Diversity at two genetic loci associated with the major histocompatibility complex in the golden snub-nosed monkey (Rhinopithecus roxellana)

Habitat loss via human activity has fragmented populations of the golden snub-nosed monkey (Rhinopithecus roxellana), and thus affected patterns of gene flow. We investigated in-depth a single troop in the Qinling Mountains, central China, two major histocompatibility complex (MHC) class II loci, DQA1 and DQB1, and compared the resulting data with data from troops from the wider Qinling Mountains region. We found that a novel DQB1 allele was only present in the study troop and relatively few divergent alleles at the DQA1 and DQB1 loci were present compared with the wider population. The inbreeding coefficient (F_is) at the MHC region was lower than previous measurements, which may have reflected different sampling strategies. However, R. roxellana has relatively high diversity in MHC genes, even though it has probably experienced serious past population bottlenecks and reduced gene flow between populations. We also found that some alleles present in the wider population had been lost in the study troop, and suggest that conservation management strategies be implemented to increase gene flow between troops in order to increase genetic variation.     

The genetic diversity and population structure of wild soybean evaluated by chloroplast and nuclear gene sequences

Glycine soja, also called wild soybean, is the wild ancestor of domesticated soybean (Glycine max), and one of the world's major cultivated crops. Wild soybean is a valuable resource for the breeding of cultivated soybean and harbors useful genes or agronomic traits. To use and conserve this valuable resource, we conducted a study to evaluate the genetic diversity and population structure of wild soybean using the sequencing data of two nuclear loci (AF105221 and PhyB) and one chloroplast locus (trnQ-rps16) of more than 600 individuals representing 53 populations throughout the natural distribution range. The results showed that most of the variation was found within the populations and groups, but significant genetic differentiation was also detected among different eco-geographical groups. Correlations between genetic and geographical distance at all the loci were consistent with the isolation by distance gene flow model. G. soja exhibited the highest genetic diversity in middle and downstream of Yangzi River (MDYR) region, followed by North East China (NEC), and was the lowest in North West China (NWC). We concluded that both in situ and ex situ conservation strategies required for wild soybean populations, especially which are native to MDYR and NEC regions.     

Insight into genetic relation and diversity of cultivated and semi-domesticated under-utilized Crotalaria species gained using start codon targeted (SCoT) markers

To augment conventional crop improvement approaches in cultivated sunnhemp (Crotalaria juncea L.) and other under-utilized Crotalaria species, genetic diversity of 94 genotypes from seven Crotalaria species was studied using 20 Start Codon Targeted (SCoT) markers. High allele number (1.32), polymorphism information content (0.37) and resolving power (6.59) established SCoT as a reliable marker system for genetic analysis in Crotalaria. All the species except Crotalaria retusa L. exhibited high number of SCoT amplicons. Analysis of molecular variance revealed significant variability between (24.0%) the species as well as within species (76.0%). A cluster analysis identified distinct groups corresponding to the seven species and also identified sub-groups within the species. The sunnhemp cultivars were distant from the landraces, suggesting the need of population improvement using distantly related genotypes. Species relationship identified Crotalaria pallida Aiton to be a close relative of C. juncea. The results of principal coordinate analysis were comparable to that of cluster analysis, revealing high genetic variability in sunnhemp and other semi-domesticated Crotalaria species. The study further suggests some measure for conservation of genetic resources and genetic improvement of these species based on the results of diversity analysis.     

Genetic diversity of wild population of Pyropia haitanensis based on SSR analysis

Genetic diversity analysis was conducted on 80 individuals of 4 populations of non-bred Pyropia haitanensis by simple sequence repeat (SSR) method. Using 15 pairs of microsatellite primers, 37 polymorphic loci were amplified, representing 94.9% of all loci. At the population level, the percentage of polymorphic bands (P) and polymorphism information content (PIC) were 66.67–84.62% and 0.481–0.488, with average value at 73.72% and 0.483, respectively. Expected heterozygosity (H_e) and Shannon's information index (I) were 0.279 and 0.434, respectively, at the species level, and 0.233 and 0.356 at population level. According to the coefficient of gene differentiation (G_ST), a large proportion of genetic variance (83.6%) of P. haitanensis was among individuals within populations, only 16.4% genetic variance was among populations, which was identified with the moderate gene flow value (N_m = 2.542). UPGMA clustered the 4 populations into 3 groups, and no significant correlation was found between the genetic distance and the corresponding geographic distance among the populations.     

Characterization of three pro-inflammatory cytokines,TNFα1, TNFα2 and IL-1β, in cage-reared Atlantic bluefin tuna Thunnus thynnus

Atlantic bluefin tuna (BFT) (Thunnus thynnus) is of great economic significance for world aquaculture and therefore it is necessary to ensure optimal and sustainable conditions for the farming of this species. Intensive culture of fish may be limited by infectious diseases that can impact on growth performance and cause heavy losses. However, to date there are no reports of cloning and expression analysis of any major immune genes of Atlantic BFT although some immune genes are known in other BFT species. Therefore the aim of this study was to characterize the first cytokine molecules in Atlantic BFT, through: 1) Isolation of full-length cDNA and gene sequences of TNFα1, TNFα2 and IL-1β, 2) comparison of these molecules to known sequences in other vertebrates, especially teleost fish, by multiple sequence alignment, phylogenetic tree analysis and homology modeling; 3) Quantification of in vivo expression of these cytokines in selected tissues in reared BFT over the duration of the farming process. The results indicated that these three cytokines could have value for monitoring Atlantic BFT health status. Curiously, the liver seemed to be an important site of cytokine production during poor health conditions in this species, perhaps reflecting its role as an important organ involved in fish defenses.     

Morphometric,meristic and ISSR marker systems for species identification and evolutionary analysis in five Indian Channids

Snakehead species belonging to Channidae are primary group of freshwater air breathing fishes having their confined distribution in African and Asian continents. ISSR – PCR was used to investigate the phylogenetic relationship among five Channidae species viz. Channa striatus, Channa marulius, Channa punctatus, Channa diplogramme and Channa gachua. In addition, morphometric and meristic characters were subjected to principal component analysis (PCA) and the bootstrap values within the species were also calculated. The genetic identity between the species ranged from 0.5526 to 0.7632 and the genetic distance ranged from 0.2703 to 0.5931. The Nei's gene diversity (H) was calculated as 0.2653 and the Shannon's information index (I) was 0.3842. UPGMA dendrogram arrived by the morphological and molecular markers revealed the closeness between C. striatus and C. marulius among the five species.     

Evolutionary and demographic history among Maghrebian Medicago species (Fabaceae) based on the nucleotide sequences of the chloroplast DNA barcode trnH-psbA

Data from trnH-psbA intergenic spacer (cpDNA) were analyzed to elucidate molecular evolution within and among Maghrebian species of Medicago. The spacer highlighted a high interspecific variation and a low intraspecific diversity among species. Haplotype and nucleotide diversities revealed high level of variation. Parsimony and median-joining Network methods revealed (1) the segregation into 17 haplotypes; (2) the ancestral behaviour of the annual Medicago minima and (3) the clusters are independent of the geographic origin. The neutral evolution of Wright and Fisher is rejected since the Tajima's D values deviated from 0. Besides, the statistical analyses are in agreement with an evolution into stable populations' size.     

Sternal gland structures in males of bean flower thrips,Megalurothrips sjostedti,and Poinsettia thrips,Echinothrips americanus,in comparison with those of western flower thrips,Frankliniella occidentalis (Thysanoptera: Thripidae)

Sternal pores are important features for identification of male thrips, especially within the subfamily Thripinae. They vary in shape, size and distribution even between species of one genus. Their functional role is speculated to be that of sex- and/or aggregation pheromone production. Yet, sexual aggregations are not reported in Echinothrips americanus, known to have sternal pores, while we observed aggregations in Megalurothrips sjostedti, previously reported to lack them.We examined the sternal glands and pores of the thripine species E. americanus and M. sjostedti males, in comparison with those of Frankliniella occidentalis using light microscopy, as well as scanning and transmission electron microscopy. Pore plates of F. occidentalis were ellipsoid and medial on sternites III–VII, while in E. americanus they were distributed as multiple micro pore plates on sternites III–VIII. In M. sjostedti they appeared as an extremely small pore in front of the posterior margin of each of sternites IV–VII. Pore plate and pore plate area were distributed similarly on sternites III–VII in F. occidentalis. However, in E. americanus the total pore plate area increased significantly from sternites III to VIII. Ultrastructure of cells associated with sternal glands showed typical characteristics of gland cells that differ in size, shape and number. The function of sternal glands is further discussed on the basis of morphological comparisons with other thrips species.     

Development and application of 15 novel polymorphic microsatellite markers for sect. Paeonia (Paeonia L.)

Herbaceous peony, which belongs to sect. Paeonia, Paeonia L., Paeoniaceae, is not only a famous traditional flower in China; it is also highly regarded as an ornamental in Europe and the USA. On account of the abundance in germplasm resources and wide distribution ranges, herbaceous peony can be divided into three cultivar groups, the Chinese Peony Cultivar Group, the European Peony Cultivar Group and the Hybrid Peony Cultivar Group. However, most studies on genetic relationships in Paeonia are limited to the first group. This study used 15 polymorphic microsatellite markers that were developed by magnetic bead enrichment to explore the genetic diversity of 89 genotypes collected from Beijing, the USA and Canada and introduced to China. From 145 allelic loci that were detected from 15 primers, 140 (96.6%) were polymorphic. The number of allelic loci ranged from 4 to 16 with an average of 9.3, and the polymorphic index content ranged from 0.362 to 0.825 with an average of 0.678. Both cluster analysis and principal component analysis based on the 15 polymorphic primer pairs could distinguish the species from cultivars although the cluster analysis was better able to reflect the genetic relationships between all individuals. Some new insights about Paeonia L. are suggested. Furthermore, the 15 SSR markers have great significance as they will allow for furthering the studies on genetic diversity, genetic relationships and even the molecular marker-assisted breeding of Paeonia L.     

The ACBP gene family in Rhodnius prolixus: Expression,characterization and function of RpACBP-1

The acyl-CoA-binding proteins (ACBP) constitute a family of conserved proteins that bind acyl-CoA with high affinity and protect it from hydrolysis. Thus, ACBPs may have essential roles in basal cellular lipid metabolism. The genome of the insect Rhodnius prolixus encodes five ACBP genes similar to those described for other insect species. The qPCR analysis revealed that these genes have characteristic expression profiles in insect organs, suggesting that they have specific roles in insect physiology. Recombinant RpACBP-1 was able to bind acyl-CoA in an in vitro gel-shift assay. Moreover, heterologous RpACBP-1 expression in acb1Δ mutant yeast rescued the multi-lobed vacuole phenotype, indicating that RpACBP-1 acts as a bona fide acyl-CoA-binding protein. RpACBP-1 knockdown using RNAi caused triacylglycerol accumulation in the insect posterior midgut and a reduction in the number of deposited eggs. The amount of stored triacylglycerol was reduced in flight muscle, and the incorporation of fatty acids in cholesteryl esters was increased in the fat body. These results showed that RpACBP-1 participates in several lipid metabolism steps in R. prolixus.     

Molecular characterization of Lathyrus species using chloroplast DNA trnH-psbA

Lathyrus L. is an important genus contributing in human food, animal feed and fodder. The genetic variation is studied among and within six species sampled over a large geographical area: Lathyrus cicera, Lathyrus sativus, Lathyrus sylvestris, Lathyrus tuberosus, Lathyrus ochrus and Lathyrus aphaca. The phylogenetic relationship among these species was assessed using sequences of chloroplast DNA trnH-psbA (intergenic spacer). The highly polymorphic spacer' length was 330 bp. The phylogenetic analyses using Maximum Parsimony and Genetic Distances, agreed with the universal taxonomy of Kupicha. L. sativus and L. cicera could be considered as sister species, sharing a common ancestor.     

An independent occurrence of the chimeric P450 enzyme CYP337B3 of Helicoverpa armigera confers cypermethrin resistance in Pakistan

The increasing resistance level of insect pest species is a major concern to agriculture worldwide. The cotton bollworm, Helicoverpa armigera, is one of the most important pest species due to being highly polyphagous, geographically widespread, and resistant towards many chemical classes of insecticides. We previously described the mechanism of fenvalerate resistance in Australian populations conferred by the chimeric cytochrome P450 monooxygenase CYP337B3, which arose by unequal crossing-over between CYP337B1 and CYP337B2. Here, we show that this mechanism is also present in the cypermethrin-resistant FSD strain from Pakistan. The Pakistani and the Australian CYP337B3 alleles differ by 18 synonymous and three nonsynonymous SNPs and additionally in the length and sequence of the intron. Nevertheless, the activity of both CYP337B3 proteins is comparable. We demonstrate that CYP337B3 is capable of metabolizing cypermethrin (trans- and especially cis-isomers) to the main metabolite 4'-hydroxycypermethrin, which exhibits no intrinsic toxicity towards susceptible larvae. In a bioassay, CYP337B3 confers a 7-fold resistance towards cypermethrin in FSD larvae compared to susceptible larvae from the Australian TWB strain lacking CYP337B3. Linkage analysis shows that presence of CYP337B3 accounts for most of the cypermethrin resistance in the FSD strain; up-regulation of other P450s in FSD plays no detectable role in resistance. The presence or absence of CYP337B3 can be easily detected by a simple PCR screen, providing a powerful tool to rapidly distinguish resistant from susceptible individuals in the field and to determine the geographical distribution of this resistance gene. Our results suggest that CYP337B3 evolved twice independently by unequal crossing-over between CYP337B2 and two different CYP337B1 alleles.     

The effectiveness of ISSR profiling for studying genetic diversity of Aspergillus flavus from peanut-cropped soils in China

We used markers based on inter-simple sequence repeats (ISSR) to examine the genetic diversity of Aspergillus flavus from peanut-cropped soils in China. Of the 100 primers, 22 primers produced clear and reproducible ISSR bands, and the di-nucleotide accounted for 73% of those primers. The size of DNA fragments ranged from 100 to 2000 bp. The primer UBC 834 produced the largest number of polymorphic bands (10), followed by UBC 809, UBC 817, UBC 895, and UBC 899, which all amplified 7 polymorphic bands. Using the five primers, the tested strains were clearly separated based on genetic similarity coefficients (GSC). The range of GSC was from 0.59 to 0.90. In unweighted pair-group method with arithmetic averages (UPGMA) analysis, the A. flavus samples grouped in five clusters. The study showed that the ISSR technology is an effective molecular approach for studying diversity of A. flavus from peanut-cropped soils in China.     

Cadherin AdCad1 in Alphitobius diaperinus larvae is a receptor of Cry3Bb toxin from Bacillus thuringiensis

Bacillus thuringiensis (Bt) Cry proteins are used as components of biopesticides or expressed in transgenic crops to control diverse insect pests worldwide. These Cry toxins bind to receptors on the midgut brush border membrane and kill enterocytes culminating in larval mortality. Cadherin proteins have been identified as Cry toxin receptors in diverse lepidopteran, coleopteran, and dipteran species. In the present work we report a 185 kDa cadherin (AdCad1) from larvae of the lesser mealworm (Alphitobius diaperinus) larvae as the first identified receptor for Cry3Bb toxin. The AdCad1 protein contains typical structural components for Cry toxin receptor cadherins, including nine cadherin repeats (CR9), a membrane-proximal extracellular domain (MPED) and a cytosolic region. Peptides corresponding to the CR9 and MPED regions bound Cry3Bb toxin with high affinities (23 nM and 40 nM) and significantly synergized Cry3Bb toxicity against A. diperinus larvae. Silencing of AdCad1 expression through RNA interference resulted in highly reduced susceptibility to Cry3Bb in A. diperinus larvae. The CR9 peptide fed with toxin to RNAi-treated larvae restored Cry3Bb toxicity. These results are evidences that AdCad1 is a functional receptor of Cry3Bb toxin and that exogenously fed CR9 peptide can overcome the effect of reduced AdCad1expression on Cry3Bb toxicity to larvae.     

Ins and Outs of Interpreting Lipidomic Results

Membrane lipids are essential for life; however, research on how cells regulate cell lipid composition has been falling behind for quite some time. One reason was the difficulty in establishing analytical methods able to cope with the cell lipid repertoire. Development of a diversity of mass spectrometry-based technologies, including imaging mass spectrometry, has helped to demonstrate beyond doubt that the cell lipidome is not only greatly cell type dependent but also highly sensitive to any pathophysiological alteration such as differentiation or tumorigenesis. Interestingly, the current popularization of metabolomic studies among numerous disciplines has led many researchers to rediscover lipids. Hence, it is important to underscore the peculiarities of these metabolites and their metabolism, which are both radically different from protein and nucleic acid metabolism. Once differences in lipid composition have been established, researchers face a rather complex scenario, to investigate the signaling pathways and molecular mechanisms accounting for their results. Thus, a detail often overlooked, but of crucial relevance, is the complex networks of enzymes involved in controlling the level of each one of the lipid species present in the cell. In most cases, these enzymes are redundant and promiscuous, complicating any study on lipid metabolism, since the modification of one particular lipid enzyme impacts simultaneously on many species. Altogether, this review aims to describe the difficulties in delving into the regulatory mechanisms tailoring the lipidome at the activity, genetic, and epigenetic level, while conveying the numerous, stimulating, and sometimes unexpected research opportunities afforded by this type of studies.     

The silkworm glutathione S-transferase gene noppera-bo is required for ecdysteroid biosynthesis and larval development

Insect molting and metamorphosis are tightly controlled by ecdysteroids, which are important steroid hormones that are synthesized from dietary sterols in the prothoracic gland. One of the ecdysteroidogenic genes in the fruit fly Drosophila melanogaster is noppera-bo (nobo), also known as GSTe14, which encodes a member of the epsilon class of glutathione S-transferases. In D. melanogaster, nobo plays a crucial role in utilizing cholesterol via regulating its transport and/or metabolism in the prothoracic gland. However, it is still not known whether the orthologs of nobo from other insects are also involved in ecdysteroid biosynthesis via cholesterol transport and/or metabolism in the prothoracic gland. Here we report genetic evidence showing that the silkworm Bombyx mori ortholog of nobo (nobo-Bm; GSTe7) is essential for silkworm development. nobo-Bm is predominantly expressed in the prothoracic gland. To assess the functional importance of nobo-Bm, we generated a B. mori genetic mutant of nobo-Bm using TALEN-mediated genome editing. We show that loss of nobo-Bm function causes larval arrest and a glossy cuticle phenotype, which are rescued by the application of 20-hydroxyecdysone. Moreover, the prothoracic gland cells isolated from the nobo-Bm mutant exhibit an abnormal accumulation of 7-dehydrocholesterol, a cholesterol metabolite. These results suggest that the nobo family of glutathione S-transferases is essential for development and for the regulation of sterol utilization in the prothoracic gland in not only the Diptera but also the Lepidoptera. On the other hand, loss of nobo function mutants of D. melanogaster and B. mori abnormally accumulates different sterols, implying that the sterol utilization in the PG is somewhat different between these two insect species.     

Essential oil yield,composition, bioactivity and leaf morphology of Juniperus oxycedrus L. from Bulgaria and Serbia

Juniperus oxycedrus L. (Cupressaceae Bartlett) is widely distributed in countries with a Mediterranean climate. All plant parts contain highly aromatic essential oil (EO) and recently there have been efforts to introduce it as a cultivated crop. The species is known for its large morphological and chemical variation and its debatable taxonomic status. This study aimed to (1) compare content, composition, and antimicrobial activity of J. oxycedrus EO samples from plants growing in Bulgaria and Serbia, and (2) quantify morphological variations of leaves. Тhe EO content (yield) in dried juniper leaves varied from 0.06% (Кopaonik, Serbia) to 0.24% (Markovo, Bulgaria). We identified 51 EO constituents, belonging to monoterpenes, sesquiterpenes, and diterpenes. The class monoterpenes (monoterpene hydrocarbons and oxygenated monoterpenes) were the predominant compounds, representing 38.6–65.4% of the total EO, consisting primarily of α-pinene, limonene, sabinene, β-pinene, and β-myrcene. In addition, α-pinene was the major oil constituent in plants from all locations. Sesquiterpenes (sesquiterpene hydrocarbons and oxygenated sesquiterpenes) were the second largest class of constituents, which represented 19.3% tо 33.6% of the total EO. γ-Elemene was found only in the EO of J. oxycedrus from Bulgaria, while a high concentration of α-curcumene was found only in samples from Serbia (7.5–7.8%). Significant differences in antimicrobial activity of the EO were found in bacterial strains Bacillus cereus and Streptococcus pneumoniae. There was no significant difference among the mean leaf width of the six combinations location x sex, and the overall leaf mean width was 1.24 mm. However, there was a significant difference between the mean leaf lengths. In this study, none of the studied populations had a higher concentration of limonene than of α-pinene, indicating that the flora of the two countries include J. oxycedrus and not the previously reported J. deltoides. The results revealed significant variation in EO profile that may contribute to the development of new cultivars of J. oxycedrus.