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.

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.

Omicron Waves in Argentina: Dynamics of SARS-CoV-2 Lineages BA.1, BA.2 and the Emerging BA.2.12.1 and BA.4/BA.5.

The COVID-19 pandemic has lately been driven by Omicron. This work aimed to study the dynamics of SARS-CoV-2 Omicron lineages during the third and fourth waves of COVID-19 in Argentina. Molecular surveillance was performed on 3431 samples from Argentina, between EW44/2021 and EW31/2022. Sequencing, phylogenetic and phylodynamic analyses were performed. A differential dynamic between the Omicron waves was found. The third wave was associated with lineage BA.1, characterized by a high number of cases, very fast displacement of Delta, doubling times of 3.3 days and a low level of lineage diversity and clustering. In contrast, the fourth wave was longer but associated with a lower number of cases, initially caused by BA.2, and later by BA.4/BA.5, with doubling times of about 10 days. Several BA.2 and BA.4/BA.5 sublineages and introductions were detected, although very few clusters with a constrained geographical distribution were observed, suggesting limited transmission chains. The differential dynamic could be due to waning immunity and an increase in population gatherings in the BA.1 wave, and a boosted population (for vaccination or recent prior immunity for BA.1 infection) in the wave caused by BA2/BA.4/BA.5, which may have limited the establishment of the new lineages.

ddRADseq-mediated detection of genetic variants in sugarcane.

KEY MESSAGE: The article presents an optimization of the key parameters for the identification of SNPs in sugarcane using a GBS protocol based on two Illumina NextSeq and NovaSeq platforms. Sugarcane (Saccharum sp.), a world-wide known feedstock for sugar production, bioethanol, and energy, has an extremely complex genome, being highly polyploid and aneuploid. A double-digestion restriction site-associated DNA sequencing protocol (ddRADseq) was tested in four commercial sugarcane hybrids and one high-fibre biotype for the detection of single nucleotide polymorphisms (SNPs). In this work we tested two Illumina sequencing platforms, read size (70 vs. 150 bp), different sequencing coverage per individual (medium and high coverage), and single-reads versus paired-end reads. We also explored different variant calling strategies (with and without reference genome) and filtering schemes [combining two minor allele frequencies (MAFs) with three depth of coverage thresholds]. For the discovery of a large number of novel SNPs in sugarcane, we recommend longer size and paired-end reads, medium sequencing coverage per individual and Illumina platform NovaSeq6000 for a cost-effective approach, and filter parameters of lower MAF and higher depth coverages thresholds. Although the de novo analysis retrieved more SNPs, the reference-based method allows downstream characterization of variants. For the two best performing matrices, the number of SNPs per chromosome correlated positively with chromosome length, demonstrating the presence of variants throughout the genome. Multivariate comparisons, with both matrices, showed closer relationships among commercial hybrids than with the high-fibre biotype. Functional analysis of the SNPs demonstrated that more than half of them landed within regulatory regions, whereas the other half affected coding, intergenic and intronic regions. Allelic distances values were lower than 0.07 when analysing two replicated genotypes, confirming the protocol robustness.

Assessing the hidden diversity underlying consensus sequences of SARS-CoV-2 using VICOS, a novel bioinformatic pipeline for identification of mixed viral populations.

INTRODUCTION: Coinfection with two SARS-CoV-2 viruses is still a very understudied phenomenon. Although next generation sequencing methods are very sensitive to detect heterogeneous viral populations in a sample, there is no standardized method for their characterization, so their clinical and epidemiological importance is unknown. MATERIAL AND METHODS: We developed VICOS (Viral COinfection Surveillance), a new bioinformatic algorithm for variant calling, filtering and statistical analysis to identify samples suspected of being mixed SARS-CoV-2 populations from a large dataset in the framework of a community genomic surveillance. VICOS was used to detect SARS-CoV-2 coinfections in a dataset of 1,097 complete genomes collected between March 2020 and August 2021 in Argentina. RESULTS: We detected 23 cases (2%) of SARS-CoV-2 coinfections. Detailed study of VICOS's results together with additional phylogenetic analysis revealed 3 cases of coinfections by two viruses of the same lineage, 2 cases by viruses of different genetic lineages, 13 were compatible with both coinfection and intra-host evolution, and 5 cases were likely a product of laboratory contamination. DISCUSSION: Intra-sample viral diversity provides important information to understand the transmission dynamics of SARS-CoV-2. Advanced bioinformatics tools, such as VICOS, are a necessary resource to help unveil the hidden diversity of SARS-CoV-2.

Phenotypic and genetic characterization of Africanized Apis mellifera colonies with natural tolerance to Varroa destructor and contrasting defensive behavior.

Africanized Apis mellifera colonies with promising characteristics for beekeeping have been detected in northern Argentina (subtropical climate) and are considered of interest for breeding programs. Integral evaluation of this feral material revealed high colony strength and resistance/tolerance to brood diseases. However, these Africanized honeybees (AHB) also showed variable negative behavioral traits for beekeeping, such as defensiveness, tendency to swarm and avoidance behavior. We developed a protocol for the selection of AHB stocks based on defensive behavior and characterized contrasting colonies for this trait using NGS technologies. For this purpose, population and behavioral parameters were surveyed throughout a beekeeping season in nine daughter colonies obtained from a mother colony (A1 mitochondrial haplotype) with valuable characteristics (tolerance to the mite Varroa destructor, high colony strength and low defensiveness). A Defensive Behavior Index was developed and tested in the colonies under study. Mother and two daughter colonies displaying contrasting defensive behavior were analyzed by ddRADseq. High-quality DNA samples were obtained from 16 workers of each colony. Six pooled samples, including two replicates of each of the three colonies, were processed. A total of 12,971 SNPs were detected against the reference genome of A. mellifera, 142 of which showed significant differences between colonies. We detected SNPs in coding regions, lncRNA, miRNA, rRNA, tRNA, among others. From the original data set, we also identified 647 SNPs located in protein-coding regions, 128 of which are related to 21 genes previously associated with defensive behavior, such as dop3 and dopR2, CaMKII and ADAR, obp9 and obp10, and members of the 5-HT family. We discuss the obtained results by considering the influence of polyandry and paternal lineages on the defensive behavior in AHB and provide baseline information to use this innovative molecular approach, ddRADseq, to assist in the selection and evaluation of honey bee stocks showing low defensive behavior for commercial uses.

Anthropogenic Infection of Domestic Cats With SARS-CoV-2 Alpha Variant B.1.1.7 Lineage in Buenos Aires.

SARS-CoV-2 reverse zoonosis, particularly to domestic animals, and the potential role of infected animals in perpetuating the spread of the virus is an issue of increasing concern. In this case report, we identified the natural infection of two cats by SARS-CoV-2, in Argentina, whose owner had been previously infected by SARS-CoV-2. Viral genetic material was detected in feline oropharyngeal (OP) and rectal (R) swab by RT-qPCR, and sequence analysis revealed that the virus infecting the owner and one cat were genetically similar. The alpha variant (B.1.1.7 lineage) was identified with a unique additional mutation, strongly suggesting human-to-cat route of transmission. This study reinforces the One Health concept and the importance of integrating human, animal, and environmental perspectives to promptly address relevant health issues.

Cost-Effective Method to Perform SARS-CoV-2 Variant Surveillance: Detection of Alpha, Gamma, Lambda, Delta, Epsilon, and Zeta in Argentina.

SARS-CoV-2 variants with concerning characteristics have emerged since the end of 2020. Surveillance of SARS-CoV-2 variants was performed on a total of 4,851 samples from the capital city and 10 provinces of Argentina, during 51 epidemiological weeks (EWs) that covered the end of the first wave and the ongoing second wave of the COVID-19 pandemic in the country (EW 44/2020 to EW 41/2021). The surveillance strategy was mainly based on Sanger sequencing of a Spike coding region that allows the identification of signature mutations associated with variants. In addition, whole-genome sequences were obtained from 637 samples. The main variants found were Gamma and Lambda, and to a lesser extent, Alpha, Zeta, and Epsilon, and more recently, Delta. Whereas, Gamma dominated in different regions of the country, both Gamma and Lambda prevailed in the most populated area, the metropolitan region of Buenos Aires. The lineages that circulated on the first wave were replaced by emergent variants in a term of a few weeks. At the end of the ongoing second wave, Delta began to be detected, replacing Gamma and Lambda. This scenario is consistent with the Latin American variant landscape, so far characterized by a concurrent increase in Delta circulation and a stabilization in the number of cases. The cost-effective surveillance protocol presented here allowed for a rapid response in a resource-limited setting, added information on the expansion of Lambda in South America, and contributed to the implementation of public health measures to control the disease spread in Argentina.

Development of novel SSR molecular markers using a Next-Generation Sequencing approach (ddRADseq) in Stetsonia coryne (Cactaceae).

The Cactaceae family is native to the American continent with several centers of diversity. In South America, one of these centers is the Central Andes and many species are considered to be threatened or vulnerable according to the International Union for Conservation of Nature (IUCN). Stetsonia coryne is an emblematic giant columnar cacti of the Chaco phytogeographic province. It has an extensive geographical distribution in many countries of the continent. However, to date there are no specific molecular markers for this species, neither reports of population genetic variability studies, such as for many cactus species. The lack of information is fundamentally due to the lack of molecular markers that allow these studies. In this work, by applying a Genotyping by Sequencing (GBS) technique, we developed polymorphic SSR markers for the Stetsonia coryne and evaluated their transferability to phylogenetically close species, in order to account for a robust panel of molecular markers for multispecies-studies within Cactaceae.

The ER-mitochondria Ca2+ signaling in cancer progression: Fueling the monster.

Cancer is a leading cause of death worldwide. All major tumor suppressors and oncogenes are now recognized to have fundamental connections with metabolic pathways. A hallmark feature of cancer cells is a reprogramming of their metabolism even when nutrients are available. Increasing evidence indicates that most cancer cells rely on mitochondrial metabolism to sustain their energetic and biosynthetic demands. Mitochondria are functionally and physically coupled to the endoplasmic reticulum (ER), the major calcium (Ca2+) storage organelle in mammalian cells, through special domains known as mitochondria-ER contact sites (MERCS). In this domain, the release of Ca2+ from the ER is mainly regulated by inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), a family of Ca2+ release channels activated by the ligand IP3. IP3R mediated Ca2+ release is transferred to mitochondria through the mitochondrial Ca2+ uniporter (MCU). Once in the mitochondrial matrix, Ca2+ activates several proteins that stimulate mitochondrial performance. The role of IP3R and MCU in cancer, as well as the other proteins that enable the Ca2+ communication between these two organelles is just beginning to be understood. Here, we describe the function of the main players of the ER mitochondrial Ca2+ communication and discuss how this particular signal may contribute to the rise and development of cancer traits.

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.

Elucidating diversity in the class composition of the minicircle hypervariable region of Trypanosoma cruzi: New perspectives on typing and kDNA inheritance.

BACKGROUND: Trypanosoma cruzi, the protozoan causative of Chagas disease, is classified into six main Discrete Typing Units (DTUs): TcI-TcVI. This parasite has around 105 copies of the minicircle hypervariable region (mHVR) in their kinetoplastic DNA (kDNA). The genetic diversity of the mHVR is virtually unknown. However, cross-hybridization assays using mHVRs showed hybridization only between isolates belonging to the same genetic group. Nowadays there is no methodologic approach with a good sensibility, specificity and reproducibility for direct typing on biological samples. Due to its high copy number and apparently high diversity, mHVR becomes a good target for typing. METHODOLOGY/PRINCIPAL FINDINGS: Around 22 million reads, obtained by amplicon sequencing of the mHVR, were analyzed for nine strains belonging to six T. cruzi DTUs. The number and diversity of mHVR clusters was variable among DTUs and even within a DTU. However, strains of the same DTU shared more mHVR clusters than strains of different DTUs and clustered together. In addition, hybrid DTUs (TcV and TcVI) shared similar percentages (1.9-3.4%) of mHVR clusters with their parentals (TcII and TcIII). Conversely, just 0.2% of clusters were shared between TcII and TcIII suggesting biparental inheritance of the kDNA in hybrids. Sequencing at low depth (20,000-40,000 reads) also revealed 95% of the mHVR clusters for each of the analyzed strains. Finally, the method revealed good correlation in cluster identity and abundance between different replications of the experiment (r = 0.999). CONCLUSIONS/SIGNIFICANCE: Our work sheds light on the sequence diversity of mHVRs at intra and inter-DTU level. The mHVR amplicon sequencing workflow described here is a reproducible technique, that allows multiplexed analysis of hundreds of strains and results promissory for direct typing on biological samples in a future. In addition, such approach may help to gain knowledge on the mechanisms of the minicircle evolution and phylogenetic relationships among strains.

The first complete genomic structure of Butyrivibrio fibrisolvens and its chromid.

Butyrivibrio fibrisolvens forms part of the gastrointestinal microbiome of ruminants and other mammals, including humans. Indeed, it is one of the most common bacteria found in the rumen and plays an important role in ruminal fermentation of polysaccharides, yet, to date, there is no closed reference genome published for this species in any ruminant animal. We successfully assembled the nearly complete genome sequence of B. fibrisolvens strain INBov1 isolated from cow rumen using Illumina paired-end reads, 454 Roche single-end and mate pair sequencing technology. Additionally, we constructed an optical restriction map of this strain to aid in scaffold ordering and positioning, and completed the first genomic structure of this species. Moreover, we identified and assembled the first chromid of this species (pINBov266). The INBov1 genome encodes a large set of genes involved in the cellulolytic process but lacks key genes. This seems to indicate that B. fibrisolvens plays an important role in ruminal cellulolytic processes, but does not have autonomous cellulolytic capacity. When searching for genes involved in the biohydrogenation of unsaturated fatty acids, no linoleate isomerase gene was found in this strain. INBov1 does encode oleate hydratase genes known to participate in the hydrogenation of oleic acids. Furthermore, INBov1 contains an enolase gene, which has been recently determined to participate in the synthesis of conjugated linoleic acids. This work confirms the presence of a novel chromid in B. fibrisolvens and provides a new potential reference genome sequence for this species, providing new insight into its role in biohydrogenation and carbohydrate degradation.

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.

Sunflower: a potential fructan-bearing crop?

Grain filling in sunflower (Helianthus annuus L.) mainly depends on actual photosynthesis, being the contribution of stored reserves in stems (sucrose, hexoses, and starch) rather low. Drought periods during grain filling often reduce yield. Increasing the capacity of stem to store reserves could help to increase grain filling and yield stability in dry years. Fructans improve water uptake in soils at low water potential, and allow the storage of large amount of assimilates per unit tissue volume that can be readily remobilized to grains. Sunflower is a close relative to Jerusalem artichoke (H. tuberosus L.), which accumulates large amounts of fructan (inulin) in tubers and true stems. The reason why sunflower does not accumulate fructans is obscure. Through a bioinformatics analysis of a sunflower transcriptome database, we found sequences that are homologous to dicotyledon and monocotyledon fructan synthesis genes. A HPLC analysis of stem sugar composition revealed the presence of low amounts of 1-kestose, while a drastic enhancement of endogenous sucrose levels by capitulum removal did not promote 1-kestose accumulation. This suggests that the regulation of fructan synthesis in this species may differ from the currently best known model, mainly derived from research on Poaceae, where sucrose acts as both a signaling molecule and substrate, in the induction of fructan synthesis. Thus, sunflower might potentially constitute a fructan-bearing species, which could result in an improvement of its performance as a grain crop. However, a large effort is needed to elucidate how this up to now unsuspected potential could be effectively expressed.

Draft Genome Sequences of Escherichia coli O157:H7 Strains Rafaela_II (Clade 8) and 7.1_Anguil (Clade 6) from Cattle in Argentina.

Escherichia coli O157:H7 is a major etiologic agent of diseases in humans that cause diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. Here, we report the draft genome sequences of two strains isolated from cattle that had high levels of Shiga toxin 2 and high lethality in mice.

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.

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.

Functional characterization of a sucrose:fructan 6-fructosyltransferase of the cold-resistant grass Bromus pictus by heterelogous expression in Pichia pastoris and Nicotiana tabacum and its involvement in freezing tolerance.

We have previously reported the molecular characterization of a putative sucrose:fructan 6-fructosyltransferase (6-SFT) of Bromus pictus, a graminean species from Patagonia, tolerant to cold and drought. Here, this enzyme was functionally characterized by heterologous expression in Pichia pastoris and Nicotiana tabacum. Recombinant P. pastoris Bp6-SFT showed comparable characteristics to barley 6-SFT and an evident fructosyltransferase activity synthesizing bifurcose from sucrose and 1-kestotriose. Transgenic tobacco plants expressing Bp6-SFT, showed fructosyltransferase activity and fructan accumulation in leaves. Bp6-SFT plants exposed to freezing conditions showed a significantly lower electrolyte leakage in leaves compared to control plants, indicating less membrane damage. Concomitantly these transgenic plants resumed growth more rapidly than control ones. These results indicate that Bp6-SFT transgenic tobacco plants that accumulate fructan showed enhanced freezing tolerance compared to control plants.

Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in the cold tolerant Patagonian species Bromus pictus.

Fructans are fructose polymers synthesized in a wide range of species such as bacteria, fungi and plants. Fructans are synthesized by fructosyltransferases (FTs) and depolymerized by fructan exohydrolases (FEHs). Bromus pictus is a graminean decaploid species from the Patagonian region of Argentina, which accumulates large amounts of fructans even at temperate temperatures. The first gene isolated from B. pictus fructan metabolism was a putative sucrose:fructan 6-fructosyltransferase (6-SFT). Here, a complete cDNA of the first fructan exohydrolase (FEH) from B. pictus (Bp1-FEHa) was isolated using RT-PCR strategies. The Bp1-FEHa encoding gene is present as a single copy in B. pictus genome. Functional characterization in Pichia pastoris confirmed Bp1-FEHa is a fructan exohydrolase with predominant activity towards beta-(2-1) linkages. Its expression was analyzed in different leaf sections, showing the highest expression levels in the second section of the sheath and the tip of the blade. Bp1-FEHa expression was studied along with FEH and FT activities and fructan accumulation profile in response to chilling conditions during a 7-day time course experiment. Bp1-FEHa expression and FEH activity followed a similar pattern in response to low temperatures, especially in basal sections of the sheaths. In these sections the FEH and FT activities were particularly high and they were significantly correlated to fructan accumulation profile, along with cold treatment.