Methods for Transcriptome Assembly in the Allopolyploid Brassica napus

Canada is the world’s largest producer of canola and the trend of production is ever increasing with an annual growth rate of 9.38% according to FAOSTAT. In 2017, canola acreage surpassed wheat in Saskatchewan, the highest producer of both crops in Canada. Country-wide, the total farming area of canola increased by 9.9% to 22.4 million acres while wheat area saw a slight decline to 23.3 million acres. While Canada is the highest producer of the crop, yields are lower than other countries. To maximize the benefit of this market, canola cultivation could be made more efficient with further characterization of the organism’s genes and their involvement in plant robustness. Such studies using transcriptome analysis have been successful in organisms with relatively small and simple genomes. However, such analyses in B. napus are complicated by the allopolyploid genome structure resulting from ancestral whole genome duplications in the species’ evolutionary history. Homeologous gene pairs originating from the orthology between the two B. napus progenitor species complicate the process of transcriptome assembly. Modern assemblers: Trinity, Oases and SOAPdenovo-Trans were used to generate several de novo transcriptome assemblies for B. napus. A variety of metrics were used to determine the impact that the complex genome structure has on transcriptome studies. In particular, the most important questions for transcriptome assembly in B. napus were how does varying the k-mer parameter effect assembly quality, and to what extent do similar genes resulting from homeology within B. napus complicate the process of assembly. These metrics used for evaluating the assemblies include basic assembly statistics such as the number of contigs and contig lengths (via N25, N50 and N75 statistics); as well as more involved investigation via comparison to annotated coding DNA sequences; evaluation softwares scores for de novo transcriptome assemblies and finally; quantification of homeolog differentiation by alignment to previously identified pairs of homeologous genes. These metrics provided a picture of the trade-offs between assembly softwares and the k-parameter determining the length of subsequences used to build de Bruijn graphs for de novo transcriptome assembly. It was shown that shorter k-mer lengths produce fewer, and more complete contigs due to the shorter required overlap between read sequences; while longer k-mer lengths increase the sensitivity of an assembler to sequence variation between similar gene sequences. The Trinity assembler outperformed Oases and SOAPdenovo-Trans when considering the total breadth of evaluation metrics, generating longer transcripts with fewer chimers between homeologous gene pairs

Seedling, early vegetative, and adult plant growth of oilseed rapes (Brassica napus L.) under saline stress

Salinity is a major limiting factor for early crop establishment and yield. In this study, 131 B. napus genotypes were evaluated for germination and early seedling growth in Murashige Skoog medium supplemented with NaCl. Eight genotypes were then evaluated for adult plant tolerance to salinity in a semi-hydroponic system using 1.4, 5, 10, 15, 20, and 28 dS m-1 salt stress. Relative salt tolerance (RST) was calculated and compared with genotype performance under no salt stress (control). The area under the germination progress curve (AUGPC) varied from 53-90 in the control and from 6-89 under 200 mM NaCl stress. The seedling vigor index (SVI) ranged from 200-1606 and 10-736 in the control and 200 mM salt stress, respectively. The RST for germination, root length, shoot length, and SVI ranged from 8-97.7%, 2-98.3%, 6.5-70.8%, and 1.9-83%, respectively. Root length was most severely affected by saline conditions, followed by shoot length and AUGPC when RST percentages of these traits were compared among responses of the 131 genotypes. Genotypes showed varied levels of proline and glucosinolate accumulation under different levels of saline stress. Greater accumulation of proline and glucosinolates was recorded with increased salinity level. This study indicates that variation exists in seedling and adult plant responses to saline stress in the B. napus genotypes and that improvement for salinity tolerance requires selection at the seedling, vegetative, and adult plant stages.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Genes associated with chloroplasts and hormone-signaling, and transcription factors other than CBFs are associated with differential survival after low temperature treatments of Camelina sativa biotypes.

Winter annual biotypes of Camelina sativa regularly survive after winter conditions experienced in northern regions of the U.S., whereas summer annual biotypes do not. To determine potential molecular mechanisms associated with these biotype differences in survival after low temperature treatments, we examined genetic and transcript variations in both a winter- (Joelle) and a summer- (CO46) biotype. It was determined that as few as one or two dominant genes may control differential survival after low temperature treatments. Of the 1797 genes that were differentially expressed in response to cold in both the winter and summer biotypes many COR genes were identified, indicating that the CBF regulon is functional in both. However, only 153 and 76 genes from Joelle and CO46, respectively, were either differentially expressed or not expressed at all in one biotype versus the other following cold acclimation. We hypothesize that these 229 genes play a significant role in, or are primarily responsive to, differences in survival after freezing between these two biotypes. Promoter analysis provided few clues as to the regulation or these genes; however, genes that were down-regulated specifically in the winter biotype Joelle were enriched with the sequence TGGCCCTCGCTCAC, which is over-represented among genes associated with chloroplasts in Arabidopsis. Additionally, several genes involved in auxin signaling were down-regulated specifically in Joelle. A transcription factor with strong similarity to MYB47, known to be up-regulated by salt, drought, and jasmonic acid, but not cold in Arabidopsis, was essentially off in the freezing sensitive biotype CO46, but was cold-induced in the winter biotype Joelle. Several other transcription factors genes including three with similarity to WRKY70, that may be involved in SA/JA-dependent responses, a HOMEOBOX 6 gene involved in ABA signaling, and two others (NUCLEAR FACTOR Y and CONSTANS-like 2) known to be implicated in photoperiodic flowering were also differentially expressed between the two biotypes