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.     

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.     

When physical oceanography meets population genetics: The case study of the genetic/evolutionary discontinuity in the endangered goliath grouper (Epinephelus itajara; Perciformes: Epinephelidae) with comments on the conservation of the species

Epinephelus itajara is one of the marine fish species most threatened for extinction and it is considered to be “critically endangered” by the IUCN. The present study evaluated the genetic diversity of the species and the genetic/evolutionary relationships of its populations along the Atlantic coast of South America. The results indicate relatively reduced genetic variation, re-emphasizing the low adaptive potential of the species. One of the populations presented relatively high degrees of genetic diversity and it is evolutionary isolated from the all other populations. The evidences indicate the existence of two Evolutionarily Significant Units comprising E. itajara in the Atlantic coast of South America and the conservation prospects for the species must take these evidences into account.     

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.     

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.     

Analysis of the genetic diversity of garlic (Allium sativum L.) by simple sequence repeat and inter simple sequence repeat analysis and agro-morphological traits

The genetic diversity of 39 garlic accessions was investigated using eight simple sequence repeat (SSR) primer combinations and 17 inter-simple sequence repeat (ISSR) primer combinations. A total of 109 polymorphic loci were detected among these accessions, with an average of 4.63 polymorphic loci per SSR primer combination and 4.29 polymorphic loci per ISSR primer combination. The mean effective number of alleles, the mean Nei's genetic diversity, and the mean Shannon's information index for SSR were 1.4799, 0.2870, and 0.4378, respectively; and those for ISSR were 1.4847, 0.2898 and 0.4415, respectively. Cluster analysis, using the unweighted pair-group method with arithmetic averages (UPGMA) based on the allele frequency data, classified the accessions into three groups. The results of principal component analysis (PCA) were consistent with those of the cluster analysis. PCA showed that each of these three groups exhibited significant variation in agro-morphological traits. These findings suggest that the eight SSR and 17 ISSR primers identified could define valuable markers for genetic diversity for use by plant breeders.     

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.     

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.     

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.     

LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes,fibH, fibL and fhx,in the silk gland of the silkworm,Bombyx mori

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors.     

Transposition burst of mariner-like elements in the sequenced genome of Rhodnius prolixus

Transposable elements (TEs) are widespread in insect's genomes. However, there are wide differences in the proportion of the total DNA content occupied by these repetitive sequences in different species. We have analyzed the TEs present in R. prolixus (vector of the Chagas disease) and showed that 3.0% of this genome is occupied by Class II TEs, belonging mainly to the Tc1-mariner superfamily (1.65%) and MITEs (1.84%). Interestingly, most of this genomic content is due to the expansion of two subfamilies belonging to: irritans himar, a well characterized subfamily of mariners, and prolixus1, one of the two novel subfamilies here described. The high amount of sequences in these subfamilies suggests that bursts of transposition occurred during the life cycle of this family. In an attempt to characterize these elements, we performed an in silico analysis of the sequences corresponding to the DDD/E domain of the transposase gene. We performed an evolutionary analysis including network and Bayesian coalescent-based methods in order to infer the dynamics of the amplification, as well as to estimate the time of the bursts identified in these subfamilies. Given our data, we hypothesized that the TE expansions occurred around the time of speciation of R. prolixus around 1.4 mya. This suggestion lays on the “Transposon Model” of TE evolution, in which the members of a TE population that are replicative active are present at multiple loci in the genome, but their replicative potential varies, and of the “Life Cycle Model” that states that when present-day TEs have been involved in amplification bursts, they share an ancestral copy that dates back to this initial amplification.     

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.     

Describing the role of Drosophila melanogaster ABC transporters in insecticide biology using CRISPR-Cas9 knockouts

ABC transporters have a well-established role in drug resistance, effluxing xenobiotics from cells and tissues within the organism. More recently, research has been dedicated to understanding the role insect ABC transporters play in insecticide toxicity, but progress in understanding the contribution of specific transporters has been hampered by the lack of functional genetic tools. Here, we report knockouts of three Drosophila melanogaster ABC transporter genes, Mdr49, Mdr50, and Mdr65, that are homologous to the well-studied mammalian ABCB1 (P-glycoprotein). Each knockout mutant was created in the same wild type background and tested against a panel of insecticides representing different chemical classes. Mdr65 knockouts were more susceptible to all neuroactive insecticides tested, but Mdr49 and Mdr50 knockouts showed increased susceptibility or resistance depending on the insecticide used. Mdr65 was chosen for further analysis. Calculation of LC₅₀ values for the Mdr65 knockout allowed the substrate specificity of this transporter to be examined. No obvious distinguishing structural features were shared among MDR65 substrates. A role for Mdr65 in insecticide transport was confirmed by testing the capacity of the knockout to synergize with the ABC inhibitor verapamil and by measuring the levels of insecticide retained in the body of knockout flies. These data unambiguously establish the influence of ABC transporters on the capacity of wild type D. melanogaster to tolerate insecticide exposure and suggest that both tissue and substrate specificity underpin this capacity.     

Phenotypic and genotypic intra-diversity among Anatolian durum wheat “Kunduru” landraces

Kunduru is an important Anatolian landrace having peculiar traits that are appreciated by farmers and breeders. 33 accessions known as Kunduru collected by ICARDA from six geographical provinces of Turkey, were used to study the phenotypic and genotypic intra-diversity. Kunduru landraces exhibited high intra-diversity for most of the studied morphological traits. GPC (12.10–14.90%), vitreousness (75–100%), TKW (31.80–56.70 g), YP (4.70–8.00 ppm), b*-value (14.30–19.50), ash content (1.60–2.0%) and gluten strength (14–60 ml) showed marked variations. Gliadin and glutenin banding patterns showed high polymorphism. 65 alleles were detected with 14 SSR markers, giving a mean of 6.77 alleles per locus. The average PIC value was 0.44 and ranged from 0.11 to 0.70. The average genetic distance between pairs of landraces was 0.47 and ranged between 0.11 and 0.72. This study showed that Kunduru landraces maintains high allelic variation. PCoA indicated that eco-geographical variables have a significant effect on SSR diversity as well as morphological traits. Many of the landraces studied are in danger of disappearing from the local farmers' fields; this study demonstrates the importance of maintaining and conserving this precious genetic resources.     

Increased potential for wound activated production of Prostaglandin E2 and related toxic compounds in non-native populations of Gracilaria vermiculophylla

The capacity of the East Asian seaweed Gracilaria vermiculophylla (“Ogonori”) for production of prostaglandin E₂ from arachidonic acid occasionally causes food poisoning after ingestion. During the last two decades the alga has been introduced to Europe and North America. Non-native populations have been shown to be generally less palatable to marine herbivores than native populations. We hypothesized that the difference in palatability among populations could be due to differences in the algal content of prostaglandins. We therefore compared the capacity for wound-activated production of prostaglandins and other eicosatetraenoid oxylipins among five native populations in East Asia and seven non-native populations in Europe and NW Mexico, using a targeted metabolomics approach. In two independent experiments non-native populations exhibited a significant tendency to produce more eicosatetraenoids than native populations after acclimation to identical conditions and subsequent artificial wounding. Fourteen out of 15 eicosatetraenoids that were detected in experiment I and all 19 eicosatetraenoids that were detected in experiment II reached higher mean concentrations in non-native than in native specimens. Wounding of non-native specimens resulted on average in 390% more 15-keto-PGE₂, in 90% more PGE₂, in 37% more PGA₂ and in 96% more 7,8-di-hydroxy-eicosatetraenoic acid than wounding of native specimens. Not only PGE₂, but also PGA₂ and dihydroxylated eicosatetraenoic acid are known to deter various biological enemies of G. vermiculophylla that cause tissue or cell wounding, and in the present study the latter two compounds also repelled the mesograzer Littorina brevicula. Non-native populations of G. vermiculophylla are thus more defended against herbivory than native populations. This increased capacity for activated chemical defense may have contributed to their invasion success and at the same time it poses an elevated risk for human food safety.     

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.