Anete Pereira de Souza

Comparison of RAPD, RFLP, AFLP and SSR markers for diversity studies in tropical maize inbred lines

In order to compare their relative efficiencies as markers and to find the most suitable marker for maize diversity studies we evaluated 18 inbred tropical maize lines using a number of different loci as markers. The loci used were: 774 amplified fragment length polymorphisms (AFLPs); 262 random amplified polymorphic DNAs (RAPDs); 185 restriction fragment length polymorphisms (RFLPs); and 68 simple sequence repeats (SSR). For estimating genetic distance the AFLP and RFLP markers gave the most correlated results, with a correlation coefficient of r = 0.87. Bootstrap analysis were used to evaluate the number of loci for the markers and the coefficients of variation (CV) revealed a skewed distribution. The dominant markers (AFLP and RAPD) had small CV values indicating a skewed distribution while the codominant markers gave high CV values. The use of maximum values of genetic distance CVs within each sample size was efficient in determining the number of loci needed to obtain a maximum CV of 10%. The number of RFLP and AFLP loci used was enough to give CV values of below 5%, while the SSRs and RAPD loci gave higher CV values. Except for the RAPD markers, all the markers correlated genetic distance with single cross performance and heterosis which showed that they could be useful in predicting single cross performance and heterosis in intrapopulation crosses for broad-based populations. Our results indicate that AFLP seemed to be the best-suited molecular assay for fingerprinting and assessing genetic relationships among tropical maize inbred lines with high accuracy.

Comparison of similarity coefficients used for cluster analysis with dominant markers in maize (Zea mays L)

The objective of this study was to evaluate whether different similarity coefficients used with dominant markers can influence the results of cluster analysis, using eighteen inbred lines of maize from two different populations, BR-105 and BR-106. These were analyzed by AFLP and RAPD markers and eight similarity coefficients were calculated: Jaccard, Sorensen-Dice, Anderberg, Ochiai, Simple-matching, Rogers and Tanimoto, Ochiai II and Russel and Rao. The similarity matrices obtained were compared by the Spearman correlation, cluster analysis with dendrograms (UPGMA, WPGMA, Single Linkage, Complete Linkage and Neighbour-Joining methods), the consensus fork index between all pairs of dendrograms, groups obtained through the Tocher optimization procedure and projection efficiency in a two-dimensional space. The results showed that for almost all methodologies and marker systems, the Jaccard, Sorensen-Dice, Anderberg and Ochiai coefficient showed close results, due to the fact that all of them exclude negative co-occurrences. Significant alterations in the results for the Simple Matching, Rogers and Tanimoto, and Ochiai II coefficients were not observed either, probably due to the fact that they all include negative co-occurrences. The Russel and Rao coefficient presented very different results from the others in almost all the cases studied and should not be used, because it excludes the negative co-occurrences in the numerator and includes them in the denominator of their expression. Due to the fact that the negative co-occurrences do not necessarily mean that the regions of the DNA are identical, the use of coefficients that do not include negative co-occurrences was suggested.

Relationship of intra- and interpopulation tropical maize single cross hybrid performance and genetic distances computed from AFLP and SSR markers

AbstractTwo sets of tropical maize inbred lines, one derived from the BR-105 population and another derived from the BR-106 population, were assayed for Amplified Fragment Length Polymorphisms (AFLP) and for Simple Sequence Repeat (SSR), in order to investigate genetic distances among lines and their relationship to heterotic group assignment and single cross yield performance. Genetic distances were on average greater for interpopulation than intrapopulation crosses for both AFLP and SSR. Cluster analysis was in agreement with the original assignment for heterotic groups. Inbred line 16, derived from BR-106, was assigned to the BR-105 set, in agreement with single cross yield performance from intra- and interpopulation crosses. However, the same pattern was not observed for SSR where another two lines from BR-106 were also assigned to the BR-105 set. Correlation coefficients of genetic distances (GD) with F1 grain yield and heterosis were high for BR-106 ×BR-106 crosses (0.91** and 0.82** for AFLP and SSR, respectively), moderate for BR-105 × BR-105 crosses (0.52* for AFLP and SSR) and low for BR-105 × BR-106 crosses (0.29 and 0.16 for AFLP and SSR, respectively). The lower correlation at interpopulation level was due to the smaller range of GD caused probably by a previous selection for combining ability. General results showed that the AFLP molecular marker is efficient in assigning maize lines to heterotic groups and that AFLP-based GD is suitable for predicting the maize single cross performance for intrapopulation crosses of broad-based populations. The efficiency of SSR in assigning lines to heterotic groups and for predicting single cross performance was smaller than AFLP.

Development, characterization, and comparative analysis of polymorphism at common bean SSR loci isolated from genic and genomic sources

Microsatellites or SSRs (single sequence repeats) have been used to construct and integrate genetic maps in crop species, including Phaseolus vulgaris. In the present study, 3 cDNA libraries generated by the Bean EST project (http://lgm.esalq.usp.br/BEST/), comprising a unigene collection of 3126 sequences and a genomic microsatellite-enriched library, were analyzed for the presence of SSRs. A total of 219 expressed sequence tags (ESTs) were found to carry 240 SSRs (named EST-SSR), whereas 714 genomic sequences contained 471 SSRs (named genomic-SSR). A subset of 80 SSRs, 40 EST-SSRs, and 40 genomic-SSRs were evaluated for molecular polymorphism in 23 genotypes of cultivated beans from the Mesoamerican and Andean genetic pools, including Brazilian cultivars and 2 related species. Of the common bean genotypes, 31 EST-SSR loci were polymorphic, yielding 2-12 alleles as compared with 26 polymorphic genomic-SSRs, accounting for 2-7 alleles. Cluster analysis from data using both genic and genomic-SSR revealed a clear separation between Andean and Mesoamerican beans. The usefulness of these loci for distinguishing bean genotypes and genetic mapping is discussed.

Mapping QTL for Grain Yield and Plant Traits in a Tropical Maize Population

The vast majority of reported QTL mapping for maize (Zea mays L.) traits are from temperate germplasm and, also, QTL by environment interaction (QTL × E) has not been thoroughly evaluated and analyzed in most of these papers. The maize growing areas in tropical regions are more prone to environmental variability than in temperate areas, and, therefore, genotype by environment interaction is of great concern for maize breeders. The objectives of this study were to map QTL and to test their interaction with environments for several traits in a tropical maize population. Two-hundred and fifty-six F2:3 families evaluated in five environments, a genetic map with 139 microsatellites markers, and the multiple-environment joint analysis (mCIM) were used to map QTL and to test QTL × E interaction. Sixteen, eight, six, six, nine, and two QTL were mapped for grain yield, ears per plant, plant lodging, plant height, ear height, and number of leaves, respectively. Most of these QTL interacted significantly with environments, most of them displayed overdominance for all traits, and genetic correlated traits had a low number of QTL mapped in the same genomic regions. Few of the QTL mapped had already been reported in both temperate and tropical germplasm. The low number of stable QTL across environments imposes additional challenges to design marker-assisted selection in tropical areas, unless the breeding programs could be directed towards specific target areas.

Structure of genetic diversity among common bean ( Phaseolus vulgaris L.) varieties of Mesoamerican and Andean origins using new developed microsatellite markers

A common bean genomic library was constructed using the ‘IAC-UNA’ variety enriched for (CT) and (GT) for microsatellite motifs. From 1,209 sequenced clones, 714 showed microsatellites distributed over 471 simple and 243 compound motifs. GA/CT and GT/CA were the most frequent motifs found among these sequences. A total of 123 microsatellites has been characterized. Out of these, 87 were polymorphic (73.7%), 33 monomorphic (26.8%), and 3 (2.4%) did not amplify at all. In a sample of 20 common bean materials selected from the Agronomic Institute Germplasm Bank, the number of alleles per locus varied 2–9, with an average of 2.82. The polymorphic information content (PIC) of each marker varied from 0.05 to 0.83, with a 0.45 average value. Cluster and principal coordinate analysis of the microsatellite data were consistent with the original assignment of the germplasm accessions into the Andean and Mesoamerican gene pools of common bean. Low polymorphism levels detected could be associated with the domestication process. These microsatellites could be a valuable resource for the bean community because of their use as new markers for genetic studies.

Building the sugarcane genome for biotechnology and identifying evolutionary trends.

BackgroundSugarcane is the source of sugar in all tropical and subtropical countries and is becoming increasingly important for bio-based fuels. However, its large (10 Gb), polyploid, complex genome has hindered genome based breeding efforts. Here we release the largest and most diverse set of sugarcane genome sequences to date, as part of an on-going initiative to provide a sugarcane genomic information resource, with the ultimate goal of producing a gold standard genome.ResultsThree hundred and seventeen chiefly euchromatic BACs were sequenced. A reference set of one thousand four hundred manually-annotated protein-coding genes was generated. A small RNA collection and a RNA-seq library were used to explore expression patterns and the sRNA landscape. In the sucrose and starch metabolism pathway, 16 non-redundant enzyme-encoding genes were identified. One of the sucrose pathway genes, sucrose-6-phosphate phosphohydrolase, is duplicated in sugarcane and sorghum, but not in rice and maize. A diversity analysis of the s6pp duplication region revealed haplotype-structured sequence composition. Examination of hom(e)ologous loci indicate both sequence structural and sRNA landscape variation. A synteny analysis shows that the sugarcane genome has expanded relative to the sorghum genome, largely due to the presence of transposable elements and uncharacterized intergenic and intronic sequences.ConclusionThis release of sugarcane genomic sequences will advance our understanding of sugarcane genetics and contribute to the development of molecular tools for breeding purposes and gene discovery.

SNP genotyping allows an in-depth characterisation of the genome of sugarcane and other complex autopolyploids

Many plant species of great economic value (e.g., potato, wheat, cotton, and sugarcane) are polyploids. Despite the essential roles of autopolyploid plants in human activities, our genetic understanding of these species is still poor. Recent progress in instrumentation and biochemical manipulation has led to the accumulation of an incredible amount of genomic data. In this study, we demonstrate for the first time a successful genetic analysis in a highly polyploid genome (sugarcane) by the quantitative analysis of single-nucleotide polymorphism (SNP) allelic dosage and the application of a new data analysis framework. This study provides a better understanding of autopolyploid genomic structure and is a sound basis for genetic studies. The proposed methods can be employed to analyse the genome of any autopolyploid and will permit the future development of high-quality genetic maps to assist in the assembly of reference genome sequences for polyploid species.

The Biotechnology Roadmap for Sugarcane Improvement

Due to the strategic importance of sugarcane to Brazil, FAPESP, the main São Paulo state research funding agency, launched in 2008 the FAPESP Bioenergy Research Program (BIOEN, http://bioenfapesp.org). BIOEN aims to generate new knowledge and human resources for the improvement of the sugarcane and ethanol industry. As part of the BIOEN program, a Workshop on Sugarcane Improvement was held on March 18th and 19th 2009 in São Paulo, Brazil. The aim of the workshop was to explore present and future challenges for sugarcane improvement and its use as a sustainable bioenergy and biomaterial feedstock. The workshop was divided in four sections that represent important challenges for sugarcane improvement: a) gene discovery and sugarcane genomics, b) transgenics and controlled transgene expression, c) sugarcane physiology (photosynthesis, sucrose metabolism, and drought) and d) breeding and statistical genetics. This report summarizes the roadmap for the improvement of sugarcane.

De Novo Assembly and Transcriptome Analysis of Contrasting Sugarcane Varieties

Sugarcane is an important crop and a major source of sugar and alcohol. In this study, we performed de novo assembly and transcriptome annotation for six sugarcane genotypes involved in bi-parental crosses. The de novo assembly of the sugarcane transcriptome was performed using short reads generated using the Illumina RNA-Seq platform. We produced more than 400 million reads, which were assembled into 72,269 unigenes. Based on a similarity search, the unigenes showed significant similarity to more than 28,788 sorghum proteins, including a set of 5,272 unigenes that are not present in the public sugarcane EST databases; many of these unigenes are likely putative undescribed sugarcane genes. From this collection of unigenes, a large number of molecular markers were identified, including 5,106 simple sequence repeats (SSRs) and 708,125 single-nucleotide polymorphisms (SNPs). This new dataset will be a useful resource for future genetic and genomic studies in this species.