary dominant sequence-tagged site (STS) markers, POD-3A1 and POD-3A2, were developed based on single nucleotide polymorphisms (SNPs) between two alleles at the TaPod-A1 locus, amplifying 291- and 766-bp fragments in cultivars with lower and higher POD activities, respectively. The two gene-specific markers were mapped on chromosome 3AL using a set of Chinese Spring (CS) nulli-tetrasomic lines, and ditelosomic lines 3AL and 3AS. QTL analysis indicated that QPod.caas-3AL co-segregated with the gene-specific markers POD-3A1 and POD-3A2. POD-3A1 and POD-3A2 were verified on 281 wheat cultivars and advanced lines, and showed significant (P < 0.05) associations with POD activities. POD-3A1 and POD-3A2 may be useful as markers for improving color attributes in wheat breeding programs.

An SCD gene from the Mollusca and its upregulation in carotenoid-enriched scallops

Carotenoids are a diverse group of red, orange, and yellow pigments that act as vitamin A precursors and antioxidants. Animals can only obtain carotenoids through their diets. Amongst the carotenoids identified in nature, over one third are of marine origin, but current research on carotenoid absorption in marine species is limited. Bivalves possess an adductor muscle, which is normally white in scallops. However, a new variety of Yesso scallop (Patinopecten yessoensis), the ‘Haida golden scallop’, can be distinguished by its adductor muscle's orange colour, which is caused by carotenoid accumulation. Studying the genes related to carotenoid accumulation in this scallop could benefit our understanding of the mechanisms underlying carotenoid absorption in marine organisms, and it could further improve scallop breeding for carotenoid content. Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in the production of monounsaturated fatty acids, which enhance carotenoid absorption. Here, the full-length cDNA and genomic DNA sequences of the SCD gene from the Yesso scallop (PySCD) were obtained. The PySCD gene consisted of four exons and three introns, and it contained a 990-bp open reading frame encoding 329 amino acids. It was ubiquitously expressed in adult tissues, embryos and larvae of both white Yesso scallops and ‘Haida golden’ scallops. Although the expression pattern of PySCD in both types of scallops was similar, significantly more PySCD transcripts were detected in the ‘Haida golden’ scallops than in the white scallops. Elevated PySCD expression was found in tissues including the adductor muscle, digestive gland, and gonad, as well as in veliger larvae. This study represents the first characterisation of an SCD gene from the Mollusca. Our data imply that PySCD functions in multiple biological processes, and it might be involved in carotenoid accumulation.

Sequence analysis of MIPS-1s gene isolated from the low-phytic-acid inbred line of maize Qi319 and characterization of its expression patterns

Myo-inositol 1-phosphate synthase (MIPS) is a pivotal enzyme that initiates the first step of phytic acid synthesis. In the present study the MIPS-1s gene isolated was from an inbred line of maize Qi319 containing low contents of phytic acid (lpa). Its sequence and expression patterns were also characterized. Results showed that the full-length genomic DNA sequence of the MIPS-1s gene is comprised of ten exons and nine introns, with a length of 3596 bp, and an open reading frame of 1533 bp that encodes a protein with 510 amino acids. A single amino acid substitution was found, compared to the sequence of the inbred maize line B73. In silico promoter analysis revealed various ciselements that might contribute to the differential regulation of MIPS-1s. The expression patterns of MIPS-1s proteins in leaves, roots, flowers and developing seeds of maize were also investigated using western blot. It showed similar patterns compared to the control line (Chang 7–2), suggesting that the MIPS-1s protein plays similar roles in the process of maize development and growth. Notably, it was found that the control line had a dramatically higher abundance of MIPS-1s protein than did Qi319, in the developing seeds and especially during the early period. This strongly indicates that higher levels of MIPS expression should precede and/or accompany synthesis and accumulation of phytate.

Molecular cloning of BnZEP and its expression in petals of different colors inBrassica napus L.

C?????arotenoids are a class of pigment elements present in photosynthetic organisms. T??he transcripts of specific carotenoid biosynthetic genes are probably important for the formation of different colors of flower petals. Zeaxanthin epoxidase (ZEP) enzyme activity is important for the high content of carotenoids in petals. In the present study, the zeaxanthin epoxidase gene in Brassica napus L. (BnZEP) was cloned and bioinformatically analyzed. The expression levels of the BnZEP gene in different plant organs and petals of different colors were investigated and compared in B. Napus. The results showed that the full-length cDNA sequence of BnZEP was 2228 bp, containing an open reading frame of 2010 bp and encoding a putative protein of 669 amino acids, with a molecular weight of 73.8 kDa and an isoelectric point (pI) of 6.16. The full-length genomic DNA sequence of BnZEP was 3268 bp, containing 16 exons and 15 introns, the same as in Arabidopsis thaliana (AtZEP). The predicted amino acid sequence of BnZEP revealed a close phylogenetic relationship among the cruciferous species, including Arabidopsis thaliana. The expression levels of BnZEP were remarkably different in different organs and in petals of different colors. Obviously higher expression levels of BnZEP were found in flower petals than in leaves and buds. Yellow petals had markedly higher expression levels of BnZEP than white petals. The results showed that the expression level of BnZEP was positively correlated with yellow petal color in B. Napus L. However, the highest expression level of BnZEP was observed in the pale yellow flower petals of the F1 hybrid (HW243 × ZHONGYOU 821). This implied that the expression level of BnZEP was not completely related to the degree of yellow petal color in B. Napus L. Based on the roles of ZEP enzyme in the xanthophyll cycle, it was suggested that the yellow flower color in Brassica napus L. Was probably associated with the accumulation of antheraxanthin and violaxanthin in the petals. However, further studies are necessary to identify the specific key carotenoid(s) for the yellow color in the petals of Brassica napus L.

Characterization of E3 ubiquitin ligase neuregulin receptor degradation protein-1 (Nrdp1) in the large yellow croaker (Larimichthys crocea) and its immune responses to Cryptocaryon irritans

Neuregulin receptor degradation protein-1 (Nrdp1) was recently identified in humans as an important immune factor responding to the challenge of virus, LPS or cytokine. Its role in fish immune defense and whether it is involved in anti-parasite immunity have not been proven yet. In this report, the full-length cDNA sequence and genomic structure of Nrdp1 in the large yellow croaker Larimichthys crocea (LcNrdp1) were identified and characterized. The full-length cDNA of LcNrdp1 was 1248bp, including a 5′ untranslated region (UTR) of 32bp, a 3′ UTR of 259bp and an open reading frame (ORF) of 937bp, encoding a polypeptide of 318 amino acid residues. The full-length genomic DNA sequence of LcNrdp1 was composed of 2635 nucleotides, including four exons and three introns. The putative LcNrdp1 protein had no signal peptide sequence and contained a characteristic Nrdp1 consensus motif C3HC3D ring finger and a Coiled-coil domain. Phylogenetic analysis showed that Nrdp1 in fish was closer with that in other vertebrates (79%–90% amino acid identity) than in invertebrates and bacteria (27%–65%). In fishes, Nrdp1 in large yellow croaker was closer with that in Takifugu rubripes. The expression profile showed that LcNrdp1 was constitutively expressed in all tested tissues, especially highly expressed in brain, muscle and kidney. Post-infection (PI) with Cryptocaryon irritans, an increased expression of LcNrdp1 was induced in infection sites (skin and gill), whereas in immune organs, the expression of LcNrdp1 was up-regulated in spleen (except the 1st d and 10th d PI) but suppressed in head kidney. These results suggested that LcNrdp1 might play an important immune role in the finfish L. crocea in the defense against the parasite C. irritans.