Synergistic effects of carbon cycle metabolism and photosynthesis in Chinese cabbage under salt stress

Chinese cabbage(Brassica rapa ssp. pekinensis) has a long cultivation history and is one of the vegetable crops with the largest cultivation area in China. However, salt stress severely damages photosynthesis and hormone metabolism, nutritional balances, and results in ion toxicity in plants. To better understand the mechanisms of salt-induced growth inhibition in Chinese cabbage, RNA-seq and physiological index determination were conducted to explore the impacts of salt stress on carbon cycle metabolism and photosynthesis in Chinese cabbage. Here, we found that the number of thylakoids and grana lamellae and the content of starch granules and chlorophyll in the leaves of Chinese cabbage under salt stress showed a time-dependent response, first increasing and then decreasing. Chinese cabbage increased the transcript levels of genes related to the photosynthetic apparatus and carbon metabolism under salt stress, probably in an attempt to alleviate damage to the photosynthetic system and enhance CO_(2) fixation and energy metabolism. The transcription of genes related to starch and sucrose synthesis and degradation were also enhanced;this might have been an attempt to maintain intracellular osmotic pressure by increasing soluble sugar concentrations. Soluble sugars could also be used as potential reactive oxygen species(ROS) scavengers, in concert with peroxidase(POD)enzymes, to eliminate ROS that accumulate during metabolic processes. Our study characterizes the synergistic response network of carbon metabolism and photosynthesis under salt stress.

Molecular mechanism of flowering time regulation in Brassica rapa:similarities and differences with Arabidopsis

Properly regulated flowering time is pivotal for successful plant reproduction.The floral transition from vegetative growth to reproductive growth is regulated by a complex gene regulatory network that integrates environmental signals and internal conditions to ensure that flowering takes place under favorable conditions.Brassica rapa is a diploid Cruciferae species that includes several varieties that are cultivated as vegetable or oil crops.Flowering time is one of the most important agricultural traits of B.rapa crops because of its influence on yield and quality.The transition to flowering in B.rapa is regulated by several environmental and developmental cues,which are perceived by several signaling pathways,including the vernalization pathway,the autonomous pathway,the circadian clock,the thermosensory pathway,and gibberellin(GA)signaling.These signals are integrated to control the expression of floral integrators BrFTs and BrSOC1s to regulate flowering.In this review,we summarized current research advances on the molecular mechanisms that govern flowering time regulation in B.rapa and compare this to what is known in Arabidopsis.

Hormones and carbohydrates synergistically regulate the formation of swollen roots in a Chinese cabbage translocation line

The genus Brassica contains a rich diversity of species and morphological types,including leaf,root,and oil crops,all of which show substantial phenotypic variation.Both Chinese cabbage and cabbage are typical leaf-type crops with normal roots.We created translocation lines based on interspecific crosses between Chinese cabbage and cabbage and identified qdh225,which exhibited a swollen-root phenotype.The swollen root of qdh225 contained a large number of granular substances,and the formation of its irregular morphological tissue was caused by a thickening of the phloem.Transcriptomic and metabolomic data suggested that differential expression of genes encoding nine types of enzymes involved in starch and sucrose metabolism caused changes in starch synthesis and degradation in the swollen root.These genes jointly regulated sucrose and starch levels,leading to significant enrichment of starch and soluble proteins in the swollen root and a reduction in the content of soluble sugars such as d-glucose and trehalose 6-phosphate.A significant increase in auxin(IAA)and abscisic acid(ABA)contents and a decrease in gibberellin(GA)content in the swollen root likely promoted the differential expression of genes associated with hormone signal transduction,thereby regulating the development of the swollen root.Taken together,our data suggest that accumulation of IAA and ABA and reduction in GA promote swollen root formation by regulating hormone-mediated signaling,leading to a thickening of phloem,root enlargement,and substantial accumulation of starch and soluble proteins.The latter provide materials,energy,and nutrient sources for the development of swollen roots.

Molecular and genetic regulations of fleshy fruit shape and lessons from Arabidopsis and rice

Fleshy fruit shape is an important external quality trait influencing the usage of fruits and consumer preference.Thus,modification of fruit shape has become one of the major objectives for crop improvement.However,the underlying mechanisms of fruit shape regulation are poorly understood.In this review we summarize recent progress in the genetic basis of fleshy fruit shape regulation using tomato,cucumber,and peach as examples.Comparative analyses suggest that the OFP-TRM(OVATE Family Protein-TONNEAU1 Recruiting Motif)and IQD(IQ67 domain)pathways are probably conserved in regulating fruit shape by primarily modulating cell division patterns across fleshy fruit species.Interestingly,cucumber homologs of FRUITFULL(FUL1),CRABS CLAW(CRC)and 1-aminocyclopropane-1-carboxylate synthase 2(ACS2)were found to regulate fruit elongation.We also outline the recent progress in fruit shape regulation mediated by OFP-TRM and IQD pathways in Arabidopsis and rice,and propose that the OFP-TRM pathway and IQD pathway coordinate regulate fruit shape through integration of phytohormones,including brassinosteroids,gibberellic acids,and auxin,and microtubule organization.In addition,functional redundancy and divergence of the members of each of the OFP,TRM,and IQD families are also shown.This review provides a general overview of current knowledge in fruit shape regulation and discusses the possible mechanisms that need to be addressed in future studies.

Genome-wide development and utilization of Simple Sequence Repeats in Chinese cabbage(Brassica rapa L.ssp.pekinensis)

With the complementation of whole-genome sequencing of Chinese cabbage,it is necessary to develop genome-wide Simple Sequence Repeat(SSR)markers and analyse their characteristics,which will bring a revolution in the molecular marker-assisted breeding of Chinese cabbage.In this study distribution and characteristics of SSR loci in the genome of Chinese cabbage'Chiifu'was analyzed.Finally,a total of 173,892 SSR markers that occurred in 10 chromosomes were identified,and Perfect Repeat Motifs(P type)which is a main form of SSR loci with a mean distance of 1.89 kb/SSR was identified.Among them,mono-,di-,and tri-nucleotide repeat motifs were equally distributed across chromosomes compared with tetra-,penta-,and hexa-nucleotide repeat motifs.The largest proportion of SSR loci consisted of mono-and dinucleotide repeat motifs,which accounted for 93.04%of the total SSR loci.A/T and AT/TA were the most abundant motifs,accounting for 97.61%and 64.05%of the mono-and dinucleotide repeats,respectively.Furthermore,200 SSR markers were used for the amplification of 12 Chinese cabbage germplasm,in which 30 were highly polymorphic.These SSR markers were further used for the association analyses of flowering time of 190 Chinese cabbage accessions.The results revealed that 9 and 10 alleles were associated with bolting and flowering time,respectivley.The results show that development of SSR markers is feasible and useful in marker assisted selection of Chinese cabbage.

Gene characterization and molecular pathway analysis of reverse thermosensitive genic male sterility in eggplant(Solanum melongena L.)

The naturally occurring mutant eggplant line 05ms was identified with reverse thermosensitive genic male sterility(rTGMS),but its temperature-responsive fertility mechanisms remain largely unknown.Here,we studied the flower morphology,anther cellular structure,and genome-wide gene expression of this rTGMS line.Candidate genes for thermosensitive male sterility during the microspore development of 05ms and the temperature-insensitive line S63 under low-temperature(LT)and high-temperature(HT)conditions were identified.Under LT,tapetum cells were vacuolated and had delayed disintegration in 05ms.RNA-seq analysis indicated that DEGs were enriched in the KEGG pathways‘plant hormone signal transduction’,‘starch and sucrose metabolism’,and‘phenylpropanoid biosynthesis’.We identified two genes,4CLL1(Sme2.5_00368.1_g00010.1)and CKI1(Sme2.5_10056.1_g00002.1),which could potentially regulate eggplant anther development and may be candidate genes for rTGMS.Finally,we propose a working model of anther abortion for rTGMS in eggplant.CKI1 responds to LT stress and causes expression changes in genes related to anther development,such as 4CLL1,and the cellular structure of the tapetum becomes abnormal,causing male sterility.The findings of this study explain the underlying molecular mechanisms of male sterility in eggplant rTGMS lines.

What makes turnips:anatomy,physiology and transcriptome during early stages of its hypocotyl-tuber development

Brassica species are characterized by their tremendous intraspecific diversity,exemplified by leafy vegetables,oilseeds,and crops with enlarged inflorescences or above ground storage organs.In contrast to potato tubers that are edible storage organs storing energy as starch and are the vegetative propagation modules,the storage organs of turnips,grown from true seed,are swollen hypocotyls with varying degrees of root and stem that mainly store glucose and fructose.To highlight their anatomical origin,we use the term“hypocotyl-tuber”for these turnip vegetative storage organs.We combined cytological,physiological,genetic and transcriptomic approaches,aiming to identify the initial stages,molecular pathways and regulatory genes for hypocotyl-tuber induction in turnips(B.rapa subsp.rapa).We first studied the development of the hypocotyl zone of turnip and Pak choi and found that 16 days after sowing(DAS)morphological changes occurred in the xylem which indicated the early tuberization stage.Tissue culture experiments showed a clear effect of auxin on hypocotyl-tuber growth.Differentially expressed genes between 1 and 6 weeks after sowing in turnip hypocotyls,located in genomic regions involved in tuber initiation and/or tuber growth defined by QTL and selective sweeps for tuber formation,were identified as candidate genes that were studied in more detail for their role in hypocotyl-tuber formation.This included a Bra-FLOR1 paralogue with increased expression 16 DAS,when the hypocotyl starts swelling,suggesting dual roles for duplicated flowering time genes in flowering and hypocotyltuber induction.Bra-CYP735A2 was identified for its possible role in tuber growth via trans-zeatin.Weigthed Coexpression Network Analysis(WGCNA)identified 59 modules of co-expressed genes.Bra-FLOR1 and Bra-CYP735A2 were grouped in a module that included several genes involved in carbohydrate transport and metabolism,cell-wall growth,auxin regulation and secondary metabolism that serve as starting points to illuminate the transcriptional regulation of hypocotyl-tuber formation and development.

Genetic analysis of the “head top shape” quality trait of Chinese cabbage and its association with rosette leaf variation

The agricultural and consumer quality of Chinese cabbage is determined by its shape.The shape is defined by the folding of the heading leaves,which defines the head top shape(HTS).The overlapping HTS,in which the heading leaves curve inward and overlap at the top,is the shape preferred by consumers.To understand the genetic regulation of HTS,we generated a large segregating F 2 population from a cross between pak choi and Chinese cabbage,with phenotypes ranging from nonheading to heading with either outward curving or inward curving overlapping heading leaves.HTS was correlated with plant height,outer/rosette leaf length,and petiole length.A high-density genetic map was constructed.Quantitative trait locus(QTL)analysis resulted in the identification of 22 QTLs for leafy head-related traits,which included five HTS QTLs.Bulked segregant analysis(BSA)was used to confirm HTS QTLs and identify candidate genes based on informative single-nucleotide polymorphisms.Interestingly,the HTS QTLs colocalized with QTLs for plant height,outer/rosette leaf,and petiole length,consistent with the observed phenotypic correlations.Combined QTL analysis and BSA laid a foundation for molecular marker-assisted breeding of Chinese cabbage HTS and directions for further research on the genetic regulation of this trait.

Comparative transcriptome analysis reveals defense responses against soft rot in Chinese cabbage

Pectobacterium carotovorum ssp.carotovorum(Pcc)is a necrotrophic bacterial species that causes soft rot disease in Chinese cabbage.In this study,plants harboring the resistant mutant sr gene,which confers resistance against Pcc,were screened from an 800 M2 population mutated by ethyl methane sulfonate(EMS)and scored in vitro and in vivo for lesion size.The transcript profiles showed~512 differentially expressed genes(DEGs)between sr and WT plants occurring between 6 and 12 h postinoculation(hpi),which corresponded to the important defense regulation period(resistance)to Pcc in Chinese cabbage.The downstream defense genes(CPK,CML,RBOH MPK3,and MPK4)of pathogen pattern-triggered immunity(PTI)were strongly activated during infection at 12 hpi in resistant mutant sr;PTI appears to be central to plant defense against Pcc via recognition by three putative pattern recognition receptors(PRRs;BrLYM1-BrCERK1,BrBKK1/SERK4-PEPR1,BrWAKs).Pcc triggered the upregulation of the jasmonic acid(JA)and ethylene(ET)biosynthesis genes in mutant sr,but auxins and other hormones may have affected some negative signals.Endogenous hormones(auxins,JAs,and SA),as well as exogenous auxins(MEJA and BTH),were also verified as functioning in the immune system.Concurrently,the expression of glucosinolate and lignin biosynthesis genes was increased at 12 hpi in resistant mutant sr,and the accumulation of glucosinolate and lignin also indicated that these genes have a functional defensive role against Pcc.Our study provides valuable information and elucidates the resistance mechanism of Chinese cabbage against Pcc infection.

Genome-wide transcriptome analysis reveals molecular pathways involved in leafy head formation of Chinese cabbage(Brassica rapa)

Chinese cabbage plants go through seedling and rosette stages before forming their leafy head.Chinese cabbage plants resemble pak-choi plants at their seedling stage,but in their rosette stage the leaves of Chinese cabbage differentiate,as they increase in size with shorter petioles.In order to understand the molecular pathways that play a role in leafy head formation,transcript abundance of young emerging leaves was profiled during development of two Chinese cabbage genotypes and a single pak-choi genotype.The two Chinese cabbages differed in many aspects,among others earliness,leaf size and shape,leaf numbers,and leafy head shape.Genome-wide transcriptome analysis clearly separated the seedling stages of all three genotypes together with the later stages from pak-choi,from the later developmental stages of both Chinese cabbages(rosette,folding,and heading).Weighted correlation network analysis and hierarchical clustering using Euclidean distances resulted in gene clusters with transcript abundance patterns distinguishing the two Chinese cabbages from pak-choi.Three clusters included genes with transcript abundance affected by both genotype and developmental stage,whereas two clusters showed only genotype effects.This included a genotype by developmental stage cluster highly enriched with the MapMan category photosynthesis,with high expression during rosette and folding in Chinese cabbages and low expression in the heading inner leaves that are not exposed to light.The other clusters contained many genes in the MapMan categories Cell,showing again differences between pak-choi and both Chinese cabbages.We discuss how this relates to the differences in leaf blade growth between Chinese cabbage and pak-choi,especially at the rosette stage.Overall,comparison of the transcriptome between leaves of two very different Chinese cabbages with pak-choi during plant development allowed the identification of specific gene categories associated with leafy head formation.

Exogenous melatonin improves the chilling tolerance and preharvest fruit shelf life in eggplant by affecting ROS-and senescence-related processes

Low temperature is the most common abiotic stress factors during the eggplant cultivation in solar greenhouses.Melatonin plays important roles in plant resistance to low temperature.However,the role of melatonin in regulating chilling tolerance and extending the preharvest shelf life of eggplant fruits is still unknown.In this study,we investigated the effects of exogenous melatonin on eggplant plants and fruits in response to low temperature.Under simulated low-temperature conditions,exogenous melatonin significantly relieved the chilling symptoms of seedlings by reducing reactive oxygen species (ROS) and malondialdehyde (MDA) levels and relative leakage rates.These reductions were caused by higher superoxide dismutase (SOD) and catalase (CAT) activities and increased endogenous polyamine and melatonin levels compared with those in untreated seedlings.Notably,the expression levels of SOD,CAT1/2,and polyamine synthesis genes (ADC and ODC) were also increased by 100μmol·L~(-1)melatonin,as well as those of genes involved in melatonin synthesis (TDC,T5H,SNAT,ASMT,and COMT) and cold regulation (COR1,CBFa/b,and ZAT2/6/12).To further investigate the effects of melatonin on eggplant leaves and fruits under natural low temperature conditions,100μmol·L~(-1)melatonin was sprayed on the functional leaves at three days before commodity maturation.Melatonin significantly alleviated chilling injury in the leaves and pericarp and extended the preharvest shelf life of the fruit by increasing the expression of COR1,CBF,ZAT2/6/12,and API5 and decreasing the expression of senescence-related genes (NCED1/2 and SAG12).Therefore,100μmol·L~(-1)melatonin improved chilling tolerance and fruit shelf life by upregulating ZAT2/6/12 to affect ROS-and senescence-related processes,which provides a reference for alleviating cold stress and extending the preharvest fruit shelf life in eggplant.

Identiffcation of soft rot resistance loci in Brassica rapa with SNP markers

Soft rot caused by Pectobacterium carotovorum(Pc)is a devastating disease of Brassica rapa,causing substantial reductions in crop yield and quality.Identifying genes related to soft rot resistance is the key to solving this problem.To characterize soft rot resistance,we screened a soft rot-susceptible Chinese cabbage(A03),a resistant pakchoi(‘Huaguan’),and a resistant mutant(sr).An F_(2) population was generated by crossing susceptible Chinese cabbage A03and resistant pakchoi‘Huaguan’to identify quantitative trait loci(QTLs)that confer soft rot resistance.A high-density genetic map was constructed and the three QTLs identified contain 166 genes.Based on available transcriptome data,we analyzed the expression of the 166 genes during an important defense regulatory period in Pc infection in both A03and the resistant mutant sr.Among the 166 genes,six candidate genes were related to the soft rot defense response in B.rapa.TIFY10B(JAZ2,BraA07g038660.3C)was located in the major soft rot resistance QTL,DRQTL-3 on A07,and we speculate that this gene may play an important role in the defense mechanism against soft rot in B.rapa.This study lays the foundation for further investigations on the mechanism of soft rot resistance in B.rapa crops.

Genetic regulators of leaf size in Brassica crops

Leaf size influences plant development and biomass and is also an important agricultural trait in Brassica crops,in which leaves are the main organ produced for consumption.Leaf size is determined by the coordinated regulation of cell proliferation and cell expansion during leaf development,and these processes are strictly controlled by various integrated signals from the intrinsic regulatory network and the growth environment.Understanding the molecular mechanism of leaf size control is a prerequisite for molecular breeding for crop improvement purposes.Although research on leaf size control is just beginning in Brassica,recent studies have identified several genes and QTLs that are important in leaf size regulation.These genes have been proposed to influence leaf growth through different pathways and mechanisms,including phytohormone biosynthesis and signaling,transcription regulation,small RNAs,and others.In this review,we summarize the current findings regarding the genetic regulators of leaf size in Brassica and discuss future prospects for this research.

Fast tracking alien gene discovery by molecular markers in a late flowering Chinese cabbage-cabbage translocation line‘AT7–4'

Flowering time is an important agronomic trait of Chinese cabbage with late flowering being a primary breeding objective.In our previous work,we obtained Chinese cabbage-cabbage translocation lines that contained several beneficial cabbage genes.Cabbage-specific molecular markers show that these genes were coming from chromosome C01 of cabbage.In this study,we investigated the inheritance of flowering time in a couple of translocation lines and analyzed the transmission rate of molecular markers in the offspring.Consequently,we obtained the late flowering Chinese cabbage-cabbage translocation line‘AT7–4’in which the flowering time was later than that of‘85–1’by about 7 days under 4-week vernalization.Based on previous studies of the genomes of Chinese cabbage and cabbage,we located the cabbage-specific molecular markers that were closely linked at the top of the chromosome A01 in the F2mapping population generated by self-crossing F1s derived from a cross between the translocation line‘AT7–4’and Chinese cabbage‘14–36’.Five flowering-related genes in the alien fragment were found by functional annotation and their molecular markers were developed.This study lays the foundation for the future improvement of Chinese cabbage varieties using A-C translocation lines.

The application of CRISPR/Cas technologies to Brassica crops:current progress and future perspectives

Brassica species are a global source of nutrients and edible vegetable oil for humans.However,all commercially important Brassica crops underwent a whole-genome triplication event,hindering the development of functional genomics and breeding programs.Fortunately,clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated(Cas)technologies,by allowing multiplex and precise genome engineering,have become valuable genome-editing tools and opened up new avenues for biotechnology.Here,we review current progress in the use of CRISPR/Cas technologies with an emphasis on the latest breakthroughs in precise genome editing.We also summarize the application of CRISPR/Cas technologies to Brassica crops for trait improvements.Finally,we discuss the challenges and future directions of these technologies for comprehensive application in Brassica crops.Ongoing advancement in CRISPR/Cas technologies,in combination with other achievements,will play a significant role in the genetic improvement and molecular breeding of Brassica crops.

Fungal diversity notes 1387-1511:taxonomic and phylogenetic contributions on genera and species of fungal taxa

This article is the 13th contribution in the Fungal Diversity Notes series,wherein 125 taxa from four phyla,ten classes,31 orders,69 families,92 genera and three genera incertae sedis are treated,demonstrating worldwide and geographic distri-bution.Fungal taxa described and illustrated in the present study include three new genera,69 new species,one new com-bination,one reference specimen and 51 new records on new hosts and new geographical distributions.Three new genera,Cylindrotorula(Torulaceae),Scolecoleotia(Leotiales genus incertae sedis)and Xenovaginatispora(Lindomycetaceae)are introduced based on distinct phylogenetic lineages and unique morphologies.Newly described species are Aspergillus lan-naensis,Cercophora dulciaquae,Cladophialophora aquatica,Coprinellus punjabensis,Cortinarius alutarius,C.mammil-latus,C.quercoflocculosus,Coryneum fagi,Cruentomycena uttarakhandina,Cryptocoryneum rosae,Cyathus uniperidiolus,Cylindrotorula indica,Diaporthe chamaeropicola,Didymella azollae,Diplodia alanphillipsii,Dothiora coronicola,Efibula rodriguezarmasiae,Erysiphe salicicola,Fusarium queenslandicum,Geastrum gorgonicum,G.hansagiense,Helicosporium sexualis,Helminthosporium chiangraiensis,Hongkongmyces kokensis,Hydrophilomyces hydraenae,Hygrocybe boertmannii,Hyphoderma australosetigerum,Hyphodontia yunnanensis,Khaleijomyces umikazeana,Laboulbenia divisa,Laboulbenia triarthronis,Laccaria populina,Lactarius pallidozonarius,Lepidosphaeria strobelii,Longipedicellata megafusiformis,Lophiotrema lincangensis,Marasmius benghalensis,M.jinfoshanensis,M.subtropicus,Mariannaea camelliae,Mel-anographium smilaxii,Microbotryum polycnemoides,Mimeomyces digitatus,Minutisphaera thailandensis,Mortierella solitaria,Mucor harpali,Nigrograna jinghongensis,Odontia huanrenensis,O.parvispina,Paraconiothyrium ajrekarii,Par-afuscosporella niloticus,Phaeocytostroma yomensis,Phaeoisaria synnematicus,Phanerochaete hainanensis,Pleopunctum thailandicum,Pleurotheciella dimorphospora,Pseudochaetosphaeronema chiangraiense,Pseudodactylaria albicolonia,Rhexoacrodictys nigrospora,Russula paravioleipes,Scolecoleotia eriocamporesi,Seriascoma honghense,Synandromyces makranczyi,Thyridaria aureobrunnea,Torula lancangjiangensis,Tubeufia longihelicospora,Wicklowia fusiformispora,Xenovaginatispora phichaiensis and Xylaria apiospora.One new combination,Pseudobactrodesmium stilboideus is pro-posed.A reference specimen of Comoclathris permunda is designated.New host or distribution records are provided for Acrocalymma fici,Aliquandostipite khaoyaiensis,Camarosporidiella laburni,Canalisporium caribense,Chaetoscutula juniperi,Chlorophyllum demangei,C.globosum,C.hortense,Cladophialophora abundans,Dendryphion hydei,Diaporthe foeniculina,D.pseudophoenicicola,D.pyracanthae,Dictyosporium pandanicola,Dyfrolomyces distoseptatus,Ernakula-mia tanakae,Eutypa flavovirens,E.lata,Favolus septatus,Fusarium atrovinosum,F.clavum,Helicosporium luteosporum,Hermatomyces nabanheensis,Hermatomyces sphaericoides,Longipedicellata aquatica,Lophiostoma caudata,L.clematidis-vitalbae,Lophiotrema hydei,L.neoarundinaria,Marasmiellus palmivorus,Megacapitula villosa,Micropsalliota globocys-tis,M.gracilis,Montagnula thailandica,Neohelicosporium irregulare,N.parisporum,Paradictyoarthrinium diffractum,Phaeoisaria aquatica,Poaceascoma taiwanense,Saproamanita manicata,Spegazzinia camelliae,Submersispora variabi-lis,Thyronectria caudata,T.mackenziei,Tubeufia chiangmaiensis,T.roseohelicospora,Vaginatispora nypae,Wicklowia submersa,Xanthagaricus necopinatus and Xylaria haemorrhoidalis.The data presented herein are based on morphological examination of fresh specimens,coupled with analysis of phylogenetic sequence data to better integrate taxa into appropriate taxonomic ranks and infer their evolutionary relationships.

An efficient and universal protoplast-based transient gene expression system for genome editing in Brassica crops

Protoplast-based transient gene expression system has been widely used in plant genome editing because of its simple operation and less time-consuming.In order to establish a universal protoplast-based transient transfection system for verifying activities of genome editing vectors containing targets in Brassica,we systematically optimized factors affecting protoplast isolation and transient gene expression.We established an efficient protoplast-based transient gene expression system(PTGE)in Chinese cabbage,achieving high protoplast yield of 4.9×10^(5)·g^(-1)FW,viability over 95%,and transfection efficiency of 76%.We showed for the first time that pretreatment of protoplasts with a hypotonic MMG could significantly enhance the transfection efficiency.Furthermore,protoplasts incubated at 37℃ for 6 min improved the transfection efficiency to 86%.We also demonstrated that PTGE worked well(more than 50%transfection efficiency)in multiple Brassica species including cabbage,Pak Choi,Chinese kale,and turnip.Finally,PTGE was used for validating the activities of CRISPR/Cas9 vectors containing targets in Chinese cabbage,cabbage,and pak choi,demonstrating the broad applicability of the established PTGE for genome editing in Brassica crops.

Construction of a high-density mutant population of Chinese cabbage facilitates the genetic dissection of agronomic traits

Chinese cabbage(Brassica rapa ssp.pekinensis)is an economically important vegetable crop throughout the world,especially in Asia.High-quality genome sequences are available for Chinese cabbage,but gene functional studies remain challenging.To promote functional genomic studies of Chinese cabbage,we generated an ethyl methane sulfonate(EMS)mutant population of~8000 M_(2) plants using the double haploid inbred line A03 as the parent.The genome of A03 was sequenced and used as a reference for high-throughput functional characterization of gene mutations at the whole-genome level.A total of 300 M_(2) to M_(5) EMS mutants were phenotypically screened and then sequenced,revealing 750629 SNPs and 46272 InDel mutations that cover 98.27%of all predicted genes in the A03 genome.A forward-genetics approach was successfully used to identify two genes with chloroplast-related functions that are responsible for the yellow leaf mutant trait.A reverse-genetics approach was also used to identify associations between mutations in five genes of the glucosinolate biosynthetic pathway and variations in glucosinolate content of the mutant plants.In addition,we built the Chinese cabbage EMS mutation database(CCEMD,www.bioinformaticslab.cn/EMSmutation/home)to increase the usability of this mutant population resource.In summary,we performed large-scale screening of a heading Chinese cabbage EMS mutant collection at the phenotypic and genotypic levels,which will facilitate gene mining of Chinese cabbage and might also be useful for the study of other Brassica crops.

Glucosinolates in Self-crossed Progenies of Monosomic Cabbage Alien Addition Lines in Chinese Cabbage

Brassica species have been reported to possess cancer preventive activity due to glucosinolates(GLS) and their derived properties. Many studies on GLS have focused on Brassica oleracea and Brassica rapa. However, information on GLS in progeny between Chinese cabbage(B. rapa ssp. pekinensis) and cabbage(B. oleracea var. capitata) remains limited. In this study, eight GLS were detected in the self-crossed progenies of monosomic cabbage alien addition lines in Chinese cabbage(Chinese cabbage–cabbage MAALs) and parental Chinese cabbage, and nine GLS were detected in the parental cabbage. The variation of GLS content ranges was greater in the progeny than in the parental Chinese cabbage. The nine GLS identified were subjected to PCA to evaluate the differences among progeny and parents. Eight progeny samples had a comprehensive principal component score closer to or greater than that of cabbage, and four of them exhibited glucoraphanin(GRA) and total GLS contents greater than that of Chinese cabbage with the relative content of total indolic GLS was greater than 50%. These results offered new opportunity to improve GLS-containing of Chinese cabbage using genes from cabbage.

Delimiting species in Basidiomycota:a review

Species delimitation is one of the most fundamental processes in biology.Biodiversity undertakings,for instance,require explicit species concepts and criteria for species delimitation in order to be relevant and translatable.However,a perfect species concept does not exist for Fungi.Here,we review the species concepts commonly used in Basidiomycota,the second largest phylum of Fungi that contains some of the best known species of mushrooms,rusts,smuts,and jelly fungi.In general,best practice is to delimitate species,publish new taxa,and conduct taxonomic revisions based on as many independent lines of evidence as possible,that is,by applying a so-called unifying(or integrative)conceptual framework.However,the types of data used vary considerably from group to group.For this reason we discuss the different classes of Basidiomycota,and for each provide:(i)a general introduction with difficulties faced in species recognition,(ii)species concepts and methods for species delimitation,and(iii)community recommendations and conclusions.