Integrative Physiological and Transcriptome Analysis Reveals the Mechanism of Cd Tolerance in Sinapis alba.

Recently, pollution caused by the heavy metal Cd has seriously affected the environment and agricultural crops. While Sinapis alba is known for its edible and medicinal value, its tolerance to Cd and molecular response mechanism remain unknown. This study aimed to analyze the tolerance of S. alba to Cd and investigate its molecular response mechanism through transcriptomic and physiological indicators. To achieve this, S. alba seedlings were treated with different concentrations of CdCl2 (0.25 mmol/L, 0.5 mmol/L, and 1.0 mmol/L) for three days. Based on seedling performance, S. alba exhibited some tolerance to a low concentration of Cd stress (0.25 mmol/L CdCl2) and a strong Cd accumulation ability in its roots. The activities and contents of several antioxidant enzymes generally exhibited an increase under the treatment of 0.25 mmol/L CdCl2 but decreased under the treatment of higher CdCl2 concentrations. In particular, the proline (Pro) content was extremely elevated under the 0.25 and 0.5 mmol/L CdCl2 treatments but sharply declined under the 1.0 mmol/L CdCl2 treatment, suggesting that Pro is involved in the tolerance of S. alba to low concentration of Cd stress. In addition, RNA sequencing was utilized to analyze the gene expression profiles of S. alba exposed to Cd (under the treatment of 0.25 mmol/L CdCl2). The results indicate that roots were more susceptible to disturbance from Cd stress, as evidenced by the detection of 542 differentially expressed genes (DEGs) in roots compared to only 37 DEGs in leaves. GO and KEGG analyses found that the DEGs induced by Cd stress were primarily enriched in metabolic pathways, plant hormone signal transduction, and the biosynthesis of secondary metabolites. The key pathway hub genes were mainly associated with intracellular ion transport and cell wall synthesis. These findings suggest that S. alba is tolerant to a degree of Cd stress, but is also susceptible to the toxic effects of Cd. Furthermore, these results provide a theoretical basis for understanding Cd tolerance in S. alba.

Identification and Genome-Wide Gene Expression Perturbation of a Trisomy in Chinese Kale (Brassica oleracea var. alboglabra).

Trisomy harbouring an extra copy of the chromosome generally causes a variety of physical and intellectual disabilities in mammals but is an extremely rare and important genetic stock in plants. In this study, a spontaneous trisomy plant in a Chinese kale accession (Brassica oleracea var. alboglabra, CC, 2n = 18) that showed significantly smaller plant architecture when compared to other normal plants was found and subsequently confirmed by cytological analysis in which the chromosome set of 2n = 19 and abnormal chromosome behaviour were observed. Then, based on the gene expression deviation determined by RNA-seq, the extra chromosome copy in this trisomy was identified as chromosome C2 (TC2). Compared to normal plants, TC2 not only showed generally upregulated differentially expressed genes (DEGs) on chromosome C2 (97.21% of 573 DEGs in chromosome C2) but also exhibited a whole-genome expression perturbation, in which 1329 DEGs (69.87% of total DEGs) were observed along two-copy chromosomes (trans-effect). The genes in the high (gene expression value > 100) and medium (100 > gene expression value > 10) groups were more prone to decreased gene expression, but the genes in the low group (10 > gene expression value > 0.1) showed upregulated expression deviation. In addition, GO (Gene ontology) annotation analysis revealed that the upregulated DEGs in the trans-effect group were overrepresented by the genes involved in the response to stress category, while the downregulated DEGs in the trans-effect group were mostly enriched in pathways related to DNA synthesis. In conclusion, we think our results can provide important resources for genetic analysis in B. oleracea and show some novel insights for understanding trisomy plant biology.

Identification, Characterization, and Cytological Analysis of Several Unexpected Hybrids Derived from Reciprocal Crosses between Raphanobrassica and Its Diploid Parents.

Interspecific hybridization and accompanying backcross between crops and relatives have been recognized as a powerful method to broaden genetic diversity and transfer desirable adaptive traits. Crosses between radish (Raphanus sativus, RR, 2n = 18) and Brassica oleracea (CC, 2n = 18), which formed allotetraploid Raphanobrassica (RRCC, 2n = 36), initiated the construction of resynthetic allopolyploids. However, these progenies from the backcrosses between Raphanobrassica and the two diploid parents have not been well deciphered. Herein, thousands of backcrosses using both Raphanobrassica and the two diploid parents as pollen donors were employed. Several hybrids with expected (2n = 27) and unexpected chromosome numbers (2n = 26 and 2n = 36) were obtained. Fluorescence in situ hybridization (FISH) analysis with R-genome-specific sequences as probes demonstrated that the genome structures of the two expected hybrids were RRC and CCR, and the genome structures of the three unexpected hybrids were RRRC, CCCR, and RRC' (harbouring an incomplete C genome). The unexpected hybrids with extra R or C genomes showed similar phenotypic characteristics to their expected hybrids. FISH analysis with C-genome-specific sequences as probes demonstrated that the unexpected allotetraploid hybrids exhibited significantly more intergenomic chromosome pairings than the expected hybrids. The expected and unexpected hybrids provide not only novel germplasm resources for the breeding of radish and B. oleracea but also very important genetic material for genome dosage analysis.

The complete chloroplast genome of a purple Ethiopian rape (Brassica carinata: Brassicaceae) from Guizhou Province, China and its phylogenetic analysis.

Brassica carinata A. Braun (Ethiopian rape), which was derived from the interspecific hybridization between B. nigra and B. oleracea, is used as both an oilseed and a leafy vegetable. The complete chloroplast (cp) genome of a purple B. carinata was obtained. This cp genome has a typical quadripartite structure and is 153,641 bp in length. The GC content of the cp genome is 36.39%. A total of 113 genes were predicted on this cp genome, including 79 protein coding, 4 rRNA, and 30 tRNA genes. Among these genes, 18 genes were duplicated (7 tRNAs, 4 rRNAs, and 7 protein coding genes). Sixty-eight SSR loci, including 11 compound SSRs, were identified in this cp genome by MISA. The phylogenetic tree analysis fully resolved B. carinata in a clade with B. nigra. This study provides important information for future evolution, genetic and molecular biology studies of B. carinata.

Complete chloroplast genome features and phylogenetic analysis of Eruca sativa (Brassicaceae).

Eruca sativa Mill. (Brassicaceae) is an important edible vegetable and a potential medicinal plant due to the antibacterial activity of its seed oil. Here, the complete chloroplast (cp) genome of E. sativa was de novo assembled with a combination of long PacBio reads and short Illumina reads. The E. sativa cp genome had a quadripartite structure that was 153,522 bp in size, consisting of one large single-copy region of 83,320 bp and one small single-copy region of 17,786 bp which were separated by two inverted repeat (IRa and IRb) regions of 26,208 bp. This complete cp genome harbored 113 unique genes: 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. Forty-nine long repetitive sequences and 69 simple sequence repeats were identified in the E. sativa cp genome. A codon usage analysis of the E. sativa cp genome showed a bias toward codons ending in A/T. The E. sativa cp genome was similar in size, gene composition, and linearity of the structural region when compared with other Brassicaceae cp genomes. Moreover, the analysis of the synonymous (Ks) and non-synonymous (Ka) substitution rates demonstrated that protein-coding genes generally underwent purifying selection pressure, expect ycf1, ycf2, and rps12. A phylogenetic analysis determined that E. sativa is evolutionarily close to important Brassica species, indicating that it may be possible to transfer favorable E. sativa alleles into other Brassica species. Our results will be helpful to advance genetic improvement and breeding of E. sativa, and will provide valuable information for utilizing E. sativa as an important resource to improve other Brassica species.

Chloroplast genome features of an important medicinal and edible plant: Houttuynia cordata (Saururaceae).

Houttuynia cordata (Saururaceae), an ancient and relic species, has been used as an important medicinal and edible plant in most parts of Asia. However, because of the lack of genome information and reliable molecular markers, studies on its population structure, or phylogenetic relationships with other related species are still rare. Here, we de novo assembled the complete chloroplast (cp) genome of H. cordata using the integration of the long PacBio and short Illumina reads. The cp genome of H. cordata showed a typical quadripartite cycle of 160,226 bp. This included a pair of inverted repeats (IRa and IRb) of 26,853 bp, separated by a large single-copy (LSC) region of 88,180 bp and a small single-copy (SSC) region of 18,340 bp. A total of 112 unique genes, including 79 protein-coding genes, 29 tRNA genes, and four rRNA genes, were identified in this cp genome. Eighty-one genes were located on the LSC region, 13 genes were located on the SSC region, and 17 two-copy genes were located on the IR region. Additionally, 48 repeat sequences and 86 SSR loci, which can be used as genomic markers for population structure analysis, were also detected. Phylogenetic analysis using 21 cp genomes of the Piperales family demonstrated that H. cordata had a close relationship with the species within the Aristolochia genus. Moreover, the results of mVISTA analysis and comparisons of IR regions demonstrated that the cp genome of H. cordata was conserved with that of the Aristolochia species. Our results provide valuable information for analyzing the genetic diversity and population structure of H. cordata, which can contribute to further its genetic improvement and breeding.

The complete chloroplast genome sequence of garden cress (Lepidium sativum L.) and its phylogenetic analysis in Brassicaceae family.

Garden cress, Lepidium sativum L., is not only an important vegetable which is cultivated in the entire world, but also a widely used folk medicine for the treatment of hyperactive airways disorders. However, as a member of Brassicaceae, few studies have been carried out on its phylogenetic relationship with other Brassicaceae members. Herein, the complete chloroplast (cp) genome of garden cress wa deciphered by the combination of Illumina Hiseq and PacBio Hiseq Platform after extracting of its cp DNA. The cp genome showed a typically quadripartite cycle with 154997 bp, including a pair of inverted repeats (IRa and IRb) of 26491 bp intersected by a large single copy (LSC) region of 84007 bp and a small single copy (SSC) region of 18008 bp. Totally, 128 unique genes were assembled in this cp genome, including 83 protein genes, 37 tRNAs and 8 rRNAs. A total of 73 simple sequence repeats (SSRs) with a length of at least 10 bp were detected. Phylogenetic analysis based on 30 cp genome of Brassicaceae family showed that the L. sativum was closely related to L. virginicum. This study provides important information for future evolution, genetic and molecular biology studies of L. sativum.

High-throughput multiplex cpDNA resequencing clarifies the genetic diversity and genetic relationships among Brassica napus, Brassica rapa and Brassica oleracea.

Brassica napus (rapeseed) is a recent allotetraploid plant and the second most important oilseed crop worldwide. The origin of B. napus and the genetic relationships with its diploid ancestor species remain largely unresolved. Here, chloroplast DNA (cpDNA) from 488 B. napus accessions of global origin, 139 B. rapa accessions and 49 B. oleracea accessions were populationally resequenced using Illumina Solexa sequencing technologies. The intraspecific cpDNA variants and their allelic frequencies were called genomewide and further validated via EcoTILLING analyses of the rpo region. The cpDNA of the current global B. napus population comprises more than 400 variants (SNPs and short InDels) and maintains one predominant haplotype (Bncp1). Whole-genome resequencing of the cpDNA of Bncp1 haplotype eliminated its direct inheritance from any accession of the B. rapa or B. oleracea species. The distribution of the polymorphism information content (PIC) values for each variant demonstrated that B. napus has much lower cpDNA diversity than B. rapa; however, a vast majority of the wild and cultivated B. oleracea specimens appeared to share one same distinct cpDNA haplotype, in contrast to its wild C-genome relatives. This finding suggests that the cpDNA of the three Brassica species is well differentiated. The predominant B. napus cpDNA haplotype may have originated from uninvestigated relatives or from interactions between cpDNA mutations and natural/artificial selection during speciation and evolution. These exhaustive data on variation in cpDNA would provide fundamental data for research on cpDNA and chloroplasts.

Characterization of FAE1 in the zero erucic acid germplasm of Brassica rapa L.

The modification of erucic acid content in seeds is one of the major goals for quality breeding in oil-yielding Brassica species. However, few low erucic acid (LEA) resources are available, and novel LEA genetic resources are being sought. Fatty acid elongase 1 (FAE1) is the key gene that controls erucic acid synthesis. However, the mechanism for erucic acid synthesis in B. rapa lacks systematic study. Here, we isolated zero erucic acid lines from 1981 Chinese landraces of B. rapa and found that the formation of LEA is not attributable to variations in FAE1 coding sequences, as reported for B. napus, but may be attributable to the decrease in FAE1 expression. Moreover, the FAE1 promoter sequences of LEA and high erucic acid materials shared 95% similarity. Twenty-eight bases deletions (containing a 24-base AT-rich region) were identified approximately 1300 bp upstream from the FAE1 start codon in the LEA accessions. The genotype with the deletions co-segregated with the LEA trait in the segregating population. This study isolated an LEA B. rapa resource that can be exploited in Brassica cultivation. The promoter variations might modify the expression level of FAE1, and the results shed light on novel regulation mechanisms for erucic acid synthesis.