V. V. Lia

Behaviour of ring bivalents in holokinetic systems: alternative sites of spindle attachment in Pachylis argentinus and Nezara viridula (Heteroptera).

Heteropteran chromosomes are holokinetic; during mitosis, sister chromatids segregate parallel to each other but, during meiosis, kinetic activity is restricted to one pair of telomeric regions. This meiotic behaviour has been corroborated for all rod bivalents. For ring bivalents, we have previously proposed that one of the two chiasmata releases first, and a telokinetic activity is also achieved.In the present work we analyse the meiotic behaviour of ring bivalents in Pachylis argentinus (Coreidae) and Nezara viridula (Pentatomidae) and we describe for the first time the chromosome complement and male meiosis of the former (2n=12+2m+X0, pre-reduction of the X). Both species possess a large chromosome pair with a secondary constriction which is a nucleolus organizer region as revealed by in-situ hybridization. Here we propose a new mode of segregation for ring bivalents: when the chromosome pair bears a secondary constriction, it is not essential that one of the chiasmata releases first since these regions or repetitive DNA sequences adjacent to them become functional as alternative sites for microtubule attachment and they undertake chromosome segregation to the poles during anaphase I.

Identification of Single Nucleotide Polymorphisms and analysis of Linkage Disequilibrium in sunflower elite inbred lines using the candidate gene approach

BackgroundAssociation analysis is a powerful tool to identify gene loci that may contribute to phenotypic variation. This includes the estimation of nucleotide diversity, the assessment of linkage disequilibrium structure (LD) and the evaluation of selection processes. Trait mapping by allele association requires a high-density map, which could be obtained by the addition of Single Nucleotide Polymorphisms (SNPs) and short insertion and/or deletions (indels) to SSR and AFLP genetic maps. Nucleotide diversity analysis of randomly selected candidate regions is a promising approach for the success of association analysis and fine mapping in the sunflower genome. Moreover, knowledge of the distance over which LD persists, in agronomically meaningful sunflower accessions, is important to establish the density of markers and the experimental design for association analysis.ResultsA set of 28 candidate genes related to biotic and abiotic stresses were studied in 19 sunflower inbred lines. A total of 14,348 bp of sequence alignment was analyzed per individual. In average, 1 SNP was found per 69 nucleotides and 38 indels were identified in the complete data set. The mean nucleotide polymorphism was moderate (θ = 0.0056), as expected for inbred materials. The number of haplotypes per region ranged from 1 to 9 (mean = 3.54 ± 1.88). Model-based population structure analysis allowed detection of admixed individuals within the set of accessions examined. Two putative gene pools were identified (G1 and G2), with a large proportion of the inbred lines being assigned to one of them (G1). Consistent with the absence of population sub-structuring, LD for G1 decayed more rapidly (r2 = 0.48 at 643 bp; trend line, pooled data) than the LD trend line for the entire set of 19 individuals (r2 = 0.64 for the same distance).ConclusionKnowledge about the patterns of diversity and the genetic relationships between breeding materials could be an invaluable aid in crop improvement strategies. The relatively high frequency of SNPs within the elite inbred lines studied here, along with the predicted extent of LD over distances of 100 kbp (r2~0.1) suggest that high resolution association mapping in sunflower could be achieved with marker densities lower than those usually reported in the literature.

Microsatellite typing of ancient maize: insights into the history of agriculture in southern South America.

Archaeological maize specimens from Andean sites of southern South America, dating from 400 to 1400 years before present, were tested for the presence of ancient DNA and three microsatellite loci were typed in the specimens that gave positive results. Genotypes were also obtained for 146 individuals corresponding to modern landraces currently cultivated in the same areas and for 21 plants from Argentinian lowland races. Sequence analysis of cloned ancient DNA products revealed a high incidence of substitutions appearing in only one clone, with transitions prevalent. In the archaeological specimens, there was no evidence of polymorphism at any one of the three microsatellite loci: each exhibited a single allelic variant, identical to the most frequent allele found in contemporary populations belonging to races Amarillo Chico, Amarillo Grande, Blanco and Altiplano. Affiliation between ancient specimens and a set of races from the Andean complex was further supported by assignment tests. The striking genetic uniformity displayed by the ancient specimens and their close relationship with the Andean complex suggest that the latter gene pool has predominated in the western regions of southern South America for at least the past 1400 years. The results support hypotheses suggesting that maize cultivation initially spread into South America via a highland route, rather than through the lowlands.

Complex mutational patterns and size homoplasy at maize microsatellite loci

Microsatellite markers have become one of the most popular tools for germplasm characterization, population genetics and evolutionary studies. To investigate the mutational mechanisms of maize microsatellites, nucleotide sequence information was obtained for ten loci. In addition, Single-Strand Conformation Polymorphism (SSCP) analysis was conducted to assess the occurrence of size homoplasy. Sequence analysis of 54 alleles revealed a complex pattern of mutation at 8/10 loci, with only 2 loci showing allele variation strictly consistent with stepwise mutations. The overall allelic diversity resulted from changes in the number of repeat units, base substitutions, and indels within repetitive and non-repetitive segments. Thirty-one electromorphs sampled from six maize landraces were considered for SSCP analysis. The number of conformers per electromorph ranged from 1 to 7, with 74.2% of the electromorphs showing more than one conformer. Size homoplasy was apparent within landraces and populations. Variation in the amount of size homoplasy was observed within and between loci, although no differences were detected among populations. The results of the present study provide useful information on the interpretation of genetic data derived from microsatellite markers. Further efforts are still needed to determine the impact of these findings on the estimation of population parameters and on the inference of phylogenetic relationships in maize investigations.

Genetic diversity in maize landraces from indigenous settlements of Northeastern Argentina

In South America, native maize germplasm has been extensively studied particularly for the Andean region. However, relatively few genetic diversity studies include materials from the eastern region of the continent. Herein we present a genetic diversity characterization of four Popcorn maize landraces, maintained in indigenous settlements, from Northeastern Argentina (NEA). In addition, one Popcorn landrace from Northwestern Argentina (NWA) was incorporated for comparison. We characterized these landraces using ten microsatellite markers. For the whole data set, a total of 65 alleles were found, with an average of 7.22 alleles per locus. The average gene diversity was 0.370. Global fit to Hardy–Weinberg proportions was observed in all landraces. Global estimates of FST revealed a significant differentiation among the populations. Individual Neighbor-joining clustering and Bayesian analyses allowed the recognition of most populations studied. Two main groups were distinguished by the Neighbor-joining clustering of populations. This grouping pattern would be consistent with a hypothesis of successive introductions of Popcorn in South America. The results presented will be useful to design strategies that maximize the utility of maize genetic resources.

Microsatellite variation in maize landraces from Northwestern Argentina: genetic diversity, population structure and racial affiliations.

The highland region or Northwestern Argentina (NWA) is one of the southernmost areas of native maize cultivation and constitutes an expansion of the peruvian Andes sphere of influence. To examine the genetic diversity and racial affiliations of the landraces cultivated in this area, 18 microsatellite markers were used to characterize 147 individuals from 6 maize races representative of traditional materials. For the whole data set, a total of 184 alleles were found, with an average of 10.2 alleles per locus. The average gene diversity was 0.571. The observed patterns of genetic differentiation suggest that historical association is probably the main factor in shaping population structure for the landraces studied here. In agreement with morphological and cytogenetic data, Bayesian analysis of NWA landraces revealed the occurrence of three main gene pools. Assessment of racial affiliations using a combined dataset including previous data on American landraces showed a clear relationship between one of these gene pools and typical Andean races, whereas the remaining two gene pools exhibited a closer association to Caribbean accessions and native germplasm from the United States, respectively. These results highlight the importance of integrating regional genetic studies if a deeper understanding of maize diversification and dispersal is to be achieved.

Association mapping in sunflower for sclerotinia head rot resistance

BackgroundSclerotinia Head Rot (SHR) is one of the most damaging diseases of sunflower in Europe, Argentina, and USA, causing average yield reductions of 10 to 20 %, but leading to total production loss under favorable environmental conditions for the pathogen. Association Mapping (AM) is a promising choice for Quantitative Trait Locus (QTL) mapping, as it detects relationships between phenotypic variation and gene polymorphisms in existing germplasm without development of mapping populations. This article reports the identification of QTL for resistance to SHR based on candidate gene AM.ResultsA collection of 94 sunflower inbred lines were tested for SHR under field conditions using assisted inoculation with the fungal pathogen Sclerotinia sclerotiorum. Given that no biological mechanisms or biochemical pathways have been clearly identified for SHR, 43 candidate genes were selected based on previous transcript profiling studies in sunflower and Brassica napus infected with S. sclerotiorum. Associations among SHR incidence and haplotype polymorphisms in 16 candidate genes were tested using Mixed Linear Models (MLM) that account for population structure and kinship relationships. This approach allowed detection of a significant association between the candidate gene HaRIC_B and SHR incidence (P < 0.01), accounting for a SHR incidence reduction of about 20 %.ConclusionsThese results suggest that AM will be useful in dissecting other complex traits in sunflower, thus providing a valuable tool to assist in crop breeding.

Single nucleotide polymorphism genotyping by heteroduplex analysis in sunflower (Helianthus annuus L.)

Single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) are increasingly used for cultivar identification, construction of genetic maps, genetic diversity assessment, association mapping and marker-assisted breeding. Although there are several highly sensitive methods for the detection of polymorphisms, most of them are often beyond the budget of medium-throughput academic laboratories or seed companies. Heteroduplex analysis by enzymatic cleavage (CEL1CH) or denaturing high-performance liquid chromatography (dHPLC) has been successfully used to examine genetic variation in several plant and animal species. In this work, we assess and compare the performance of both methods in sunflower by genotyping SNPs from a set of 24 selected polymorphic candidate genes. The CEL1CH method allowed us to accurately detect allele differences in 10 out of 24 regions using an in-house prepared CEL1 enzyme (celery single strand endonuclease 1, Apium graveolens L.). Similarly, a total of 11 regions were successfully optimized for dHPLC analysis. As a scaling-up approach, both strategies were tested to genotype either 42 SNPs/indels in 22 sunflower accessions from the local germplasm bank or 33 SNPs/indels in 90 recombinant inbred lines (RILs) for genetic mapping purposes. Summarizing, a total of 601 genotypes were efficiently analyzed either with CEL1CH (110) or dHPCL (491). In conclusion, CEL1CH and dHPLC proved to be robust, complementary methods, allowing medium-scale laboratories to scale up the number of both SNPs and individuals to be included in genetic studies and targeted germplasm diversity characterization (EcoTILLING).

B Chromosome Polymorphism in Maize Landraces: Adaptive vs. Demographic Hypothesis of Clinal Variation

Cytogenetic analysis of maize landraces from northwestern Argentina has revealed an altitudinal cline in the mean number of B chromosomes (Bs) per plant, with cultivars growing at higher altitudes exhibiting a higher number of Bs. Altitudinal and longitudinal clines are frequently interpreted as evidence of selection, however, they can also be produced by the interplay between drift and spatially restricted gene flow or by admixture between previously isolated populations that have come into secondary contact. Here, we test the adaptive significance of the observed altitudinal gradient by comparing the levels of differentiation in the mean number of Bs to those obtained from 18 selectively neutral loci [simple sequence repeats (SSRs)] among seven populations of the cline. The adequacy of alternative genetic-differentiation measures was determined, and associations between cytogenetic, genetic, and altitudinal distances were assessed by means of matrix- correspondence tests. No evidence for association between pairwise FST and altitudinal distance or B-chromosome differentiation was found. The contrasting pattern of altitudinal divergence between the mean number of Bs per plant and the genetic differentiation at SSR loci indicates that demographic processes cannot account for the observed levels of divergence in the mean number of Bs.

Sunflower germin-like protein HaGLP1 promotes ROS accumulation and enhances protection against fungal pathogens in transgenic Arabidopsis thaliana

Key messageThe novel sunflower gene HaGLP1 is the first germin-like protein characterized from the family Asteraceae. It alters the host redox status and confers protection againstSclerotiniasclerotiorumandRhizoctonia solani.AbstractGermin-like proteins (GLPs) are a large, diverse and ubiquitous family of plant glycoproteins belonging to the Cupin super family. These proteins have been widely studied because of their diverse roles in important plant processes, including defence. The novel sunflower gene HaGLP1 encodes the first germin-like protein characterized from the family Asteraceae. To analyse whether constitutive in vivo expression of the HaGLP1 gene may lead to disease tolerance, we developed transgenic Arabidopsis plants that were molecularly characterized and biologically assessed after inoculation with Sclerotinia sclerotiorum or Rhizoctonia solani.HaGLP1 expression in Arabidopsis plants conferred tolerance to S. sclerotiorum at the first stages of disease and interfered with R. solani infection, thus giving rise to significant protection against the latter. Furthermore, HaGLP1 expression in Arabidopsis plants elevated endogenous ROS levels. HaGLP1-induced tolerance does not appear to be related to a constitutive induction of the plant defence or the ROS-related genes examined here. In conclusion, our data suggest that HaGLP1 is an interesting candidate for the engineering of plants with increased fungal tolerance and that this gene could also be useful for the selection of naturally overexpressing sunflower genotypes for conventional breeding purposes.