Unveiling the defensive role of Snakin-3, a member of the subfamily III of Snakin/GASA peptides in potatoes.

KEY MESSAGE: The first in-depth characterization of a subfamily III Snakin/GASA member was performed providing experimental evidence on promoter activity and subcellular localization and unveiling a role of potato Snakin-3 in defense Snakin/GASA proteins share 12 cysteines in conserved positions in the C-terminal region. Most of them were involved in different aspects of plant growth and development, while a small number of these peptides were reported to have antimicrobial activity or participate in abiotic stress tolerance. In potato, 18 Snakin/GASA genes were identified and classified into three groups based on phylogenetic analysis. Snakin-1 and Snakin-2 are members of subfamilies I and II, respectively, and were reported to be implicated not only in defense against pathogens but also in plant development. In this work, we present the first in-depth characterization of Snakin-3, a member of the subfamily III within the Snakin/GASA gene family of potato. Transient co-expression of Snakin-3 fused to the green fluorescent protein and organelle markers revealed that it is located in the endoplasmic reticulum. Furthermore, expression analyses via pSnakin-3::GUS transgenic plants showed GUS staining mainly in roots and vascular tissues of the stem. Moreover, GUS expression levels were increased after inoculation with Pseudomonas syringae pv. tabaci or Pectobacterium carotovorum subsp. carotovorum and also after auxin treatment mainly in roots and stems. To gain further insights into the function of Snakin-3 in planta, potato overexpressing lines were challenged against P. carotovorum subsp. carotovorum showing enhanced tolerance to this bacterial pathogen. In sum, here we report the first functional characterization of a Snakin/GASA gene from subfamily III in Solanaceae. Our findings provide experimental evidence on promoter activity and subcellular localization and reveal a role of potato Snakin-3 in plant defense.

Novel B2 mitogenomes from Continental southern Patagonia's Late Holocene: New insights into the peopling of the Southern Cone.

OBJECTIVES: The main aim of this study is to discuss the migratory processes and peopling dynamics that shaped the genetic variability of populations during the settlement of the Southern Cone, through the analysis of complete mitogenomes of individuals from southern Patagonia. MATERIALS AND METHODS: Complete mitogenomes were sequenced through massively parallel sequencing from two late Holocene individuals (SAC 1-1-3 and SAC 1-1-4) buried in the same chenque at Salitroso Lake Basin (Santa Cruz province, Argentina). To evaluate matrilineal phylogenetic affinities with other haplotypes, maximum likelihood and Bayesian phylogenetic reconstructions were performed, as well as a haplotype median-joining network. RESULTS: The mitogenomes were assigned to haplogroups B2 and B2b, exhibiting an average depth of 54X and 89X (≥1X coverage of 98.6% and 100%), and a high number of nucleotide differences among them. The phylogenetic analyses showed a relatively close relationship between the haplotype found in SAC 1-1-4 and those retrieved from a Middle Holocene individual from Laguna Chica (Buenos Aires province), and from a group of individuals from the Peruvian coast. For the SAC 1-1-3, no clear affiliations to any other haplotype were established. DISCUSSION: The large divergence between the haplotypes presented in this study suggests either a highly variable founder gene pool, or a later enrichment by frequent biological contact with other populations. Our results underline the persistence of genetic signals related to the first waves of peopling in South America, suggesting that the regional settlement of the southern end of the continent has been much more complex than initially thought.

OBJETIVOS: El objetivo principal de este estudio es discutir los procesos migratorios y la dinámica de poblamiento que moldearon la variabilidad genética de las poblaciones durante el poblamiento del Cono Sur, a través del análisis de mitogenomas completos de individuos del sur de Patagonia. MATERIALES Y MÉTODOS: Se obtuvieron mitogenomas completos mediante secuenciación masiva de dos individuos del Holoceno tardío (SAC 1-1-3 y SAC 1-1-4) enterrados en el mismo chenque en la Cuenca del Lago Salitroso (provincia de Santa Cruz, Argentina). Para evaluar las afinidades matrilineales con otros haplotipos, se realizaron reconstrucciones filogenéticas de máxima verosimilitud y bayesianas, así como una red mediana de haplotipos. RESULTADOS: Los mitogenomas fueron asignados a los haplogrupos B2 y B2b, exhibiendo una profundidad de secuenciación promedio de 54X y 89X (cobertura ≥1X de 98,6% y 100%), y un elevado número de diferencias nucleotídicas entre ellos. Los análisis filogenéticos mostraron una relación relativamente estrecha entre el haplotipo encontrado en el SAC 1-1-4 y los recuperados de un individuo del Holoceno Medio de Laguna Chica (provincia de Buenos Aires), y de un grupo de individuos de la costa peruana. Para el SAC 1-1-3, no se establecieron relaciones claras con ningún otro haplotipo. DISCUSIÓN: La gran divergencia entre los haplotipos presentados en este estudio sugiere gran variabilidad en el acervo genético fundador, o bien un enriquecimiento posterior por contacto biológico frecuente con otras poblaciones. Nuestros resultados destacan la persistencia de señales genéticas relacionadas con las primeras oleadas de poblamiento de Sudamérica, lo que sugiere que el poblamiento regional del extremo sur del continente ha sido mucho más complejo de lo que se pensaba inicialmente.

Co-Expression Networks in Sunflower: Harnessing the Power of Multi-Study Transcriptomic Public Data to Identify and Categorize Candidate Genes for Fungal Resistance.

Fungal plant diseases are a major threat to food security worldwide. Current efforts to identify and list loci involved in different biological processes are more complicated than originally thought, even when complete genome assemblies are available. Despite numerous experimental and computational efforts to characterize gene functions in plants, about ~40% of protein-coding genes in the model plant Arabidopsis thaliana L. are still not categorized in the Gene Ontology (GO) Biological Process (BP) annotation. In non-model organisms, such as sunflower (Helianthus annuus L.), the number of BP term annotations is far fewer, ~22%. In the current study, we performed gene co-expression network analysis using eight terabytes of public transcriptome datasets and expression-based functional prediction to categorize and identify loci involved in the response to fungal pathogens. We were able to construct a reference gene network of healthy green tissue (GreenGCN) and a gene network of healthy and stressed root tissues (RootGCN). Both networks achieved robust, high-quality scores on the metrics of guilt-by-association and selective constraints versus gene connectivity. We were able to identify eight modules enriched in defense functions, of which two out of the three modules in the RootGCN were also conserved in the GreenGCN, suggesting similar defense-related expression patterns. We identified 16 WRKY genes involved in defense related functions and 65 previously uncharacterized loci now linked to defense response. In addition, we identified and classified 122 loci previously identified within QTLs or near candidate loci reported in GWAS studies of disease resistance in sunflower linked to defense response. All in all, we have implemented a valuable strategy to better describe genes within specific biological processes.

Double Digest Restriction-Site Associated DNA Sequencing (ddRADseq) Technology.

Double digest restriction-site associated DNA sequencing (ddRADseq) technology combines genome reduced representation by digestion with two restriction enzymes and next generation sequencing (NGS) to obtain thousands of markers (SNP, SSR, and InDels) and genotype tens to hundreds of samples simultaneously. In this chapter, we describe a 96-plex derived ddRADseq protocol that can be set up to obtain different depth of coverage per locus and can be exploited to model and non-model plant species.

Genome-Wide Association Studies in Sunflower: Towards Sclerotinia sclerotiorum and Diaporthe/Phomopsis Resistance Breeding.

Diseases caused by necrotrophic fungi, such as the cosmopolitan Sclerotinia sclerotiorum and the Diaporthe/Phomopsis complex, are among the most destructive diseases of sunflower worldwide. The lack of complete resistance combined with the inefficiency of chemical control makes assisted breeding the best strategy for disease control. In this work, we present an integrated genome-wide association (GWA) study investigating the response of a diverse panel of sunflower inbred lines to both pathogens. Phenotypic data for Sclerotinia head rot (SHR) consisted of five disease descriptors (disease incidence, DI; disease severity, DS; area under the disease progress curve for DI, AUDPCI, and DS, AUDPCS; and incubation period, IP). Two disease descriptors (DI and DS) were evaluated for two manifestations of Diaporthe/Phomopsis: Phomopsis stem canker (PSC) and Phomopsis head rot (PHR). In addition, a principal component (PC) analysis was used to derive transformed phenotypes as inputs to a univariate GWA (PC-GWA). Genotypic data comprised a panel of 4269 single nucleotide polymorphisms (SNP), generated via genotyping-by-sequencing. The GWA analysis revealed 24 unique marker-trait associations for SHR, 19 unique marker-trait associations for Diaporthe/Phomopsis diseases, and 7 markers associated with PC1 and PC2. No common markers were found for the response to the two pathogens. Nevertheless, epistatic interactions were identified between markers significantly associated with the response to S. sclerotiorum and Diaporthe/Phomopsis. This suggests that, while the main determinants of resistance may differ for the two pathogens, there could be an underlying common genetic basis. The exploration of regions physically close to the associated markers yielded 364 genes, of which 19 were predicted as putative disease resistance genes. This work presents the first simultaneous evaluation of two manifestations of Diaporthe/Phomopsis in sunflower, and undertakes a comprehensive GWA study by integrating PSC, PHR, and SHR data. The multiple regions identified, and their exploration to identify candidate genes, contribute not only to the understanding of the genetic basis of resistance, but also to the development of tools for assisted breeding.

Morphological and genetic diversity of maize landraces along an altitudinal gradient in the Southern Andes.

Maize (Zea mays ssp. mays) is a major cereal crop worldwide and is traditionally or commercially cultivated almost all over the Americas. The North-Western Argentina (NWA) region constitutes one of the main diversity hotspots of the Southern Andes, with contrasting landscapes and a large number of landraces. Despite the extensive collections performed by the "Banco Activo de Germoplasma INTA Pergamino, Argentina" (BAP), most of them have not been characterized yet. Here we report the morphological and molecular evaluation of 30 accessions collected from NWA, along an altitudinal gradient between 1120 and 2950 meters above sea level (masl). Assessment of morphological variation in a common garden allowed the discrimination of two groups, which differed mainly in endosperm type and overall plant size. Although the groups retrieved by the molecular analyses were not consistent with morphological clusters, they showed a clear pattern of altitudinal structuring. Affinities among accessions were not in accordance with racial assignments. Overall, our results revealed that there are two maize gene pools co-existing in NWA, probably resulting from various waves of maize introduction in pre-Columbian times as well as from the adoption of modern varieties by local farmers. In conclusion, the NWA maize landraces preserved at the BAP possess high morphological and molecular variability. Our results highlight their potential as a source of diversity for increasing the genetic basis of breeding programs and provide useful information to guide future sampling and conservation efforts.

Characterization and expression analysis of WRKY genes during leaf and corolla senescence of Petunia hybrida plants.

Several families of transcription factors (TFs) control the progression of senescence. Many key TFs belonging to the WRKY family have been described to play crucial roles in the regulation of leaf senescence, mainly in Arabidopsis thaliana. However, little is known about senescence-associated WRKY members in floricultural species. Delay of senescence in leaves and petals of Petunia hybrida, a worldwide ornamental crop are highly appreciated traits. In this work, starting from 28 differentially expressed WRKY genes of A. thaliana during the progression of leaf senescence, we identified the orthologous in P. hybrida and explored the expression profiles of 20 PhWRKY genes during the progression of natural (age-related) leaf and corolla senescence as well as in the corollas of flowers undergoing pollination-induced senescence. Simultaneous visualization showed consistent and similar expression profiles of PhWRKYs during natural leaf and corolla senescence, although weak expression changes were observed during pollination-induced senescence. Comparable expression trends between PhWRKYs and the corresponding genes of A. thaliana were observed during leaf senescence, although more divergence was found in petals of pollinated petunia flowers. Integration of expression data with phylogenetics, conserved motif and cis-regulatory element analyses were used to establish a list of candidates that could regulate more than one senescence process. Our results suggest that several members of the WRKY family of TFs are tightly linked to the regulation of senescence in P. hybrida. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01243-y.

Diversity of the BoLA-DRB3 gene in cattle breeds from tropical and subtropical regions of Argentina.

Bovine leukocyte antigens (BoLA) have been widely studied because of their primary function in the recognition of pathogens by the immune system. To date, however, the characterization of the BoLA-DRB3 gene in Latin American Zebu and mixed zebuine breeds is scarce. By a sequence-based typing method, here we sequenced exon 2 of BoLA class II DRB3 gene in 264 animals from the five most commonly used breeds in northern Argentina (Creole, Brahman, Braford, Brangus, and Nellore).The Bos taurus, Bos indicus, and mixed breeds analyzed here contained 61 previously reported alleles. Genetic diversity was high at both allelic and nucleotide sequence levels, particularly in the mixed breeds Braford and Brangus. In contrast to previous reports on DRB3 diversity, no evidence of balancing selection was found in our data. Differentiation among breeds was highly significant, as shown by FST (FST = 0.052, P < 0.001) and cluster analyses. In accordance with historical origin of the breeds, UPGMA trees and metric multidimensional scaling (MDS) analyses showed that Creole is distantly related to the other zebuine breeds. Among them, Brahman, Braford, and Brangus exhibited the closest affiliations. Despite the overall differentiation of the breeds, analysis of the peptide binding regions at the aminoacid level revealed that the key aminoacids involved in peptide recognition are greatly conserved suggesting little influence of domestication and breeding in functional MHC variability. In sum, this is the first report of BoLA-DRB3 diversity in pure and mixed Bos indicus cattle breeds from Argentina. Knowledge of BoLA-DRB3 variability in breeds adapted to tropical and subtropical environments contributes not only to the characterization of MHC diversity but also to the design of peptide-based vaccines.

On-field phenotypic evaluation of sunflower populations for broad-spectrum resistance to Verticillium leaf mottle and wilt.

Sunflower Verticillium Wilt and Leaf Mottle (SVW), caused by Verticillium dahliae (Kleb.; Vd), is a soil-borne disease affecting sunflower worldwide. A single dominant locus, known as V1, was formerly effective in controlling North-American Vd races, whereas races from Argentina, Europe and an emerging race from USA overcome its resistance. This emphasizes the need for identifying broad-spectrum genetic resistance (BSR) sources. Here we characterize two sunflower mapping populations (MPs) for SVW resistance: a biparental MP and the association MP from the National Institute of Agricultural Technology (INTA), under field growing conditions. Nine field-trials (FTs) were conducted in highly infested fields in the most SVW-affected region of Argentina. Several disease descriptors (DDs), including incidence and severity, were scored across four phenological stages. Generalized linear models were fitted according to the nature of each variable, adjusting mean phenotypes for inbred lines across and within FTs. Comparison of these responses allowed the identification of novel BSR sources. Furthermore, we present the first report of SVW resistance heritability, with estimates ranging from 35 to 45% for DDs related to disease incidence and severity, respectively. This study constitutes the largest SVW resistance characterization reported to date in sunflower, identifying valuable genetic resources for BSR-breeding to cope with a pathogen of increasing importance worldwide.

Plastome genomics in South American maize landraces: chloroplast lineages parallel the geographical structuring of nuclear gene pools.

BACKGROUND AND AIMS: The number of plastome sequences has increased exponentially during the last decade. However, there is still little knowledge of the levels and distribution of intraspecific variation. The aims of this study were to estimate plastome diversity within Zea mays and analyse the distribution of haplotypes in connection with the landrace groups previously delimited for South American maize based on nuclear markers. METHODS: We obtained the complete plastomes of 30 South American maize landraces and three teosintes by means of next-generation sequencing (NGS) and used them in combination with data from public repositories. After quality filtering, the curated data were employed to search for single-nucleotide polymorphisms, indels and chloroplast simple sequence repeats. Exact permutational contingency tests were performed to assess associations between plastome and nuclear variation. Network and Bayesian phylogenetic analyses were used to infer evolutionary relationships among haplotypes. KEY RESULTS: Our analyses identified a total of 124 polymorphic plastome loci, with the intergenic regions psbE-rps18, petN-rpoB, trnL_UAG-ndhF and rpoC2-atpI exhibiting the highest marker densities. Although restricted in number, these markers allowed the discrimination of 27 haplotypes in a total of 51 Zea mays individuals. Andean and lowland South American landraces differed significantly in haplotype distribution. However, overall differentiation patterns were not informative with respect to subspecies diversification, as evidenced by the scattered distribution of maize and teosinte plastomes in both the network and Bayesian phylogenetic reconstructions. CONCLUSIONS: Knowledge of intraspecific plastome variation provides the framework for a more comprehensive understanding of evolutionary processes at low taxonomic levels and may become increasingly important for future plant barcoding efforts. Whole-plastome sequencing provided useful variability to contribute to maize phylogeographic studies. The structuring of haplotype diversity in the maize landraces examined here clearly reflects the distinction between the Andean and South American lowland gene pools previously inferred based on nuclear markers.

Exploring sunflower responses to Sclerotinia head rot at early stages of infection using RNA-seq analysis.

Sclerotinia head rot (SHR), caused by the necrotrophic fungus Sclerotinia sclerotiorum, is one of the most devastating sunflower crop diseases. Despite its worldwide occurrence, the genetic determinants of plant resistance are still largely unknown. Here, we investigated the Sclerotinia-sunflower pathosystem by analysing temporal changes in gene expression in one susceptible and two tolerant inbred lines (IL) inoculated with the pathogen under field conditions. Differential expression analysis showed little overlapping among ILs, suggesting genotype-specific control of cell defense responses possibly related to differences in disease resistance strategies. Functional enrichment assessments yielded a similar pattern. However, all three ILs altered the expression of genes involved in the cellular redox state and cell wall remodeling, in agreement with current knowledge about the initiation of plant immune responses. Remarkably, the over-representation of long non-coding RNAs (lncRNA) was another common feature among ILs. Our findings highlight the diversity of transcriptional responses to SHR within sunflower breeding lines and provide evidence of lncRNAs playing a significant role at early stages of defense.

Genetic Diversity, Population Structure and Linkage Disequilibrium Assessment among International Sunflower Breeding Collections.

Sunflower germplasm collections are valuable resources for broadening the genetic base of commercial hybrids and ameliorate the risk of climate events. Nowadays, the most studied worldwide sunflower pre-breeding collections belong to INTA (Argentina), INRA (France), and USDA-UBC (United States of America-Canada). In this work, we assess the amount and distribution of genetic diversity (GD) available within and between these collections to estimate the distribution pattern of global diversity. A mixed genotyping strategy was implemented, by combining proprietary genotyping-by-sequencing data with public whole-genome-sequencing data, to generate an integrative 11,834-common single nucleotide polymorphism matrix including the three breeding collections. In general, the GD estimates obtained were moderate. An analysis of molecular variance provided evidence of population structure between breeding collections. However, the optimal number of subpopulations, studied via discriminant analysis of principal components (K = 12), the bayesian STRUCTURE algorithm (K = 6) and distance-based methods (K = 9) remains unclear, since no single unifying characteristic is apparent for any of the inferred groups. Different overall patterns of linkage disequilibrium (LD) were observed across chromosomes, with Chr10, Chr17, Chr5, and Chr2 showing the highest LD. This work represents the largest and most comprehensive inter-breeding collection analysis of genomic diversity for cultivated sunflower conducted to date.

Identification and expression analysis of NAC transcription factors potentially involved in leaf and petal senescence in Petunia hybrida.

Progression of leaf senescence depends on several families of transcription factors. In Arabidopsis, the NAC family plays crucial roles in the modulation of leaf senescence; however, the mechanisms involved in this NAC-mediated regulation have not been extensively explored in agronomic species. Petunia hybrida is an ornamental plant that is commonly found worldwide. Decreasing the rate of leaf and petal senescence in P. hybrida is essential for maintaining plant quality. In this study, we examined the NAC-mediated networks involved in regulating senescence in this species. From 41 NAC genes, the expression of which changed in Arabidopsis during leaf senescence, we identified 29 putative orthologs in P. hybrida. Analysis using quantitative real-time-PCR indicated that 24 genes in P. hybrida changed their transcript levels during natural leaf senescence. Leaf-expressed genes were subsequently assessed in petals undergoing natural and pollination-induced senescence. Expression data and phylogenetic analysis were used to generate a list of 10-15 candidate genes; 7 of these were considered key regulatory candidates in senescence because of their consistent upregulation in the three senescence processes examined. Altogether, we identified common and distinct patterns of gene expression at different stages of leaf and petal development and during progression of senescence. The results obtained in this study will contribute to the understanding of NAC-mediated regulatory networks in petunia.

Main and epistatic QTL analyses for Sclerotinia Head Rot resistance in sunflower.

Sclerotinia Head Rot (SHR), a disease caused by Sclerotinia sclerotiorum, is one of the most limiting factors in sunflower production. In this study, we identified genomic loci associated with resistance to SHR to support the development of assisted breeding strategies. We genotyped 114 Recombinant Inbred Lines (RILs) along with their parental lines (PAC2 -partially resistant-and RHA266 -susceptible-) by using a 384 single nucleotide polymorphism (SNP) Illumina Oligo Pool Assay to saturate a sunflower genetic map. Subsequently, we tested these lines for SHR resistance using assisted inoculations with S. sclerotiorum ascospores. We also conducted a randomized complete-block assays with three replicates to visually score disease incidence (DI), disease severity (DS), disease intensity (DInt) and incubation period (IP) through four field trials (2010-2014). We finally assessed main effect quantitative trait loci (M-QTLs) and epistatic QTLs (E-QTLs) by composite interval mapping (CIM) and mixed-model-based composite interval mapping (MCIM), respectively. As a result of this study, the improved map incorporates 61 new SNPs over candidate genes. We detected a broad range of narrow sense heritability (h2) values (1.86-59.9%) as well as 36 M-QTLs and 13 E-QTLs along 14 linkage groups (LGs). On LG1, LG10, and LG15, we repeatedly detected QTLs across field trials; which emphasizes their putative effectiveness against SHR. In all selected variables, most of the identified QTLs showed high determination coefficients, associated with moderate to high heritability values. Using markers shared with previous Sclerotinia resistance studies, we compared the QTL locations in LG1, LG2, LG8, LG10, LG11, LG15 and LG16. This study constitutes the largest report of QTLs for SHR resistance in sunflower. Further studies focusing on the regions in LG1, LG10, and LG15 harboring the detected QTLs are necessary to identify causal alleles and contribute to unraveling the complex genetic basis governing the resistance.

Dissecting maize diversity in lowland South America: genetic structure and geographic distribution models.

BACKGROUND: Maize landraces from South America have traditionally been assigned to two main categories: Andean and Tropical Lowland germplasm. However, the genetic structure and affiliations of the lowland gene pools have been difficult to assess due to limited sampling and the lack of comparative analysis. Here, we examined SSR and Adh2 sequence variation in a diverse sample of maize landraces from lowland middle South America, and performed a comprehensive integrative analysis of population structure and diversity including already published data of archaeological and extant specimens from the Americas. Geographic distribution models were used to explore the relationship between environmental factors and the observed genetic structure. RESULTS: Bayesian and multivariate analyses of population structure showed the existence of two previously overlooked lowland gene pools associated with Guaraní indigenous communities of middle South America. The singularity of this germplasm was also evidenced by the frequency distribution of microsatellite repeat motifs of the Adh2 locus and the distinct spatial pattern inferred from geographic distribution models. CONCLUSION: Our results challenge the prevailing view that lowland middle South America is just a contact zone between Andean and Tropical Lowland germplasm and highlight the occurrence of a unique, locally adapted gene pool. This information is relevant for the conservation and utilization of maize genetic resources, as well as for a better understanding of environment-genotype associations.

Population structure and genetic diversity characterization of a sunflower association mapping population using SSR and SNP markers.

BACKGROUND: Argentina has a long tradition of sunflower breeding, and its germplasm is a valuable genetic resource worldwide. However, knowledge of the genetic constitution and variability levels of the Argentinean germplasm is still scarce, rendering the global map of cultivated sunflower diversity incomplete. In this study, 42 microsatellite loci and 384 single nucleotide polymorphisms (SNPs) were used to characterize the first association mapping population used for quantitative trait loci mapping in sunflower, along with a selection of allied open-pollinated and composite populations from the germplasm bank of the National Institute of Agricultural Technology of Argentina. The ability of different kinds of markers to assess genetic diversity and population structure was also evaluated. RESULTS: The analysis of polymorphism in the set of sunflower accessions studied here showed that both the microsatellites and SNP markers were informative for germplasm characterization, although to different extents. In general, the estimates of genetic variability were moderate. The average genetic diversity, as quantified by the expected heterozygosity, was 0.52 for SSR loci and 0.29 for SNPs. Within SSR markers, those derived from non-coding regions were able to capture higher levels of diversity than EST-SSR. A significant correlation was found between SSR and SNP- based genetic distances among accessions. Bayesian and multivariate methods were used to infer population structure. Evidence for the existence of three different genetic groups was found consistently across data sets (i.e., SSR, SNP and SSR + SNP), with the maintainer/restorer status being the most prevalent characteristic associated with group delimitation. CONCLUSION: The present study constitutes the first report comparing the performance of SSR and SNP markers for population genetics analysis in cultivated sunflower. We show that the SSR and SNP panels examined here, either used separately or in conjunction, allowed consistent estimations of genetic diversity and population structure in sunflower breeding materials. The generated knowledge about the levels of diversity and population structure of sunflower germplasm is an important contribution to this crop breeding and conservation.

Development of a multilocus sequence typing scheme for the study of Anaplasma marginale population structure over space and time.

Bovine Anaplasmosis caused by Anaplasma marginale is a worldwide disease prevalent in tropical and subtropical regions where Rhipicephalus microplus is considered the most significant biological vector. Molecular markers previously applied for A. marginale typing are efficient for isolate discrimination but they are not a suitable tool for studying population structure and dynamics. Here we report the development of an MLST scheme based on the study of seven genes: dnaA, ftsZ, groEl, lipA, recA, secY and sucB. Five annotated genomes (Saint Maries, Florida, Mississippi, Puerto Rico and Virginia) and 53 bovine blood samples from different world regions were analyzed. High nucleotide diversity and a large proportion of synonymous substitutions, indicative of negative selection resulted from DnaSP 5.00.02 package application. Recombination events were detected in almost all genes, this evidence together with the coexistence of more than one A. marginale strain in the same sample might suggest the superinfection phenomena as a potential source of variation. The allelic profile analysis performed through GoeBURST shown two main CC that did not support geography. In addition, the AMOVA test confirmed the occurrence of at least two main genetically divergent groups. The composition of the emergent groups reflected the impact of both historical and environmental traits on A. marginale population structure. Finally, a web-based platform "Galaxy MLST-Pipeline" was developed to automate DNA sequence editing and data analysis that together with the Data Base are freely available to users. The A. marginale MLST scheme developed here is a valuable tool with a high discrimination power, besides PCR based strategies are still the better choice for epidemiological intracellular pathogens studies. Finally, the allelic profile describe herein would contribute to uncover the mechanisms in how intracellular pathogens challenge virulence paradigm.

SNP genotyping by heteroduplex analysis.

Heteroduplex-based genotyping methods have proven to be technologically effective and economically efficient for low- to medium-range throughput single-nucleotide polymorphism (SNP) determination. In this chapter we describe two protocols that were successfully applied for SNP detection and haplotype analysis of candidate genes in association studies. The protocols involve (1) enzymatic mismatch cleavage with endonuclease CEL1 from celery, associated with fragment separation using capillary electrophoresis (CEL1 cleavage), and (2) differential retention of the homo/heteroduplex DNA molecules under partial denaturing conditions on ion pair reversed-phase liquid chromatography (dHPLC). Both methods are complementary since dHPLC is more versatile than CEL1 cleavage for identifying multiple SNP per target region, and the latter is easily optimized for sequences with fewer SNPs or small insertion/deletion polymorphisms. Besides, CEL1 cleavage is a powerful method to localize the position of the mutation when fragment resolution is done using capillary electrophoresis.

Population genetics structure of glyphosate-resistant Johnsongrass (Sorghum halepense L. Pers) does not support a single origin of the resistance.

Single sequence repeats (SSR) developed for Sorghum bicolor were used to characterize the genetic distance of 46 different Sorghum halepense (Johnsongrass) accessions from Argentina some of which have evolved toward glyphosate resistance. Since Johnsongrass is an allotetraploid and only one subgenome is homologous to cultivated sorghum, some SSR loci amplified up to two alleles while others (presumably more conserved loci) amplified up to four alleles. Twelve SSR providing information of 24 loci representative of Johnsongrass genome were selected for genetic distance characterization. All of them were highly polymorphic, which was evidenced by the number of different alleles found in the samples studied, in some of them up to 20. UPGMA and Mantel analysis showed that Johnsongrass glyphosate-resistant accessions that belong to different geographic regions do not share similar genetic backgrounds. In contrast, they show closer similarity to their neighboring susceptible counterparts. Discriminant Analysis of Principal Components using the clusters identified by K-means support the lack of a clear pattern of association among samples and resistance status or province of origin. Consequently, these results do not support a single genetic origin of glyphosate resistance. Nucleotide sequencing of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from glyphosate-resistant and susceptible accessions collected from different geographic origins showed that none presented expected mutations in aminoacid positions 101 and 106 which are diagnostic of target-site resistance mechanism.