<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.     

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.

     

Comparative Genomics and Synteny Analysis of <Emphasis Type="BoldItalic">KCS17</Emphasis>-<Emphasis Type="BoldItalic">KCS18</Emphasis> Cluster Across Different Genomes and Sub-genomes of Brassicaceae for Analysis of Its Evolutionary History

Comparative genomics-based synteny analysis has proved to be an effective strategy to understand evolution of genomic regions spanning a single gene (micro-unit) to large segments encompassing hundreds of kilobases to megabases. Brassicaceae is in a unique position to contribute to understanding genome and trait evolution through comparative genomics because whole genome sequences from as many as nine species have been completed and are available for analysis. In the present work, we compared genomic loci surrounding the KCS17-KCS18 cluster across these nine genomes. KCS18 or FAE 1 gene encodes beta-ketoacyl synthase, (β-KCS) a membrane-bound enzyme that catalyses the key rate-limiting step during synthesis of VLCFAs such as erucic acid (C22) present in seed oil in Brassicaceae by elongating carbon chain from C18 to C22; knowledge on role of KCS17 in plant development is however lacking. Synteny across the genomic segments harbouring FAE1 showed variable levels of gene retention ranging between 26% (Arabidopsis thaliana and Brassica napus C03) and 89% (between A. thaliana and Brassica rapa A01), and gene density ranged between 1 gene/2.86 kb and 1 gene/4.88 kb. Interestingly, in diploid Brassica species, FAE1 was retained in only one of the sub-genomes in spite of the presence of three sub-genomes created as a result of genome triplication; in contrast, FAE1 was present at three loci, with four copies in Camellina sativa which is also known to have experienced a recent genome triplication revealing contrasting fates upon duplication. The organization of KCS17 and KCS18 as head-to-tail cluster was conserved across most of the species, except the C genome containing Brassicas, namely B. oleracea and B. napus, where disruptions because of other genes were observed. Even in the conserved blocks, the distance between KCS17 and KCS18 varied; the functional implication of the organization of KCS17-KCS18 as a cluster vis-à-vis fatty acid biosynthesis needs to be dissected, as the cis-regulatory region is expected to be present in the intergenic space. Phylogenetic analysis of KCS gene family along with KCS17-KCS18 from members of Brassicaceae reveals their ancestral relationship with KCS8-KCS9 block. Further comparative functional analysis between KCS8, KCS9, KCS16, KCS17 and KCS18 across evolutionary time-scale will be required to understand the conservation or diversification of roles of these members of KCS family in fatty acid biosynthesis during course of evolution.     

Gene loss/retention and evolutionary pattern of ascorbic acid biosynthesis and recycling genes in <Emphasis Type="Italic">Brassica rapa</Emphasis> following whole genome triplication

Ascorbic acid (AsA) is an inevitable antioxidant found abundantly in higher plants. Despite the importance of AsA in plants, how AsA biosynthesis (ABGs; d-mannose/l-galactose pathway) and AsA recycling genes (ARGs) evolved through polyploidization has not been addressed to date. Here, we evaluated the impacts of whole genome triplication (WGT) on ABGs and ARGs in Chinese cabbage (Brassica rapa ssp. pekinensis), which diverged from Arabidopsis thaliana before the WGT event. Twenty-three ABGs coded in 13 loci representing nine different enzyme classes and 29 ARGs coded in 19 loci representing five different enzyme classes were identified in the B. rapa genome by whole-genome screening through comparative genomic analyses. Five of these loci maintained three gene copies, 10 loci maintained two gene copies and the majority of the loci (n = 17) maintained single gene copies. Segmental (62 %) and tandem duplication (6 %), and fragment (21 %) and large-scale recombination (10 %) events accelerated the diversification of ABGs and ARGs. Thirteen of the 52 (25 %) identified genes experienced intron losses and two (4 %) experienced intron gains implying that intron losses outnumbered intron gains. The expansion and the retention of ABGs and ARGs were similar to the whole genome gene expansion and retention (P > 0.05). These findings provide new insights into the structural characteristics and evolutionary trends of ABGs and ARGs. In addition, our data could become a useful resource to further the functional characterization of these genes.     

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.

     

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.     

Inheritance and gene mapping of the white flower trait in <Emphasis Type="Italic">Brassica juncea</Emphasis>

Despite being a unique marker trait, white flower inheritance in Brassica juncea remains poorly understood at the gene level. In this study, we investigated a B. juncea landrace with white petal in China. The white petal phenotype possessed defective chromoplasts with less plastoglobuli than the yellow petal phenotype. Genetic analysis confirmed that two independent recessive genes (Bjpc1 and Bjpc2) controlled the white flower trait. We then mapped the BjPC1 gene in a BC₄ population comprising 2295 individuals. We identified seven AFLP (amplified fragment length polymorphism) markers closely linked to the white flower gene. BLAST search revealed the sequence of AFLP fragments were highly homologous with the Scaffold000085 and Scaffold000031 sequences on the A02 chromosome in the Brassica rapa genome. Based on this sequence homology, we developed simple sequence repeat (SSR) primer pairs and identified 13 SSRs linked to the BjPC1 gene, including two that were co-segregated (SSR9 and SSR10). The two closest markers (SSR4 and SSR11) were respectively 0.9 and 0.4 cM on either side of BjPC1. BLAST analysis revealed that these marker sequences corresponded highly to A02 in B. juncea. They were mapped within a 33 kb genomic region on B. rapa A02 (corresponds to a 40 kb genomic region on B. juncea A02) that included three genes. Sequence BjuA008406, homologous to AtPES2 in Arabidopsis thaliana and Bra032956 in B. rapa, was the most likely candidate for BjPC1. These results should accelerate BjPC1 cloning and facilitate our understanding of the molecular mechanisms controlling B. juncea petal color.     

Natural genetic variation in <Emphasis Type="Italic">Brassica</Emphasis> homologs of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> and characterization of its expression domains

FLOWERING LOCUS T (FT), a major effect gene, regulates flowering time in Arabidopsis. We analyzed evolutionary changes distinguishing two FT homeologous loci in B. rapa, described genetic variation in homologs isolated and reported expression pattern of FT in B. juncea. Synteny analysis confirmed presence of two FT genomic copies in B. rapa ssp. pekinensis and resolved pre-existing anomalies regarding copy number in “AA” genome. Synteny analysis of B. rapa homeologous regions CR1 (129 kb) and CR2 (232 kb) revealed differential gene fractionation and wide-spread re-arrangements. Seven genomic DNA (gDNA) variants (2.1–2.2 kb) and 10 complementary DNA (cDNA) variants (528 bp) were isolated from 6 Brassica species. The gDNA variants shared 72–99 % similarity within Brassica and 58–60 % between Arabidopsis and Brassica. FT cDNA variants shared 92–100 % similarity within Brassica and 87 % between Arabidopsis and Brassica. Phylogenetic analysis of FT gDNA, cDNA and protein sequences revealed two major clades, differentiating homologs derived from species containing shared “BB” and “CC” genomes. Phylogram based on Brassica FT gDNA differentiated homeologs derived from AA-LF (Least fractioned) and AA-MF1 (Moderately fractioned) sub-genomes. Analysis of FT expression pattern in B. juncea revealed increasing levels correlating with attainment of physiological maturity; highest levels were detected in older leaves implying conservation in spatio-temporal expression pattern vis-à-vis Arabidopsis. In conclusion, our study reveals that polyploidy in Brassicas resulted in expansion of FT gene copies with homologs charting independent evolutionary course through accumulation of mutations. However, expression domains of FT remained conserved across Brassicaceae to preserve the critical function of FT in controlling flowering time.     

Genomic location and expression analysis of expansin gene family reveals the evolutionary and functional significance in <Emphasis Type="Italic">Triticum aestivum</Emphasis>

The plant-specific expansin proteins constitute an ancient and major gene family known to have roles in regulating diverse biological processes in plants. Although the functions of many expansin genes have been identified in wheat and other species, little is known about the evolution and genomic locations of the expansin genes in wheat (Triticum aestivum). In this study, a total of 87 expansin genes were identified in the wheat genome, including 52 EXPAs, 42 EXPBs and 4 EXLAs. The EXLB gene was not found in the wheat genome. Phylogenetic tree and comparative analysis revealed amplification of the EXPBs in rice, maize and wheat. The predicted wheat expansins were distributed across 14 of 21 chromosomes with different densities, 3 tightly co-located clusters and 15 paralogous pairs, indicating that tandem duplication and segmental duplication events also played roles in the evolution of expansins in wheat. In addition, the gene structures and conserved protein domains of wheat expansins suggest high levels of conservation within the phylogenetic subgroups. Analysis of a published microarray database showed that most wheat expansin genes exhibit different expression levels in different tissues and developmental stages. To our knowledge, this is the first report of a genome-wide analysis of the wheat expansin gene family, which should provide valuable information for further elucidating the classification and putative functions of the entire gene family.     

Genome-wide dissection of the chalcone synthase gene family in <Emphasis Type="Italic">Oryza sativa</Emphasis>

Enzymes of the chalcone synthase (CHS) family catalyze the generation of multiple secondary metabolites in fungi, plants, and bacteria. These metabolites have played key roles in antimicrobial activity, UV protection, flower pigmentation, and pollen fertility during the evolutionary process of land plants. We performed a genome-wide investigation about CHS genes in rice (Oryza sativa). The phylogenetic relationships, gene structures, chromosomal locations, and functional predictions of the family members were examined. Twenty-seven CHS family genes (OsCHS01–27) were identified in the rice genome and were found to cluster into six classes according to their phylogenetic relationships. The 27 OsCHS genes were unevenly distributed on six chromosomes, and 17 genes were found in the genome duplication zones with two segmental duplication and five tandem duplication events that may have played key roles in the expansion of the rice CHS gene family. In addition, the OsCHS genes exhibited diverse expression patterns under salicylic acid treatment. Our results revealed that the OsCHS genes exhibit both diversity and conservation in many aspects, which will contribute to further studies of the function of the rice CHS gene family and provide a reference for investigating this family in other plants.     

Effect of colchicine-induced polyploidy on morphological characteristics and essential oil composition of ajowan (<Emphasis Type="Italic">Trachyspermum ammi</Emphasis> L.)

Cyclophilins (CYPs) belong to the immunophilin superfamily, having the peptidyl prolyl cis/trans isomerase activity that can catalyze the cis/trans isomerisation process of proline residues. Previous studies have shown their importance in plants, but no comprehensive analysis of maize CYP family has been reported. In the present study, a whole-genome-wide analysis of maize CYP family was performed and 39 ZmCYP genes (ZmCYP1 to ZmCYP39) were identified from maize genome, which were unequally distributed on maize ten chromosomes. Phylogenetic analysis revealed a weak relationship among these ZmCYP genes. Furthermore, their gene structure and motif patterns also displayed variant within the gene family. Four segmental and one tandem duplicated gene pairs were found from 39 ZmCYP genes, respectively, indicating their roles in the expansion of maize CYP family. Expression analysis of 39 ZmCYP genes in maize tissues showed their differential tissue specific expression patterns. Quantitative real-time PCR analysis of 19 selected ZmCYP genes under salinity stress indicated their stress-inducible expression profile. Heterologous expression of ZmCYP15 in E. coli enhanced tolerance against abiotic stress. Subcellular localization analysis indicated ZmCYP15 was located in nucleus and cytoplasm. Our study describes the importance of the maize CYP gene family in stress response, and provides a reference for future study and application for maize genetic improvement.