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.

In vitro Culture of Unfertilized Ovary of Strawberry(Fragaria spp.)

[Objective] This study was to investigate the in vitro culture of unfertilized ovary of strawberry.[Methods] Employing single factor experiment,we investigated six key factors including genotype,extrinsic hormone,sucrose concentration,low temperature pretreatment,growth environment and development status,and illumination condition on induction of gynogenesis in vitro of unfertilized ovary,on the induction of gynogenesis in vitro of unfertilized ovary.[Results] The optimal conditions for in vitro culture of unfertilized ovary of strawberry were as follows：the primary flower buds cultured on bare land as explants,selection of appropriate genotype,2,4-D as external hormone,sucrose at the concentration of 6%,low temperature pretreatment for 48 hours and dark culture under alternated temperature.[Conclusion] The research provided reference for ploidy breeding in strawberry.

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.

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。

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(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。

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。

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。

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。

The telomere-to-telomere genome of Fragaria vesca reveals the genomic evolution of Fragaria and the origin of cultivated octoploid strawberry

Fragaria vesca,commonly known as wild or woodland strawberry,is the most widely distributed diploid Fragaria species and is native to Europe and Asia.Because of its small plant size,low heterozygosity,and relative ease of genetic transformation,F.vesca has been a model plant for fruit research since the publication of its Illumina-based genome in 2011.However,its genomic contribution to octoploid cultivated strawberry remains a long-standing question.Here,we de novo assembled and annotated a telomere-to-telomere,gap-free genome of F.vesca‘Hawaii 4’,with all seven chromosomes assembled into single contigs,providing the highest completeness and assembly quality to date.The gap-free genome is 220785082 bp in length and encodes 36173 protein-coding gene models,including 1153 newly annotated genes.All 14 telomeres and seven centromeres were annotated within the seven chromosomes.Among the three previously recognized wild diploid strawberry ancestors,F.vesca,F.iinumae,and F.viridis,phylogenomic analysis showed that F.vesca and F.viridis are the ancestors of the cultivated octoploid strawberry F.×ananassa,and F.vesca is its closest relative.Three subgenomes of F.×ananassa belong to the F.vesca group,and one is sister to F.viridis.We anticipate that this high-quality,telomere-to-telomere,gap-free F.vesca genome,combined with our phylogenomic inference of the origin of cultivated strawberry,will provide insight into the genomic evolution of Fragaria and facilitate strawberry genetics and molecular breeding.

Genome-wide identification and expression analysis of auxin response factor(ARF) gene family in strawberry(Fragaria vesca)

Auxin signaling plays a significant role in the whole process of plant growth and development from embryogenesis to senescence.Auxin response factors(ARFs) are reported to regulate the expression of auxin response genes by binding to auxin response elements.ARF is the most critical transcription factor family which has been released in most species,but few reports in strawberry.In this study,the structure characterization of 12 FvARF genes in strawberry,their expression patterns at different development stages,different organizations,and different indole-3-acetic acid(IAA) treatments were analyzed.The expression of 12 FvARFs was found in all experiment tissues and showed almost the same trend during fruit development.All FvARFs respond to the treatment of IAA.Our study provides comprehensive information on ARF family in strawberry,including gene structures,chromosome locations,phylogenetic relationships and expression patterns.The information on FvARF genes paves the way for future research on strawberry ARF genes.

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.

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.

Updated annotation of the wild strawberry Fragaria vesca V4 genome

The diploid strawberry Fragaria vesca serves as an ideal model plant for cultivated strawberry(Fragaria×ananassa,8x)and the Rosaceae family.The F.vesca genome was initially published in 2011 using older technologies.Recently,a new and greatly improved F.vesca genome,designated V4,was published.However,the number of annotated genes is remarkably reduced in V4(28,588 genes)compared to the prior annotations(32,831 to 33,673 genes).Additionally,the annotation of V4(v4.0.a1)implements a new nomenclature for gene IDs(FvH4_XgXXXXX),rather than the previous nomenclature(geneXXXXX).Hence,further improvement of the V4 genome annotation and assigning gene expression levels under the new gene IDs with existing transcriptome data are necessary to facilitate the utility of this high-quality F.vesca genome V4.Here,we built a new and improved annotation,v4.0.a2,for F.vesca genome V4.The new annotation has a total of 34,007 gene models with 98.1%complete Benchmarking Universal Single-Copy Orthologs(BUSCOs).In this v4.0.a2 annotation,gene models of 8,342 existing genes are modified,9,029 new genes are added,and 10,176 genes possess alternatively spliced isoforms with an average of 1.90 transcripts per locus.Transcription factors/regulators and protein kinases are globally identified.Interestingly,the transcription factor family FAr-red-impaired Response 1(FAR1)contains 82 genes in v4.0.a2 but only two members in v4.0.a1.Additionally,the expression levels of all genes in the new annotation across a total of 46 different tissues and stages are provided.Finally,miRNAs and their targets are reanalyzed and presented.Altogether,this work provides an updated genome annotation of the F.vesca V4 genome as well as a comprehensive gene expression atlas with the new gene ID nomenclature,which will greatly facilitate gene functional studies in strawberry and other evolutionarily related plant species.

Author Correction:Updated annotation of the wild strawberry Fragaria vesca V4 genome

Since the publication of this article,the authors have noticed that the GeneIDs from new and original genome annotations don’t match in Table S6,the correct Table S6 is given here.The authors would like to apologize for this error.

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.

Additive QTLs on three chromosomes control flowering time in woodland strawberry(Fragaria vesca L.)

Flowering time is an important trait that affects survival,reproduction and yield in both wild and cultivated plants.Therefore,many studies have focused on the identification of flowering time quantitative trait locus(QTLs)in different crops,and molecular control of this trait has been extensively investigated in model species.Here we report the mapping of QTLs for flowering time and vegetative traits in a large woodland strawberry mapping population that was phenotyped both under field conditions and in a greenhouse after flower induction in the field.The greenhouse experiment revealed additive QTLs in three linkage groups(LG),two on both LG4 and LG7,and one on LG6 that explain about half of the flowering time variance in the population.Three of the QTLs were newly identified in this study,and one co-localized with the previously characterized FvTFL1 gene.An additional strong QTL corresponding to previously mapped PFRU was detected in both field and greenhouse experiments indicating that gene(s)in this locus can control the timing of flowering in different environments in addition to the duration of flowering and axillary bud differentiation to runners and branch crowns.Several putative flowering time genes were identified in these QTL regions that await functional validation.Our results indicate that a few major QTLs may control flowering time and axillary bud differentiation in strawberries.We suggest that the identification of causal genes in the diploid strawberry may enable fine tuning of flowering time and vegetative growth in the closely related octoploid cultivated strawberry.

Molecular characteristics of S-RNase alleles as the determ inant of self-i ncompatibility in the style of Fragaria viridi

Strawberry(Fragaria spp.)is a member of the Rosoideae subfamily in the family Rosaceae.The self-incompatibility(SI)of some diploid species is a key agronomic trait that acts as a basic pollination barrier;however,the genetic mechanism underlying SI control in strawberry remains unclear.Two candidate S-RNases(S a-and S^-RNase)identi fi ed in the transcriptome of the styles of the self-incompatible Fragaria viridis 42 were con fi rmed to be SI determinants at the S locus following genotype identi fi cation and intraspeci fi c hybridization using sel fi ng progenies.Whole-genome collinearity and RNase T2 family analysis revealed that only an S locus exists in Fragaria;however,none of the compatible species contained S-RNase.Although the results of interspeci fi c hybridization experiments showed that F.viridis(SI)styles could accept pollen from F.mandshurica(self-compatible),the reciprocal cross was incompatible.S a and S b-RNase contain large introns,and their noncoding sequences(promotors and introns)can be transcribed into long noncoding RNAs(lncRNAs).Overall,the genus Fragaria exhibits S-RNase-based gametophytic SI,and S-RNase loss occurs at the S locus of compatible germplasms.In addition,a type of SI-independent unilateral incompatibility exists between compatible and incompatible Fragaria species.Furthermore,the large introns and neighboring lncRNAs in S-RNase in Fragaria could offer clues about S-RNase expression strategies.

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.