Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.     

Transcriptome and physiological analyses reveal that AM1 as an ABA-mimicking ligand improves drought resistance in <Emphasis Type="Italic">Brassica napus</Emphasis>

Abscisic acid (ABA) is the most important stress hormone in the regulation of plant adaptation to drought. Owing to the chemical instability and rapid catabolism of ABA, ABA mimic 1 (AM1) is frequently applied to enhance drought resistance in plants, but the molecular mechanisms governed by AM1 on improving drought resistance in Brassica napus are not entirely understood. To investigate the effect of AM1 on drought resistance at the physiological and molecular levels, exogenous ABA and AM1 were applied to the leaves of two B. napus genotypes (Q2 and Qinyou 8) given progressive drought stress. The results showed that the leaves of 50 µM ABA- and AM1-treated plants shared over 60% differential expressed genes and 90% of the enriched functional pathways in Qinyou 8 under drought. AM1 affected the expression of the genes involved in ABA signaling; they down-regulated pyrabactin resistance/PYR1-like (PYR/PYLs), up-regulated type 2C protein phosphatases (PP2Cs), partially up-regulated sucrose non-fermenting 1-related protein kinase 2s (SnRK2s), and down-regulated ABA-responsive element (ABRE)-binding protein/ABRE-binding factors (AREB/ABFs). Additionally, AM1 treatment repressed the expression of photosynthesis-related genes, those mainly associated with the light reaction process. Moreover, AM1 decreased the stomatal conductance, the net photosynthetic rate, and the transpiration rate, but increased the relative water content in leaves and increased survival rates of two genotypes under drought stress. Our findings suggest that AM1 has a potential to improve drought resistance in B. napus by triggering molecular and physiological responses to reduce water loss and impair growth, leading to increased survival rates.     

Salt tolerance conferred by expression of a global regulator IrrE from <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in oilseed rape

As salinity is a major threat to sustainable agriculture worldwide, cultivation of salt-tolerant crops becomes increasingly important. IrrE acts as a global regulator and a general switch for stress resistance in Deinococcus radiodurans. In this study, to determine whether the irrE gene can improve the salt tolerance of Brassica napus, we introduced the irrE gene into B. napus by the Agrobacterium tumefaciens-mediated transformation method. Forty-two independent transgenic plants were regenerated. Polymerase chain reaction (PCR) analyses confirmed that the irrE gene had integrated into the plant genome. Northern as well as Western blot analyses revealed that the transgene was expressed at various levels in transgenic plants. Analysis for the T₁ progenies derived from four independent transformants showed that irrE had enhanced the salt tolerance of T₁ in the presence of 350 mM NaCl. Furthermore, under salt stress, transgenic plants accumulated more compatible solutes (proline) and a lower level of malondialdehyde (MDA), and they had higher activities of catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD). However, agronomic traits were not affected by irrE gene overexpression in the transgenic B. napus plants. This study indicates that the irrE gene can improve the salt tolerance of B. napus and represents a promising candidate for the development of crops with enhanced salt tolerance by genetic engineering.     

<Emphasis Type="Italic">BcMF11</Emphasis> and its homologous sequences may form a lncRNA family in <Emphasis Type="Italic">Brassica</Emphasis> diploids

BcMF11 is a long non-coding RNA that has been identified in Brassica rapa and shown to be involved in pollen development. Here, when re-cloned the gene sequence, multiple paralogous copies of BcMF11 were identified in B. rapa (A genome). Multiple paralogous copies of BcMF11 were also found in B. nigra (B genome) and Brassica oleracea (C genome), the other two primary diploids of Brassica U triangle. While in the early diverging Brassicaceae lineage including Arabidopsis thaliana, no BcMF11 homolog was found. Phylogenetic analysis showed that the BcMF11 homologous sequences cloned from A genome or C genome could be clustered into a separate branch, respectively. However, there was no distinct cluster defined for BcMF11 homologous sequences cloned from B genome. The expression of BcMF11 in B. rapa was investigated and revealed a different result in the previous study. In addition, 12 expressed sequence tags from B. napus and B. rapa showing high similarities with BcMF11 were identified in the NCBI database, which further verified that rather than the useless repeat fragments in the genome, the BcMF11 homologous genes could transcribe. It is possible that BcMF11 and its homologous sequences may form a large gene family which might be originated in the recent ancestral lineage of Brassica.     

Small RNA changes in synthetic <Emphasis Type="Italic">Brassica napus</Emphasis>

Main conclusion

Small RNAs and microRNAs were found to vary extensively in synthetic Brassica napus and subsequent generations, accompanied by the activation of transposable elements in response to hybridization and polyploidization.

Abstract

Resynthesizing B. napus by hybridization and chromosome doubling provides an approach to create novel polyploids and increases the usable genetic variability in oilseed rape. Although many studies have shown that small RNAs (sRNAs) act as important factor during hybridization and polyploidization in plants, much less is known on how sRNAs change in synthetic B. napus, particularly in subsequent generations after formation. We performed high-throughput sequencing of sRNAs in S₁–S₄ generations of synthetic B. napus and in the homozygous B. oleracea and B. rapa parent lines. We found that the number of small RNAs (sRNAs) and microRNAs (miRNAs) doubled in synthetic B. napus relative to the parents. The proportions of common sRNAs detected varied from the S₁ to S₄ generations, suggesting sRNAs are unstable in synthetic B. napus. The majority of miRNAs (67.2 %) were non-additively expressed in the synthesized Brassica allotetraploid, and 33.3 % of miRNAs were novel in the resynthesized B. napus. The percentage of miRNAs derived from transposable elements (TEs) also increased, indicating transposon activation and increased transposon-associated miRNA production in response to hybridization and polyploidization. The number of target genes for each miRNA in the synthesized Brassica allotetraploid was doubled relative to the parents, enhancing the complexity of gene expression regulation. The potential roles of miRNAs and their targets are discussed. Our data demonstrate generational changes in sRNAs and miRNAs in synthesized B. napus.

     

Molecular genetics and functional genomics of abiotic stress-responsive genes in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.): a review of recent advances and future

Abiotic stresses are the key factors which negatively influence plant development and productivity and are the main cause of extensive agricultural production losses worldwide. Brassica napus is an oilseed crop of global economic significance and major contributor to the total oilseed production, quite often encounters abiotic stresses, resulting in reduced agricultural productivity. Hence, there is an immediate need being felt to raise B. napus cultivars which would be more suitable for various abiotic stress conditions presently and in the years to come. Biotechnology and molecular plant breeding has emerged as an important tool for molecular understanding of plant response to various abiotic stresses. Currently, various stress-responsive genes and mechanisms have been identified and functionally characterized in model plant Arabidopsis and other major crop plants such as Oryza sativa and Zea mays. However, very inadequate success has been achieved in this direction in a major oilseed crop such as B. napus. In this review, we present the latest methods and approaches of studying abiotic stress in B. napus. In this review, we describe the genes functioning as markers for crop breeding and discuss the recent progress and advances in genome editing by break through CRISPR/Cas9 multigene–multiplex approaches for developing multiple abiotic stress tolerance with our on-going research as a scheme. We also throw some light on molecular genetics, plant breeding and abiotic stress biotechnology of B. napus which offer a new prospective on the research directions for the practical plant breeding and functional genomics of B. napus in response to different abiotic stress conditions.     

<Emphasis Type="Italic">LMI1</Emphasis>-like genes involved in leaf margin development of <Emphasis Type="Italic">Brassica napus</Emphasis>

In rapeseed (Brassica napus L.), leaf margins are variable and can be entire, serrate, or lobed. In our previous study, the lobed-leaf gene (LOBED-LEAF 1, BnLL1) was mapped to a 32.1 kb section of B. napus A10. Two LMI1-like genes, BnaA10g26320D and BnaA10g26330D, were considered the potential genes that controlled the lobed-leaf trait in rapeseed. In the present study, these two genes and another homologous gene (BnaC04g00850D) were transformed into Arabidopsis thaliana (L.) Heynh. plants to identify their functions. All three LMI1-like genes of B. napus produced serrate leaf margins. The expression analysis indicated that the expression level of BnaA10g26320D determined the difference between lobed- and entire-leaved lines in rapeseed. Therefore, it is likely that BnaA10g26320D corresponds to BnLL1.     

Identification of candidate genes of QTLs for seed weight in <Emphasis Type="Italic">Brassica napus</Emphasis> through comparative mapping among <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Brassica</Emphasis>species

Background

Map-based cloning of quantitative trait loci (QTLs) in polyploidy crop species remains a challenge due to the complexity of their genome structures. QTLs for seed weight in B. napus have been identified, but information on candidate genes for identified QTLs of this important trait is still rare.

Results

In this study, a whole genome genetic linkage map for B. napus was constructed using simple sequence repeat (SSR) markers that covered a genetic distance of 2,126.4 cM with an average distance of 5.36 cM between markers. A procedure was developed to establish colinearity of SSR loci on B. napus with its two progenitor diploid species B. rapa and B. oleracea through extensive bioinformatics analysis. With the aid of B. rapa and B. oleracea genome sequences, the 421 homologous colinear loci deduced from the SSR loci of B. napus were shown to correspond to 398 homologous loci in Arabidopsis thaliana. Through comparative mapping of Arabidopsis and the three Brassica species, 227 homologous genes for seed size/weight were mapped on the B. napus genetic map, establishing the genetic bases for the important agronomic trait in this amphidiploid species. Furthermore, 12 candidate genes underlying 8 QTLs for seed weight were identified, and a gene-specific marker for BnAP2 was developed through molecular cloning using the seed weight/size gene distribution map in B. napus.

Conclusions

Our study showed that it is feasible to identify candidate genes of QTLs using a SSR-based B. napus genetic map through comparative mapping among Arabidopsis and B. napus and its two progenitor species B. rapa and B. oleracea. Identification of candidate genes for seed weight in amphidiploid B. napus will accelerate the process of isolating the mapped QTLs for this important trait, and this approach may be useful for QTL identification of other traits of agronomic significance.

     

Overexpression of <Emphasis Type="Italic">MeDREB1D</Emphasis> confers tolerance to both drought and cold stresses in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Cassava (Manihot esculenta) is an important tropical crop with extraordinary tolerance to drought stress but few reports on it. In this study, MeDREB1D was significantly and positively induced by drought stress. Two allelic variants of the gene named MeDREB1D(R-2) and MeDREB1D(Y-3) were identified. Overexpressing MeDREB1D(R-2) and MeDREB1D(Y-3) in Arabidopsis resulted in stronger tolerance to drought and cold stresses. Under drought stress, transgenic plants had more biomass, higher survival rates and less MDA content than wild-type plants. Under cold stress, transgenic plants also had higher survival rates than wild-type plants. To further characterize the molecular function of MeDREB1D, we conducted an RNA-Seq analysis of transgenic and wild-type Arabidopsis plants. The results showed that the Arabidopsis plants overexpressing MeDREB1D led to changes in downstream genes. Several POD genes, which may play a vital role in drought and cold tolerance, were up-regulated in transgenic plants. In brief, these results suggest that MeDREB1D can simultaneously improve plant tolerance to drought and cold stresses.     

Molecular and cytological analyses of A and C genomes at meiosis in synthetic allotriploid <Emphasis Type="Italic">Brassica</Emphasis> hybrids (ACC) between <Emphasis Type="Italic">B.napus</Emphasis> (AACC) and <Emphasis Type="Italic">B.oleracea</Emphasis> (CC)

Success of interspecific hybridization relies mostly on the adequate similarity between the implicated genomes to ensure synapsis, pairing and recombination between appropriate chromosomes during meiosis in allopolyploid species. Allotetraploid Brassica napus (AACC) is a model of natural hybridization between Brassica rapa (AA) and Brassica oleracea (CC), which are originally derived from a common ancestor, but genomic constitution of the same chromosomes probably varied among these species through time after establishment, giving rise to cytogenetic difference in the synthetic hybrids. Herein we investigated meiotic behaviors of A and C chromosomes of synthetic allotriploid Brassica hybrids (ACC) at molecular and cytological levels, which result from the interspecific cross between natural B. napus (AACC) and B.oleracea (CC), and the results showed that meiosis course was significantly aberrant in allotriploid Brassica hybrids, and chromosomes aligned chaotically at metaphase I, chromosome bridges and lags were frequently observed from later metaphase I to anaphase II during meiosis. Simultaneously, we also noticed that meiosis-related genes were abruptly down-regulated in allotriploid Brassica hybrids, which likely accounted for irregular scenario of meiosis observed in these synthetic hybrids. Therefore, these results indicated that inter-genomic exchanges of A and C chromosomes could occur frequently in synthetic Brassica hybrids, and provided an efficient approach for genetic changes of homeologous chromosomes during meiosis in polyploid B.napus breeding program.     

Preferential retention,expression profile and potential functional diversity analysis of HD-Zip gene family in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Homeodomain-Leu zipper (HD-Zip) gene family performs important biological functions related to organ development, photomorphogenesis and abiotic stress response in higher plants. However, systematic analysis of HD-Zip genes in Brassica rapa has not been performed. In the present study, a bioinformatics approach was used to identify and characterize the BraHD-Zip gene family in B. rapa. A total of 88 members were identified. All putative BraHD-Zip proteins contained a clear HD and LZ combined domain. Eighty-seven BraHD-Zips were non-randomly located on ten chromosomes. This gene family was mainly expanded following the whole genome triplication event and was preferentially over-retained relative to its neighboring genes in B. rapa. On phylogenetic analysis, the BraHD-Zips could be categorized into four distinct major groups (I–IV). Each group exhibited variant gene structures and motif distributions. Some syntenic orthologous gene pairs presented diverse expression profiles, which indicate that these gene pairs may be involved in the development of new functions during evolution. In summary, our analysis provided genome-wide insights into the expansion, preferential retention, expression profiles and functional diversity of BraHD-Zip genes following whole genome triplication in B. rapa.     

Genome-wide analysis and stress-responsive expression of CCCH zinc finger family genes in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Background

Ubiquitous CCCH nucleic acid-binding motif is found in a wide-variety of organisms. CCCH genes are involved in plant developmental processes and biotic and abiotic stress responses. Brassica rapa is a vital economic crop and classical model plant of polyploidy evolution, but the functions of CCCH genes in B. rapa are unclear.

Results

In this study, 103 CCCH genes in B. rapa were identified. A comparative analysis of the chromosomal position, gene structure, domain organization and duplication event between B. rapa and Arabidopsis thaliana were performed. Results showed that CCCH genes could be divided into 18 subfamilies, and segmental duplication might mainly contribute to this family expansion. C-X_7/8-C-X₅-C₃-H was the most commonly found motif, but some novel CCCH motifs were also found, along with some loses of typical CCCH motifs widespread in other plant species. The multifarious gene structures and domain organizations implicated functional diversity of CCCH genes in B. rapa. Evidence also suggested functional redundancy in at least one subfamily due to high conservation between members. Finally, the expression profiles of subfamily-IX genes indicated that they are likely involved in various stress responses.

Conclusion

This study provides the first genome-wide characterization of the CCCH genes in B. rapa. The results suggest that B. rapa CCCH genes are likely functionally divergent, but mostly involved in plant development and stress response. These results are expected to facilitate future functional characterization of this potential RNA-binding protein family in Brassica crops.