Genome-wide characterization and expression analysis of WRKY family genes during development and resistance to Colletotrichum fructicola in cultivated strawberry(Fragaria×ananassa Duch.)

Based on the recently published whole-genome sequence of cultivated strawberry ’Camarosa’, in this study, 222FaWRKY genes were identified in the ’Camarosa’ genome. Phylogenetic analysis showed that the 222 FaWRKY candidate genes were classified into three groups, of which 41 were in group Ⅰ, 142 were in group Ⅱ, and 39 were in group Ⅲ. The 222 FaWRKY genes were evenly distributed among the seven chromosomes. The exon–intron structures and motifs of the WRKY genes had evolutionary diversity in different cultivated strawberry genomes. Regarding differential expression, the expression of FaWRKY133 was relatively high in leaves, while FaWRKY63 was specifically expressed in roots. FaWRKY207, 59, 46, 182, 156, 58, 39, 62 and 115 were up-regulated during achene development from the green to red fruit transition. FaWRK181, 166 and 211 were highly expressed in receptacles at the ripe fruit stage. One interesting finding was that Fa WRKY179 and 205 were significantly repressed after Colletotrichum fructicola inoculation in both ’Benihoppe’ and ’Sweet Charlie’ compared with Mock. The data reported here provide a foundation for further comparative genomics and analyses of the distinct expression patterns of FaWRKY genes in various tissues and in response to C. fructicola inoculation.

The transcriptional landscape of cultivated strawberry(Fragaria×ananassa)and its diploid ancestor(Fragaria vesca)during fruit development

Cultivated strawberry(Fragaria×ananassa)originated from four diploid ancestors:F.vesca,F.viridis,F.iinumae and F.nipponica.Among them,F.vesca is the dominant subgenome for cultivated strawberry.It is not well understood how differences in gene expression between diploid and octoploid strawberry contribute to differences during fruit development.In this study,we used comprehensive transcriptomic analyses of F.vesca and F.×ananassa to investigate gene expression at the different stages of fruit development.In total,we obtained 3508(turning stage)and 3958(red stage)differentially expressed genes with pairwise comparisons between diploid and octoploid.The genes involved in flavonoid biosynthesis were almost upregulated in the turning stages of octoploid,and we also discovered a ripe fruit-specific module associated with several flavonoid biosynthetic genes,including FveMYB10,FveMYB9/11,and Fve RAP,using weighted gene coexpression network analysis(WGCNA).Furthermore,we identified the species-specific regulated networks in the octoploid and diploid fruit.Notably,we found that the WAK and F-box genes were enriched in the octoploid and diploid fruits,respectively.This study elucidates new findings on flavonoid biosynthesis and fruit size of strawberry with important implications for future molecular breeding in cultivated strawberry.

Study on Ornamental Cultivation Techniques of Strawberry(Fragaria×ananassa Duch)

Strawberry(Fragaria×ananassa Duch)is a very popular fruit,which is very common for productive cultivation,but not for ornamental cultivation.In order to improve the ornamental quality of strawberry and study its ornamental cultivation techniques,the variety selection,cultivation methods,flower pot selection,soil,fertilizer and water management,temperature,humidity and light management,and flower,fruit and pollination management of strawberry were investigated,especially its precision fertilization.The optimum combination fertilization scheme was A_(3)B_(2)C_(1),namely‘Lvbao’6 g,‘Weibao’6 g,‘Double carbon’2 g,which was obtained by an orthogonal test with three factors and three levels.This combined fertilization method overcomes the deficiency of single fertilization and improves the fertilization effect,and provides a reference for ornamental cultivation of strawberry.

Clarifying sub-genomic positions of QTLs for flowering habit and fruit quality in U.S. strawberry (Fragaria×ananassa) breeding populations using pedigree-based QTL analysis

The cultivated strawberry(Fragaria×ananassa)is consumed worldwide for its flavor and nutritional benefits.Genetic analysis of commercially important traits in strawberry are important for the development of breeding methods and tools for this species.Although several quantitative trait loci(QTL)have been previously detected for fruit quality and flowering traits using low-density genetic maps,clarity on the sub-genomic locations of these QTLs was missing.Recent discoveries in allo-octoploid strawberry genomics led to the development of the IStraw90 single-nucleotide polymorphism(SNP)array,enabling high-density genetic maps and finer resolution QTL analysis.In this study,breeder-specified traits were evaluated in the Eastern(Michigan)and Western(Oregon)United States for a common set of breeding populations during 2 years.Several QTLs were validated for soluble solids content(SSC),fruit weight(FWT),pH and titratable acidity(TA)using a pedigree-based QTL analysis approach.For fruit quality,a QTL for SSC on linkage group(LG)6A,a QTL for FWT on LG 2BII,a QTL for pH on LG 4CII and two QTLs for TA on LGs 2A and 5B were detected.In addition,a large-effect QTL for flowering was detected at the distal end of LG 4A,coinciding with the FaPFRU locus.Marker haplotype analysis in the FaPFRU region indicated that the homozygous recessive genotype was highly predictive of seasonal flowering.SNP probes in the FaPFRU region may help facilitate marker-assisted selection for this trait.

Mapping QTL associated with Verticillium dahliae resistance in the cultivated strawberry(Fragaria×ananassa)

A biparental cross of octoploid strawberry segregating for resistance to Verticillium dahliae,the causative agent of Verticillium wilt,was screened under field conditions for three seasons.Average wilt scores were significantly associated with multiple QTL,which were mostly significant across all years.Markers significantly associated with the traits were used to screen material with known wilt resistance and susceptibility phenotypes.A clear and statistically significant relationship was observed between resistant,tolerant and susceptible material and the total number of markers present in the different resistance classes.In field situations resistance QTL appear to behave in an additive manner.These markers are abundant in the cultivated strawberry germplasm indicating that,despite the large number of markers,clear genetic gain is possible through marker-assisted breeding.

Virus-induced gene silencing and virusinduced flowering in strawberry(Fragaria×ananassa)using apple latent spherical virus vectors

Apple latent spherical virus(ALSV)vector is a convenient alternative to genetic transformation in horticultural plants,especially in species recalcitrant to genetic transformation.ALSV,an RNA virus,can infect a wide variety of plant species including major horticultural plants without inducing symptoms.Here,methodologies were developed for infection of ALSV vectors to strawberry seedlings and plantlets cultured in vitro.A seed-propagated F1 hybrid strawberry cultivar'Yotsuboshi'was aseptically grown on half-strength Murashige–Skoog medium for 1 month and true leaves were inoculated with an ALSV RNA preparation by particle bombardment.ALSV vector infection rates varied from 58 to 100%according to the insertion sequences,in‘Yotsuboshi’seedlings.Plantlets(‘Dover’)propagated in vitro could also be infected with ALSV vector at a similar infection rate.For virus-induced gene silencing(VIGS),we prepared an ALSV vector carrying a 201 nucleotide segment of the strawberry phytoene desaturase gene.‘Yotsuboshi’and‘Dover’plants infected by this vector generated completely white leaves at fifth or sixth true leaves and above.For virus-induced flowering(VIF),we used an ALSV vector expressing the Arabidopsis thaliana flowering locus T gene.Strawberry seedlings infected by this vector started to flower from about 2 months post inoculation and bore fruits with viable seeds.The ALSV vector was no longer detected in any of the seedlings from early-flowered strawberries.Thus,the ALSV vector may be beneficial for examination of gene functions by VIGS in strawberry,and VIF using ALSV vector constitutes an effective new plant breeding technique for the promotion of cross-breeding in strawberry.

Evolution of the MLO gene families in octoploid strawberry(Fragaria×ananassa)and progenitor diploid species identified potential genes for strawberry powdery mildew resistance

Powdery mildew(PM)caused by Podosphaera aphanis is a major fungal disease of cultivated strawberry.Mildew Resistance Locus O(MLO)is a gene family described for having conserved seven-transmembrane domains.Induced loss-of-function in specific MLO genes can confer durable and broad resistance against PM pathogens.However,the genomic structure and potential role of MLO genes for PM resistance have not been characterized yet in the octoploid cultivated strawberry.In the present study,MLO gene families were characterized in four diploid progenitor species(Fragaria vesca,F.iinumae,F.viridis,and F.nipponica)and octoploid cultivated(Fragaria×ananassa)strawberry,and potential sources of MLO-mediated susceptibility were identified.Twenty MLO sequences were identified in F.vesca and 68 identified in F.×ananassa.Phylogenetic analysis divided diploid and octoploid strawberry MLO genes into eight different clades,in which three FveMLO(MLO10,MLO17,and MLO20)and their twelve orthologs of FaMLO were grouped together with functionally characterized MLO genes conferring PM susceptibility.Copy number variations revealed differences in MLO composition among homoeologous chromosomes,supporting the distinct origin of each subgenome during the evolution of octoploid strawberry.Dissecting genomic sequence and structural variations in candidate FaMLO genes revealed their potential role associated with genetic controls and functionality in strawberry against PM pathogen.Furthermore,the gene expression profiling and RNAi silencing of putative FaMLO genes in response to the pathogen indicate the function in PM resistance.These results are a critical first step in understanding the function of strawberry MLO genes and will facilitate further genetic studies of PM resistance in cultivated strawberry.

The sugar transporter system of strawberry:genome-wide identification and expression correlation with fruit soluble sugar-related traits in a Fragaria×ananassa germplasm collection

Sugar from plant photosynthesis is a basic requirement for life activities.Sugar transporters are the proteins that mediate sugar allocation among or within source/sink organs.The transporters of the major facilitator superfamily(MFS)targeting carbohydrates represent the largest family of sugar transporters in many plants.Strawberry(Fragaria×ananassa Duchesne)is an important crop appreciated worldwide for its unique fruit flavor.The involvement of MFS sugar transporters(STs)in cultivated strawberry fruit sugar accumulation is largely unknown.In this work,we characterized the genetic variation associated with fruit soluble sugars in a collection including 154 varieties.Then,a total of 67 ST genes were identified in the v4.0 genome integrated with the v4.0.a2 protein database of F.vesca,the dominant subgenome provider for modern cultivated strawberry.Phylogenetic analysis updated the nomenclature of strawberry ST homoeologs.Both the chromosomal distribution and structural characteristics of the ST family were improved.Semi-RT-PCR analysis in nine tissues from cv.Benihoppe screened 34 highly expressed ST genes in fruits.In three varieties with dramatically differing fruit sugar levels,qPCR integrated with correlation analysis between ST transcript abundance and sugar content identified 13 sugar-correlated genes.The correlations were re-evaluated across 19 varieties,including major commercial cultivars grown in China.Finally,a model of the contribution of the sugar transporter system to subcellular sugar allocation in strawberry fruits was proposed.Our work highlights the involvement of STs in controlling strawberry fruit soluble sugars and provides candidates for the future functional study of STs in strawberry development and responses and a new approach for strawberry genetic engineering and molecular breeding.

Quantitative trait loci controlling Phytophthora cactorum resistance in the cultivated octoploid strawberry(Fragaria×ananassa)

The cultivated strawberry,Fragaria×ananassa(Fragaria spp.)is the most economically important global soft fruit.Phytophthora cactorum,a water-borne oomycete causes economic losses in strawberry production globally.A biparental cross of octoploid cultivated strawberry segregating for resistance to P.cactorum,the causative agent of crown rot disease,was screened using artificial inoculation.Multiple putative resistance quantitative trait loci(QTL)were identified and mapped.Three major effect QTL(FaRPc6C,FaRPc6D and FaRPc7D)explained 37%of the variation observed.There were no epistatic interactions detected between the three major QTLs.Testing a subset of the mapping population progeny against a range of P.cactorum isolates revealed no significant interaction(p=0.0593).However,some lines showed higher susceptibility than predicted,indicating that additional undetected factors may affect the expression of some quantitative resistance loci.Using historic crown rot disease score data from strawberry accessions,a preliminary genome-wide association study(GWAS)of 114 individuals revealed an additional locus associated with resistance to P.cactorum.Mining of the Fragaria vesca Hawaii 4 v1.1 genome revealed candidate resistance genes in the QTL regions.

草莓(Fragaria ananassa Duch)茎段组织培养的研究

实验在草莓茎段的组织培养过程中,对外植体不同消毒时间,最佳诱导培养基、分化培养基与生根培养基进行了筛选。结果表明,0.1%升汞对茎段消毒2min时效果最好;诱导愈伤的最佳培养基为:MS+1.5mg·L-16-BA+0.1mg·L-1NAA;最佳分化的培养基为:MS+2.0mg·L-16-BA+0.2mg·L-1NAA;最佳生根培养基为:MS+0.25mg·L-16-BA。

The complexity of the Fragaria x ananassa(octoploid)transcriptome by singlemolecule long-read sequencing

Strawberry(Fragaria x ananassa)is an allopolyploid species with diverse and complex transcripts.The regulatory mechanisms of fruit development and maturation have been extensively studied;however,little is known about the signaling mechanisms that direct this process in octoploid strawberry(Fragaria x ananassa).Here,we used long-read sequencing(LRS)technology and RNA-seq analysis to investigate the diversity and complexity of the polyploid transcriptome and differentially expressed transcripts along four successive fruit developmental stages of cultivated strawberry.We obtained a reference transcriptome with 119,897 unique full-length isoforms,including 2017 new isoforms and 2510 long noncoding RNAs.Based on the genome of the plausible progenitor(Fragaria vesca),20,229 alternative splicing(AS)events were identified.Using this transcriptome,we found 17,485 differentially expressed transcripts during strawberry fruit development,including 527 transcription factors(TFs)belonging to 41 families.The expression profiles of all members of the auxin,ABA pathway,and anthocyanin biosynthesis gene families were also examined,and many of them were highly expressed at the ripe fruit stage,strongly indicating that the role of those genes is in the regulation of fruit ripening.We produce a high-quality reference transcriptome for octoploid strawberry,including much of the full-length transcript diversity,to help understand the regulatory mechanisms of fruit development and maturation of polyploid species,particularly via elucidation of the biochemical pathways involved in auxin,ABA,and anthocyanin biosynthesis.

Identification and substrate prediction of new Fragaria x ananassa aquaporins and expression in different tissues and during strawberry fruit development

The newly identified aquaporin coding sequences presented here pave the way for further insights into the plant–water relations in the commercial strawberry(Fragaria x ananassa).Aquaporins are water channel proteins that allow water to cross(intra)cellular membranes.In Fragaria x ananassa,few of them have been identified hitherto,hampering the exploration of the water transport regulation at cellular level.Here,we present new aquaporin coding sequences belonging to different subclasses:plasma membrane intrinsic proteins subtype 1 and subtype 2(PIP1 and PIP2)and tonoplast intrinsic proteins(TIP).The classification is based on phylogenetic analysis and is confirmed by the presence of conserved residues.Substrate-specific signature sequences(SSSSs)and specificity-determining positions(SDPs)predict the substrate specificity of each new aquaporin.Expression profiling in leaves,petioles and developing fruits reveals distinct patterns,even within the same(sub)class.Expression profiles range from leaf-specific expression over constitutive expression to fruit-specific expression.Both upregulation and downregulation during fruit ripening occur.Substrate specificity and expression profiles suggest that functional specialization exists among aquaporins belonging to a different but also to the same(sub)class.

An effector-reporter system to study cellular signal transduction in strawberry fruit (Fragaria ananassa)

An effector-reporter system is a powerful tool used to study cellular signal transduction,but this technique has been traditionally used in protoplasts.A similar system to study cellular signal transduction in fruits has not yet been established.In this study,we aimed to establish an effector–reporter system for strawberry fruit,a model nonclimacteric fruit.We first investigated the characteristics of transient gene expression in strawberry fruits and found marked variation in gene expression levels among individual fruits,and this variation has complicated the establishment of a technical system.To overcome this difficulty,we investigated a sampling strategy based on a statistical analysis of the activity pattern of four different reporters(GUS,GFP,FLuc,and RLuc)among individual fruits and combinations of pairs of reporters(GUS/GFP and RLuc/FLuc).Based on an optimized sampling strategy,we finally established a step-by step protocol for the effector/reporter assay.Using FaMYB10 and FaWRKY71 as the effectors and GUS driven by the FaCHS promoter as the reporter,we demonstrated that this effector/reporter system was practical and reliable.This effector/reporter technique will contribute to an in-depth exploration of the signaling mechanism for the regulation of strawberry fruit ripening.

A novel transcription factor FnMYB4 regulates pigments metabolism of yellow leaf mutants in Fragaria nilgerrensis

The strawberry species Fragaria nilgerrensis Schlechtendal ex J.Gay,renowned for its distinctive white,fragrant peach-like fruits and strong disease resistance,is an exceptional research material.In a previous study,an ethyl methane sulfonate(EMS)mutant library was established for this species,resulting in various yellow leaf mutants.Leaf yellowing materials are not only the ideal materials for basic studies on photosynthesis mechanism,chloroplast development,and molecular regulation of various pigments,but also have important utilization value in ornamental plants breeding.The present study focused on four distinct yellow leaf mutants:mottled yellow leaf(MO),yellow green leaf(YG),light green leaf(LG),and buddha light leaf(BU).The results revealed that the flavonoid content and carotenoid-to-chlorophyll ratio exhibited a significant increase among these mutants,while experiencing a significant decrease in chlorophyll and carotenoid contents compared to the wild type(WT).To clarify the regulatory mechanisms and network relationships underlying these mutants,the RNA-seq and weighted gene coexpression network(WGCNA)analyses were employed.The results showed flavonoid metabolism pathway was enriched both in MO and YG mutants,while the chlorophyll biosynthesis pathway and carotenoid degradation pathway were only enriched in MO and YG mutants,respectively.Subsequently,key structural genes and transcription factors were identified on metabolic pathways of three pigments through correlation analyses and quantitative experiments.Furthermore,a R2R3-MYB transcription factor,FnMYB4,was confirmed to be positively correlated with flavonoid synthesis through transient overexpression,virus-induced gene silencing(VIGS),and RNA interference(RNAi),accompanying by reoccurrence and attenuation of mutant phenotype.Finally,dual-luciferase(LUC)and yeast one-hybrid assays confirmed the binding of FnMYB4 to the FnFLS and FnF3H promoters,indicating that FnMYB4 positively regulates flavonoid synthesis.In addition,correlation analyses suggested that FnMYB4 also might be involved in chlorophyll and carotenoid metabolisms.These findings demonstrated the pivotal regulatory role of FnMYB4 in strawberry leaf coloration.

Strawberry (Fragaria spp.): Cultivation, Production, Consumption, and Marketing in Cameroon

Strawberry (Fragaria spp.) is one of the most important fruits classified as exotic fruits imported into Cameroon. To have an inventory of its cultivation in Cameroon, a survey study was carried out among eight farms of Fragaria spp. from January 2021 to February 2022. The plant was introduced in Cameroon in 2018. There are 13 varieties of Fragaria spp. currently cultivated. Among these 13 varieties, eleven are hybrids of Fragaria x ananassa (“Amiga”, “Amine”, “Camarosa”, “Chandler”, “Charlotte”, “Elsanta”, “Gariguette”, “Madame Moutot”, “Ostara”, “Ruby gem” and “San Andreas”), and two of the hybrids of Fragaria vesca (“Maestro” and “Mara des bois”). The cropping system, irrigation system, and type of fertilizers applied differ from one strawberry farm to another. Biofertilizers (such as mycorrhizal), inorganic and organic fertilizers are actually used to improve production. The potential annual production of strawberries from January 2021 to February 2022, estimated based on the survey data, was 21.216 tons for all growers. Among these eight production farms, the Lolodorf BIO Farm presents 6000 kg (six tons) of strawberries and 100,000 stolons (seedlings) produced, from seven varieties of Fragaria spp. cultivated, with 6 varieties which are hybrids variety Fragaria x ananassa (“Amiga”, “Amine”, “Chandler”, “Gariguette”, “Madame Moutot”, and “Ruby gem”), and one which is a hybrid of Fragaria vesca (“Mara des bois”). Certain diseases were also observed and recorded depending on the growing areas.

中国草莓属(Fragaria)植物的分类研究

中国是世界上野生草莓种类最丰富的国家。近20年来,从国内外收集保存了103份野生草莓资源,鉴定出我国自然分布有11个种,约占世界草莓属植物20个种的一半。这11个种包括8个二倍体种:森林草莓(Fragaria vescaL.)、黄毛草莓(F.nilgrrensisSchlecht.)、五叶草莓(F.pentaphyllaLozin-sk.)、纤细草莓(F.gracilisLozinsk.)、西藏草莓(F.nubicolaL ind l.)、绿色草莓(F.viridisDuch.)、裂萼草莓(F.daltonianaGay)、东北草莓(F.mandschuricaStaudt)和3个四倍体种:东方草莓(F.oriental-isLozinsk.)、西南草莓〔F.m oupinensis(Franch.)Card.〕、伞房草莓(F.corym bosaLozinsk.)。对我国原产野生草莓种类进行了较系统的性状描述和分类研究,并列出了我国野生草莓分种检索表。提出我国分布有11个种的结论比以前记载的8个种更全面和完整。

西藏色季拉山野生西南草莓(Fragaria moupinensis)遗传多样性的初步研究

采用RAPD分子标记技术,从42个引物种筛选出10个引物对11份西藏色季拉山野生西南草莓的遗传多样性进行评价。结果表明,草莓植株的相似系数变化范围为0.91～0.99,平均相似系数为0.95,说明色季拉山野生西南草莓亲缘关系比较近,遗传分异程度较低。利用RAPD技术所获得西藏不同海拔的遗传相似性较高,很难在它们之间划分出明显的类群。

新疆天山野生草莓与绿色草莓(Fragariaviridis Duch.)同一性的鉴定

通过植物学性状观察和RAPD技术鉴定证明新疆天山野生草莓为绿色草莓 (Fra gariaviridisDuch .) ,表明中国自然分布的草莓野生种达到 9个。绿色草莓在分类学上的典型特征为匍匐茎上从第二节开始每节均形成幼苗 ;花序直立、高于叶面 ,花瓣显露时呈黄绿色 ,雄蕊花丝细长 ,高于雌蕊 ;果实圆形或扁圆形 ,淡绿色 ,阳面略红 ,果硬 ,成熟时萼片紧贴 ,脱萼难。

草莓属二倍体种东北草莓(Fragariamandschurica Staudt)研究

从中国吉林、黑龙江、内蒙古收集到5份形态相近的二倍体野生草莓,经多年植物分类学性状观察鉴定为东北草莓(FragariamandschuricaStaudt),该中文名为首次赋予,此前国内尚无该种的记载。“Manchuria”意为满州,是对中国东北的旧称。该种植株绒毛多少及其着生方式、花器官、种子等诸多性状明显不同于森林草莓(F.vescaL.)。RAPD(RandomAmplifiedPolymorphicDNA)鉴定技术也对这一结果给予有力支持,而且表明二倍体东北草莓与四倍体东方草莓(F.orientalisLozinsk)、六倍体麝香草莓(F.moschataDuch.)的亲缘关系最近,东北草莓可能是它们的原始种。

绿色草莓(Fragaria viridis Duch.)自交后代的性状观察及评价

本研究对绿色草莓(Fragaria viridis Duch.)及其自交后代5个株系的植物学性状进行了观察,发现与亲本均存在一定程度差异,在观察的67个性状中,可溶性固形物等42个性状存在显著差异。利用SPSS软件对其中15个差异明显的性状进行主成分分析和变异系数分析,结果表明植株高度、冠径、小花梗长、小花梗粗、花序梗粗及花粉生活力是区分绿色草莓及其自交后代株系的主要指标。5个自交一代株系自交坐果率差异明显,其中株系Ls-S1-4坐果率最低。本研究为绿色草莓自交亲和/不亲和系的创建奠定基础。