Identification of long noncoding RNAs involved in resistance to downy mildew in Chinese cabbage

Brassica downy mildew,a severe disease caused by Hyaloperonospora brassicae,can cause enormous economic losses in Chinese cabbage(Brassica rapa L.ssp.pekinensis)production.Although some research has been reported recently concerning the underlying resistance to this disease,no studies have identified or characterized long noncoding RNAs involved in this defense response.In this study,using high-throughput RNA sequencing,we analyzed the disease-responding mRNAs and long noncoding RNAs in two resistant lines(T12–19 and 12–85)and one susceptible line(91–112).Clustering and Gene Ontology analysis of differentially expressed genes(DEGs)showed that more DEGs were involved in the defense response in the two resistant lines than in the susceptible line.Different expression patterns and proposed functions of differentially expressed long noncoding RNAs among T12–19,12–85,and 91–112 indicated that each has a distinct disease response mechanism.There were significantly more cis-and trans-functional long noncoding RNAs in the resistant lines than in the susceptible line,and the genes regulated by these RNAs mostly participated in the disease defense response.Furthermore,we identified a candidate resistance-related long noncoding RNA,MSTRG.19915,which is a long noncoding natural antisense transcript of a MAPK gene,BrMAPK15.Via an agroinfiltration-mediated transient overexpression system and virus-induced gene silencing technology,BrMAPK15 was indicated to have a greater ability to defend against pathogens.MSTRG.19915-silenced seedlings showed enhanced resistance to downy mildew,probably because of the upregulated expression of BrMAPK15.This research identified and characterized long noncoding RNAs involved in resistance to downy mildew,laying a foundation for future in-depth studies of disease resistance mechanisms in Chinese cabbage.

Genome-wide analysis of changes in miRNA and target gene expression reveals key roles in heterosis for chinese cabbage biomass

Heterosis is a complex phenomenon in which hybrids show better phenotypic characteristics than their parents do.Chinese cabbage(Brassica rapa L.spp.pekinensis)is a popular leafy crop species,hybrids of which are widely used in commercial production;however,the molecular basis of heterosis for biomass of Chinese cabbage is poorly understood.We characterized heterosis in a Chinese cabbage hybrid cultivar and its parental lines from the seedling stage to the heading stage;marked heterosis of leaf weight and biomass yield were observed.Small RNA sequencing revealed 63 and 50 differentially expressed microRNAs(DEMs)at the seedling and early-heading stages,respectively.The expression levels ofthe majority of miRNA clusters in the hybrid were lower than the mid-parent values(MPVs).Using degradome sequencing,we identi fied 1,819 miRNA target genes.Gene ontology(GO)analyses demonstrated that the target genes ofthe MPV-DEMs and low parental expression level dominance(ELD)miRNAs were signi ficantly enriched in leaf morphogenesis,leaf development,and leaf shaping.Transcriptome analysis revealed that the expression levels of photosynthesis and chlorophyll synthesis-related MPV-DEGs(differentially expressed genes)were signi ficantly different in the F_(1) hybrid compared to the parental lines,resulting in increased photosynthesis capacity and chlorophyll content in the former.Furthermore,expression of genes known to regulate leaf development was also observed at the seedling stage.Arabidopsis plants overexpressing BrGRF4.2 and bra-miR396 presented increased and decreased leaf sizes,respectively.These results provide new insight into the regulation of target genes and miRNA expression patterns in leaf size and heterosis for biomass of B.rapa.

The kinase OsSK41/OsGSK5 negatively regulates amylose content in rice endosperm by affecting the interaction between OsEBP89 and OsBP5

Amylose content(AC) is the main factor determining the palatability, viscosity, transparency, and digestibility of rice(Oryza sativa)grains. AC in rice grains is mainly controlled by different alleles of the Waxy(Wx) gene. The AP2/EREBP transcription factor OsEBP89 interacts with the MYC-like protein OsBP5 to synergistically regulate the expression of Wx.Here, we determined that the GLYCOGEN SYNTHASE KINASE 5(OsGSK5, also named SHAGGY-like kinase 41 [OsSK41]) inhibits the transcriptional activation activity of OsEBP89 in rice grains during amylose biosynthesis. The loss of OsSK41 function enhanced Wx expression and increased AC in rice grains. By contrast, the loss of function of OsEBP89 reduced Wx expression and decreased AC in rice grains. OsSK41 interacts with OsEBP89 and phosphorylates four of its sites(Thr-28,Thr-30, Ser-238, and Thr-257), which makes OsEBP89 unstable and attenuates its interaction with OsBP5. Wx promoter activity was relatively weak when regulated by the phosphomimicvariantOsEBP89E–OsBP5but relatively strong when regulated by the nonphosphorylatable variant OsEBP89A–OsBP5.Therefore, OsSK41-mediated phosphorylation of OsEBP89 represents an additional layer of complexity in the regulation of amylose biosynthesis during rice grain development. In addition, our findings provide four possible sites for regulating rice grain AC via precise gene editing.

A Novel QTL qTGW3 Encodes the GSK3/ SHAGGY-Like Kinase OsGSK5/OsSK41 that Interacts with OsARF4 to Negatively Regulate Grain Size and Weight in Rice

Grain size and shape are important determinants of grain weight and yield in rice. Here, we report a new major quantitative trait locus （QTL）, qTGW3, that controls grain size and weight in rice. This locus, qTGW3, encodes OsSK41 （also known as OsGSK5）, a member of the GLYCOGEN SYNTHASE KINASE 3/SHAGGY-like family. Rice near-isogenic lines carrying the loss-of-function allele of OsSK41 have increased grain length and weight. We demonstrate that OsSK41 interacts with and phosphorylates AUXIN RESPONSE FACTOR 4 （OsARF4）. Co-expression of OsSK41 with OsARF4 increases the accumulation of OsARF4 in rice protoplasts. Loss of function of OsARF4 results in larger rice grains. RNA-sequencing analysis suggests that OsARF4 and OsSK41 repress the expression of a common set of downstream genes, including some auxin-responsive genes, during rice grain development. The loss-of-function form of OsSK41 at qTGW3 represents a rare allele that has not been extensively utilized in rice breeding. Suppression of OsSK41 function by either targeted gene editing or QTL pyramiding enhances rice grain size and weight. Thus, our study reveals the important role of OsSK41 in rice grain development and provides new candidate genes for genetic improvement of grain yield in rice and perhaps in other cereal crops.

A Kelch Motif-Containing Serine/Threonine Protein Phosphatase Determines the Large Grain QTL Trait in Rice

A thorough understanding of the genetic basis of rice grain traits is critical for the improvement of rice （Oryza sativa L.） varieties. In this study, we generated an F2 population by crossing the large-grain japonica cultivar CW23 with Peiai 64 （PA64）, an elite indica small-grain cultivar. Using QTL analysis, 17 QTLs for five grain traits were detected on four different chromosomes. Eight of the QTLs were newly-identified in this study. In particular, qGL3-1, a newly-identified grain length QTL with the highest LOD value and largest phenotypic variation, was fine-mapped to the 17 kb region of chromosome 3. A serine/threonine protein phosphatase gene encoding a repeat domain containing two Kelch motifs was identified as the unique candidate gene corresponding to this QTL. A comparison of PA64 and CW23 sequences revealed a single nucleotide substitution （C→A） at position 1092 in exon 10, resulting in replacement of Asp （D） in PA64 with Glu （E） in CW23 for the 364th amino acid. This variation is located at the D position of the conserved sequence motif AVLDT of the Kelch repeat. Genetic analysis of a near-isogenic line （NIL） for qGL3-1 revealed that the allele qGL3-1 from CW23 has an additive or partly dominant effect, and is suitable for use in molecular marker-assisted selection.

A Genomic Variation Map Provides Insights into the Genetic Basis of Spring Chinese Cabbage (Brassica rapa ssp.pekinensis)Selection

Chinese cabbage is the most consumed leafy crop in East Asian countries.However,premature bolting induced by continuous low temperatures severely decreases the yield and quality of the Chinese cabbage, and therefore restricts its planting season and geographic distribution.In the past 40years,spring Chinese cabbage with strong winterness has been selected to meet the market demand.Here,we report a genome variation map of Chinese cabbage generated from the resequencing data of 194 geographically diverse accessions of three ecotypes.In-depth analyses of the selection sweeps and genome-wide patterns revealed that spring Chinese cabbage was selected from a specific population of autumn Chinese cabbage around the area of Shandong peninsula in northern China.We identified 23 genomic loci that underwent intensive selection,and further demonstrated by gene expression and haplotype analyses that the incorporation of elite alleles of VERNALISATION INSENTIVE 3.1(BrVIN3.1)and FLOWER LOCUS C 1(BrFLC1)is a determinant genetic source of variation during selection.Moreover,we showed that the quantitative response of BrVIN3.1 to cold due to the sequence variations in the cis elements of the BrVlN3.1 promoter significantly contributes to bolting-time variation in Chinese cabbage.Collectively, our study provides valuable insights into the genetic basis of spring Chinese cabbage selection and will facilitate the breeding of bolting-resistant Varieties by molecular-marker-assisted selection,transgenic or gene editingapproaches.

Genetic Analysis and Fine Mapping of a Novel Semidominant Dwarfing Gene LB4D in Rice

A dwarf mutant,designated LB4D,was obtained among the progeny of backcrosses to a wild rice introgression line.Genetic analysis of LB4D indicated that the dwarf phenotype was controlled by a single semidominant dwarfing gene,which was named LB4D.The mutants were categorized as dn-type dwarf mutants according to the pattern of internode reduction.In addition,gibberellin（GA） response tests showed that LB4D plants were neither deficient nor insensitive to GA.This study found that tiller formation by LB4D plants was decreased by 40%compared with the wild type,in contrast to other dominant dwarf mutants that have been identified,indicating that a different dwarfing mechanism might be involved in the LB4D dominant mutant.The reduction of plant height in F1 plants ranged from 27.9%to 38.1%in different genetic backgrounds,showing that LB4D exerted a stronger dominant dwarfing effect. Using large F2 and F3 populations derived from a cross between heterozygous LB4D and the japonica cultivar Nipponbare,the LB4D gene was localized to a 46 kb region between the markers Indel 4 and Indel G on the short arm of chromosome 11,and four predicted genes were identified as candidates in the target region.