Single nucleotide polymorphism discovery and linkage mapping of lipoxygenase-2 gene (Lx 2) in soybean

Lipoxygenase-2 (Lx ₂) in soybean seed is mainly responsible for generation of grassy-beany and bitter flavors. Genetic elimination of this flavor can be accelerated by the development of single nucleotide polymorphism (SNP) markers linked to Lx ₂. A frame map based on simple sequence repeat (SSR) markers was constructed first using a recombinant inbred line (RIL) population of Pureunkong × Jinpumkong 2. Sixty-five SSR markers were incorporated into 13 linkage groups (LGs) spanning a total of 737 cM. Among five primer pairs designed from the Lx ₂ gene sequence, one produced an amplicon with sequence variations between Pureunkong and Jinpumkong 2. Three SNPs, T/C, G/A and C/A, were identified at 251,367 and 420 bp, respectively, in the intron region of the 804 bp amplified product. Using single base chain extension based on the capture probe sequence in the 5' region of the T/C SNP, the 90 RILs were genotyped for each allele of Lx ₂. The allelic segregation for the SNP linked toLx ₂ was in accordance with the expected ratio of 1:1 in the RIL population. Based on the results of linkage analysis between Lx ₂ and the SSR markers, Lx ₂ was found to be positioned on one end of LG F in the frame map, flanked by the SSR markers Satt522 and Sat074. This study demonstrates that SNP markers closely linked to Lx ₂can be developed to facilitate marker-assisted selection and fine mapping of the region around this locus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of light on endogenous levels of gibberellin and abscisic acid in seed germination of photoblastic weedy rice (Oryza sativa L.)

The effect of red (R) and R/far-red (FR) lights on endogenous gibberellin (GA) and abscisic acid (ABA) content was first investigated during the germination of photoblastic black-hulled weedy rice (PBWR) seeds. The R light-treated PBWR seeds germinated after 36–48 h and germination was increased to 63% at 72 h. However, the FR light-treated seeds after R light treatment, suppressed the R light effect showing only 11% germination even at 72 h after the light treatment. The PBWR seed treated with R light rapidly increased the endogenous level of GA₁ to about 200 times at 12 h before seed germination as compared with R/FR (control) which suppressed the effect of R light. The contents of other GAs like GA₁₂, GA₅₃, GA₁₉, GA₂₀, and GA₈ were not affected by the R light irradiation. These results showed that the major biosynthetic pathway of GAs in PBWR seeds is the early 13-hydroxylation pathway leading to GA₁, which was suggested to be physiologically active in the PBWR seed germination. The decrease in the level of ABA in the R light-treated seeds was greater than the R/FR light-treated seeds, indicating that the balance of endogenous GA1 and ABA is responsible for the induction of germination in the PBWR seed.     

Haplotype analysis of CMS-associated DNA markers in sweet peppers

Cytoplasmic male sterility (CMS)/restorer-of-fertility (Rf) is an economical and efficient system to produce F₁ hybrid seeds. Although the CMS/Rf system has been used to produce hybrid seeds of hot peppers, this system has never been used for sweet pepper seed production, presumably due to the inability to select stable restorer lines during the breeding process. To test the feasibility of the CMS/Rf system in sweet pepper breeding, we investigated the distribution of haplotypes of previously developed, CMS-associated markers (orf456, ψ atp6-2, CRF-SCAR, OPP13-CAPS, PR-CAPS, and PR-SNP) in 27 commercial sweet pepper F₁ hybrids and 12 breeding lines. When CMS-associated cytoplasmic markers orf456 and ψ atp6-2 were applied, male sterile cytoplasm was not detected in commercial sweet pepper cultivars. When nuclear haplotype markers linked to Rf were applied, all sweet pepper cultivars showed haplotype 3, haplotype 1, and the rf genotype for OPP13-CAPS, PR-CAPS, and CRF-SCAR, respectively. In contrast, we were able to detect male sterile cytoplasm in some breeding lines, and we were also able to detect polymorphisms for PR-CAPS between stable and unstable maintainer lines. The 17T7-SNP also showed polymorphisms between unstable and stable maintainer (or restorer) lines. In conclusion, we expect that it will be possible to develop stable A, B, and C sweet pepper lines using CMS-associated markers and that this will eventually lead to successful implementation of the CMS/Rf system to produce F₁ hybrid sweet pepper seeds.     

Mapping wound-response genes involved in post-harvest physiological deterioration (PPD) of cassava (Manihot esculenta Crantz)

The genome locations of the wound-response genes that were expressedduring the post-harvest physiological deterioration (PPD) of cassava, suchas phenylalanine ammonia lyase, -1.3 glucanase, hydroxyprolinerich glycoprotein, catalase, 1-aminocyclopropane 1-carboxylate, cysteineprotease inhibitor, aspartic protease, a partial cDNA for serine/threonineprotein kinase and peroxidase, have been identified on the frameworkmolecular genetic map of cassava. Also, molecular markers linked toputative quantitative trait loci (QTLs) influencing PPD of cassava weremapped using an F₁mapping population derived from elite parentallines (TMS 30572 × cm 2177-2). A molecular linkage map previouslyconstructed based on the segregation of 240 RFLP, 100 RAPD, 85microsatellite and five isoenzyme markers on 144 F₁ individuals wasused for the QTL mapping.A set of 10 molecular markers with a significant association with putativeQTLs for PPD were identified based on probability values < 0.005in order to minimize the detection of false positives. Based on single-markerregression, eight putative QTLs located on the linkage groups G, P, L, U,and X of the female-derived framework map were found to explain between 5–12% of the phenotypic variance of the PPD. In the male-derived frameworkmap, two putative QTLs on linkage groups C and L explained 13% and11% of this variance, respectively. This study thus identified the majorgenome regions of cassava related to physiological post-harvestdeterioration, thereby providing tools for the identification of gene(s)controlling this trait.     

Identification of QTLs for fruit quality traits in Japanese apples: QTLs for early ripening are tightly related to preharvest fruit drop

Many important apple (Malus × domestica Borkh.) fruit quality traits are regulated by multiple genes, and more information about quantitative trait loci (QTLs) for these traits is required for marker-assisted selection. In this study, we constructed genetic linkage maps of the Japanese apple cultivars ‘Orin’ and ‘Akane’ using F₁ seedlings derived from a cross between these cultivars. The ‘Orin’ map consisted of 251 loci covering 17 linkage groups (LGs; total length 1095.3 cM), and the ‘Akane’ map consisted of 291 loci covering 18 LGs (total length 1098.2 cM). We performed QTL analysis for 16 important traits, and found that four QTLs related to harvest time explained about 70% of genetic variation, and these will be useful for marker-assisted selection. The QTL for early harvest time in LG15 was located very close to the QTL for preharvest fruit drop. The QTL for skin color depth was located around the position of MYB1 in LG9, which suggested that alleles harbored by ‘Akane’ are regulating red color depth with different degrees of effect. We also analyzed soluble solids and sugar component contents, and found that a QTL for soluble solids content in LG16 could be explained by the amount of sorbitol and fructose.     

Regiospecific flavonoid 7-O-methylation with Streptomyces avermitilis O-methyltransferase expressed in Escherichia coli

O-Methylation, commonly found in synthesis of secondary metabolites of plants and micro-organisms, appears to transfer a methyl group to the hydroxyl group of the recipient which increases the hydrophobicity of the recipient. O-Methyltransferase (OMT), , was isolated and characterized from Streptomyces avermitilis MA-4680. Its amino acid sequence showed 68% similarity with antibiotic C-1027 OMT and 53% similarity with the carminomycin 4-OMT. was expressed in E. coli as a His-tag fusion protein and showed that the methyl was transferred onto the 7-hydroxyl group of the isoflavones, daidzein and genistein, and the flavones, kaempferol and quercetin, as well as the flavanone naringenin. NMR and liquid chromatography-mass spectrometry were used to confirm the location of the methyl group on the recipient compound of naringenin, which was biotransformed into sakuranetin by E. coli transformant expressing (E. coli Sa-2). Therefore, E. coli Sa-2 would be used for the synthesis of the antifungal flavonoid, sakuranetin, through biotransformation.     

Quantitative structure-activity relationship study of bitter peptides

A database consisting of 224 di- to tetradecapeptides and five amino acids was compiled to study quantitative structure-activity relationships of bitter peptides. Partial least-squares regression-1 analysis was conducted using the amino acid three z-scores and/or three parameters (total hydrophobicity, residue number, and log mass values) as X-variables and bitterness values (log 1/T where T is the bitterness threshold) as Y-variables. Using the three parameters only, significant models (p < 0.001) were obtained describing the entire data set as well as data subsets, except that comprised only of octa- to tetradecapeptides. For data sets comprising different peptide lengths, the models were improved by including the three z-scores at the N-terminal and C-terminal positions. Correlation coefficients for bitterness prediction of 48 dipeptides and 12 pentapeptides were 0.75 (RMSEP = 0.53) and 0.90 (RMSEP = 0.48), respectively. Bulky hydrophobic amino acids at the C terminus and bulky basic amino acids at the N terminus were highly correlated to bitterness.     

Enzymatic synthesis and properties of highly branched rice starch amylose and amylopectin cluster

We enzymatically modified rice starch to produce highly branched amylopectin and amylose and analyzed the resulting structural changes. To prepare the highly branched amylopectin cluster (HBAPC), we first treated waxy rice starch with Thermus scotoductus alpha-glucanotransferase (TSalphaGT), followed by treatment with Bacillus stearothermophilus maltogenic amylase (BSMA). Highly branched amylose (HBA) was prepared by incubating amylose with Bacillus subtilis 168 branching enzyme (BBE) and subsequently treating it with BSMA. The molecular weight of TSalphaGT-treated waxy rice starch was reduced from 8.9 x 10(8) to 1.2 x 10(5) Da, indicating that the alpha-1,4 glucosidic linkage of the segment between amylopectin clusters was hydrolyzed. Analysis of the amylopectin cluster side chains revealed that a rearrangement in the side-chain length distribution occurred. Furthermore, HBAPC and HBA were found to contain significant numbers of branched maltooligosaccharide side chains. In short, amylopectin molecules of waxy rice starch were hydrolyzed into amylopectin clusters by TSalphaGT in the enzymatic modification process, and then further branched by transglycosylation using BSMA. HBAPC and HBA showed higher water solubility and stability against retrogradation than amylopectin clusters or branched amylose. The hydrolysis rates of HBAPC and HBA by glucoamylase and alpha-amylase greatly decreased. The k cat/ K m value of glucoamylase acting on the amylopectin cluster was 45.94 s(-1)(mg/mL)(-1) and that for glucoamylase acting on HBAPC was 11.10 s(-1)(mg/mL)(-1), indicating that HBAPC was 4-fold less susceptible to glucoamylase. The k cat/ K m value for HBA was 15.90 s(-1)(mg/mL)(-1), or about three times less than that for branched amylose. The k cat/ K m values of porcine pancreatic alpha-amylase for HBAPC and HBA were 496 and 588 s(-1)(mg/mL)(-1), respectively, indicating that HBA and HBAPC are less susceptible to hydrolysis by glucoamylase and alpha-amylase. HBAPC and HBA show potential as novel glucan polymers with low digestibility and high water solubility.     

Ammonia oxidizers and denitrifiers in response to reciprocal elevation translocation in an alpine meadow on the Tibetan Plateau

Purpose

Global climate change, in particular temperature variation, is likely to alter soil microbial abundance and composition, with consequent impacts on soil biogeochemical cycling and ecosystem functioning. However, responses of belowground nitrogen transformation microorganisms to temperature changes in high-elevation terrestrial ecosystems are not well understood.

Materials and methods

Here, the effects of simulated cooling and warming on the abundance and community composition of ammonia-oxidizing archaea (AOA) and bacteria (AOB), as well as the abundance of denitrifiers, were investigated using quantitative polymerase chain reaction and clone library approaches, on the basis of a 2-year reciprocal elevation translocation experiment along an elevation gradient from 3,200 to 3,800 m above sea level on the Tibetan Plateau.

Results and discussion

We found that, compared with the temperature variations caused by elevation translocation, the soil origin exerted a much stronger influence on AOA abundance. There were significant effects of both soil origin and elevation translocation on AOB abundance, which was particularly decreased by elevation-enhanced (simulated cooling) and increased by elevation-decreased (simulated warming) treatments. Altered temperature affected the abundance of nirK rather than nirS and nosZ genes, and the latter two seemed to be associated tightly with the soil origin. Furthermore, the results showed that temperature changes had obvious influences on the community structure and diversity of AOB, but not AOA. More apparent response of AOB to warming than in other studies on grassland and forest ecosystems may be attributed to higher elevation and lower mean annual temperature in this study.

Conclusions

Our findings thus suggest that, in comparison with AOA and denitrifying populations, AOB may respond more sensitively to natural temperature variation caused by elevation translocation in this alpine grassland ecosystem on the Tibetan Plateau.     

Antimicrobial activity of p-hydroxyphenyl acrylate derivatives

To estimate the antimicrobial effect of p-hydroxyphenyl acrylate (H5) derivatives on the basis of their molecular structure, the hydroxy and acryl groups of p-hydroxyphenyl acrylate were modified. The antimicrobial activity of the resulting compounds was assessed against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Pseudomonas aeruginosa), and fungi (Aspergillus fumigatus and Penicillium pinphilum) by the halo zone and the shake flask test. The antimicrobial activity of H5 was ascribed mainly to the acryl group. Compounds with acryl or acryloxy groups bound to the phenyl moiety were found to exhibit particularly high antimicrobial activities. The activities of phenyl acrylate and phenyl vinyl ketone were excellent as compared to aliphatic acrylates such as cyclohexyl acrylate and hexyl acrylate, indicating that the stereoelectronic effect of the phenyl group was important to the antimicrobial activity.