Development of a Yellow-Seeded Stable Allohexaploid Brassica Through Inter-Generic Somatic Hybridization With a High Degree of Fertility and Resistance to Sclerotinia sclerotiorum

Abstract

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.

Development of de-novo transcriptome assembly and SSRs in allohexaploid Brassica with functional annotations and identification of heat-shock proteins for thermotolerance

“…Many GO terms (e.g., response to abiotic and biotic stimulus, immune response, stress response, and response to other organisms) have substantial significance to allohexaploid's wide pathogen tolerance and high temperature and present new avenues for future research. The allohexaploid brassica (H1) and S. alba (one of their parents) have been reported to be resistant to a variety of fungal diseases, insect pest, drought, and heat tolerance (Kumari Frontiers in Genetics frontiersin.org 13 Kumari et al, 2020a;Kumari et al, 2020c). The genetic mechanism underlying all of these resistance responses, however, is still unknown.…”

“…However, wild diploid Brassicaceae members should be added to cultivated species as a source of unique genes for resistance/tolerance to a variety of fungal diseases, insects, pests, nematodes, heat, and drought (Sjodin and Glimelius, 1989;Kirti et al, 1995;Li et al, 2009;Garg et al, 2010;Singh et al, 2021). With this in mind, we developed stable allohexaploid Brassicas through somatic hybridization involving diploid S. alba (a Brassica coenospecies) and tetraploid B. juncea (an amphidiploid crop species); the resulting stable allohexaploids had a somatic chromosome number of 2n = 60 (AABBSS) and a high level of male and female fertility (Kumari et al, 2018;2020c). Two allohexaploids (H1 and H2) showed resistance to Alternaria blight and Sclerotinia stem rot and had high-temperature tolerance (upto 40 °C).…”

Protoplasts: From Isolation to CRISPR/Cas Genome Editing Application

“…This strategy became a solution for crop breeders in cases where sexual incompatibility was a barrier or as a means to incorporate traits from wild species into related crops without the need for sexual reproduction (Louzada et al, 1993). Variations on this method have been used to introduce a variety of desired traits into crops, including stress resistance (Hennig et al, 2015), pathogen resistance (Kumari et al, 2020), seedlessness (Wu et al, 2005), male sterility (Bruznican et al, 2021), and increased photosynthetic efficiency (Takahata and Takeda, 1990). This can be performed to create a symmetric cell fusion, in which the complete nuclear genomes of the two species are combined (Narasimhulu et al, 1992;Laiq et al, 1994;Ling and Iwamasa, 1994;Desprez et al, 1995;Kirti et al, 1995); an asymmetric cell fusion, in which DNA fragments or partial chromosomes from one species are introduced into the other (Zhou and Xia 2005;Sigeno et al, 2009); or a cybrid, in which chloroplast or mitochondrial genomes from one species are introduced into cells of another (Kochevenko et al, 2000;Guo et al, 2004).…”

Current Status of the Disease-Resistant Gene(s)/QTLs, and Strategies for Improvement in Brassica juncea

“…The wild members of the Brassicaceae family such as S . alba ( Kolte, 1985 ; Brun et al, 1987 ; Ripley et al, 1992 ; Sharma and Singh, 1992 ; Hansen and Earle, 1995 , 1997 ; Kumari et al, 2018 , 2020b ), Camelina sativa , Capsella bursa-pastoris , Eruca sativa, Neslia paniculata , Alliaria petiolata, Barbarea vulgaris, Brassica elongate, B. desnottessi, B. fruticulosa , B. maurorum , B. nigra , B. souliei , B. spinescens , Camelina sativa , Capsella bursa-pastoris , Coincya spp., Diplotaxis catholica , D. berthautii , D. creacea , D. erucoides , D. tenuifolia , Erucastrum gallicum, Eruca vesicaria subsp. sativa , Hemicrambe fruticulosa , H. matronalis , Neslia paniculata, Raphanus sativus , and S. arvensis ( Conn and Tewari, 1986 ; Brun et al, 1987 ; Conn et al, 1988 ; Tewari, 1991a ; Zhu and Spanier, 1991 ; Tewari and Conn, 1993 ; Sharma et al, 2002 ; Warwick, 2011 ) reportedly possess the highest level of resistance against A. brassicae .…”