Numerous regulatory genes participate in plant thermotolerance. In Arabidopsis, HEAT-INDUCED TAS1 TARGET2 (HTT2) is an important thermotolerance gene that is silenced by ta-siR255, a trans-acting siRNA. ta-siR255 is absent from heading Chinese cabbage (Brassica rapa ssp. pekinensis). Our previous attempt to overexpress the endogenous BrpHTT2 gene of heading Chinese cabbage (B. rapa ssp. pekinensis) failed because of cosuppression. In theory, heading Chinese cabbage can overexpress Arabidopsis HTT2 to improve thermotolerance in the absence of ta-siR255-mediated gene silencing and the weak potential of coexpression. To test the potential application of HTT2 in improving crop thermotolerance, we transferred p35S::HTT2 to heading Chinese cabbage. We tested the leaf electrical conductivity, hypocotyl elongation, and survival percentage of p35S::HTT2 plants subjected to high-temperature (38 °C) and heat-shock (46 °C) treatment. The leaf electrical conductivity of p35S::HTT2 seedlings under high temperature decreased but did negligibly change under heat shock. The hypocotyl length of p35S::HTT2 seedlings increased under high temperature and heat shock. The survival rate of p35S::HTT2 seedlings increased under heat shock. BrpHsfs, a subset of heat-shock factor genes, were upregulated in p35S::HTT2 plants under high-temperature and heat shock conditions. In the field, transgenic plants with HTT2 appeared greener and formed leafy heads earlier than wild-type plants. Exogenous HTT2 increased the survival rates of heat-shocked heading Chinese cabbage by promoting thermotolerance through decreasing electrical conductivity and extending hypocotyl length. Our work provides a new approach to the genetic manipulation of thermotolerance in crops through the introduction of exogenous thermotolerance genes.

Background: Brassica napus is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been used successfully for rapeseed hybrid production in China. MicroRNAs (miRNAs) play crucial regulatory roles in various plant growth, development, and stress response processes. However, reports on miRNAs that regulate the pollen development of GMS lines in B. napus are few. Results: In this study, 12 small RNA and transcriptome libraries were constructed and sequenced for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines, namely, “6251AB” and “6284AB”. At the same time, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two dominant GMS (DGMS) lines, namely, “4001AB” and “4006AB”. Based on the results, 46 known miRNAs, 27 novel miRNAs on the other arm of known pre-miRNAs, and 44 new conserved miRNAs were identified. Thirty-five pairs of novel miRNA-3p/miRNA-5p were found. Among all the identified miRNAs, fifteen differentially expressed miRNAs with over 1.5-fold change between flower buds of sterile and fertile lines were identified, including six differentially expressed miRNAs between “4001A” and “4001B”, two differentially expressed miRNAs between “4006A” and “4006B”, four differentially expressed miRNAs between “6251A” and “6251B”, and ten differentially expressed miRNAs between “6284A” and “6284B”. The correlation analysis of small RNA and transcriptome sequencing was conducted. And 257 candidate target genes were predicted for the 15 differentially expressed miRNAs. The results of 5′ modified RACE indicated that BnaA09g48720D, BnaA09g11120D, and BnaCnng51960D were cleaved by bna-miR398a-3p, bna-miR158-3p and bna-miR159a, respectively. Among the differentially expressed miRNAs, miR159 was chosen to analyze its function. Overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate the fertility and silique development in rapeseed. Conclusions: Our findings provide an overview of miRNAs that are potentially involved in GMS and pollen development. New information on miRNAs and their related target genes are provided to exploit the GMS mechanism and reveal the miRNA networks in B. napus.

Rapeseed is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been widely used successfully for rapeseed hybrid production. The physiological and molecular mechanism of pollen development in GMS lines of rapeseed (Brassica napus L.) need to be determined for the creation of hybrids and cultivation of new varieties. However, limited studies have focused on systematically mining genes that regulate the pollen development of GMS lines in B. napus. In the present study, to determine the stage at which pollen development begins to show abnormality in the GMS lines, we performed semi-thin section analysis of the anthers with five pollen development stages. The results indicated that the abnormal pollen development in DGMS lines might start at the meiotic stage, and abnormal pollen development in RGMS lines probably occurred before the tetrad stage. To investigate the critical genes and pathways involved in pollen development in GMS lines, we constructed and sequenced 24 transcriptome libraries for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines (6251AB and 6284AB) and two dominant GMS (DGMS) lines (4001AB and 4006AB). A total of 23,554 redundant DEGs with over two-fold change between sterile and fertile lines were obtained. A total of 346 DEGs were specifically related to DGMS, while 1,553 DEGs were specifically related to RGMS. A total of 1,545 DEGs were shared between DGMS and RGMS. And 253 transcription factors were found to be differentially expressed between the sterile and fertile lines of GMS. In addition, 6,099 DEGs possibly related to anther, pollen, and microspore development processes were identified. Many of these genes have been reported to be involved in anther and microspore developmental processes. Several DEGs were speculated to be key genes involved in the regulation of fertility. Three differentially expressed genes were randomly selected and their expression levels were verified by quantitative PCR (qRT-PCR). The results of qRT-PCR largely agreed with the transcriptome sequencing results. Our findings provide a global view of genes that are potentially involved in GMS occurrence. The expression profiles and function analysis of these DEGs were provided to expand our understanding of the complex molecular mechanism in pollen and sterility development in B. napus.

MicroRNAs (miRNAs) and their target genes play vital roles in crops. However, the genetic variations in miRNA-targeted sites that affect miRNA cleavage efficiency and their correlations with agronomic traits in crops remain unexplored. On the basis of a genome-wide DNA re-sequencing of 210 elite rapeseed (Brassica napus) accessions, we identified the single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) in miRNA-targeted sites complementary to miRNAs. Variant calling revealed 7.14 million SNPs and 2.89 million INDELs throughout the genomes of 210 rapeseed accessions. Furthermore, we detected 330 SNPs and 79 INDELs in 357 miRNA target sites, of which 33.50% were rare variants. We also analyzed the correlation between the genetic variations in miRNA target sites and 12 rapeseed agronomic traits. Eleven SNPs in miRNA target sites were significantly correlated with phenotypes in three consecutive years. More specifically, three correlated SNPs within the miRNA-binding regions of BnSPL9-3, BnSPL13-2, and BnCUC1-2 were in the loci associated with the branch angle, seed weight, and silique number, respectively; expression profiling suggested that the variation at these 3 miRNA target sites significantly affected the expression level of the corresponding target genes. Taken together, the results of this study provide researchers and breeders with a global view of the genetic variations in miRNA-targeted sites in rapeseed and reveal the potential effects of these genetic variations on elite agronomic traits.

Plant cuticular wax is the covering of the outer layer of the plant. It forms a protective barrier on the epidermis of plants and plays a vital role like a safeguard from abiotic and biotic stresses. In the present study, Brassica napus L. materials with and without wax powder were observed. Genetic analysis showed that the separation ratio of waxy plants to waxless plants was 15:1 in the F₂ population, which indicated that the wax powder formation was controlled by two pairs of genes. In order to identify the candidate genes associated with the wax powder trait of B. napus L., bulked segregant analysis (BSA) was performed. The homozygous waxy plants, the homozygous waxless plants, and plants from three parents were selected for establishing five DNA pools for genome-wide resequencing. The results of the resequencing showed that the site associated with wax powder trait was located in the region of 590,663–1,657,546 bp on chromosome A08. And 48 single nucleotide polymorphisms (SNPs) were found between the DNA sequences of waxy plants and waxless plants in this region. These SNPs were distributed across 16 gene loci. qRT-PCR analysis was conducted for the 16 candidate genes and three genes (BnaA08g01070D, BnaA08g02130D, and BnaA08g00890D) showed significantly differential expression between waxy and waxless parents. BnaA08g01070D and BnaA08g02130D were significantly down-regulated in the waxless parent, while BnaA08g00890D was significantly up-regulated in the waxless parent. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the BnaA08g02130D gene was enriched in lipid biosynthetic or metabolic processes. All the results in our study would provide valuable clues for exploring the genes involved in wax powder development.

Background: Brassica napus is the third leading source of edible oil in the world. Genic male sterility (GMS) lines provide crucial material for harnessing heterosis for rapeseed. GMS lines have been used successfully for rapeseed hybrid production in China. MicroRNAs (miRNAs) play crucial regulatory roles in various plant growth, development, and stress response processes. However, reports on miRNAs that regulate the pollen development of GMS lines in B. napus are few.Results: In this study, 12 small RNA and transcriptome libraries were constructed and sequenced for the flower buds from the fertile and sterile lines of two recessive GMS (RGMS) lines, namely, “6251AB” and “6284AB”. At the same time, 12 small RNA and transcriptome libraries were also constructed and sequenced for the flower buds from the fertile and sterile lines of two dominant GMS (DGMS) lines, namely, “4001AB” and “4006AB”. Based on the results, 46 known miRNAs, 27 novel miRNAs on the other arm of known pre-miRNAs, and 44 new conserved miRNAs were identified. Thirty-five pairs of novel miRNA-3p/miRNA-5p were found. Among all the identified miRNAs, 21 differentially expressed miRNAs with over 1.5-fold change between flower buds of sterile and fertile lines were identified, including six differentially expressed miRNAs between “4001A” and “4001B”, two differentially expressed miRNAs between “4006A” and “4006B”, six differentially expressed miRNAs between “6251A” and “6251B”, and 14 differentially expressed miRNAs between “6284A” and “6284B”. The correlation analysis of small RNA and transcriptome sequencing was conducted. The analysis results indicated that 360 genes were predicted to be the candidate targets of 21 differentially expressed miRNAs. BnaA09g48720D, BnaA09g11120D, and BnaCnng51960D were demonstrated to be cleaved by bna-miR398a-3p, bna-miR158-3p and bna-miR159a, respectively. Among the differentially expressed miRNAs, miR159 was chosen to analyze its function in reproduction development and male sterility. Overexpression of bna-miR159 in Arabidopsis resulted in decreased seed setting rate, and shortened siliques, illustrating that miR159 may regulate the fertility and silique development in rapeseed.Conclusions: Our findings provide an overview of miRNAs that are potentially involved in GMS and pollen development. New information on miRNAs and their related target genes are provided to exploit the GMS mechanism and reveal the miRNA networks in B. napus.