Luciana Rossini Pinto

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.

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.

Genetic-diversity assessed by microsatellites in tropical maize populations submitted to a high-intensity reciprocal recurrent selection

BR-105 and BR-106 are important tropical maize populations, which were submitted to a high-intensity reciprocal recurrent selection, generating the IG-3 and IG-4synthetics. Using 30-microsatellite loci,we measured and compared the genetic diversity of these populations and their synthetics. The populations did not differ significantly regarding the amount of genetic diversity. As a consequence of selection, genetic variability losses, in terms of mean number of alleles per locus,proportion of polymorphic loci, and gene diversity did occur and were greater in the synthetic IG-3 than IG-4. In the synthetics, the number of loci in adherence to Hardy-Weinberg proportions was superior to that observed in the populations. The Wrights mean fixation index was higher than the mean value expected for outcrossing species (5%) indicating as light excess of homozygotic individuals in both populations. The genetic distances confirmed the favourable effects of one cycle of recurrent selection, as the synthetics became more isolated in comparison to the original populations.BR-105 and BR-106 are important tropical maize populations, which were submitted to a high-intensity reciprocal recurrent selection, generating the IG-3 and IG-4synthetics. Using 30-microsatellite loci,we measured and compared the genetic diversity of these populations and their synthetics. The populations did not differ significantly regarding the amount of genetic diversity. As a consequence of selection, genetic variability losses, in terms of mean number of alleles per locus,proportion of polymorphic loci, and gene diversity did occur and were greater in the synthetic IG-3 than IG-4. In the synthetics, the number of loci in adherence to Hardy-Weinberg proportions was superior to that observed in the populations. The Wrights mean fixation index was higher than the mean value expected for outcrossing species (5%) indicating as light excess of homozygotic individuals in both populations. The genetic distances confirmed the favourable effects of one cycle of recurrent selection, as the synthetics became more isolated in comparison to the original populations.

Analysis of genomic and functional RFLP derived markers associated with sucrose content, fiber and yield QTLs in a sugarcane (Saccharum spp.) commercial cross

Expressed sequence tags derived markers have a great potential to be used in functional map construction and QTL tagging. In the present work, sugarcane genomic probes and expressed sequence tags having homology to genes, mostly involved in carbohydrate metabolism were used in RFLP assays to identify putative QTLs as well as their epistatic interactions for fiber content, cane yield, pol and tones of sugar per hectare, at two crop cycles in a progeny derived from a bi-parental cross of sugarcane elite materials. A hundred and twenty marker trait associations were found, of which 26 at both crop cycle and 32 only at first ratoon cane. A sucrose synthase derived marker was associated with a putative QTL having a high negative effect on cane yield and also with a QTL having a positive effect on Pol at both crop cycles. Fifty digenic epistatic marker interactions were identified for the four traits evaluated. Of these, only two were observed at both crop cycles.

Functional markers for gene mapping and genetic diversity studies in sugarcane.

BackgroundThe database of sugarcane expressed sequence tags (EST) offers a great opportunity for developing molecular markers that are directly associated with important agronomic traits. The development of new EST-SSR markers represents an important tool for genetic analysis. In sugarcane breeding programs, functional markers can be used to accelerate the process and select important agronomic traits, especially in the mapping of quantitative traits loci (QTL) and plant resistant pathogens or qualitative resistance loci (QRL). The aim of this work was to develop new simple sequence repeat (SSR) markers in sugarcane using the sugarcane expressed sequence tag (SUCEST database).FindingsA total of 365 EST-SSR molecular markers with trinucleotide motifs were developed and evaluated in a collection of 18 genotypes of sugarcane (15 varieties and 3 species). In total, 287 of the EST-SSRs markers amplified fragments of the expected size and were polymorphic in the analyzed sugarcane varieties. The number of alleles ranged from 2-18, with an average of 6 alleles per locus, while polymorphism information content values ranged from 0.21-0.92, with an average of 0.69. The discrimination power was high for the majority of the EST-SSRs, with an average value of 0.80. Among the markers characterized in this study some have particular interest, those that are related to bacterial defense responses, generation of precursor metabolites and energy and those involved in carbohydrate metabolic process.ConclusionsThese EST-SSR markers presented in this work can be efficiently used for genetic mapping studies of segregating sugarcane populations. The high Polymorphism Information Content (PIC) and Discriminant Power (DP) presented facilitate the QTL identification and marker-assisted selection due the association with functional regions of the genome became an important tool for the sugarcane breeding program.

Reciprocal recurrent selection effects on the genetic structure of tropical maize populations assessed at microsatellite loci

A modified reciprocal recurrent selection (RRS) method, which employed one cycle of high-intensity selection, was applied to two tropical maize (Zea mays L.) populations, BR-105 and BR-106, originating the improved synthetics IG-3 and IG-4, respectively. In the present study the effects of this kind of selection on the genetic structure of these populations and their synthetics were investigated at 30 microsatellite (SSR) loci. A total of 125 alleles were revealed. A reduction in the number of alleles was observed after selection, as well as changes in allele frequencies. In nearly 13% (BR-105) and 7% (BR-106) of the loci evaluated, the changes in allele frequencies were not explained, exclusively due to the effects of genetic drift. The effective population sizes estimated for the synthetics using 30 SSR loci were similar to those theoretically expected after selection. The genetic differentiation (GST) between the synthetics increased to 77% compared with the original populations. The estimated RST values, a genetic differentiation measure proper for microsatellite data, were similar to those obtained for GST. Despite the high level of selection applied, the total gene diversity found in the synthetics allows them to be used in a new RRS cycle.

Constitutive expression of pea Lhcb1–2 in tobacco affects plant development, morphology and photosynthetic capacity

Lhcb1–2 from pea was constitutively expressed in transgenic tobacco plants and assessed for functional impact. The successful assembly of the encoded proteins into LHCII trimers was confirmed by electrospray tandem mass spectrometry. Constitutive production of LHCb1–2 led to increased number of thylakoid membranes per chloroplast, increased grana stacking, higher chloroplast numbers per palisade cell and increased photosynthetic capacity at low irradiance, both on a chlorophyll and leaf area basis. The transgenic plants also displayed increased cell volume, larger leaves, higher leaf number per plant at flowering, increased biomass and increased seed weight, when grown under low irradiance levels. Under high irradiance, both transgenic and wild type plants displayed similar photosynthetic rates when tested at 25 °C; however, the non-photochemical quenching (NPQ) and qE values increased in the transgenic plants. The exposure of transgenic plants to a photoinhibitory treatment (4 °C for 4 h, under continuous illumination) resulted in more detrimental impairment of photosynthesis, since recovery was slower than the non-transgenic plants. These data indicate that constitutive expression of additional Lhcb1–2 transgenes led to a series of changes at all levels of the plant (cellular, leaf and whole organism), and a delay in flowering and senescence. The additional production of the pea protein appears to be accommodated by increasing cellular structures such as the number of thylakoids per chloroplast, organelle volume, organelles per cell, and leaf expansion. The presence of the trimeric pea protein in the tobacco LHCII, however, caused a possible change in the organization of the associated super-complex, that in turn limited photosynthesis at low temperature.

Genetic variability among sugarcane genotypes based on polymorphisms in sucrose metabolism and drought tolerance genes

Target region amplification polymorphism (TRAP) markers were used to estimate the genetic similarity (GS) among 53 sugarcane varieties and five species of the Saccharum complex. Seven fixed primers designed from candidate genes involved in sucrose metabolism and three from those involved in drought response metabolism were used in combination with three arbitrary primers. The clustering of the genotypes for sucrose metabolism and drought response were similar, but the GS based on Jaccard’s coefficient changed. The GS based on polymorphism in sucrose genes estimated in a set of 46 Brazilian varieties, all of which belong to the three Brazilian breeding programs, ranged from 0.52 to 0.9, and that based on drought data ranged from 0.44 to 0.95. The results suggest that genetic variability in the evaluated genes was lower in the sucrose metabolism genes than in the drought response metabolism ones.

Comparison of AFLP, TRAP and SSRs in the estimation of genetic relationships in sugarcane

The Sugarcane Breeding Programme of the Instituto Agronômico de Campinas has been using routinely different types of molecular markers for the characterization of their breeding clones and varieties. In the present work we compared the genetic relationships among 82 sugarcane clones/varieties based on three types of molecular markers; AFLP, TRAP and Microsatellites (SSRs). Five AFLP selective primer combinations, 10 SSRs and four TRAP fixed primers, designed from candidate genes involved in the drought tolerance response metabolism, were used in combination with three arbitrary primers. The pair-wise genetic similarity based on the Jaccard’s coefficient, the dedrogram and matrix comparison were done using NTSYS Software. A total of 410 polymorphic markers were obtained: 145 AFLPs, 103 SSRs, and 160 TRAPs. Although the average genetic similarity estimates based on AFLP (0,675) and TRAP (0,655) was closer to each other than to SSRs (0,522), the correlation between TRAP and SSRs was higher (r= 0.24). The coefficient of variation was lower for AFLP and TRAP (∼6.55%) than for SSRs (13%). These results indicate that the choice of molecular markers should be considered carefully, based on the purpose of the application in the breeding programme, as it is not possible to select a marker system that fits all the requirements for germplasm characterization.

Isoenzymatic variability in tropical maize populations under reciprocal recurrent selection

Maize (Zea mays L.) is one of the crops in which the genetic variability has been extensively studied at isoenzymatic loci. The genetic variability of the maize populations BR-105 and BR-106, and the synthetics IG-3 and IG-4, obtained after one cycle of a high-intensity reciprocal recurrent selection (RRS), was investigated at seven isoenzymatic loci. A total of twenty alleles were identified, and most of the private alleles were found in the BR-106 population. One cycle of reciprocal recurrent selection (RRS) caused reductions of 12% in the number of alleles in both populations. Changes in allele frequencies were also observed between populations and synthetics, mainly for the Est 2 locus. Populations presented similar values for the number of alleles per locus, percentage of polymorphic loci, and observed and expected heterozygosities. A decrease of the genetic variation values was observed for the synthetics as a consequence of genetic drift effects and reduction of the effective population sizes. The distribution of the genetic diversity within and between populations revealed that most of the diversity was maintained within them, i.e. BR-105 x BR-106 (GST = 3.5%) and IG-3 x IG-4 (GST = 4.0%). The genetic distances between populations and synthetics increased approximately 21%. An increase in the genetic divergence between the populations occurred without limiting new selection procedures.