SLAF-based high-density genetic map construction and QTL mapping for major economic traits in sea urchin Strongylocentrotus intermedius.

Sea urchin (Strongylocentrotus intermedius) has long been a model species for developmental and evolutionary research, but only a few studies have focused on gene mapping. Here, we reported a high-density genetic map containing 4,387 polymorphism specific-length amplified fragment (SLAF) markers spanning 21 linkage groups (LG) for sea urchin. Based on this genetic map and phenotyping data for eight economic traits, 33 potentially significant QTLs were detected on ten different LGs with explanations ranging from 9.90% to 46.30%, partly including 10 QTLs for test diameter, six QTLs for body weight and eight QTLs for Aristotle's lantern weight. Moreover, we found a QTL enrichment LG, LG15, gathering QTLs for test diameter, body weight, gonad weight, light orange-yellow color difference (≥E1) and light yellow color difference (≥E2). Among all QTLs, we genotyped four QTLs for test diameter, Aristotle's lantern weight and body weight using High Resolution Melting (HRM) technology. Finally, we used the verified SNP marker (detected using SLAF sequencing) to explore their marker-assisted selection (MAS) breeding application potential and found that SNP-29 associated tightly with body weight and that heterozygous genotype was a dominant genotype, indicating that SNP-29 was a promising marker for MAS.

Detection of the Single Nucleotide Polymorphism at Position rs2735940 in the Human Telomerase Reverse Transcriptase Gene by the Introduction of a New Restriction Enzyme Site for the PCR-RFLP Assay.

It has been shown that the single nucleotide polymorphism (SNP) of the rs2735940 site in the human telomerase reverse transcriptase (hTERT) gene is associated with increased cancer risk. The traditional method to detect SNP genotypes is polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). However, there is a limitation to utilizing PCR-RFLP due to a lack of proper restriction enzyme sites at many polymorphic loci. This study used an improved PCR-RFLP method with a mismatched base for detection of the SNP rs2735940. A new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and in addition, the restriction enzyme MspI for CRS-PCR was cheaper than other enzymes. We used this novel assay to determine the allele frequencies in 552 healthy Chinese Han individuals, and found the allele frequencies to be 63% for allele C and 37% for allele T In summary, the modified PCR-RFLP can be used to detect the SNP of rs2735940 with low cost and high efficiency.

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment.

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (Aß) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

BACKGROUND: It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. RESULTS: Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. CONCLUSION: MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

AFLP assessment of genetic variability and relationships in an Asian wild germplasm collection of Dactylis glomerata L.

Orchardgrass (Dactylis glomerata L.), an excellent perennial and cool season forage species distributed in most temperate regions, has been cultivated widely in Western China. Amplified fragment length polymorphism markers were employed to determine the genetic variability and population structure among 41 indigenous orchardgrass accessions from Central Asia and Western China. On the basis of 531 polymorphic fragments resulted from eight primer combinations, polymorphic information content (PIC), marker index (MI) and resolving power (RP) averaged 0.252, 16.34 and 25.27 per primer combination, respectively, demonstrating the high efficiency and reliability of the markers used. We found relatively low differentiation (Fst=0.135) for three geographical groups, where Central Asia (CA) and Southwest China (SWC) group exhibited higher intra-population diversity (He=0.20 and 0.21) than that of the Xinjiang (XJ) group (He=0.14). We also did not detect a clear pattern of isolation by distance with a low value of r=0.301 in the Mantel test. STRUCTURE, FLOCK, UPGMA clustering and PCoA analyses showed that CA group is more related to the SWC Group rather than to the XJ Group. In addition, this study strongly suggests that geographical and ecological environmental factors together could better explain the genetic differentiation between different geographical regions than geographic isolation alone, especially for Xinjiang accessions. The present study also could support that Southwest China might be the internal diversity center of D. glomerata in China. The knowledge about the genetic variability of the Asian accessions examined contributes to rapid characterization, defining gene pools of wild accessions, and selecting appropriate germplasms for plant improvement.

Landscape genetic approaches to guide native plant restoration in the Mojave Desert.

Restoring dryland ecosystems is a global challenge due to synergistic drivers of disturbance coupled with unpredictable environmental conditions. Dryland plant species have evolved complex life-history strategies to cope with fluctuating resources and climatic extremes. Although rarely quantified, local adaptation is likely widespread among these species and potentially influences restoration outcomes. The common practice of reintroducing propagules to restore dryland ecosystems, often across large spatial scales, compels evaluation of adaptive divergence within these species. Such evaluations are critical to understanding the consequences of large-scale manipulation of gene flow and to predicting success of restoration efforts. However, genetic information for species of interest can be difficult and expensive to obtain through traditional common garden experiments. Recent advances in landscape genetics offer marker-based approaches for identifying environmental drivers of adaptive genetic variability in non-model species, but tools are still needed to link these approaches with practical aspects of ecological restoration. Here, we combine spatially explicit landscape genetics models with flexible visualization tools to demonstrate how cost-effective evaluations of adaptive genetic divergence can facilitate implementation of different seed sourcing strategies in ecological restoration. We apply these methods to Amplified Fragment Length Polymorphism (AFLP) markers genotyped in two Mojave Desert shrub species of high restoration importance: the long-lived, wind-pollinated gymnosperm Ephedra nevadensis, and the short-lived, insect-pollinated angiosperm Sphaeralcea ambigua. Mean annual temperature was identified as an important driver of adaptive genetic divergence for both species. Ephedra showed stronger adaptive divergence with respect to precipitation variability, while temperature variability and precipitation averages explained a larger fraction of adaptive divergence in Sphaeralcea. We describe multivariate statistical approaches for interpolating spatial patterns of adaptive divergence while accounting for potential bias due to neutral genetic structure. Through a spatial bootstrapping procedure, we also visualize patterns in the magnitude of model uncertainty. Finally, we introduce an interactive, distance-based mapping approach that explicitly links marker-based models of adaptive divergence with local or admixture seed sourcing strategies, promoting effective native plant restoration.

Development of new SNP derived cleaved amplified polymorphic sequence marker set and its successful utilization in the genetic analysis of seed color variation in barley.

The aim of the present study was to develop a new cost effective PCR based CAPS marker set using advantages of high-throughput SNP genotyping. Initially, SNP survey was made using 20 diverse barley genotypes via 9k iSelect array genotyping that resulted in 6334 polymorphic SNP markers. Principle component analysis using this marker data showed fine differentiation of barley diverse gene pool. Till this end, we developed 200 SNP derived CAPS markers distributed across the genome covering around 991cM with an average marker density of 5.09cM. Further, we genotyped 68 CAPS markers in an F2 population (Cheri×ICB181160) segregating for seed color variation in barley. Genetic mapping of seed color revealed putative linkage of single nuclear gene on chromosome 1H. These findings showed the proof of concept for the development and utility of a newer cost effective genomic tool kit to analyze broader genetic resources of barley worldwide.

Assessment of diversity and genetic relationships of Neonectria ditissima: the causal agent of fruit tree canker.

BACKGROUND: Neonectria ditissima is one of the most important fungal pathogens of apple trees, where it causes fruit tree canker. Information about the amount and partitioning of genetic variation of this fungus could be helpful for improving orchard management strategies and for breeding apple cultivars with high levels of genetically determined resistance. In this study single-spore Neonectria isolates originating from both the same and from different perithecia, apple cultivars and apple orchards in Sweden and Belgium, were evaluated for AFLP- and SSR-based genetic similarity and for mating system. RESULTS: Seven SSR loci produced a total of 31 alleles with an average of 4 alleles per locus, while 11 AFLP primer combinations produced an average of 35 fragments per primer combination and 71 % polymorphic fragments. An AFLP-based analysis of molecular variance (AMOVA) revealed that 89 % of the variation was found within orchards and 11 % between orchards. Genetic similarity among the studied isolates was illustrated with a principal coordinate analyseis (PCoA) and a dendrogram. AFLP-based Jaccard's similarity coefficients were the highest when single-ascospore isolates obtained from the same perithecium were compared, medium-high for isolates from different perithecia on the same tree, and lowest when isolates from different trees were compared. CONCLUSIONS: Based on the results of PCoA and AMOVA analysis, isolates from the same or geographically close orchards did not group together. Since AFLP profiles differed also when single-ascospore isolates from the same perithecium were compared, the mating system of N. ditissima is most likely heterothallic.

A direct assessment of realized seed and pollen flow within and between two isolated populations of the food-deceptive orchid Orchis mascula.

Gene flow can counteract the loss of genetic diversity caused by genetic drift in small populations. For this reason, clearly understanding gene flow patterns is of the highest importance across fragmented landscapes. However, gene flow patterns are not only dependent upon the degree of spatial isolation of fragmented populations, but are also dependent upon the life-history traits of the species. Indeed, habitat fragmentation effects appear especially unpredictable for food-deceptive orchid species, because of their highly specialised seed and pollen dispersal mechanisms. In this study we used amplified fragment length polymorphism markers and subsequent parentage and spatial autocorrelation analysis to quantify the extent and the patterns of realized gene flow within and between two adjacent fragmented populations of the food-deceptive Orchis mascula. We observed considerable gene flow between both populations, occurring mainly through pollen dispersal. Seed dispersal, on the other hand, was mainly limited to the first few meters from the mother plant in both populations, although at least one among-population seed dispersal event was observed. This, in turn, resulted in a significant spatial genetic structure for both populations. Although genetic diversity was high in both populations and mainly outcrossing occurred, reproductive output was strongly skewed toward a limited number of successful adult plants. These observed patterns are likely due to the different pollinator behaviour associated with food-deceptive plants. We conclude that these populations can be considered viable under their current fragmented state.

ASSESSMENT OF MOLECULAR GENETIC STABILITY BETWEEN LONG-TERM CRYOPRESERVED AND TISSUE CULTURED WASABI (Wasabia japonica) PLANTS.

BACKGROUND: Maintaining the genetic integrity in long-term tissue cultured and cryopreserved plants is important for the conservation of plant genetic resources. OBJECTIVE: In this study, the genetic stability of cryopreserved wasabi shoot tips stored for 10 years at -150 degree C was visualized using Amplified Fragment Length Polymorphism (AFLP) and Methylation Sensitive Amplified Polymorphism (MSAP). MATERIALS AND METHODS: The study included plants derived from cryopreserved shoot tips after 10.5 years storage at -150 degree C (LN10yr), after 2 h storage at -196 degree C (LN2hr), cryopreservation controls (No LN cooling (TC)) and non-treated controls without LN cooling (LC). The donor plants for LN2hr, TC and LC were also maintained in vitro at 20 degree C for the same period. RESULTS: Neither technique detected genetic variations in either control or cryopreserved plants. Some mutations were noted in plants maintained in tissue culture for 10 years. Comparison of genome stability for TC and LN2hr plants showed only a minor change in DNA. However, when comparing the LC and Ln10yr, many differences were found. CONCLUSION: We conclude that cryopreservation is a superior conservation method compared to tissue culture in maintaining genetic stability for a long-term storage of wasabi germplasm.

Construction of a dense genetic linkage map and mapping quantitative trait loci for economic traits of a doubled haploid population of Pyropia haitanensis (Bangiales, Rhodophyta).

BACKGROUND: Pyropia haitanensis is one of the most economically important mariculture crops in China. A high-density genetic map has not been published yet and quantitative trait locus (QTL) mapping has not been undertaken for P. haitanensis because of a lack of sufficient molecular markers. Specific length amplified fragment sequencing (SLAF-seq) was developed recently for large-scale, high resolution de novo marker discovery and genotyping. In this study, SLAF-seq was used to obtain mass length polymorphic markers to construct a high-density genetic map for P. haitanensis. RESULTS: In total, 120.33 Gb of data containing 75.21 M pair-end reads was obtained after sequencing. The average coverage for each SLAF marker was 75.50-fold in the male parent, 74.02-fold in the female parent, and 6.14-fold average in each double haploid individual. In total, 188,982 SLAFs were detected, of which 6731 were length polymorphic SLAFs that could be used to construct a genetic map. The final map included 4550 length polymorphic markers that were combined into 740 bins on five linkage groups, with a length of 874.33 cM and an average distance of 1.18 cM between adjacent bins. This map was used for QTL mapping to identify chromosomal regions associated with six economically important traits: frond length, width, thickness, fresh weight, growth rates of frond length and growth rates of fresh weight. Fifteen QTLs were identified for these traits. The value of phenotypic variance explained by an individual QTL ranged from 9.59 to 16.61 %, and the confidence interval of each QTL ranged from 0.97 cM to 16.51 cM. CONCLUSIONS: The first high-density genetic linkage map for P. haitanensis was constructed, and fifteen QTLs associated with six economically important traits were identified. The results of this study not only provide a platform for gene and QTL fine mapping, map-based gene isolation, and molecular breeding for P. haitanensis, but will also serve as a reference for positioning sequence scaffolds on a physical map and will assist in the process of assembling the P. haitanensis genome sequence. This will have a positive impact on breeding programs that aim to increase the production and quality of P. haitanensis in the future.

Assessment of genetic diversity of bermudagrass germplasm from southwest China and Africa by using AFLP markers.

Cynodon dactylon (L.) Pers. var. dactylon (common bermudagrass) is widely distributed geographically between approximately 45°N and 45°S latitude, penetrating to approximately 53°N latitude in Europe. The extensive variation of morphological and adaptive characteristics of the taxon has been substantially documented, but information is lacking on DNA molecular variation in geographically disparate forms. The genetic diversity of 51 wild accessions of bermudagrass from southwest China (Sichuan, Chongqing, Yunnan, Guizhou, and Tibet) and 8 African bermudagrass was analyzed using amplified fragment length polymorphism molecular markers. A total of 670 polymorphic bands were detected with 11 primer combinations, of which 663 (98.74%) bands were found to be polymorphic. The genetic similarity among the accessions ranged from 0.64-0.96 with an average of 0.78. All 59 wild accessions were clustered into 5 eco-geographic groups, and nearly all accessions from the same area were classified into the same group and were found to be associated with their geographical distributions. Therefore, complex geographical and ecological environments are important factors for the genetic structure and geographical distribution of C. dactylon.

Identification of molecular markers associated with fruit traits in olive and assessment of olive core collection with AFLP markers and fruit traits.

The purpose of this study was to characterize olive core collection with amplified fragment length polymorphism (AFLP) markers and fruit traits and to determine AFLP markers significantly associated with these fruit characters in olive. A total of 168 polymorphic AFLP markers generated by five primer combinations and nine fruit traits were used to characterize relationships between 18 olive cultivars. Although all olive cultivars were discriminated from each other by either AFLP markers (<0.75 similarity level) or fruit traits, clustering based on the AFLP markers and fruit traits was not significantly correlated (r = 0.13). Partial clustering of olive cultivars by AFLP markers according to their geographical origin was observed. Associations of AFLP markers with fruits were determined using a multiple-regression analysis with stepwise addition of AFLP markers. Significant associations between eight AFLP markers and fruit traits were identified. While five AFLP markers demonstrated significant negative correlation with fruit and stone weight, width and length and total polyphenols (P < 0.05), three AFLP markers displayed significant positive correlation with α-tocopherol and γ-tocopherol (P < 0.01). This is the first report on the association of molecular markers with fruit traits in olive. Molecular markers associated with morphological and agronomic traits could be utilized for the breeding of olive cultivars. However, the association power of these markers needs to be confirmed in larger populations, and highly correlated markers should then be converted to PCR-based DNA markers such as sequence-characterized amplified region markers for better utilization.

Assessment of the genetic variability amongst mandarin (Citrus reticulata Blanco) accessions in Bhutan using AFLP markers.

BACKGROUND: Bhutan is a small Himalayan country lying within the region considered to be the origin of citrus. Diverse citrus wild types grow naturally in different climates, elevations and edaphic conditions, but only mandarin is cultivated commercially. The first report of Huanglongbing (also known as greening disease) in Bhutan in 2003, and the threat it posed to the country's citrus orchards prompted the collection of mandarin germplasm from across the country. This paper describes the genetic diversity of mandarin accessions in Bhutan using amplified fragment length polymorphic (AFLP) markers. RESULTS: Twenty three accessions of Bhutanese mandarin were analyzed using AFLP markers to assess the genetic variability that is believed to exist only in Bhutan and some parts of North East India and South China. Five primer pairs (E-ACA/M-CAG, E-ACG/M-CAT, E-ACC/M-CTT, E-AAG/M-CAA and E-ACA/M-CTC) were identified (based on the number and quality of polymorphic bands produced) and used for the analyses. A total of 244 bands were scored visually of which 126 (52%) were polymorphic with an average polymorphism information content of 0.95 per marker. A cluster dendrogram based on multiscale bootstrap sampling categorized twenty three accessions into two broad groups containing eight and 14 accessions, respectively. Group A consisted accessions (Tsirang1, Tsirang3, Sarpang1, Dagana4, Samtse4, Dagana1, and Trongsa2) from five districts (Tsirang, Sarpang, Samtse, Dagana and Trongsa) and their grouping was strongly supported by bootstrap analysis (B p-value = 96%, AU p-value = 86%). Cluster B consisted of 14 accessions divided into three sub-groups (1, 2 and 3). However, bootstrap value supported significantly for subgroup1 (containing accessions: Tsirang4, Sarpang5, and Tsirang2) and subgroup3 (with accessions - Zhemgang2, Zhemgang3 and Zhemgang4). CONCLUSION: This study indicates that Bhutanese mandarin germplasm collected from across the country are genetically diverse although the level of variability differed among the accessions assessed. The variation in genetic variability was observed irrespective of where the accessions were collected suggesting that phenotype and geographical location can serve a basis for future germplasm collection in Bhutan. Further, five primer pair combinations could separate 23 mandarins accessions considered in this study, suggesting that AFLP markers can be a useful tool for future identification.

Comparative assessment of next-generation sequencing, denaturing gradient gel electrophoresis, clonal restriction fragment length polymorphism and cloning-sequencing as methods for characterizing commercial microbial consortia.

Characterization of commercial microbial consortia products for human and environmental health risk assessment is a major challenge for regulatory agencies. As a means to develop an approach to assess the potential environmental risk of these products, research was conducted to compare four genomics methods for characterizing bacterial communities; (i) Denaturing Gradient Gel Electrophoresis (DGGE), (ii) Clonal-Restriction Fragment Length Polymorphism (C/RFLP), (iii) partial 16S rDNA amplification, cloning followed by Sanger sequencing (PRACS) and (iv) Next-Generation Sequencing (NGS) based on Ion Torrent technology. A commercially available microbial consortium, marketed as a remediation agent for degrading petroleum hydrocarbon contamination in soil and water, was assessed. The bacterial composition of the commercial microbial product was characterized using the above four methods. PCR amplification of 16S rDNA was performed targeting the variable region V6 for DGGE, C/RFLP and PRACS and V5 for Ion Torrent sequencing. Ion Torrent technology was shown to be a promising tool for initial screening by detecting the majority of bacteria in the consortium that were also detected by DGGE, C/RFLP and PRACS. Additionally, Ion Torrent sequencing detected some of the bacteria that were claimed to be in the product, while three other methods failed to detect these specific bacteria. However, the relative proportions of the microbial composition detected by Ion Torrent were found to be different from DGGE, C/RFLP and PRACS, which gave comparable results across these three methods. The discrepancy of the Ion Torrent results may be due to the short read length generated by this technique and the targeting of different variable regions on the 16S rRNA gene used in this study. Arcobacter spp. a potential pathogenic bacteria was detected in the product by all methods, which was further confirmed using genus and species-specific PCR, RFLP and DNA-based sequence analyses. However, the viability of Arcobacter spp. was not confirmed. This study suggests that a combination of two or more methods may be required to ascertain the microbial constituents of a commercial microbial consortium reliably and for the presence of potentially human pathogenic contaminants.

Application of molecular techniques for the assessment of microorganism diversity on cultural heritage objects.

As a result of their unpredictable ability to adapt to varying environmental conditions, microorganisms inhabit different types of biological niches on Earth. Owing to the key role of microorganisms in many biogeochemical processes, trends in modern microbiology emphasize the need to know and understand the structure and function of complex microbial communities. This is particularly important if the strategy relates to microbial communities that cause biodeterioration of materials that constitute our cultural heritage. Until recently, the detection and identification of microorganisms inhabiting objects of cultural value was based only on cultivation-dependent methods. In spite of many advantages, these methods provide limited information because they identify only viable organisms capable of growth under standard laboratory conditions. However, in order to carry out proper conservation and renovation, it is necessary to know the complete composition of microbial communities and their activity. This paper presents and characterizes modern techniques such as genetic fingerprinting and clone library construction for the assessment of microbial diversity based on molecular biology. Molecular methods represent a favourable alternative to culture-dependent methods and make it possible to assess the biodiversity of microorganisms inhabiting technical materials and cultural heritage objects.

Fluorescent amplified fragment length polymorphism (fAFLP) analysis of Listeria monocytogenes.

Fluorescent amplified fragment length polymorphism (fAFLP) is based on the selective PCR amplification of restriction fragments from a digest of total genomic DNA. Genomic DNA extracted from a purified bacterial isolate is completely digested with two endonucleases generating fragments which are ligated to specific double-stranded adaptors. The ligated fragments are then amplified by PCR using fluorescently labelled primers. Fluorescent amplified fragments are separated by size on an automated sequencer with a size standard. fAFLP is a rapid, highly reproducible technique which can be used to discriminate and subtype Listeria monocytogenes strains.

Revisiting AFLP fingerprinting for an unbiased assessment of genetic structure and differentiation of taurine and zebu cattle.

BACKGROUND: Descendants from the extinct aurochs (Bos primigenius), taurine (Bos taurus) and zebu cattle (Bos indicus) were domesticated 10,000 years ago in Southwestern and Southern Asia, respectively, and colonized the world undergoing complex events of admixture and selection. Molecular data, in particular genome-wide single nucleotide polymorphism (SNP) markers, can complement historic and archaeological records to elucidate these past events. However, SNP ascertainment in cattle has been optimized for taurine breeds, imposing limitations to the study of diversity in zebu cattle. As amplified fragment length polymorphism (AFLP) markers are discovered and genotyped as the samples are assayed, this type of marker is free of ascertainment bias. In order to obtain unbiased assessments of genetic differentiation and structure in taurine and zebu cattle, we analyzed a dataset of 135 AFLP markers in 1,593 samples from 13 zebu and 58 taurine breeds, representing nine continental areas. RESULTS: We found a geographical pattern of expected heterozygosity in European taurine breeds decreasing with the distance from the domestication centre, arguing against a large-scale introgression from European or African aurochs. Zebu cattle were found to be at least as diverse as taurine cattle. Western African zebu cattle were found to have diverged more from Indian zebu than South American zebu. Model-based clustering and ancestry informative markers analyses suggested that this is due to taurine introgression. Although a large part of South American zebu cattle also descend from taurine cows, we did not detect significant levels of taurine ancestry in these breeds, probably because of systematic backcrossing with zebu bulls. Furthermore, limited zebu introgression was found in Podolian taurine breeds in Italy. CONCLUSIONS: The assessment of cattle diversity reported here contributes an unbiased global view to genetic differentiation and structure of taurine and zebu cattle populations, which is essential for an effective conservation of the bovine genetic resources.

Towards a typing strategy for Arcobacter species isolated from humans and animals and assessment of the in vitro genomic stability.

Arcobacter species have a widespread distribution with a broad range of animal hosts and environmental reservoirs, and are increasingly associated with human illness. To elucidate the routes of infection, several characterization methods such as pulsed-field gel electrophoresis (PFGE), amplified fragment-length polymorphism, and enterobacterial repetitive intergenic consensus (ERIC)-PCR have already been applied, but without proper validation or comparison. At present, no criterion standard typing method or strategy has been proposed. Therefore, after the validation of PFGE, those commonly applied typing methods were compared for the characterization of six human- and animal-associated Arcobacter species. With a limited number of isolates to be characterized, PFGE with restriction by KpnI is proposed as the first method of choice. However, ERIC-PCR represents a more convenient genomic fingerprinting technique when a large number of isolates is involved. Therefore, a first clustering of similar patterns obtained after ERIC-PCR, with a subsequent typing of some representatives per ERIC cluster by PFGE, is recommended. As multiple genotypes are commonly isolated from the same host and food, genomic plasticity has been suggested. The in vitro genomic stability of Arcobacter butzleri and A. cryaerophilus was assessed under two temperatures and two oxygen concentrations. Variability in the genomic profile of A. cryaerophilus was observed after different passages for different strains at 37°C under microaerobic conditions. The bias due to these genomic changes must be taken into account in the evaluation of the relationship of strains.

A milestone in the doubled haploid pathway of cassava: a milestone in the doubled haploid pathway of cassava (Manihot esculenta Crantz): cellular and molecular assessment of anther-derived structures.

This study was aimed at inducing androgenesis in cultured anthers of cassava (Manihot esculenta Crantz) to develop a protocol for the production of doubled haploids. Microspore reprogramming was induced in cassava by cold or heat stress of anthers. Since the anthers contain both haploid microspores and diploid somatic cells, it was essential to verify the origin of anther-derived calli. The origin of anther-derived calli was assessed by morphological screening followed by histological analysis and flow cytometry (FCM). Additionally, simple sequence repeat (SSR) and amplified fragmented length polymorphism (AFLP) assays were used for the molecular identification of the microspore-derived calli. The study clearly demonstrated the feasibility of producing microspore-derived calli using heat- or cold-pretreated anthers. Histological studies revealed reprogramming of the developmental pathway of microspores by symmetrical division of the nucleus. Flow cytometry analysis revealed different ploidy level cell types including haploids, which confirmed their origin from the microspores. The SSR and AFLP marker assays independently confirmed the histological and FCM results of a haploid origin of the calli at the DNA level. The presence of multicellular microspores in the in vitro system indicated a switch of developmental program, which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and plants. This is the first detailed report of calli, embryos, and abnormal shoots originated from the haploid cells in cassava, leading to the development of a protocol for the production of doubled haploid plants in cassava.