Detection of genetic diversity among Indian strains of _Xanthomonas campestris_ pv. _mangiferaeindicae_ using PCR-RAPD

The randomly amplified polymorphic DNA (RAPD) technique was used to investigate the genetic diversity in 6 strains of _Xanthomonas campestris_ pv. _mangiferaeindicae_ (_Xcmi_), the causal pathogen of mango bacterial canker disease (MBCD). The RAPD analysis was also intended to identify molecular markers, specific to the species to develop PCR-based markers for detection of _Xcmi_ in mango field and planting materials. Twenty RAPD primers (CP 1-CP 20) were evaluated to establish molecular characters and genetic variability in the genome of _Xcmi_. Among these, only 4 were found efficient for development of reproducible banding pattern. It has been observed that the largest and smallest amplified RAPD products were of 2.036 and 0.201 kbp. A total of 136 bands were scored against 6 strains of _Xcmi_. There was 7.66 per cent polymorphism in individual isolates which indicates significant polymorphism among the evaluated strains, with mean difference of 0.33 (_Xcmi_ 2 vs. _Xcmi_ 8) and 0.29 (_Xcmi_ 10 vs. _Xcmi_ 12). However, the single linkage euclidean distances were statistically significant (P>0.05), i.e., 0.58. The markers CP 5, 10, 16 and 19 were amplified in all the strains with polymorphic alleles, which indicates that these markers could be used for rapid detection of genetic variability in _Xcmi_ strains

<i>Trypanosoma evansi</i>: Genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar (‘type A’) whereas 2 isolates differed substantially (‘type B’). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades

2b-RAD genotyping for population genomic studies of Chagas disease vectors: Rhodnius ecuadoriensis in Ecuador

Background: Rhodnius ecuadoriensis is the main triatomine vector of Chagas disease, American trypanosomiasis, in Southern Ecuador and Northern Peru. Genomic approaches and next generation sequencing technologies have become powerful tools for investigating population diversity and structure which is a key consideration for vector control. Here we assess the effectiveness of three different 2b restriction site-associated DNA (2b-RAD) genotyping strategies in R. ecuadoriensis to provide sufficient genomic resolution to tease apart microevolutionary processes and undertake some pilot population genomic analyses. Methodology/Principal findings: The 2b-RAD protocol was carried out in-house at a non-specialized laboratory using 20 R. ecuadoriensis adults collected from the central coast and southern Andean region of Ecuador, from June 2006 to July 2013. 2b-RAD sequencing data was performed on an Illumina MiSeq instrument and analyzed with the STACKS de novo pipeline for loci assembly and Single Nucleotide Polymorphism (SNP) discovery. Preliminary population genomic analyses (global AMOVA and Bayesian clustering) were implemented. Our results showed that the 2b-RAD genotyping protocol is effective for R. ecuadoriensis and likely for other triatomine species. However, only BcgI and CspCI restriction enzymes provided a number of markers suitable for population genomic analysis at the read depth we generated. Our preliminary genomic analyses detected a signal of genetic structuring across the study area. Conclusions/Significance: Our findings suggest that 2b-RAD genotyping is both a cost effective and methodologically simple approach for generating high resolution genomic data for Chagas disease vectors with the power to distinguish between different vector populations at epidemiologically relevant scales. As such, 2b-RAD represents a powerful tool in the hands of medical entomologists with limited access to specialized molecular biological equipment. Author summary: Understanding Chagas disease vector (triatomine) population dispersal is key for the design of control measures tailored for the epidemiological situation of a particular region. In Ecuador, Rhodnius ecuadoriensis is a cause of concern for Chagas disease transmission, since it is widely distributed from the central coast to southern Ecuador. Here, a genome-wide sequencing (2b-RAD) approach was performed in 20 specimens from four communities from Manabí (central coast) and Loja (southern) provinces of Ecuador, and the effectiveness of three type IIB restriction enzymes was assessed. The findings of this study show that this genotyping methodology is cost effective in R. ecuadoriensis and likely in other triatomine species. In addition, preliminary population genomic analysis results detected a signal of population structure among geographically distinct communities and genetic variability within communities. As such, 2b-RAD shows significant promise as a relatively low-tech solution for determination of vector population genomics, dynamics, and spread

Diversity, genetic mapping, and signatures of domestication in the carrot (Daucus carota L.) genome, as revealed by Diversity Arrays Technology (DArT) markers

Carrot is one of the most economically important vegetables worldwide, but genetic and genomic resources supporting carrot breeding remain limited. We developed a Diversity Arrays Technology (DArT) platform for wild and cultivated carrot and used it to investigate genetic diversity and to develop a saturated genetic linkage map of carrot. We analyzed a set of 900 DArT markers in a collection of plant materials comprising 94 cultivated and 65 wild carrot accessions. The accessions were attributed to three separate groups: wild, Eastern cultivated and Western cultivated. Twenty-seven markers showing signatures for selection were identified. They showed a directional shift in frequency from the wild to the cultivated, likely reflecting diversifying selection imposed in the course of domestication. A genetic linkage map constructed using 188 F2 plants comprised 431 markers with an average distance of 1.1 cM, divided into nine linkage groups. Using previously anchored single nucleotide polymorphisms, the linkage groups were physically attributed to the nine carrot chromosomes. A cluster of markers mapping to chromosome 8 showed significant segregation distortion. Two of the 27 DArT markers with signatures for selection were segregating in the mapping population and were localized on chromosomes 2 and 6. Chromosome 2 was previously shown to carry the Vrn1 gene governing the biennial growth habit essential for cultivated carrot. The results reported here provide background for further research on the history of carrot domestication and identify genomic regions potentially important for modern carrot breeding

High Genetic Diversity and Low Differentiation of Michelia coriacea (Magnoliaceae), a Critically Endangered Endemic in Southeast Yunnan, China

Michelia coriacea, a critically endangered tree, has a restricted and fragmented distribution in Southeast Yunnan Province, China. The genetic diversity, genetic structure and gene flow in the three extant populations of this species were detected by 10 inter-simple sequence repeat (ISSR) markers and 11 simple sequence repeat (SSR) markers. Examination of genetic diversity revealed that the species maintained a relatively high level of genetic diversity at the species level (percentage of polymorphic bands) PPB = 96.36% from ISSRs; PPL (percentage of polymorphic loci) = 95.56% from SSRs, despite several fragmental populations. Low levels of genetic differentiation among the populations of M. coriacea were detected by Nei’s Gst = 0.187 for ISSR and Wright’s Fst = 0.090 for SSR markers, which is further confirmed by Bayesian model-based STRUCTURE and PCoA analysis that could not reveal a clear separation between populations, although YKP was differentiated to other two populations by ISSR markers. Meanwhile, AMOVA analysis also indicated that 22.84% and 13.90% of genetic variation existed among populations for ISSRs and SSRs, respectively. The high level of genetic diversity, low genetic differentiation, and the population, structure imply that the fragmented habitat and the isolated population of M. coriacea may be due to recent over-exploitation. Conservation and management of M. coriacea should concentrate on maintaining the high level of genetic variability through both in and ex-situ conservation actions

Molecular Genetic Diversity Study of Forest Coffee Tree (Coffea arabica L.) Populations in Ethiopia: Implications for Conservation and Breeding

Coffee provides one of the most widely drunk beverages in the world, and is a very important source of foreign exchange income for many countries. Coffea arabica, which contributes over 70 percent of the world's coffee productions, is characterized by a low genetic diversity, attributed to its allopolyploidy origin, reproductive biology and evolution. C. arabica has originated in the southwest rain forests of Ethiopia, where it is grown under four different systems, namely forest coffee, small holders coffee, semi plantation coffee and plantation coffee. Genetic diversity of the forest coffee (C. arabica) gene pool in Ethiopia is being lost at an alarming rate because of habitat destruction (deforestation), competition from other cash crops and replacement by invariable disease resistant coffee cultivars. This study focused on molecular genetic diversity study of forest coffee populations in Ethiopia using PCR based DNA markers such as random amplified polymorphic DNA (RAPD), inverse sequence-tagged repeat (ISTR), inter-simple sequence repeats (ISSR) and simple sequence repeat (SSR) or microsatellites. The objectives of the study are to estimate the extent and distribution of molecular genetic diversity of forest coffee and to design conservation strategies for it’s sustainable use in future coffee breeding. In this study, considerable samples of forest coffee collected from four coffee growing regions (provinces) of Ethiopia were analysed. The results indicate that moderate genetic diversity exists within and among few forest coffee populations, which need due attention from a conservation and breeding point of view. The cluster analysis revealed that most of the samples from the same region (province) were grouped together which could be attributed to presence of substantial gene flow between adjacent populations in each region in the form of young coffee plants through transplantation by man. In addition wild animals such as monkeys also play a significant role in coffee trees gene flow between adjacent populations. The overall variation of the forest coffee is found to reside in few populations from each region. Therefore, considering few populations from each region for either in situ or ex situ conservation may preserve most of the variation within the species. For instance, Welega-2, Ilubabor-2, Jima-2 and Bench Maji-2 populations should be given higher priority. In addition, some populations or genotypes have displayed unique amplification profiles particularly for RAPD and ISTR markers. Whether these unique bands are linked to any of the important agronomic traits and serve in marker assisted selections in future coffee breeding requires further investigations

Development and characterization of tri- and tetra-nucleotide polymorphic microsatellite markers for skipjack tuna (Katsuwonus pelamis)

Skipjack tuna (katsuwonus pelamis) (SJT) is the largest tuna fishery in all the major oceans around the world, and the largest marine fishery in Sri Lanka. Knowledge of genetic population structure and effective population size of SJT in the Indian Ocean and other major oceans, however, is still lacking for better management practices and conservation strategies. We developed microsatellite genetic markers using SJT around Sri Lanka in the Indian Ocean, and characterise one tri- and seven tetra-nucleotide microsatellite loci isolated from enriched genomic libraries from SJT, to provide tools for addressing both conservation and fisheries management questions. An analysis of these eight microsatellite markers in two populations of SJT from eastern Sri Lanka (n = 44) and the Maldives Islands (n = 53) showed that all eight microsatellites were polymorphic with an average number of alleles per locus of 11.80 (range 5-27). Expected heterozygosities at marker loci ranged from 0.450 to 0.961. These markers are being used currently to characterise population structure and extent of natural gene flow in SJT populations from the eastern and western Indian Ocean. No significant linkage disequilibrium was detected among any loci pairs

Disease progression in Plasmodium knowlesi malaria is linked to variation in invasion gene family members.

Emerging pathogens undermine initiatives to control the global health impact of infectious diseases. Zoonotic malaria is no exception. Plasmodium knowlesi, a malaria parasite of Southeast Asian macaques, has entered the human population. P. knowlesi, like Plasmodium falciparum, can reach high parasitaemia in human infections, and the World Health Organization guidelines for severe malaria list hyperparasitaemia among the measures of severe malaria in both infections. Not all patients with P. knowlesi infections develop hyperparasitaemia, and it is important to determine why. Between isolate variability in erythrocyte invasion, efficiency seems key. Here we investigate the idea that particular alleles of two P. knowlesi erythrocyte invasion genes, P. knowlesi normocyte binding protein Pknbpxa and Pknbpxb, influence parasitaemia and human disease progression. Pknbpxa and Pknbpxb reference DNA sequences were generated from five geographically and temporally distinct P. knowlesi patient isolates. Polymorphic regions of each gene (approximately 800 bp) were identified by haplotyping 147 patient isolates at each locus. Parasitaemia in the study cohort was associated with markers of disease severity including liver and renal dysfunction, haemoglobin, platelets and lactate, (r = ≥ 0.34, p =  <0.0001 for all). Seventy-five and 51 Pknbpxa and Pknbpxb haplotypes were resolved in 138 (94%) and 134 (92%) patient isolates respectively. The haplotypes formed twelve Pknbpxa and two Pknbpxb allelic groups. Patients infected with parasites with particular Pknbpxa and Pknbpxb alleles within the groups had significantly higher parasitaemia and other markers of disease severity. Our study strongly suggests that P. knowlesi invasion gene variants contribute to parasite virulence. We focused on two invasion genes, and we anticipate that additional virulent loci will be identified in pathogen genome-wide studies. The multiple sustained entries of this diverse pathogen into the human population must give cause for concern to malaria elimination strategists in the Southeast Asian region

Genetic diversity of peanut (Arachis hypogaea L.) and its wild relatives based on the analysis of hypervariable regions of the genome

BACKGROUND: The genus Arachis is native to a region that includes Central Brazil and neighboring countries. Little is known about the genetic variability of the Brazilian cultivated peanut (Arachis hypogaea, genome AABB) germplasm collection at the DNA level. The understanding of the genetic diversity of cultivated and wild species of peanut (Arachis spp.) is essential to develop strategies of collection, conservation and use of the germplasm in variety development. The identity of the ancestor progenitor species of cultivated peanut has also been of great interest. Several species have been suggested as putative AA and BB genome donors to allotetraploid A. hypogaea. Microsatellite or SSR (Simple Sequence Repeat) markers are co-dominant, multiallelic, and highly polymorphic genetic markers, appropriate for genetic diversity studies. Microsatellite markers may also, to some extent, support phylogenetic inferences. Here we report the use of a set of microsatellite markers, including newly developed ones, for phylogenetic inferences and the analysis of genetic variation of accessions of A. hypogea and its wild relatives. RESULTS: A total of 67 new microsatellite markers (mainly TTG motif) were developed for Arachis. Only three of these markers, however, were polymorphic in cultivated peanut. These three new markers plus five other markers characterized previously were evaluated for number of alleles per locus and gene diversity using 60 accessions of A. hypogaea. Genetic relationships among these 60 accessions and a sample of 36 wild accessions representative of section Arachis were estimated using allelic variation observed in a selected set of 12 SSR markers. Results showed that the Brazilian peanut germplasm collection has considerable levels of genetic diversity detected by SSR markers. Similarity groups for A. hypogaea accessions were established, which is a useful criteria for selecting parental plants for crop improvement. Microsatellite marker transferability was up to 76% for species of the section Arachis, but only 45% for species from the other eight Arachis sections tested. A new marker (Ah-041) presented a 100% transferability and could be used to classify the peanut accessions in AA and non-AA genome carriers. CONCLUSION: The level of polymorphism observed among accessions of A. hypogaea analyzed with newly developed microsatellite markers was low, corroborating the accumulated data which show that cultivated peanut presents a relatively reduced variation at the DNA level. A selected panel of SSR markers allowed the classification of A. hypogaea accessions into two major groups. The identification of similarity groups will be useful for the selection of parental plants to be used in breeding programs. Marker transferability is relatively high between accessions of section Arachis. The possibility of using microsatellite markers developed for one species in genetic evaluation of other species greatly reduces the cost of the analysis, since the development of microsatellite markers is still expensive and time consuming. The SSR markers developed in this study could be very useful for genetic analysis of wild species of Arachis, including comparative genome mapping, population genetic structure and phylogenetic inferences among species