Discovery of Pod Shatter-Resistant Associated SNPs by Deep Sequencing of a Representative Library Followed by Bulk Segregant Analysis in Rapeseed

Background: Single nucleotide polymorphisms (SNPs) are an important class of genetic marker for target gene mapping. As of yet, there is no rapid and effective method to identify SNPs linked with agronomic traits in rapeseed and other crop species. Methodology/Principal Findings: We demonstrate a novel method for identifying SNP markers in rapeseed by deep sequencing a representative library and performing bulk segregant analysis. With this method, SNPs associated with rapeseed pod shatter-resistance were discovered. Firstly, a reduced representation of the rapeseed genome was used. Genomic fragments ranging from 450–550 bp were prepared from the susceptible bulk (ten F2 plants with the silique shattering resistance index, SSRI,0.10) and the resistance bulk (ten F2 plants with SSRI.0.90), and also Solexa sequencingproduced 90 bp reads. Approximately 50 million of these sequence reads were assembled into contigs to a depth of 20-fold coverage. Secondly, 60,396 ‘simple SNPs ’ were identified, and the statistical significance was evaluated using Fisher’s exact test. There were 70 associated SNPs whose –log10p value over 16 were selected to be further analyzed. The distribution of these SNPs appeared a tight cluster, which consisted of 14 associated SNPs within a 396 kb region on chromosome A09. Our evidence indicates that this region contains a major quantitative trait locus (QTL). Finally, two associated SNPs from this region were mapped on a major QTL region

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases

Genome-wide delineation of natural variation for pod shatter resistance in Brassica napus

Resistance to pod shattering (shatter resistance) is a target trait for global rapeseed (canola, Brassica napus L.), improvement programs to minimise grain loss in the mature standing crop, and during windrowing and mechanical harvest. We describe the genetic basis of natural variation for shatter resistance in B. napus and show that several quantitative trait loci (QTL) control this trait. To identify loci underlying shatter resistance, we used a novel genotyping-by-sequencing approach DArT-Seq. QTL analysis detected a total of 12 significant QTL on chromosomes A03, A07, A09, C03, C04, C06, and C08; which jointly account for approximately 57% of the genotypic variation in shatter resistance. Through Genome-Wide Association Studies, we show that a large number of loci, including those that are involved in shattering in Arabidopsis, account for variation in shatter resistance in diverse B. napus germplasm. Our results indicate that genetic diversity for shatter resistance genes in B. napus is limited; many of the genes that might control this trait were not included during the natural creation of this species, or were not retained during the domestication and selection process. We speculate that valuable diversity for this trait was lost during the natural creation of B. napus. To improve shatter resistance, breeders will need to target the introduction of useful alleles especially from genotypes of other related species of Brassica, such as those that we have identified. © 2014 Raman et al

Identification and characterization of a proline-rich mRNA that accumulates during pod development in oilseed rape (Brassica napus L.)

Pod development in oilseed rape (Brassica napus) culminates in a process known as dehiscence (shatter) which can result in the loss of seed before the crop is harvested. In order to investigate the biochemical and the genetic basis controlling this process, a cDNA library was constructed from the dehiscence zone of developing pods. This resulted in the isolation of a cDNA clone (SAC51). The mRNA encoded by SAC51 had a transcript size of ca. 700 nucleotides and was found, by northern analysis, to accumulate preferentially in the dehiscence zone of the pod and in no other part of the plant analysed. The predicted polypeptide is rich in the amino acids proline (14.2%) and leucine (14.2%). The sequence of the polypeptide has more than 40% amino acid sequence identity with polypeptides isolated from carrot embryos, maize roots, soybean seeds and young tomato fruit. The function of these proteins is unknown. Genomic Southern analysis suggests that SAC51 is encoded by a single gene or small gene family. The role of the peptide in the development of pods of oilseed rape is discussed

Preventing unwanted breakups: Using polygalacturonases to regulate cell separation

Cell separation is an important biological process in plants that is precisely regulated both spatially and temporally. Key separation events include abscission of organs such as leaves and fruit and dehiscence events such as pod shatter in canola and other Brassicas. Polygalacturonases (PGs) are enzymes essential for the degradation of pectin, an important component of the adhesive material between cells. Although there are around 70 PG genes with overlapping expression domains, recent analysis has revealed the function of several PGs in specific aspects of Arabidopsis reproductive development. Upstream regulators that control the expression domain of some of these PGs during reproductive development have also been identified. This information provides new strategies to control unwanted cell separation events in various crops