The Brassica coenospeceis have treasure troves of genes that could be beneficial if introgressed into cultivated Brassicas to combat the current conditions of climate change. Introducing genetic variability through plant speciation with polyploidization is well documented, where ploidy augmentation of inter-generic allohexaploids using somatic hybridization has significantly contributed to genetic base broadening. Sinapis alba is a member of the Brassicaceae family that possesses valuable genes, including genes conferring resistance to Sclerotinia sclerotiorum, Alternaria brassicae, pod shattering, heat, and drought stress. This work aimed to synthesize stable allohexaploid (AABBSS) Brassica while incorporating the yellow-seed trait and resistance to S. sclerotiorum stem rot. The two fertile and stable allohexaploids were developed by polyethylene glycol mediated protoplast fusions between Brassica juncea (AABB) and S. alba (SS) and named as JS1 and JS2. These symmetric hybrids (2n = 60) were validated using morphological and molecular cytology techniques and were found to be stable over consecutive generations. The complete chromosome constitution of the three genomes was determined through genomic in situ hybridization of mitotic cells probed with S. alba genomic DNA labeled with fluorescein isothiocyanate. These two allohexaploids showed 24 hybridization signals demonstrating the presence of complete diploid chromosomes from S. alba and 36 chromosomes from B. juncea. The meiotic pollen mother cell showed 30 bivalent sets of all the 60 chromosomes and none of univalent or trivalent observed during meiosis. Moreover, the backcross progeny 1 plant revealed 12 hybridization signals out of a total of 48 chromosome counts. Proper pairing and separation were recorded at the meiotic metaphase and anaphase, which proved the stability of the allohexaploid and their backcross progeny. When screening, the allohexaploid (JS2) of B. juncea and S. alba displayed a high degree of resistance to S. sclerotiorum rot along with a half-yellow and half-brown (mosaic) seed coat color, while the B. juncea and S. alba allohexaplopid1 (JS1) displayed a yellow seed coat color with the same degree of resistance to Sclerotinia rot.

Crop Brassicas contain monogenomic and digenomic species, with no evidence of a trigenomic Brassica in nature. Through somatic fusion (Sinapis alba+B. juncea), a novel allohexaploid trigenomic Brassica (H1 = AABBSS; 2n= 60) was produced and used for transcriptome analysis to uncover genes for thermotolerance, annotations, and microsatellite markers for future molecular breeding. Illumina Novaseq 6000 generated a total of 76,055,546 paired-end raw reads, which were used forde-novoassembly, resulting in the development of 486,066 transcripts. A total of 133,167 coding sequences (CDSs) were predicted from transcripts with a mean length of 507.12 bp and 46.15% GC content. The BLASTX search of CDSs against public protein databases showed a maximum of 126,131 (94.72%) and a minimum of 29,810 (22.39%) positive hits. Furthermore, 953,773 gene ontology (GO) terms were found in 77,613 (58.28%) CDSs, which were divided into biological processes (49.06%), cellular components (31.67%), and molecular functions (19.27%). CDSs were assigned to 144 pathways by a pathway study using the KEGG database and 1,551 pathways by a similar analysis using the Reactome database. Further investigation led to the discovery of genes encoding over 2,000 heat shock proteins (HSPs). The discovery of a large number of HSPs in allohexaploid Brassica validated our earlier findings for heat tolerance at seed maturity. A total of 15,736 SSRs have been found in 13,595 CDSs, with an average of one SSR per 4.29 kb length and an SSR frequency of 11.82%. The first transcriptome assembly of a meiotically stable allohexaploid Brassica has been given in this article, along with functional annotations and the presence of SSRs, which could aid future genetic and genomic studies.