Comparative transcriptome and metabolome analysis revealed diversity in the response of resistant and susceptible rose (Rosa hybrida) varieties to Marssonina rosae.

Rose black spot disease caused by Marssonina rosae is among the most destructive diseases that affects the outdoor cultivation and production of roses; however, the molecular mechanisms underlying the defensive response of roses to M. rosae have not been clarified. To investigate the diversity of response to M. rosae in resistant and susceptible rose varieties, we performed transcriptome and metabolome analyses of resistant (KT) and susceptible (FG) rose varieties and identified differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) in response to M. rosae at different time points. In response to M. rosae, DEGs and DAMs were mainly upregulated compared to the control and transcription factors were concentrated in the WRKY and AP2/ERF families. Gene Ontology analysis showed that the DEGs of FG were mainly enriched in biological processes, such as the abscisic acid-activated signaling pathway, cell wall, and defense response, whereas the DEGs of KT were mainly enriched in Golgi-mediated vesicle transport processes. Kyoto Encyclopedia of Genes and Genomes analysis showed that the DEGs of both varieties were concentrated in plant-pathogen interactions, plant hormone signal transduction, and mitogen-activated protein kinase signaling pathways, with the greatest number of DEGs associated with brassinosteroid (BR) in the plant hormone signal transduction pathway. The reliability of the transcriptome results was verified by qRT-PCR. DAMs of KT were significantly enriched in the butanoate metabolism pathway, whereas DAMs of FG were significantly enriched in BR biosynthesis, glucosinolate biosynthesis, and tryptophan metabolism. Moreover, the DAMs in these pathways were significantly positively correlated with the DEGs. Disease symptoms were aggravated when FG leaves were inoculated with M. rosae after 24-epibrassinolide treatment, indicating that the response of FG to M. rosae involves the BR signaling pathway. Our results provide new insights into the molecular mechanisms underlying rose response to M. rosae and lay a theoretical foundation for formulating rose black spot prevention and control strategies and cultivating resistant varieties.

Genome-Wide Identification and Expression Analysis of the TIR-NBS-LRR Gene Family and Its Response to Fungal Disease in Rose (Rosa chinensis).

Roses, which are one of the world's most important ornamental plants, are often damaged by pathogens, resulting in serious economic losses. As a subclass of the disease resistance gene family of plant nucleotide-binding oligomerization domain (NOD)-like receptors, TIR-NBS-LRR (TNL) genes play a vital role in identifying pathogen effectors and activating defense responses. However, a systematic analysis of the TNL gene family is rarely reported in roses. Herein, 96 intact TNL genes were identified in Rosa chinensis. Their phylogenies, physicochemical characteristics, gene structures, conserved domains and motifs, promoter cis-elements, microRNA binding sites, and intra- and interspecific collinearity relationships were analyzed. An expression analysis using transcriptome data revealed that RcTNL genes were dominantly expressed in leaves. Some RcTNL genes responded to gibberellin, jasmonic acid, salicylic acid, Botrytis cinerea, Podosphaera pannosa, and Marssonina rosae (M. rosae); the RcTNL23 gene responded significantly to three hormones and three pathogens, and exhibited an upregulated expression. Furthermore, the black spot pathogen was identified as M. rosae. After inoculating rose leaves, an expression pattern analysis of the RcTNL genes suggested that they act during different periods of pathogen infection. The present study lays the foundations for an in-depth investigation of the TNL gene function and the mining of disease resistance genes in roses.

Transposon insertions within alleles of BnaFT.A2 are associated with seasonal crop type in rapeseed.

KEY MESSAGE: We identified two new transposon insertions within the promoter of BnaFT.A2 in addition to an existing 288 bp MITE within the second intron. Each insertion event corresponds to a distinct BnaFT.A2 haplotype and is closely associated with established crop seasonal ecotypes. Florigen, encoded by FLOWERING LOCUS T (FT), plays key roles not only as a flowering hormone, but also a universal growth factor affecting several aspects of plant architecture. In rapeseed, BnaFT.A2 has been revealed as one of the major loci associated with flowering time and different ecotypes. However, it is unclear how allelic variations of BnaFT.A2 affect its function in flowering time regulation and beyond. In this study, we confirmed an existing 288 bp miniature inverted-repeat transposable element (MITE) insertion within the second intron and identified two new insertions within the promoter of BnaFT.A2-a 3971 bp CACTA and a 1079 bp Helitron. Each insertion event corresponds to a distinct BnaFT.A2 haplotype and is closely associated with established crop seasonal ecotypes. These alleles have similar tissue-specific expression patterns but discrete transcriptional patterns tightly associated with rapeseed flowering time and ecotype. RNAi lines and mutants of BnaFT.A2 flowered significantly later than controls. Differentially expressed genes (DEGs), identified in transcriptomic profiling of seedling leaves from two loss-of-function mutants (Bnaft.a2-L1 and Bnaft.a2-L2) compared with controls, indicated significant enrichment for hormone metabolic genes and roles related to plant cell wall synthesis and photosynthesis. Plants with loss-of-function BnaFT.A2 had smaller leaves and lower net photosynthetic rate compared to controls. These findings not only further clarify the genetic basis of flowering time variation and ecotype formation in B. napus, but also provide an additional toolbox for genetic improvement of seasonal adaptation and production.

Brassica evolution of essential BnaFtsH1 genes involved in the PSII repair cycle and loss of FtsH5.

The PSII repair cycle is an important part of photosynthesis and is essential for high photosynthetic efficiency. The study of essential genes in Brassica napus provides significant potential for the improvement of gene editing technology and molecular breeding design. Previously, we identified a B. napus lethal mutant (7-521Y), which was controlled by two recessive genes (cyd1 and cyd2). BnaC06.FtsH1 was identified as a CYD1 target gene through functional verification. In the present study, we employed fine-mapping, genetic complementation, and CRISPR/Cas9 experiments to identify BnaA07.FtsH1 as the target gene of CYD2, functioning similarly to BnaC06.FtsH1. By analyzing CRISPR/Cas9 T1 generation plants of the Westar variety, we found that the copy number of FtsH1 was positively correlated with its biomass accumulation. Transcriptome analysis of cotyledons revealed differences in the expression of photosynthesis antenna and structural proteins between the mutant and complementary seedlings. Phylogenetic and chromosome linear analyses, based on 15 sequenced cruciferous species, revealed that Brassica alone had lost FtsH5 during evolution. This may be related to the fact that FtsH5 was located at the end of chromosome ABK8 in the ancestor species. Cloning and identification of BnaFtsH1s provide a deeper understanding of PSII repair cycle mechanisms and offer new insights for the improvement of photosynthetic efficiency and molecular breeding design in B. napus.

QTL Mapping and Diurnal Transcriptome Analysis Identify Candidate Genes Regulating Brassica napus Flowering Time.

Timely flowering is important for seed formation and maximization of rapeseed (Brassica napus) yield. Here, we performed flowering-time quantitative trait loci (QTL) mapping using a double haploid (DH) population grown in three environments to study the genetic architecture. Brassica 60 K Illumina Infinium™ single nucleotide polymorphism (SNP) array and simple sequence repeat (SSR) markers were used for genotyping of the DH population, and a high-density genetic linkage map was constructed. QTL analysis of flowering time from the three environments revealed five consensus QTLs, including two major QTLs. A major QTL located on chromosome A03 was detected specifically in the semi-winter rapeseed growing region, and the one on chromosome C08 was detected in all environments. Ribonucleic acid sequencing (RNA-seq) was performed on the parents' leaves at seven time-points in a day to determine differentially expressed genes (DEGs). The biological processes and pathways with significant enrichment of DEGs were obtained. The DEGs in the QTL intervals were analyzed, and four flowering time-related candidate genes were found. These results lay a foundation for the genetic regulation of rapeseed flowering time and create a rapeseed gene expression library for seven time-points in a day.

Fine Mapping and Identification of BnaC06.FtsH1, a Lethal Gene That Regulates the PSII Repair Cycle in Brassica napus.

Photosystem II (PSII) is an important component of the chloroplast. The PSII repair cycle is crucial for the relief of photoinhibition and may be advantageous when improving stress resistance and photosynthetic efficiency. Lethal genes are widely used in the efficiency detection and method improvement of gene editing. In the present study, we identified the naturally occurring lethal mutant 7-521Y with etiolated cotyledons in Brassica napus, controlled by double-recessive genes (named cyd1 and cyd2). By combining whole-genome resequencing and map-based cloning, CYD1 was fine-mapped to a 29 kb genomic region using 15,167 etiolated individuals. Through cosegregation analysis and functional verification of the transgene, BnaC06.FtsH1 was determined to be the target gene; it encodes an filamentation temperature sensitive protein H 1 (FtsH1) hydrolase that degrades damaged PSII D1 in Arabidopsis thaliana. The expression of BnaC06.FtsH1 was high in the cotyledons, leaves, and flowers of B. napus, and localized in the chloroplasts. In addition, the expression of EngA (upstream regulation gene of FtsH) increased and D1 decreased in 7-521Y. Double mutants of FtsH1 and FtsH5 were lethal in A. thaliana. Through phylogenetic analysis, the loss of FtsH5 was identified in Brassica, and the remaining FtsH1 was required for PSII repair cycle. CYD2 may be a homologous gene of FtsH1 on chromosome A07 of B. napus. Our study provides new insights into lethal mutants, the findings may help improve the efficiency of the PSII repair cycle and biomass accumulation in oilseed rape.

A CACTA-like transposable element in the upstream region of BnaA9.CYP78A9 acts as an enhancer to increase silique length and seed weight in rapeseed.

Rapeseed (Brassica napus L.) is a model plant for polyploid crop research and the second-leading source of vegetable oil worldwide. Silique length (SL) and seed weight are two important yield-influencing traits in rapeseed. Using map-based cloning, we isolated qSLWA9, which encodes a P450 monooxygenase (BnaA9.CYP78A9) and functions as a positive regulator of SL. The expression level of BnaA9.CYP78A9 in silique valves of the long-silique variety is much higher than that in the regular-silique variety, which results in elongated cells and a prolonged phase of silique elongation. Plants of the long-silique variety and transgenic plants with high expression of BnaA9.CYP78A9 had a higher concentration of auxin in the developing silique; this induced a number of auxin-related genes but no genes in well-known auxin biosynthesis pathways, suggesting that BnaA9.CYP78A9 may influence auxin concentration by affecting auxin metabolism or an unknown auxin biosynthesis pathway. A 3.7-kb CACTA-like transposable element (TE) inserted in the 3.9-kb upstream regulatory sequence of BnaA9.CYP78A9 elevates the expression level, suggesting that the CACTA-like TE acts as an enhancer to stimulate high gene expression and silique elongation. Marker and sequence analysis revealed that the TE in B. napus had recently been introgressed from Brassica rapa by interspecific hybridization. The insertion of the TE is consistently associated with long siliques and large seeds in both B. napus and B. rapa collections. However, the frequency of the CACTA-like TE in rapeseed varieties is still very low, suggesting that this allele has not been widely used in rapeseed breeding programs and would be invaluable for yield improvement in rapeseed breeding.

An auxin signaling gene BnaA3.IAA7 contributes to improved plant architecture and yield heterosis in rapeseed.

Plant architecture is the key factor affecting overall yield in many crops. The genetic basis underlying plant architecture in rapeseed (Brassica napus), a key global oil crop, is elusive. We characterized an ethyl methanesulfonate (EMS)-mutagenized rapeseed mutant, sca, which had multiple phenotypic alterations, including crinkled leaves, semi-dwarf stature, narrow branch angles and upward-standing siliques. We identified the underlying gene, which encodes an Aux/IAA protein (BnaA3.IAA7). A G-to-A mutation changed the glycine at the 84th position to glutamic acid (G84E), disrupting the conserved degron motif GWPPV and reducing the affinity between BnaA3.IAA7 and TIR1 (TRANSPORT INHIBITOR RESPONSE 1) in an auxin dosage-dependent manner. This change repressed the degradation of BnaA3.IAA7 and therefore repressed auxin signaling at low levels of auxin that reduced the length of internodes. The G84E mutation reduced branch angles by enhancing the gravitropic response. The heterozygote +/sca closely resembled a proposed ideal plant architecture, displaying strong yield heterosis through single-locus overdominance by improving multiple component traits. Our findings demonstrate that a weak gain-of-function mutation in BnaA3.IAA7 contributes to yield heterosis by improving plant architecture and would be valuable for breeding superior rapeseed hybrid cultivars and such a mutation may increase the yield in other Brassica crops.