Genome‐wide association study discovered candidate genes of Verticillium wilt resistance in upland cotton (Gossypium hirsutum L.)

Verticillium wilt (VW), caused by infection by Verticillium dahliae, is considered one of the most yield‐limiting diseases in cotton. To examine the genetic architecture of cotton VW resistance, we performed a genome‐wide association study (GWAS) using a panel of 299 accessions and 85 630 single nucleotide polymorphisms (SNPs) detected using the specific‐locus amplified fragment sequencing (SLAF‐seq) approach. Trait–SNP association analysis detected a total of 17 significant SNPs at P < 1.17 × 10^–5 (P = 1/85 630, –log₁₀P = 4.93); the peaks of SNPs associated with VW resistance on A10 were continuous and common in three environments (RDIG2015, RDIF2015 and RDIF2016). Haplotype block structure analysis predicted 22 candidate genes for VW resistance based on A10_99672586 with a minimum P‐value (–log₁₀P = 6.21). One of these genes (CG02) was near the significant SNP A10_99672586 (0.26 Mb), located in a 372‐kb haplotype block, and its Arabidopsis AT3G25510 homologues contain TIR‐NBS‐LRR domains that may be involved in disease resistance response. Real‐time quantitative PCR and virus‐induced gene silencing (VIGS) analysis showed that CG02 was specific to up‐regulation in the resistant (R) genotype Zhongzhimian2 (ZZM2) and that silenced plants were more susceptible to V. dahliae. These results indicate that CG02 is likely the candidate gene for resistance against V. dahliae in cotton. The identified locus or gene may serve as a promising target for genetic engineering and selection for improving resistance to VW in cotton.     

Promotion, termination, and anti-termination in the rpsU-dnaG-rpoD macromolecular synthesis operon of E. coli K-12

Summary The regulatory regions for the rpsU-dnaG-rpoD macromolecular synthesis operon have been fused to a structural gene whose product is readily assayed (the Cm^r structural gene coding for chloramphenicol acetyl transferase, CAT). The promoters (P₁, P₂, P₃, P_a, P_b, P_hs) for the macromolecular synthesis operon have different strengths as shown by their relative abilities to drive expression of the CAT gene. Promoter occlusion by P₁ can be demonstrated within this operon. Regions 5kb upstream have a profound effect on operon gene expression. There is a thermoinducible promoter located within the dnaG structural gene. One of the macromolecular synthesis operon promoters is under lexA control. Although the operon structure allows coordinate expression of rpsU, dnaG and rpoD these additional features suggest that expression of individual genes can be independently regulated in response to altered growth conditions.Abbreviations Ap^r ampicillin resistance - CAT chloramphenicol acetyl transferase - Cm^r chloramphenicol resistance - kb kilobase pair - orf open reading frame - P promoter - T terminator - Tc^r tetracycline resistance     

Growth performance of hybrid families by crossing selfed lines of Betula pendula Roth

Hybrid breeding is an effective approach in many agricultural crops. In allogamous tree species severe inbreeding depression and long reproductive cycles generally prohibit its use. However, three generations of selfing in silver birch (Betula pendula Roth) were obtained by forcing trees to flowering under greenhouse conditions. Hybrids were produced by crossing first-, second and third-generation selfed lines. The effects of different levels of parental inbreeding on the growth performance of hybrid families were observed in a 9-year-old field progeny test. Also, provenance crosses were carried out between selfed lines from different parts of Finland and several other European countries. Observations of growth performance of the provenance hybrids were made in the same trial. The results indicated that the mean stem volumes were significantly different between classes of parental in breeding coefficients (F_P) (P<0.0001), and were positively correlated with F_P (r=0.9106, P<0.05). Within-family variation of the hybrid families decreased with an increase of F_P. The performance of the provenance crosses between parents at a relatively close distance did not depart significantly from the standard controls. However, when the cross distance was extended far to the south, hybrids grew faster, indicating either higher heterozygosity or an extended growth period.     

Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli

Oxidized polyvinyl alcohol (PVA) hydrolase (OPH) is a key enzyme in the degradation of PVA, suggesting that OPH has a great potential for application in textile desizing processes. In this study, the OPH gene from Sphingopyxis sp. 113P3 was modified, by artificial synthesis, for overexpression in Escherichia coli. The OPH gene, lacking the sequence encoding the original signal peptide, was inserted into pET-20b (+) expression vector, which was then used to transform E. coli BL21 (DE3). OPH expression was detected in culture medium in which the transformed E. coli BL21 (DE3) was grown. Nutritional and environmental conditions were investigated for improved production of OPH protein by the recombinant strain. The highest OPH activity measured was 47.54 U/mL and was reached after 84 h under optimal fermentation conditions; this level is 2.64-fold higher that obtained under sub-optimal conditions. The productivity of recombinant OPH reached 565.95 U/L/h. The effect of glycine on the secretion of recombinant OPH was examined by adding glycine to the culture medium to a final concentration of 200 mM. This concentration of glycine reduced the fermentation time by 24 h and increased the productivity of recombinant OPH to 733.17 U/L/h. Our results suggest that the recombinant strain reported here has great potential for use in industrial applications.     

CLEISTOGAMY IN ANDROPOGON L. (GRAMINEAE)

Eleven species of Andropogon produce single-stamened flowers, and all but one of these species bear some cleistogamous flowers. Nine of the species, all diploid perennials, make up the Andropogon virginicus complex. Cleistogamy in the virginicus complex results from precocious maturation of the flowers, which leaves them confined within their spikelets at anthesis. Variation in flowering mode (expressed as mean % chasmogamy) is correlated significantly (P < 0.01) with variation in peduncle length (r_s = 0.67), spikelet length (r_s = 0.49), and anther length (r_s = 0.69). The strength of the correlation (r = 0.94) between pollen grain number per anther and anther length makes the latter a good measure of pollen-ovule ratios in the single-stamened flowers of this group. Taxa with the highest frequency of cleistogamous flowering are also the most successful colonizers of disturbed habitats. It is suggested that cleistogamy is important in the reproductive isolation of the numerous, closely related subspecific taxa of the virginicus complex.     

Evaluation of physical fitness from field tests at high altitude in circumpubertal boys: comparison with laboratory data

Field tests of running and laboratory tests were performed in La Paz [high altitude (HA), 3700 m] and in Clermont-Ferrand [low altitude (LA), 300 m] to investigate their validity at HA. Prepubertal boys of mean ages 10.6 years (HA1,n = 16; LA1,n = 28) and pubertal boys of 13.7 years (HA2,n = 12; LA2,n = 41) took part in the study. All the boys performed a 30-m sprint (v _30m), a 30-s shuttle run (v _3os) and a progressive shuttle run test until their maximal aerobic velocity (v _maxsRT). Maximal oxygen consumption was extrapolated from the last test. . In the laboratory, the boys performed a force-velocity test (P _max), a Wingate test (P _Wing) and a graded test to measure maximal oxygen consumption ; direct method) on a cycle ergometer. At similar ages, there was no significant difference between HA and LA boys forv _30m andP _max. Thev _30s of HA boys was 3%–4% lower than those of LA boys (P<0.05); there was no significant difference forP _Wing. Significant relationships were observed at both altitudes betweenP _max (watts per kilogram) andv _30m (HA:r=0.76; LA:r=0.84) and betweenP _Wing andv _30s (HA:r=0.67; LA:r = 0.77); the slopes and the origins were the same at HA and LA. The ,v _maxSRT and were lower by 9%, 12% and 20%, respectively, at HA than at LA (P<0.05). However, the relationships between and (litres per minute) at HA (r=0.88) and at LA (r=0.93) were identical. In conclusion, chronic hypoxia did not modify performance in very short dash exercises. The influence of HA appeared when the exercise duration increased and, during a maximal shuttle run test, performance was reduced by 10% at HA. Moreover, it was possible to assessP _max,P _Wing and at HA as well as at LA from field tests.     

Cloning and heterologous expression of a novel arylmalonate decarboxylase gene from Alcaligenes bronchisepticus KU 1201

We have cloned and sequenced a DNA fragment that encodes the arylmalonate decarboxylase (AMDase) gene from Alcaligenes bronchisepticus KU 1201. The AMDase gene consists of an open reading frame of 720 nucleotides, which specifies a 240-amino-acid protein of relative molecular mass (M_r) 24734. The M_r deduced from the AMDase gene is in good agreement with that of the AMDase isolated from A. bronchisepticus. No TATA or TTGA sequence was observed within the cloned DNA fragment, but the fragment was expressed in Escherichia coli by the lac promoter of pUC19. The enzyme produced in E. coli has the same M_r and the same enzyme activity as the purified from A. bronchisepticus. Comparison of the DNA sequence and the deduced amino acid sequence of AMDase with available DNA and amino acid sequence data bases revealed that there are no significant sequence homologies.Correspondence to: Hiromichi Ohta     

Comparison of phenotypic and molecular distances to predict heterosis and F1 performance in Ethiopian mustard (Brassica carinata A. Braun)

Predicting heterosis and F₁ performance from the parental generation could largely enhance the efficiency of breeding hybrid or synthetic cultivars. This study was undertaken to determine the relationship between parental distances estimated from phenotypic traits or molecular markers with heterosis, F₁ performance and general combining ability (GCA) in Ethiopian mustard (Brassica carinata). Nine inbred lines representing seven different geographic regions of Ethiopia were crossed in half-diallel. The nine parents along with their 36 F₁s were evaluated in a replicated field trail at three locations in Ethiopia. Distances among the parents were calculated from 14 phenotypic traits (Euclidean distance, ED) and 182 random amplified polymorphic DNA (RAPD) markers (Jaccard’s distances, JD), and correlated with heterosis, F₁ performance and GCA sum of parents (GCA_sum). The correlation between phenotypic and molecular distances was low (r=0.34, P≤0.05). Parents with low molecular distance also had low phenotypic distance, but parents with high molecular distance had either high, intermediate or low phenotypic distance. Phenotypic distance was highly significantly correlated with mid-parent heterosis (r=0.53), F₁ performance (r=0.61) and GCA (r=0.79) for seed yield. Phenotypic distance was also positively correlated with (1) heterosis, F₁ performance and GCA for plant height and seeds plant⁻¹, (2) heterosis for number of pods plant⁻¹, and (3) F₁ performance for 1,000 seed weight. Molecular distance was correlated with GCA_sum (r=0.36, P≤0.05) but not significantly with heterosis and F₁ performance for seed yield. For each parent a mean distance was calculated by averaging the distances to the eight other parents. Likewise, mean heterosis was estimated by averaging the heterosis obtained when each parent is crossed with the other eight. For seed yield, both mean ED and JD were significantly correlated with GCA (r=0.90, P≤0.01 for ED and r=0.68, P≤0.05 for JD) and mean heterosis (r=0.79, P≤0.05 for ED and r=0.77, P≤0.05 for JD). In conclusion, parental distances estimated from phenotypic traits better predicted heterosis, F₁ performance and GCA than distances estimated from RAPD markers. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Impact of light quality on leaf and shoot hydraulic properties: a case study in silver birch (Betula pendula)

Responses of leaf and shoot hydraulic conductance to light quality were examined on shoots of silver birch (Betula pendula), cut from lower (‘shade position’) and upper thirds of the crowns (‘sun position’) of trees growing in a natural temperate forest stand. Hydraulic conductances of leaf blades (K_lb), petioles (K_P) and branches (i.e. leafless stem; K_B) were determined using a high pressure flow meter in steady state mode. The shoots were exposed to photosynthetic photon flux density of 200–250 µmol m^?2 s^?1 using white, blue or red light. K_lb depended significantly on both light quality and canopy position (P < 0.001), K_B on canopy position (P < 0.001) and exposure time (P = 0.014), and none of the three factors had effect on K_P. The highest values of K_lb were recorded under the blue light (3.63 and 3.13 × 10^?4 kg m^?2 MPa^?1 s^?1 for the sun and shade leaves, respectively), intermediate values under white light (3.37 and 2.46 × 10^?4 kg m^?2 MPa^?1 s^?1, respectively) and lowest values under red light (2.83 and 2.02 × 10^?4 kg m^?2 MPa^?1 s^?1, respectively). Light quality has an important impact on leaf hydraulic properties, independently of light intensity or of total light energy, and the specific light receptors involved in this response require identification. Given that natural canopy shade depletes blue and red light, K_lb may be decreased both by reduced fluence and shifts in light spectra, indicating the need for studies of the natural heterogeneity of K_lb within and under canopies, and its impacts on gas exchange.     

On the phytochrome phototransformation kinetics in mustard seedlings

Summary The in vivo phototransformation kinetics of mustard hook and cotyledon phytochrome exhibit a deviation from a single first order curve, quite similar to that for pumpkin hooks as reported in a previous paper (Boisard, Marmé and Schäfer, 1971). The P _frP_rkinetics can be characterized by the ratios _fr, ^I · P _fr ^I / _fr, ^II · P _fr, ^II and where P _fr ^I and P _fr ^II are two populations of phytochrome molecules which convert to P _rwith a first order half-life of and . These ratios depend on the length of time of etiolation. The ratio _fr, ^I · P _fr ^I / _fr, ^II · P _fr, ^II is independent of the amount of total P _frpresent at the beginning of the P _frP_rphototransformation after a non-saturating dose of red light. The half-lives of the two populations, however, depend on the concentration of total P _frinitially present. P _frP_rphototransformation kinetics with different light intensities show that reciprocity holds.     

Proton NMR study of the interaction of cis-dichloro-diamine-platinum(II) with poly(I) and poly(I)·poly(C)

The fixation of cis (NH₃)₂Cl₂Pt(II) to poly(I)·poly(C) leads to the formation of two complexed species. One involves coordination to a single base (accounting for about 70% of the total platinum bound over the r_b range 0.07–0.25) and the other to two bases which are not adjacent to each other but may be on the same strand and separated by a loop. Reaction of the platinum compound with poly(I) gives in addition to the above two species a minor one (about 15%, independent of r_b over the range 0.05–0.30) in which the platinum is bound to two adjacent bases. The availability of such coordination reduces the dominance of the 1:1 species, which, however, remains the major one (ca. 55%).     

Identification of QTL regions and SSR markers associated with resistance to reniform nematode in <Emphasis Type="Italic">Gossypium barbadense</Emphasis> L. accession GB713

The identification of molecular markers that are closely linked to gene(s) in Gossypium barbadense L. accession GB713 that confer a high level of resistance to reniform nematode (RN), Rotylenchulus reniformis Linford & Oliveira, would be very useful in cotton breeding programs. Our objectives were to determine the inheritance of RN resistance in the accession GB713, to identify SSR markers linked with RN resistance QTLs, and to map these linked markers to specific chromosomes. We grew and scored plants for RN reproduction in the P₁, P₂, F₁, F₂, BC₁P₁, and BC₁P₂ generations from the cross of GB713 × Acala Nem-X. The generation means analysis using the six generations indicated that one or more genes were involved in the RN resistance of GB713. The interspecific F₂ population of 300 plants was genotyped with SSR molecular markers that covered most of the chromosomes of Upland cotton (G. hirsutum L.). Results showed two QTLs on chromosome 21 and one QTL on chromosome 18. One QTL on chromosome 21 was at map position 168.6 (LOD 28.0) flanked by SSR markers, BNL 1551_162 and GH 132_199 at positions 154.2 and 177.3, respectively. A second QTL on chromosome 21 was at map position 182.7 (LOD 24.6) flanked by SSR markers BNL 4011_155 and BNL 3279_106 at positions 180.6 and 184.5, respectively. Our chromosome 21 map had 61 SSR markers covering 219 cM. One QTL with smaller genetic effects was localized to chromosome 18 at map position 39.6 (LOD 4.0) and flanked by SSR markers BNL 1721_178 and BNL 569_131 at positions 27.6 and 42.9, respectively. The two QTLs on chromosome 21 had significant additive and dominance effects, which were about equal for each QTL. The QTL on chromosome 18 showed larger additive than dominance effects. Following the precedent set by the naming of the G. longicalyx Hutchinson & Lee and G. aridum [(Rose & Standley) Skovsted] sources of resistance, we suggest the usage of Ren ^barb1 and Ren ^barb2 to designate these QTLs on chromosome 21 and Ren ^barb3 on chromosome 18.     

Enzyme antigens associated with pigeon breeder's disease. I. Isolation and characterization of basic hydrolases

A survey of the hydrolytic enzymes present in pigeon dropping extracts (PDE) has shown that this material contains a variety of proteolytic and nonproteolytic activities. These enzymes were separated into their basic and acidic components by chromatography on DEAE-cellulose. Staining of immunoprecipitates with specific chromogenic substrates demonstrated the presence of antibodies in symptomatic breeders to several of the basic enzymes in PDE. Five distinct hydrolytic activities were isolated from the basic group of enzymes. Trypsin, elastase, and two forms of collagenase were the specific proteolytic activities isolated. A phospholipase was also purified from these preparations. The purified elastase consisted of a single polypeptide chain (M _r =22,000). The purified trypsin had a molecular weight (M _r =25,000) and charge similar to those reported for elastase and, like elastase, the trypsin from PDE appeared to be composed of a single polypeptide chain. Two molecular weight forms of collagenase were found; both hydrolyzed bovine collagen. The high-molecular-weight collagenase (M _r =51,000) was shown to be a glycoprotein consisting of two polypeptides (M _r =24,000). It was readily separated from the low-molecular-weight collagenase (M _r =15,000) by gel filtration. The phospholipase (M _r =99,000) appeared to be a dimer. The relevance of these enzymes to the development of pigeon breeder's disease is discussed.     

High level recombinant protein expression in Ralstonia eutropha using T7 RNA polymerase based amplification

We report further development of a novel recombinant protein expression system based on the Gram-negative bacterium, Ralstonia eutropha. In this study, we were able to express soluble, active, organophosphohydrolase (OPH), a protein that is prone to inclusion body formation in Escherichia coli, at titers greater than 10 g/L in high cell density fermentation. This represents a titer that is approximately 100-fold greater than titers previously reported in E. coli for this enzyme. R. eutropha strains expressing OPH were generated in two cloning steps. First, the T7 RNA polymerase gene was placed under the control of the strong, inducible phaP promoter and integrated into the phaP locus of R. eutropha NCIMB 40124. Second, a single copy of the oph gene under control of the T7 promoter was randomly integrated into the chromosome using a transposon cloning vector.     

Metapopulation structure,population trends,and status of Florida Grasshopper Sparrows

ABSTRACT The federally Endangered Florida Grasshopper Sparrow (FGSP, Ammodramus savannarum floridanus) is endemic to dry prairie habitat in central Florida and is currently only found at three public management areas (Avon Park Air Force Range, APAFR; Kissimmee Prairie Preserve State Park, KPPSP; and Three Lakes Wildlife Management Area, TLWMA). We analyzed long‐term (1991–2008) point‐count data to compare population trends of FGSPs at these management areas to determine if they function as independent populations by using Spearman's rank correlation to test for independence between annual trends. We also examined banding and resighting data to infer metapopulation structure. Populations fluctuated across years at all three sites, declining significantly at APAFR (r_s=?0.89, P < 0.001, N= 13) and KPPSP (r_s=?0.78, P= 0.004, N= 11) and remaining stable at TLWMA (r_s= 0.19, P= 0.46, N= 17). Population trends among the three management areas appeared independent (absolute value of r_s≤ 0.50, P≥ 0.12). Previous studies indicated that sparrows in the three areas were not genetically differentiated, and two cases of dispersal between APAFR and KPPSP have been documented. However, dispersal rates among areas appear to be too low to influence demographic dynamics within individual areas. Within APAFR, FGSPs are aggregated into three spatially distinct habitat patches previously considered separate populations, but dispersal among these patches is more frequent than previously reported and population trends among these patches are correlated (r_s≥ 0.91, P < 0.001). These patterns suggest that a single metapopulation of FGSPs exists consisting of three distinct populations (APAFR, KPPSP, and TLWMA), and the spatially distinct aggregations at APAFR constitute a single population using several habitat patches. The population at APAFR is at risk of extirpation, and immediate action is needed if that population is to recover. Taking broader metapopulation dynamics into account will be useful for guiding management efforts aimed at conserving the FGSP in the broader central Florida landscape.     

Interspecific variation in sustained swimming ability of late pelagic stage reef fish from two families (Pomacentridae and Chaetodontidae)

 Characteristics of the pelagic stages of reef fishes have generally been investigated at the family level, which may mask important differences among species. Here the variation in sustained swimming ability of the late pelagic stages is examined among species, within two families (Pomacentridae and Chaetodontidae). The pomacentrids displayed a 7.5-fold difference in sustained swimming ability across 24 species, while the chaetodontids displayed a 2-fold difference across 10 species. The variation within the Pomacentridae was not related to pelagic larval duration, post-settlement habitat or taxonomy. There was, however, a significant correlation between sustained swimming ability and total length (TL) of individuals (r=0.435, P<0.0001). Differences in the mean distance swum by pomacentrid species, however, was most strongly related to differences in mean wet weight (r=0.814, P<0.0001). When the mean distance swum by species was scaled with respect to mean TL there was still a strong correlation with mean wet weight (r=0.644, P<0.005). Among chaetodontid individuals TL and sustained swimming ability were not correlated (r=−0.004, P=0.978). Furthermore, sustained swimming ability was not significantly related to the trans-oceanic distribution of species in either family. The variation in sustained swimming ability, however, may contribute to explanations of the observed levels of gene flow within populations. Accepted: 24 November 1997     

The Gene Expression of the Main Lipogenic Enzymes is Downregulated in Visceral Adipose Tissue of Obese Subjects

Contradictory findings regarding the gene expression of the main lipogenic enzymes in human adipose tissue depots have been reported. In this cross‐sectional study, we aimed to evaluate the mRNA expression of fatty acid synthase (FAS) and acetyl‐CoA carboxilase (ACC) in omental and subcutaneous (SC) fat depots from subjects who varied widely in terms of body fat mass. FAS and ACC gene expression were evaluated by real time‐PCR in 188 samples of visceral adipose tissue which were obtained during elective surgical procedures in 119 women and 69 men. Decreased sex‐adjusted FAS (?59%) and ACC (?49%) mRNA were found in visceral adipose tissue from obese subjects, with and without diabetes mellitus type 2 (DM‐2), compared with lean subjects (both P < 0.0001). FAS mRNA was also decreased (?40%) in fat depots from overweight subjects (P < 0.05). Indeed, FAS mRNA was significantly and positively associated with ACC gene expression (r = 0.316, P < 0.0001) and negatively with BMI (r = ?0.274), waist circumference (r = ?0.437), systolic blood pressure (r = ?0.310), serum glucose (r = ?0.277), and fasting triglycerides (r = ?0.226), among others (all P < 0.0001). Similar associations were observed for ACC gene expression levels. In a representative subgroup of nonobese (n = 4) and obese women (n = 6), relative FAS gene expression levels significantly correlated (r = 0.657, P = 0.034; n = 10) with FAS protein values. FAS protein levels were also inversely correlated with blood glucose (r = ?0.640, P = 0.046) and fasting triglycerides (r = ?0.832, P = 0.010). In conclusion, the gene expression of the main lipogenic enzymes is downregulated in visceral adipose tissue from obese subjects.     

Proton-NMR study of the interaction of trans-dichloro-diamine-platinum(II) with poly(I) and poly(I).poly(C)

The fixation of trans-(NH₃)₂Cl₂ Pt(II) to poly(I)·poly(C) at low r_b (< 0.05) leads to the formation of two complexed species. The major species (ca. 82% of bound platinum) involves coordination of platinum to a single hypoxanthine base, while the other species involves coordination of two hypoxanthine bases, which are either far apart on the same strand or on separate poly(I) strands, to the platinum. These same two species are found after reaction with poly(I), as are two other species throughout the entire r_b range studied (r_b = 0–0.30). The latter two species are assigned to trans-Pt bound to two bases on a poly(I) strand with (a) one or (b) two free bases between the two bound bases. These two species, (a) and (b), account for ca. 35% of the bound platinum, although the 1:1 species remains dominant (ca. 55%). These two additional species are observed at high r_b (>0.075) after reaction with poly(I)·poly(C) but as very minor species. They are formed by reaction with melted poly(I) loops. Also at high r_b, we have observed a shifted cytidine H⁵ resonance arising from interaction of trans-Pt with a melted loop of poly(C). Most probably, this arises from an intramolecular poly(I) to poly(C) crosslink. Results from the reaction of trans-Pt with poly(C) are presented for comparison.