Large-scale population structure and genetic architecture of agronomic traits of garlic

Garlic,an asexually propagated crop,is the second important bulb crop after the onion and is used as a vegetable and medicinal plant.Abundant and diverse garlic resources have been formed over thousands of years of cultivation.However,genome variation,population structure and genetic architecture of garlic agronomic traits were still not well elucidated.Here,1100258 single nucleotide polymorphisms(SNPs)were identified using genotyping-by-sequencing in 606 garlic accessions collected from43 countries.Population structure,principal component and phylogenetic analysis showed that these accessions were divided into five subpopulations.Twenty agronomic traits,including above-ground growth traits,bulb-related and bolt-related traits in two consecutive years were implemented in a genome-wide association study.In total,542 SNPs were associated with these agronomic traits,among which 188 SNPs were repeatedly associated with more than two traits.One SNP(chr6:1896135972)was repeatedly associated with ten traits.These associated SNPs were located within or near 858 genes,56 of which were transcription factors.Interestingly,one non-synonymous SNP(Chr4:166524085)in ribosomal protein S5 was repeatedly associated with above-ground growth and bulb-related traits.Additionally,gene ontology enrichment analysis of candidate genes for genomic selection regions between complete-bolting and non-bolting accessions showed that these genes were significantly enriched in‘vegetative to reproductive phase transition of meristem’,‘shoot system development’,‘reproductive process’,etc.These results provide valuable information for the reliable and efficient selection of candidate genes to achieve garlic genetic improvement and superior varieties.

A Meloidogyne incognita effector Minc03329 suppresses plant immunity and promotes parasitism

Meloidogyne incognita is a devastating plant-parasitic nematode.Effectors play important roles during the stages of nematodes infection and parasitism,but their molecular functions remain largely unknown.In this study,we characterized a new effector,Minc03329,which contains signal peptide for secretion and a C-type lectin domain.The yeast signal sequence trap experiments indicated that the signal peptide of Minc03329 is functional.In situ hybridization showed that Minc03329 was specifically expressed in the subventral esophageal gland.Real-time qPCR confirmed that the expression level of Minc03329 transcript was significantly increased in pre-parasitic and parasitic second-stage juveniles(pre-J2s and par-J2s).Tobacco rattle virus(TRV)-mediated gene silencing of Minc03329 in host plants largely reduced the pathogenicity of nematodes.On the contrary,ectopic expression of Minc03329 in Arabidopsis thaliana significantly increased plant susceptibility to nematodes.Transient expression of Minc03329 in Nicotiana benthamiana leaves suppressed the programmed cell death triggered by the pro-apoptotic protein BAX.Moreover,the transcriptome analysis of Minc03329-transgenic Arabidopsis and wild type revealed that many defense-related genes were significantly down-regulated.Interestingly,some different expressed genes were involved in the formation of nematode feeding sites.These results revealed that Minc03329 is an important effector for M.incognita,suppressing host defense response and promoting pathogenicity.

Map-based cloning and CRISPR/Cas9-based editing uncover BoNA1 as the causal gene for the no-anthocyanin-accumulation phenotype in curly kale (Brassica oleracea var. sabellica)

Brassica oleracea comprises several important vegetable and ornamental crops,including curly kale,ornamental kale,cabbage,broccoli,and others.The accumulation of anthocyanins,important secondary metabolites valuable to human health,in these plants varies widely and is responsible for their pink to dark purple colors.Some curly kale varieties lack anthocyanins,making these plants completely green.The genetic basis of this trait is still unknown.We crossed the curly kale inbred line BK2019(without anthocyanins)with the cabbage inbred line YL1(with anthocyanins)and the Chinese kale inbred line TO1000(with anthocyanins)to generate segregating populations.The no-anthocyanin trait was genetically controlled by a recessive gene,bona1.We generated a linkage map and mapped bona1 to a 256-kb interval on C09.We identified one candidate gene,Bo9g058630,in the target genomic region;this gene is homologous to AT5G42800,which encodes a dihydroflavonol-4-reductase-like(DFR-like)protein in Arabidopsis.In BK2019,a 1-bp insertion was observed in the second exon of Bo9g058630 and directly produced a stop codon.To verify the candidate gene function,CRISPR/Cas9 gene editing technology was applied to knock out Bo9g058630.We generated three bona1 mutants,two of which were completely green with no anthocyanins,confirming that Bo9g058630 corresponds to BoNA1.Different insertion/deletion mutations in BoNA1 exons were found in all six of the other no-anthocyanin kale varieties examined,supporting that independent disruption of BoNA1 resulted in no-anthocyanin varieties of B.oleracea.This study improves the understanding of the regulation mechanism of anthocyanin accumulation in B.oleracea subspecies.

Lack of fitness cost and inheritance of resistance to abamectin based on the establishment of a near-isogenic strain of Tetranychus urticae

Many populations of the two-spotted spider mite, Tetranychus urticae Koch, have developed high levels of resistance to the pesticide abamectin in China and other countries. This study developed a near-isogenic line to understand better the inheritance, cross-resistance, and fitness costs associated with abamectin resistance in the field population of T. urticae in China. We introduced the trait that confers extremely high abamectin resistance in a field-collected population of T. urticae into a susceptible laboratory strain(IPP-SS) to generate an abamectin-resistant near-isogenic line(NIL-Aba).This process was carried out through multiple backcrossing to IPP-SS and via parthenogenesis and abamectin screening. Compared with IPP-SS, the NIL-Aba strain had a 25 147-fold resistance to abamectin and a high level of cross-resistance to bifenthrin(288.17-fold), an intermediate level to emamectin benzoate(42.57-fold), and low levels to bifenazate, chlorfenapyr, cyflumetofen, cyenopyrafen, and cyetpyrafen with resistance ranging from 3.18-to 9.31-fold.But it had no cross-resistance to profenofos. The resistance to abamectin in NIL-Aba was autosomal, incompletely dominant, and polygenic. Based on two sex life table parameters, no fitness cost was found in NIL-Aba. Establishing the NIL-Aba strain provides a reliable basis for an in-depth study of abamectin resistance in T. urticae. New information on toxicological characteristics and fitness cost should facilitate the management of abamectin resistance in field populations of T. urticae.

Multifaceted regulatory functions of CsBPC2 in cucumber under salt stress conditions

BASIC PENTACYSTEINE(BPC)transcription factors are essential regulators of plant growth and development.However,BPC functions and the related molecular mechanisms during cucumber(Cucumis sativus L.)responses to abiotic stresses,especially salt stress,remain unknown.We previously determined that salt stress induces CsBPC expression in cucumber.In this study,Csbpc2 transgene-free cucumber plants were created using a CRISPR/Cas9-mediated editing system to explore CsBPC functions associated with the salt stress response.The Csbpc2 mutants had a hypersensitive phenotype,with increased leaf chlorosis,decreased biomass,and increased malondialdehyde and electrolytic leakage levels under salt stress conditions.Additionally,a mutated CsBPC2 resulted in decreased proline and soluble sugar contents and antioxidant enzyme activities,which led to the accumulation of hydrogen peroxide and superoxide radicals.Furthermore,the mutation to CsBPC2 inhibited salinity-induced PM-H+-ATPase and V-H+-ATPase activities,resulting in decreased Na+efflux and increased K+efflux.These findings suggest that CsBPC2 may mediate plant salt stress resistance through its effects on osmoregulation,reactive oxygen species scavenging,and ion homeostasis-related regulatory pathways.However,CsBPC2 also affected ABA signaling.The mutation to CsBPC2 adversely affected salt-induced ABA biosynthesis and the expression of ABA signaling-related genes.Our results indicate that CsBPC2 may enhance the cucumber response to salt stress.It may also function as an important regulator of ABA biosynthesis and signal transduction.These findings will enrich our understanding of the biological functions of BPCs,especially their roles in abiotic stress responses,thereby providing the theoretical basis for improving crop salt tolerance.

Multifaceted roles of LhWRKY44 in promoting anthocyanin accumulation in Asiatic hybrid lilies (Lilium spp.)

The Asiatic hybrid lily(Lilium spp.)is a horticultural crop with high commercial value and diverse anthocyanin pigmentation patterns.However,the regulatory mechanism underlying lily flower color has been largely unexplored.Here,we identified a WRKY transcription factor from lily tepals,LhWRKY44,whose expression was closely associated with anthocyanin accumulation.Functional verification indicated that LhWRKY44 positively regulated anthocyanin accumulation.LhWRKY44 physically interacted with LhMYBSPLATTER and directly bound to the LhMYBSPLATTER promoter,which enhanced the effect of the LhMYBSPLATTER-LhbHLH2 MBW complex activator on anthocyanin accumulation.Moreover,EMSA and dual-luciferase assays revealed that LhWRKY44 activated and bound to the promoters of gene LhF3H and the intracellular anthocyanin-related glutathione S-transferase gene LhGST.Interestingly,our further results showed that LhWRKY44 participated in light and drought-induced anthocyanin accumulation,and improved the drought tolerance in lily via activating stress-related genes.These results generated a multifaceted regulatory mechanism for the LhWRKY44-meditaed enhancement by the environmental signal pathway of anthocyanin accumulation and expanded our understanding of the WRKY-mediated transcriptional regulatory hierarchy modulating anthocyanin accumulation in Asiatic hybrid lilies.

Genome resequencing reveals the evolutionary history of garlic reproduction traits

The propagation of cultivated garlic relies on vegetative cloves,thus flowers become non-essential for reproduction in this species,driving the evolution of reproductive feature-derived traits.To obtain insights into the evolutionary alteration of reproductive traits in the clonally propagated garlic,the evolutionary histories of two main reproduction-related traits,bolting and flower differentiation,were explored by genome analyses using 134 accessions displaying wide diversity in these two traits.Resequencing identified 272.8 million variations in the garlic genome,198.0million of which represent novel variants.Population analysis identified five garlic groups that have evolved into two clades.Gene expression,single-cell transcriptome sequencing,and genome-wide trait association analyses have identified numerous candidates that correlate with reproductive transition and flower development,some of which display distinct selection signatures.Selective forces acting on the B-box zinc finger protein-encoding Asa2G00291.1,the global transcription factor group E protein-encoding Asa5G01527.1,and VERNALIZATION INSENSITIVE 3-like Asa3G03399.1 appear to be representative of the evolution of garlic bolting.Plenty of novel genomic variations and trait-related candidates represent valuable resources for biological studies of garlic.Numerous selective signatures from genes associated with the two chosen reproductive traits provide important insights into the evolutionary history of reproduction in this clonally propagated crop.

A novel mutation in ACS11 leads to androecy in cucumber

Sex determination in plants gives rise to unisexual flowers. A better understanding of the regulatory mechanism underlying the production of unisexual flowers will help to clarify the process of sex determination in plants and allow researchers and farmers to harness heterosis. Androecious cucumber(Cucumis sativus L.) plants can be used as the male parent when planted alongside a gynoecious line to produce heterozygous seeds, thus reducing the cost of seed production. The isolation and characterization of additional androecious genotypes in varied backgrounds will increase the pool of available germplasm for breeding. Here, we discovered an androecious mutant in a previously generated ethyl methanesulfonate(EMS)-mutagenized library of the cucumber inbred line ‘406’. Genetic analysis, whole-genome resequencing, and molecular marker-assisted verification demonstrated that a nonsynonymous mutation in the ethylene biosynthetic gene 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 11(ACS11) conferred androecy. The mutation caused an amino acid change from serine(Ser) to phenylalanine(Phe) at position 301(S301F). In vitro enzyme activity assays revealed that this S301F mutation leads to a complete loss of enzymatic activity. This study provides a new germplasm for use in cucumber breeding as the androecious male parent, and it offers new insights into the catalytic mechanism of ACS enzymes.

Developing Stable Cultivar through Microspore Mutagenesis in ×<i>Brassicoraphanus koranhort</i>, Inter-Generic Allopolyploid between <i>Brassica rapa</i>and <i>Raphanus sativus</i>

A stable progeny was developed through induced mutation, using microspore culture, of the hybrid (F1F1) produced by crossing a newly synthesized, unstable allopolyploid (F1) and a stable cultivar, BB#1(F1) in xBrassicoraphanus. An F1F1 plant was subjected to the induced mutation system established during production of BB#1. Morphological characteristics of the progeny such as color, and leaf number and length, differed from those of BB#1. The bolting time of the progeny in spring cropping was very late compared to BB#1, allowing it to be grown to an adult plant in spring. Genomic in situ hybridization analysis of pollen mother cells at prophase I identified 19 bivalents, 10 from Brassica rapa and 9 from Raphanus sativus. The glucoraphenin content was almost identical to that of BB#1. Two cultivars are available in the baemoochae crop now. These results indicate that induced mutation using microspore culture is a viable method of stabilizing intergeneric allopolyploids between B. rapa and R. sativus.

Dominance of Brassica and No Effects of Raphanus in Mature Seed Production in Intergeneric Hybrid between Brassica rapa ssp. Pekinensis and Raphanus

We succeeded in producing mature seed from a line of Brassica rapa ssp. pekinensis that had been hybridized with Raphanus sativus var. major. Our focus was on dominance of B. rapa ssp. pekinensis;radish (R. sativus var. major) had no influence. Marker tests for similarity showed that the original CR291M-64 x HwiM-2 hybrid was an inbred CR291M-64, rather than a genuine cross;this appears to have resulted from weak self-incompatibility in this strain. The plants from the mature seed bloomed with reddish flowers differently shown up to present. The intergeneric hybrid between Brassica inbred and Raphanus hybrid was very weak in strength compared to the Brassica inbred which was self-pollinated even though the cause of the weak was not identified. The hybrids between Brassica hybrid, dominant and elite recessive, and Raphanus can be developed in large quantities using mature hybrid seed without resorting to ovule culture techniques.

Several whitefly genome assemblies and an integrated whitefly gene search platform

Dear Editor,The characteristics and importance of the whitefly Bemisia tabaci as an economic pest seem to continually get more and more attention.Among the whiteflies B.tabaci including,at least 11 genetic groups and 36 morphologically indistinguishable cryptic species,invasive MEAMl(Middle East-Asia Minor 1,"B"type)and MED(the Mediterranean,"Q"type)are the 2 most destructive cryptic species of the B.

Monitoring Xylem Transport in the Stem of Lilium lancifolium Using Fluorescent Dye 5(6)-Carboxyfluorescein Diacetate

The xylem undergoes physiological changes in response to various environmental conditions during the process of plant growth.To understand these physiological changes,it is extremely important to observe the transport of xylem.In this study,the distribution and structure of vascular bundle in Lilium lancifolium were observed using the method of semithin section.Methods for introducing a fluorescent tracer into the xylem of the stems were evaluated.Then,the transport rule of 5(6)-Carboxyfluorescein diacetate(CFDA)in the xylem of the stem of L.lancifolium was studied by fluorescence dye in live cells tracer technology.The results showed that the vascular bundles of L.lancifolium were scattered in the basic tissue,the peripheral vascular bundles were smaller and densely distributed,and the closer to the center,the larger the volume of vascular bundles and the more sparsely distributed.The vascular bundles of L.lancifolium are limited external tenacity vascular bundles,which are composed of phloem and xylem.The most suitable method for CFDA labeling the xylem of isolated stem segments of L.lancifolium was solution soaking for 24 h.The running speed of CF in the isolated stem was 0.3 cm/h,which was consistent with the running speed of the material in the field.CF could be transported between the xylem and parenchyma cells,indicating that the material transport in the xylem could be through the symplastic pathway.The above results laid a foundation for the study of the xylem transport mechanism and the xylem pathogen disease of lily.

Improved genome annotation of Brassica oleracea highlights the importance of alternative splicing

Brassica oleracea has been developed into many important crops,including cabbage,kale,cauliflower,broccoli and so on.The genome and gene annotation of cabbage(cultivar JZS),a representative morphotype of B.oleracea,has been widely used as a common reference in biological research.Although its genome assembly has been updated twice,the current gene annotation still lacks information on untranslated regions(UTRs)and alternative splicing(AS).Here,we constructed a high-quality gene annotation(JZSv3)using a full-length transcriptome acquired by nanopore sequencing,yielding a total of 59452 genes and 75684 transcripts.Additionally,we re-analyzed the previously reported transcriptome data related to the development of different tissues and cold response using JZSv3 as a reference,and found that 3843 out of 11908 differentially expressed genes(DEGs)underwent AS during the development of different tissues and 309 out of 903 cold-related genes underwent AS in response to cold stress.Meanwhile,we also identified many AS genes,including BolLHCB5 and BolHSP70,that displayed distinct expression patterns within variant transcripts of the same gene,highlighting the importance of JZSv3 as a pivotal reference for AS analysis.Overall,JZSv3 provides a valuable resource for exploring gene function,especially for obtaining a deeper understanding of AS regulation mechanisms.

Gene expression,transcription factor binding and histone modification predict leaf adaxial-abaxial polarity related genes

Leaf adaxial-abaxial(ad-abaxial)polarity is crucial for leaf morphology and function,but the genetic machinery governing this process remains unclear.To uncover critical genes involved in leaf ad-abaxial patterning,we applied a combination of in silico prediction using machine learning(ML)and experimental analysis.A Random Forest model was trained using genes known to influence ad-abaxial polarity as ground truth.Gene expression data from various tissues and conditions as well as promoter regulation data derived from transcription factor chromatin immunoprecipitation sequencing(ChIP-seq)was used as input,enabling the prediction of novel ad-abaxial polarity-related genes and additional transcription factors.Parallel to this,available and newly-obtained transcriptome data enabled us to identify genes differentially expressed across leaf ad-abaxial sides.Based on these analyses,we obtained a set of 111 novel genes which are involved in leaf ad-abaxial specialization.To explore implications for vegetable crop breeding,we examined the conservation of expression patterns between Arabidopsis and Brassica rapa using single-cell transcriptomics.The results demonstrated the utility of our computational approach for predicting candidate genes in crop species.Our findings expand the understanding of the genetic networks governing leaf ad-abaxial differentiation in agriculturally important vegetables,enhancing comprehension of natural variation impacting leaf morphology and development,with demonstrable breeding applications.

Conserved noncoding sequences correlate with distant gene contacts in Arabidopsis and Brassica

Physical contact between genes distant on chromosomes is a potentially important way for genes to coordinate their expressions.To investigate the potential importance of distant contacts,we performed high-throughput chromatin conformation capture(Hi-C)experiments on leaf nuclei isolated from Brassica rapa and Brassica oleracea.We then combined our results with published Hi-C data from Arabidopsis thaliana.We found that distant genes come into physical contact and do so preferentially between the proximal promoter of one gene and the downstream region of another gene.Genes with higher numbers of conserved noncoding sequences(CNSs)nearby were more likely to have contact with distant genes.With more CNSs came higher numbers of transcription factor binding sites and more histone modifications associated with the activity.In addition,for the genes we studied,distant contacting genes with CNSs were more likely to be transcriptionally coordinated.These observations suggest that CNSs may enrich active histone modifications and recruit transcription factors,correlating with distant contacts to ensure coordinated expression.This study advances our knowledge of gene contacts and provides insights into the relationship between CNSs and distant gene contacts in plants.

Natural variation in STAYGREEN contributes to low-temperature tolerance in cucumber

Low-temperature(LT)stress threatens cucumber production globally;however,the molecular mechanisms underlying LT tolerance in cucumber remain largely unknown.Here,using a genome-wide association study(GWAS),we found a naturally occurring single nucleotide polymorphism(SNP)in the STAYGREEN(CsSGR)coding region at the gLTT5.1 locus associated with LT tolerance.Knockout mutants of CsSGR generated by clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated nuclease 9 exhibit enhanced LT tolerance,in particularly,increased chlorophyll(Chl)content and reduced reactive oxygen species(ROS)accumulation in response to LT.Moreover,the C-repeat Binding Factor 1(CsCBF1)transcription factor can directly activate the expression of CsSGR.We demonstrate that the LT-sensitive haplotype CsSGRHapA,but not the LT-tolerant haplotype CsSGR^(HapG)could interact with NON-YELLOW COLORING 1(CsNYC1)to mediate Chl degradation.Geographic distribution of the CsSGR haplotypes indicated that the CsSGR^(HapG)was selected in cucumber accessions from high latitudes,potentially contributing to LT tolerance during cucumber cold-adaptation in these regions.CsSGR mutants also showed enhanced tolerance to salinity,water deficit,and Pseudoperonospora cubensis,thus CsSGR is an elite target gene for breeding cucumber varieties with broad-spectrum stress tolerance.Collectively,our findings provide new insights into LT tolerance and will ultimately facilitate cucumber molecular breeding.