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Authors: Yasin, Muhammad
Keywords: Plant Sciences
Plant Genomics and Biotechnology
Issue Date: 2022
Publisher: The University of Agriculture, Peshawar
Abstract: Non-synchronous opening of pods leading to seed shattering remained a major challenge in canola crop yield. In the model plant Arabidopsis thaliana, which belongs to the same family as canola, the Brassicaceae, 8 genes have been found to be included in shattering cascade. Among these, 3 genes including SHATTERPROOF1, SHATTERPROOF2 and FRUITFUL (SHP1, SHP2 & FUL) belonging to a MADS-box family implicated in fruit dehiscence zone and valve margin constitute a core regulatory module. But in Brassica, the exact number of genes involved in shattering and their expression patterns analyses remained obscure. The proposed study was planned to isolate the orthologues of SHP1, SHP2 and FUL from local Brassica napus cultivars. For this purpose, two rapeseed (Brassica napus) cultivars “Punjab Sarsoon” (susceptible to shattering) and “Faisal Canola” (moderately susceptible to shattering) were selected. The morphological characterization revealed variations between the two cultivars in pod wall thicknesses, seeds per pod and pod length. PCR amplification followed by sequencing showed the occurrence of three products designated as BnSHP1-N, BnSHP2-N and BnFUL-N in Punjab Sarsoon as well as in Faisal Canola. According to the sequence analysis, BnSHP1-N, BnSHP2-N, and BnFUL-N genes are 747 bp, 735 bp, and 729 bp in length, respectively. The nucleotide alignments showed that BnSHP1-N, BnSHP2-N and BnFUL-N are 98 percent identical to the BnSHP1, BnSHP2 and BnFUL sequences, respectively. The homology of the sequences was predicted to be 98%, 98% and 97% at the amino acid level for BnSHP1- N, BnSHP2-N and BnFUL-N genes, respectively. There were no deletions of nucleotides in the newly isolated gene sequences but insertion of three and six nucleotides were observed in BnSHP1-N and BnFUL-N, respectively. Only substitution mutations were detectable in BnSHP2-N. Therefore, the size of BnSHP2-N gene x remained unchanged. All the identified mutations were non-synonymous. The M domain of these MADS-box transcription factors remained the most conserved region but surprisingly, two non-synonymous mutations were also found in this domain. The I-region and the K-box seemed to be conserved, but the most dramatic changes occurred in the C-terminal region. BnSHP1-N, BnSHP2-N and BnFUL-N were grouped into their respective clades based on phylogenetic reconstruction of core regulatory modules (SHP1, SHP2 and FUL) and from other species' homologs. The 8 shattering cascade genes did not show any conservation indicating their involvement in shattering through separate pathways. Increased number of homologs/paralogs in Brassica was due to occurrences of genome duplication or a triplication event during evolution. Exonization and intronization could be responsible for variable number and of size of the exons and introns in gene structures. Comparative genome synteny analysis of SHP1, SHP2 and FUL revealed correlation and evolutionary insights into gene region relationships in the entire Brassicaceae. Semi-quantitative and quantitative RT-PCR showed overlapping expression of both the BnSHP1-N and BnSHP2-N transcripts in flowers and siliques but no expression in the leaf tissues was observed. In comparison to other tissues of “Pakola” and “Punjab Sarsoon 3,” strong expression of the FUL gene was detected in mature siliques and siliques from the upper portion of the plant. Surprisingly, expression of SHPs was stronger in B. napus than B. juncea (BARD-1, a cultivar of mustard), a shattering resistant close relative of rapeseed except SHP2. The expression divergence seems to be correlated with their divergent promoters where the regulatory motifs, particularly CArG-boxes might have played a role in regulating expression variations in siliques. Our results provide basic information on gene expression and sequence analyses of shattering genes that might be helpful for developing shattering resistant genome edited plants to prevent yield losses in canola in future.
Gov't Doc #: 25804
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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