Genome-wide identification and evolutionary analysis of the NRAMP gene family in the AC genomes of Brassica species.

BACKGROUND: Brassica napus, a hybrid resulting from the crossing of Brassica rapa and Brassica oleracea, is one of the most important oil crops. Despite its significance, B. napus productivity faces substantial challenges due to heavy metal stress, especially in response to cadmium (Cd), which poses a significant threat among heavy metals. Natural resistance-associated macrophage proteins (NRAMPs) play pivotal roles in Cd uptake and transport within plants. However, our understanding of the role of BnNRAMPs in B. napus is limited. Thus, this study aimed to conduct genome-wide identification and bioinformatics analysis of three Brassica species: B. napus, B. rapa, and B. oleracea. RESULTS: A total of 37 NRAMPs were identified across the three Brassica species and classified into two distinct subfamilies based on evolutionary relationships. Conservative motif analysis revealed that motif 6 and motif 8 might significantly contribute to the differentiation between subfamily I and subfamily II within Brassica species. Evolutionary analyses and chromosome mapping revealed a reduction in the NRAMP gene family during B. napus evolutionary history, resulting in the loss of an orthologous gene derived from BoNRAMP3.2. Cis-acting element analysis suggested potential regulation of the NRAMP gene family by specific plant hormones, such as abscisic acid (ABA) and methyl jasmonate (MeJA). However, gene expression pattern analyses under hormonal or stress treatments indicated limited responsiveness of the NRAMP gene family to these treatments, warranting further experimental validation. Under Cd stress in B. napus, expression pattern analysis of the NRAMP gene family revealed a decrease in the expression levels of most BnNRAMP genes with increasing Cd concentrations. Notably, BnNRAMP5.1/5.2 exhibited a unique response pattern, being stimulated at low Cd concentrations and inhibited at high Cd concentrations, suggesting potential response mechanisms distinct from those of other NRAMP genes. CONCLUSIONS: In summary, this study indicates complex molecular dynamics within the NRAMP gene family under Cd stress, suggesting potential applications in enhancing plant resilience, particularly against Cd. The findings also offer valuable insights for further understanding the functionality and regulatory mechanisms of the NRAMP gene family.

Comprehensive genome-wide identification of Snf2 gene family and their expression profile under salt stress in six Brassica species of U's triangle model.

MAIN CONCLUSION: We comprehensively identified and analyzed the Snf2 gene family. Some Snf2 genes were involved in responding to salt stress based on the RNA-seq and qRT-PCR analysis. Sucrose nonfermenting 2 (Snf2) proteins are core components of chromatin remodeling complexes that not only alter DNA accessibility using the energy of ATP hydrolysis, but also play a critical regulatory role in growth, development, and stress response in eukaryotes. However, the comparative study of Snf2 gene family in the six Brassica species in U's triangle model remains unclear. Here, a total of 405 Snf2 genes were identified, comprising 53, 50, and 46 in the diploid progenitors: Brassica rapa (AA, 2n = 20), Brassica nigra (BB, 2n = 16), and Brassica oleracea (CC, 2n = 18), and 93, 91, and 72 in the allotetraploid: Brassica juncea (AABB, 2n = 36), Brassica napus (AACC, 2n = 38), and Brassica carinata (BBCC, 2n = 34), respectively. These genes were classified into six clades and further divided into 18 subfamilies based on their conserved motifs and domains. Intriguingly, these genes showed highly conserved chromosomal distributions and gene structures, indicating that few dynamic changes occurred during the polyploidization. The duplication modes of the six Brassica species were diverse, and the expansion of most Snf2 in Brassica occurred primarily through dispersed duplication (DSD) events. Additionally, the majority of Snf2 genes were under purifying selection during polyploidization, and some Snf2 genes were associated with various abiotic stresses. Both RNA-seq and qRT-PCR analysis showed that the expression of BnaSnf2 genes was significantly induced under salt stress, implying their involvement in salt tolerance response in Brassica species. The results provide a comprehensive understanding of the Snf2 genes in U's triangle model species, which will facilitate further functional analysis of the Snf2 genes in Brassica plants.

Key Advances in the New Era of Genomics-Assisted Disease Resistance Improvement of Brassica Species.

Disease resistance improvement remains a major focus in breeding programs as diseases continue to devastate Brassica production systems due to intensive cultivation and climate change. Genomics has paved the way to understand the complex genomes of Brassicas, which has been pivotal in the dissection of the genetic underpinnings of agronomic traits driving the development of superior cultivars. The new era of genomics-assisted disease resistance breeding has been marked by the development of high-quality genome references, accelerating the identification of disease resistance genes controlling both qualitative (major) gene and quantitative resistance. This facilitates the development of molecular markers for marker assisted selection and enables genome editing approaches for targeted gene manipulation to enhance the genetic value of disease resistance traits. This review summarizes the key advances in the development of genomic resources for Brassica species, focusing on improved genome references, based on long-read sequencing technologies and pangenome assemblies. This is further supported by the advances in pathogen genomics, which have resulted in the discovery of pathogenicity factors, complementing the mining of disease resistance genes in the host. Recognizing the co-evolutionary arms race between the host and pathogen, it is critical to identify novel resistance genes using crop wild relatives and synthetic cultivars or through genetic manipulation via genome-editing to sustain the development of superior cultivars. Integrating these key advances with new breeding techniques and improved phenotyping using advanced data analysis platforms will make disease resistance improvement in Brassica species more efficient and responsive to current and future demands.

The dynamics of lncRNAs transcription in interspecific F1 allotriploid hybrids between Brassica species.

Interspecific hybridization is the intrinsic forces behind genome evolution. Long non-coding RNAs (lncRNAs) are important for plant biological processes regulation. However, it is unclear that these non-coding fractions are impacted by interspecific hybridization. Here we examined the profiles of lncRNAs by comparing them with coding genes in Brassica napus, three accessions of Brassica rapa, and their F1 hybrids. 6206 high-confidential lncRNAs were identified in F 1 hybrids and their parentals, and the lncRNAs transcriptome in the F1 hybrids was reprogrammed by the genome shock. Notably, genome-wide unbalanced of lncRNAs were observed between An and Ar subgenomes, ELD (Expression Level Dominance) was biased toward the An -genome in F1 hybrids, and ELD of non-conserved lncRNAs was more than conserved lncRNAs. Our findings demonstrate that the reprogramed lncRNAs acts as important role in enhancing plant plasticity, leading to the acquisition of desirable traits in polyploid Brassica species.

Downregulation of the INDEHISCENT Gene by RNAi Resulted in Desired Pod Shatter Reduction of Lepidium campestre in Subsequent Generations.

Wild species field cress (Lepidium campestre) has favorable agronomic traits, making it a good candidate for future development as an oil and catch crop. However, the species is very prone to pod shatter, resulting in severe yield losses. This is one of the important agronomic traits that needs to be improved in order to make this species economically viable. In this study, we cloned the L. campestre INDEHISCENT (LcIND) gene and prepared two LcIND-RNAi constructs with the IND promoter (long 400 bp and short 200 bp) from Arabidopsis. A number of stable transgenic lines were developed and evaluated in terms of pod shatter resistance. The majority of the transgenic lines showed increased resistance to pod shatter compared to the wild type, and this resistance was maintained in four subsequent generations. The downregulation of the LcIND gene by RNAi in the transgenic lines was confirmed by qRT-PCR analysis on T3 lines. Southern blot analysis showed that most of the analyzed lines had a single-copy integration of the transgene, which is desirable for further use. Our results show that it is possible to generate stable transgenic lines with desirable pod shatter resistance by downregulating the LcIND gene using RNAi in field cress, and thus speeding up the domestication process of this wild species.

Exploration of Cd transformations in Cd spiked and EDTA-chelated soil for phytoextraction by Brassica species.

The study of soil cadmium (Cd) fractionation has become the need of the hour due to phytoextraction of Cd heavy metal by indigenous Brassica species of northwest India. The present study was conducted to explore the Cd speciation in soils treated with Cd (0, 5.0, 10.0, 20.0, 40.0, and 80.0 mg kg-1 soil) and synthetic chelate ethylene diamine tetraacetic acid (EDTA-0, 1.0 and 2.0 g kg-1 soil) planted under three Brassica species (Brassica juncea L., Brassica campestris L., and Brassica napus L). The studied Cd fractions viz. exchangeable and water-soluble (EX + WS), carbonate (CARB), organic matter (OM), Mn oxide (MnOX), amorphous Fe oxide (AFeOX), crystalline Fe oxide (CFeOX), and residual (RES) differed in their Cd content in soils under three investigated Brassica species. Among all plantations, B. juncea reduced the highest soil Cd content of EX + WS form which reflected its bioavailability. The Cd supplementation significantly enhanced the Cd concentration in all Cd forms with EX + WS Cd form exhibiting higher increase even at low Cd level (5.0 mg kg-1), whereas the EDTA addition did not influence Cd fractions. The application of EDTA @ 1.0 g kg-1 soil proved beneficial as it enhanced the metal mobility for plant extraction. All species positively significantly correlated (r = 0.648** to 0.747**) with all Cd fractions but except B. juncea all confronted reduction in their total biomass. In nutshell, it suggested that Brassica species having large plant biomass could be considered as a potential candidate for phytoremediation.

Genome-wide identification of R2R3-MYB gene family and association with anthocyanin biosynthesis in Brassica species.

Brassica species include important oil crops and vegetables in the world. The R2R3-MYB gene participates in a variety of plant functions, including the activation or inhibition of anthocyanin biosynthesis. Although previous studies have reported its phylogenetic relationships, gene structures, and expression patterns in Arabidopsis, the number and sequence variation of this gene family in Brassica crops and its involvement in the natural quantitative variation in anthocyanin biosynthesis regulation are still largely unknown. In this study, by using whole genome sequences and comprehensive genome-wide comparative analysis among the six cultivated Brassica species, 2120 R2R3-MYB genes were identified in six Brassica species, in total These R2R3-MYB genes were phylogenetically clustered into 12 groups. The R2R3-MYB family between A and C subgenomes showed better collinearity than between B and C and between A and B. From comparing transcriptional changes of five Brassica species with the purple and green leaves for the detection of the R2R3-MYB genes associated with anthocyanin biosynthesis, 7 R2R3-MYB genes were co-differentially expressed. The promoter and structure analysis of these genes showed that some variations between non-coding region, but they were highly conserved at the protein level and spatial structure. Co-expression analysis of anthocyanin-related genes and R2R3-MYBs indicated that MYB90 was strongly co-expressed with TT8, and they were co-expressed with structural genes F3H, LDOX, ANS and UF3GT at the same time. These results further clarified the roles of the R2R3-MYBs for leaf coloration in Brasica species, which provided new insights into the functions of the R2R3-MYB gene family in Brasica species.

Transcriptome shock in interspecific F1 allotriploid hybrids between Brassica species.

Interspecific hybridization drives the evolution of angiosperms and can be used to introduce novel alleles for important traits or to activate heterosis in crop breeding. Hybridization brings together gene expression networks from two different species, potentially causing global alterations of gene expression in the F1 plants which is called 'transcriptome shock'. Here, we explored such a transcriptome shock in allotriploid Brassica hybrids. We generated interspecific F1 allotriploid hybrids between the allotetraploid species Brassica napus and three accessions of the diploid species Brassica rapa. RNA-seq of the F1 hybrids and the parental plants revealed that 26.34-30.89% of genes were differentially expressed between the parents. We also analyzed expression level dominance and homoeolog expression bias between the parents and the F1 hybrids. The expression-level dominance biases of the Ar, An, and Cn subgenomes was genotype and stage dependent, whereas significant homoeolog expression bias was observed among three subgenomes from different parents. Furthermore, more genes were involved in trans regulation than in cis regulation in allotriploid F1 hybrids. Our findings provide new insights into the transcriptomic responses of cross-species hybrids and hybrids showing heterosis, as well as a new method for promoting the breeding of desirable traits in polyploid Brassica species.

Complex Molecular Evolution and Expression of Expansin Gene Families in Three Basic Diploid Species of Brassica.

Expansins are a kind of structural proteins of the plant cell wall, and they enlarge cells by loosening the cell walls. Therefore, expansins are involved in many growth and development processes. The complete genomic sequences of Brassica rapa, Brassica oleracea and Brassica nigra provide effective platforms for researchers to study expansin genes, and can be compared with analogues in Arabidopsis thaliana. This study identified and characterized expansin families in B. rapa, B. oleracea, and B. nigra. Through the comparative analysis of phylogeny, gene structure, and physicochemical properties, the expansin families were divided into four subfamilies, and then their expansion patterns and evolution details were explored accordingly. Results showed that after the three species underwent independent evolution following their separation from A. thaliana, the expansin families in the three species had increased similarities but fewer divergences. By searching divergences of promoters and coding sequences, significant positive correlations were revealed among orthologs in A. thaliana and the three basic species. Subsequently, differential expressions indicated extensive functional divergences in the expansin families of the three species, especially in reproductive development. Hence, these results support the molecular evolution of basic Brassica species, potential functions of these genes, and genetic improvement of related crops.

Genome-Wide Analysis of Phosphorus Transporter Genes in Brassica and Their Roles in Heavy Metal Stress Tolerance.

Phosphorus transporter (PHT) genes encode H2PO4-/H+ co-transporters that absorb and transport inorganic nutrient elements required for plant development and growth and protect plants from heavy metal stress. However, little is known about the roles of PHTs in Brassica compared to Arabidopsis thaliana. In this study, we identified and extensively analyzed 336 PHTs from three diploid (B. rapa, B. oleracea, and B. nigra) and two allotetraploid (B. juncea and B. napus) Brassica species. We categorized the PHTs into five phylogenetic clusters (PHT1-PHT5), including 201 PHT1 homologs, 15 PHT2 homologs, 40 PHT3 homologs, 54 PHT4 homologs, and 26 PHT5 homologs, which are unevenly distributed on the corresponding chromosomes of the five Brassica species. All PHT family genes from Brassica are more closely related to Arabidopsis PHTs in the same vs. other clusters, suggesting they are highly conserved and have similar functions. Duplication and synteny analysis revealed that segmental and tandem duplications led to the expansion of the PHT gene family during the process of polyploidization and that members of this family have undergone purifying selection during evolution based on Ka/Ks values. Finally, we explored the expression profiles of BnaPHT family genes in specific tissues, at various developmental stages, and under heavy metal stress via RNA-seq analysis and qRT-PCR. BnaPHTs that were induced by heavy metal treatment might mediate the response of rapeseed to this important stress. This study represents the first genome-wide analysis of PHT family genes in Brassica species. Our findings improve our understanding of PHT family genes and provide a basis for further studies of BnaPHTs in plant tolerance to heavy metal stress.

The response of transgenic Brassica species to salt stress: a review.

Salt stress is considered one of the main abiotic factors to limit crop growth and productivity by affecting morpho-physiological and biochemical processes. Genetically, a number of salt tolerant Brassica varieties have been developed and introduced, but breeding of such varieties is time consuming. Therefore, current focus is on transgenic technology, which plays an important role in the development of salt tolerant varieties. Various salt tolerant genes have been characterized and incorporated into Brassica. Therefore, such genetic transformation of Brassica species is a significant step for improvement of crops, as well as conferring salt stress resistance qualities to Brassica species. Complete genome sequencing has made the task of genetically transforming Brassica species easier, by identifying desired candidate genes. The present review discusses relevant information about the principles which should be employed to develop transgenic Brassica species, and also will recommend tools for improved tolerance to salinity.

SSR marker variations in Brassica species provide insight into the origin and evolution of Brassica amphidiploids.

BACKGROUND: Oilseed Brassica represents an important group of oilseed crops with a long history of evolution and cultivation. To understand the origin and evolution of Brassica amphidiploids, simple sequence repeat (SSR) markers were used to unravel genetic variations in three diploids and three amphidiploid Brassica species of U's triangle along with Eruca sativa as an outlier. RESULTS: Of 124 Brassica-derived SSR loci assayed, 100% cross-transferability was obtained for B. juncea and three subspecies of B. rapa, while lowest cross-transferability (91.93%) was obtained for Eruca sativa. The average % age of cross-transferability across all the seven species was 98.15%. The number of alleles detected at each locus ranged from one to six with an average of 3.41 alleles per primer pair. Neighbor-Joining-based dendrogram divided all the 40 accessions into two main groups composed of B. juncea/B. nigra/B. rapa and B. carinata/B. napus/B. oleracea. C-genome of oilseed Brassica species remained relatively more conserved than A- and B-genome. A- genome present in B. juncea and B. napus seems distinct from each other and hence provides great opportunity for generating diversity through synthesizing amphidiploids from different sources of A- genome. B. juncea had least intra-specific distance indicating narrow genetic base. B. rapa appears to be more primitive species from which other two diploid species might have evolved. CONCLUSION: The SSR marker set developed in this study will assist in DNA fingerprinting of various Brassica species cultivars, evaluating the genetic diversity in Brassica germplasm, genome mapping and construction of linkage maps, gene tagging and various other genomics-related studies in Brassica species. Further, the evolutionary relationship established among various Brassica species would assist in formulating suitable breeding strategies for widening the genetic base of Brassica amphidiploids by exploiting the genetic diversity present in diploid progenitor gene pools.

Molasses melanoidin-like products enhance phytoextraction of lead through three Brassica species.

Previously, it has been suggested that melanoidin-like products (MLP) from sugarcane molasses may accelerate copper phytoextraction. In this study, we evaluated the facilitatory effect of MLP on phytoextraction in a medium including cadmium or lead, the concentrations of which were adjusted around the regulation values of the Soil Contamination Countermeasures Act in Japan. Three Brassica species were tested based on their fast growth, high biomass productivity, and high heavy metal absorption. Both biomass and lead uptake in the nutrient medium with 1 mM lead nitrate were significantly increased by the addition of MLP, and almost all of the lead was accumulated in the root tissue. Therefore, MLP were able both to detoxify lead ions and to improve their bioavailability in Brassica species. In contrast, only these species with MLP or citric acid survived in the nutrient medium with 1 mM cadmium sulfate. The phytoextraction of cadmium using these species was therefore impractical under the Act.

Brassica biodiversity conservation: prevailing constraints and future avenues for sustainable distribution of plant genetic resources.

The past decade has seen an observable loss of plant biodiversity which can be attributed to changing climate conditions, destroying ecosystems to create farmlands and continuous selective breeding for limited traits. This loss of biodiversity poses a significant bottleneck to plant biologists across the globe working on sustainable solutions to address the current barriers of agricultural productivity. Plant genetic resources centers or genebanks that conserve plant germplasm can majorly contribute towards addressing this problem. Second only to soybean, Brassica remains the largest oil-seed crop and is cultivated across 124 countries, and FAO estimates for a combined gross production values of broccoli, cabbages, cauliflower, mustard and rape seeds stands at a staggering 67.5 billion US dollars during the year 2020. With such a global status, wide variety of uses and more recently, growing importance in the health food sector, the conservation of diverse genetic resources of Brassica appeals for higher priority. Here we review the current status of Brassica conservation across plant genebanks. At present, at least 81,752 accessions of Brassica are recorded to be conserved in 148 holding institutes spread across only 81 countries. Several aspects that need to be addressed to improve proper conservation of the Brassica diversity was well as dissemination of germplasm are discussed. Primarily, the number of accessions conserved across countries and the diversity of Brassica taxa most countries has been highly limited which may lead to biodiversity loss in the longer run. Moreover, several practical challenges in Brassica germplasm conservation especially with respect to taxonomic authorities have been discussed. The current review identifies and highlights areas for progress in Brassica conservation, which include but are not limited to, distribution of conserved Brassica biodiversity, challenges faced by conservation biologists, conservation methods, technical hurdles and future avenues for research in diverse Brassica species.

Natural Sources of Selenium as Functional Food Products for Chemoprevention.

Cancer is one of the leading causes of death worldwide, the incidence of which is increasing annually. Interest has recently grown in the anti-cancer effect of functional foods rich in selenium (Se). Although clinical studies are inconclusive and anti-cancer mechanisms of Se are not fully understood, daily doses of 100-200 µg of Se may inhibit genetic damage and the development of cancer in humans. The anti-cancer effects of this trace element are associated with high doses of Se supplements. The beneficial anti-cancer properties of Se and the difficulty in meeting the daily requirements for this micronutrient in some populations make it worth considering the use of functional foods enriched in Se. This review evaluated studies on the anti-cancer activity of the most used functional products rich in Se on the European market.

Bioactives and Technological Quality of Functional Biscuits Containing Flour and Liquid Extracts from Broccoli By-Products.

Broccoli by-products are an important source of health-promoting bioactive compounds, although they are generally underutilized. This study aimed to valorize non-compliant broccoli florets by transforming them into functional ingredients for biscuit formulation. A broccoli flour and three water/ethanol extracts (100:0, 75:25, 50:50; v/v) were obtained. The rheological properties and the content of bioactive compounds of the functional ingredients and biscuits were evaluated. The 50:50 hydroalcoholic extract was the richest in glucosinolates (9749 µg·g-1 DW); however, the addition of a small amount strongly affected dough workability. The enrichment with 10% broccoli flour resulted the best formulation in terms of workability and color compared to the other enriched biscuits. The food matrix also contributed to protecting bioactive compounds from thermal degradation, leading to the highest total glucosinolate (33 µg·g-1 DW), carotenoid (46 µg·g-1 DW), and phenol (1.9 mg GAE·g-1 DW) contents being present in the final biscuit. Therefore, broccoli flour is a promising ingredient for innovative healthy bakery goods. Hydroalcoholic extracts could be valuable ingredients for liquid or semi-solid food formulation.

Influence of rapeseed, canola meal and glucosinolate metabolite (AITC) as potential antimicrobials: effects on growth performance, and gut health in Salmonella Typhimurium challenged broiler chickens.

Poultry is the major sources of foodborne salmonellosis. Antibiotic resistance and a surge in zoonotic diseases warrant the use of natural alternatives. Glucosinolates (GLs) are naturally occurring antimicrobial compounds in rapeseed and canola. This study investigated the effect of feeding rapeseed, canola meal, and allyl isothiocyanate (AITC; Brassica secondary metabolites) on growth performance (GP), gut health, and the potential antimicrobial activity against nalidixic acid-resistant Salmonella Typhimurium (STNR) in chickens. A total of 640 one-day-old male Cobb 500 broilers were randomly allocated to 8 treatments with 8 replicated cages and 10 birds per cage. Dietary treatments were nonchallenge control (NC, corn-SBM based), challenge (Salmonella) control (CC), 10% rapeseed (10RS), 30% rapeseed (30RS), 20% canola meal (20CLM), 40% canola meal (40CLM), 500 ppm AITC (500AITC), and 1,500 ppm AITC (1500AITC). On d 1, all the birds except NC were orally challenged with STNR (7 log CFU/bird). The chickens were reared for 21 d, and their FI and BW were recorded weekly. Salmonella cecal colonization and fecal shedding were quantified, whereas organ translocation (OT) of STNR to the spleen, liver, and kidney was tested on 0, 3, 6, 13, and 20-d postchallenge (dpc). Data were subjected to one-way ANOVA, and the means were separated by Duncan's test, except mortality and OT data analyzed after transformation by square root of (n +1) (P < 0.05). Overall, feeding 30RS resulted in reduced BW (P = 0.003), BWG (P = 0.003), and FI (P = 0.001) compared to CC, 500AITC, and 1500AITC. Similarly, feeding 20CLM resulted in lower BW and BWG compared to CC (P < 0.05) and increased FCR compared to 1500AITC (P = 0.03). Feeding CC resulted in higher mortality compared to NC and 30RS (P = 0.03). Cecal colonization of STNR was reduced (P < 0.0001) for 30RS on 6 dpc and 500AITC on 6 and 13 dpc (P < 0.0001). Although no difference in gut permeability was observed 6 dpc (P > 0.05), OT of STNR population was the highest for CC in the spleen (P = 0.05). In the liver, 10RS showed reduced OT compared to 20CLM on 13 dpc (P = 0.03), whereas 30RS showed the lowest OT on 6 dpc in the kidney. Fecal shedding was lowest for 30RS on 6 dpc (P = 0.004). Histomorphology showed 30RS had the highest duodenum (P = 0.01) and jejunum (P = 0.02) villus height (VH) and VH to crypt depth (CD) ratio compared to the other treatments, whereas 1500AITC showed similar results to 30RS. Both 30RS and 1500AITC contained comparatively higher functional GL metabolites and were able to maintain gut health. Including higher levels of rapeseed or AITC in poultry feed can reduce Salmonella colonization in the feces and their translocation to other organs.

Genome-wide survey of Calcium-Dependent Protein Kinases (CPKs) in five Brassica species and identification of CPKs induced by Plasmodiophora brassicae in B. rapa, B. oleracea, and B. napus.

Calcium-dependent protein kinase (CPK) is a class of Ser/Thr protein kinase that exists in plants and some protozoa, possessing Ca2+ sensing functions and kinase activity. To better reveal the roles that Brassica CPKs played during plant response to stresses, five Brassica species, namely Brassica rapa (B. rapa), Brassica nigra (B. nigra), Brassica oleracea (B. oleracea), Brassica juncea (B. juncea), and Brassica napus (B. napus) were selected and analyzed. In total, 51 BraCPK, 56 BniCPK, 56 BolCPK, 88 BjuCPK, and 107 BnaCPK genes were identified genome wide and phylogenetics, chromosomal mapping, collinearity, promoter analysis, and biological stress analysis were conducted. The results showed that a typical CPK gene was constituted by a long exon and tandem short exons. They were unevenly distributed on most chromosomes except chromosome A08 in B. napus and B. rapa, and almost all CPK genes were located on regions of high gene density as non-tandem form. The promoter regions of BraCPKs, BolCPKs, and BnaCPKs possessed at least three types of cis-elements, among which the abscisic acid responsive-related accounted for the largest proportion. In the phylogenetic tree, CPKs were clustered into four primary groups, among which group I contained the most CPK genes while group IV contained the fewest. Some clades, like AT5G23580.1(CPK12) and AT2G31500.1 (CPK24) contained much more gene members than others, indicating a possibility that gene expansion occurred during evolution. Furthermore, 4 BraCPKs, 14 BolCPKs, and 31 BnaCPKs involved in the Plasmodiophora brassicae (P. brassicae) defense response in resistant (R) or susceptible (S) materials were derived from online databases, leading to the discovery that some R-specific induced CPKs, such as BnaC02g08720D, BnaA03g03800D, and BolC04g018270.2J.m1 might be ideal candidate genes for P. brassicae resistant research. Overall, these results provide valuable information for research on the function and evolution of CDK genes.

Multicolored Fluorescent In Situ Hybridization to Assess Pairing Configurations at Metaphase I in Brassica Hybrids.

Genetic diversity can be introduced into polyploid crop species through meiotic recombination by exchanges between homologous or homoeologous chromosomes. Fluorescent in situ hybridization (FISH) enables the characterization of these homologous and homoeologous chromosome pairs during meiosis and identification of structural rearrangements during mitosis in metaphase I. In this chapter, we describe a protocol for the multicolored fluorescent labeling of chromosome spreads. This protocol allows the characterization of each A and C homoeologous subgenomes in a polyploid species using a genome-specific BAC combined with specific chromosome labeling BAC sequences.