Mapping of QTLs and candidate genes associated with multiple phenotypic traits for Huanglongbing tolerance in citrus

Huanglongbing(HLB)is the most devastating disease for citrus worldwide.Candidatus Liberibacter asiaticus(C Las),vectored by Asian citrus psyllid(ACP,Diaphorina citri Kuwayama),is the most common pathogen causing the disease.Commercial citrus varieties are highly susceptible to HLB,whereas trifoliate orange(Poncirus trifoliata)is considered highly tolerant to HLB.An F1 segregating population and their parent trifoliate orange and sweet orange,which had been exposed to intense HLB pressure for three years,was evaluated for disease symptoms,ACP colonization,C Las titer and tree vigor repeatedly for two to three years.Trifoliate orange and sweet orange showed significant differences for most of the phenotypic traits,and the F1 population exhibited a large variation.A high-density SNP-based genetic map with 1402 markers was constructed for trifoliate orange,which exhibited high synteny and high coverage of its reference genome.A total of 26 quantitative trait locus(QTLs)were identified in four linkage groups LG-t6,LG-t7,LG-t8 and LG-t9,of which four QTL clusters exhibit a clear co-localization of QTLs associated with different traits.Through genome-wide analysis of gene expression in response to C Las infection in‘Flying Dragon’and‘Larger-Flower DPI-50-7’trifoliate orange,85 differentially expressed genes were found located within the QTL clusters.Among them,seven genes were classified as defense or immunity protein which exhibited the highest transcriptional change after C Las infection.Our results indicate a quantitative genetic nature of HLB tolerance and identified candidate genes that should be valuable for searching for genetic solutions to HLB through breeding or genetic engineering.

花叶芋Cal.11和Cal.22杂交F1代性状分离研究

本研究以阔心形叶品种C11为母本,披针形叶品种C22为父本进行杂交,对其F1代群体的性状分离进行研究。结果表明:在观赏性状方面,根据叶形,F1代群体分为阔心形和披针形2个组群,分离比率为1∶1。根据叶色,分为4个组群,分离比率为3∶2∶2∶1。第1组群,整个叶片粉色至红色,与父本相似,但叶色更加鲜艳;第2组群叶片中间粉色至白色斑点,与母本相似;第3组群,叶片中间红色,外围绿色;第4组群,整个叶片绿色,主脉红色。母本的主脉色为红色,父本为白色,获得的杂交后代主脉色为红色和绿色,分离比率为3∶1,绿色主脉与斑点色紧密连锁。母本的叶柄色为褐色,父本为粉色,杂交后代的叶柄均为红色。在数量性状方面,F1代的芽数、叶片数、块茎数和块茎直径表现为中亲优势,其中芽数和叶片数表现为高亲优势。因此,采用Cal.11和Cal.22作为亲本,产生的杂交后代变异丰富,可从中筛选出叶色更鲜艳、块茎产量更高、叶片数和芽数更多的优选单株。

彩叶芋愈伤组织的诱导、增殖及植株再生

以Tapestry彩叶芋(Caladium×hortulanum Birdsey)未展开叶片和叶柄薄层切片为外植体,研究了不同激素浓度配比对Tapestry彩叶芋愈伤组织的诱导、增殖及植株再生的影响。结果表明:在4种愈伤组织诱导培养基上均能产生愈伤组织,其中2 mg/L TDZ和1 mg/L 6-BA是彩叶芋愈伤组织诱导的最佳激素配比;叶柄薄层切片是诱导愈伤组织的理想外植体,其愈伤组织诱导率均在90%以上;愈伤组织在MS基本培养基上增殖率最高,愈伤组织质地致密,呈颗粒状;诱导的愈伤组织分化能力很强,在附加有1 mg/L 6-BA和1 mg/L NAA的MS培养基上再生了许多幼苗。

A method for the production and expedient screening of CRISPR/Cas9-mediated non-transgenic mutant plants

Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable.Here,we report a highly useful method using an Agrobacterium-mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation.We have also developed a rapid,cost-effective,and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting(HRM)analysis.Using tetraploid tobacco as a model species and the phytoene desaturase(PDS)gene as a target,we successfully created and expediently identified mutant plants,which were verified as tetra-allelic mutants.We produced pds mutant shoots at a rate of 47.5%from tobacco leaf explants,without the use of antibiotic selection.Among these pds plants,17.2%were confirmed to be non-transgenic,for an overall non-transgenic mutation rate of 8.2%.Our method is reliable and effective in creating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction.This method should be applicable to many economically important,heterozygous,perennial crop species that are more difficult to regenerate.

Genome resequencing and transcriptome profiling reveal structural diversity and expression patterns of constitutive disease resistance genes in Huanglongbing-tolerant Poncirus trifoliata and its hybrids

Huanglongbing(HLB)is the most destructive bacterial disease of citrus worldwide.While most citrus varieties are susceptible to HLB,Poncirus trifoliata,a close relative of Citrus,and some of its hybrids with Citrus are tolerant to HLB.No specific HLB tolerance genes have been identified in P.trifoliata but recent studies have shown that constitutive disease resistance(CDR)genes were expressed at much higher levels in HLB-tolerant Poncirus hybrids and the expression of CDR genes was modulated by Candidatus Liberibacter asiaticus(CLas),the pathogen of HLB.The current study was undertaken to mine and characterize the CDR gene family in Citrus and Poncirus and to understand its association with HLB tolerance in Poncirus.We identified 17 CDR genes in two citrus genomes,deduced their structures,and investigated their phylogenetic relationships.We revealed that the expansion of the CDR family in Citrus seems to be due to segmental and tandem duplication events.Through genome resequencing and transcriptome sequencing,we identified eight CDR genes in the Poncirus genome(PtCDR1-PtCDR8).The number of SNPs was the highest in PtCDR2 and the lowest in PtCDR7.Most of the deletion and insertion events were observed in the UTR regions of Citrus and Poncirus CDR genes.PtCDR2 and PtCDR8 were in abundance in the leaf transcriptomes of two HLB-tolerant Poncirus genotypes and were also upregulated in HLB-tolerant,Poncirus hybrids as revealed by real-time PCR analysis.These two CDR genes seem to be good candidate genes for future studies of their role in citrus-CLas interactions.

Integration of early disease-resistance phenotyping, histological characterization, and transcriptome sequencing reveals insights into downy mildew resistance in impatiens

Downy mildew(DM),caused by obligate parasitic oomycetes,is a destructive disease for a wide range of crops worldwide.Recent outbreaks of impatiens downy mildew(IDM)in many countries have caused huge economic losses.A system to reveal plant–pathogen interactions in the early stage of infection and quickly assess resistance/susceptibility of plants to DM is desired.In this study,we established an early and rapid system to achieve these goals using impatiens as a model.Thirty-two cultivars of Impatiens walleriana and I.hawkeri were evaluated for their responses to IDM at cotyledon,first/second pair of true leaf,and mature plant stages.All I.walleriana cultivars were highly susceptible to IDM.While all I.hawkeri cultivars were resistant to IDM starting at the first true leaf stage,many(14/16)were susceptible to IDM at the cotyledon stage.Two cultivars showed resistance even at the cotyledon stage.Histological characterization showed that the resistance mechanism of the I.hawkeri cultivars resembles that in grapevine and type II resistance in sunflower.By integrating full-length transcriptome sequencing(Iso-Seq)and RNA-Seq,we constructed the first reference transcriptome for Impatiens comprised of 48,758 sequences with an N50 length of 2060 bp.Comparative transcriptome and qRT-PCR analyses revealed strong candidate genes for IDM resistance,including three resistance genes orthologous to the sunflower gene RGC203,a potential candidate associated with DM resistance.Our approach of integrating early disease-resistance phenotyping,histological characterization,and transcriptome analysis lay a solid foundation to improve DM resistance in impatiens and may provide a model for other crops.

Genome-wide identification of quantitative trait loci for important plant and flower traits in petunia using a high-density linkage map and an interspecific recombinant inbred population derived from Petunia integrifolia and P.axillaris

Petunia is a very important flower in the global floriculture industry and has played a critical role as a model in plant genetic studies.Owing to limited genetic variability in commercial germplasm,development of novel petunia phenotypes and new varieties has become increasingly difficult.To enrich petunia germplasm and facilitate genetic improvement,it is important to explore genetic variation in progenitor species that may contain highly valuable genes/alleles.In this study,an interspecific recombinant inbred population(168 recombinant inbreds)derived from Petunia integrifolia×P.axillaris were phenotyped for days to anthesis(DTA),flower count(Flower_C),flower diameter(Flower_D),flower length(Flower_L),plant height(Plant_H),plant spread(Plant_S),and plant size(Plant_Z)in 2014 and 2015.Transgressive segregation was observed for all traits in both years.The broad-sense heritability on a 2-year basis varied from 0.38(Flower_C)to 0.82(Flower_L).Ten QTL were consistently identified in both years and by two mapping strategies[multiple QTL mapping(MQM)in MapQTL and inclusive composite interval mapping(ICIM)in IciMapping].Major QTL explained up to 30.2,35.5,and 47.1%of the total phenotypic variation for Plant_S,Flower_L,and Flower_D,respectively.These findings should be of significant values for introgression of desirable genes from wild petunias into commercial varieties and future genetic improvement of this important flower.

Construction of citrus gene coexpression networks from microarray data using random matrix theory

After the sequencing of citrus genomes,gene function annotation is becoming a new challenge.Gene coexpression analysis can be employed for function annotation using publicly available microarray data sets.In this study,230 sweet orange(Citrus sinensis)microarrays were used to construct seven coexpression networks,including one condition-independent and six condition-dependent(Citrus canker,Huanglongbing,leaves,flavedo,albedo,and flesh)networks.In total,these networks contain 37633 edges among 6256 nodes(genes),which accounts for 52.11%measurable genes of the citrus microarray.Then,these networks were partitioned into functional modules using the Markov Cluster Algorithm.Significantly enriched Gene Ontology biological process terms and KEGG pathway terms were detected for 343 and 60 modules,respectively.Finally,independent verification of these networks was performed using another expression data of 371 genes.This study provides new targets for further functional analyses in citrus.

A single gene controls leaf background color in caladium (Araceae) and is tightly linked to genes for leaf main vein color, spotting and rugosity

Modern cultivated caladiums(Caladium×hortulanum)are grown for their long-lasting and colorful leaves.Understanding the mode of inheritance for caladium leaf characteristics is critical for plant breeders to select appropriate parents,predict progeny performance,estimate breeding population sizes needed,and increase breeding efficiencies.This study was conducted to determine the mode of inheritance of two leaf background colors(lemon and green)in caladium and to understand their relationships with four other important leaf characteristics including leaf shape,main vein color,spotting,and rugosity.Seven caladium cultivars and three breeding lines were used as parents in 19 crosses,and their progeny were phenotyped for segregation of leaf traits.Results showed that the two leaf background colors are controlled by a single nuclear locus,with two alleles,LEM and lem,which control the dominant lemon and the recessive green leaf background color,respectively.The lemon-colored cultivar‘Miss Muffet’and breeding lines UF-52 and UF-53 have a heterozygous genotype LEMlem.Chi-square tests showed that the leaf background color locus LEM is independent from the leaf shape locus F,but is tightly linked to three loci(S,V and RLF)controlling leaf spotting,main vein color,and rugosity in caladium.A linkage map that consists of four loci controlling major caladium leaf characteristics and extends~15 cM was developed based on the observed recombination frequencies.This is the first report on the mode of inheritance of leaf background colors in caladium and in the Araceae family.The information gained in this study will be very useful for caladium breeding and study of the inheritance of leaf colors in other ornamental aroids,an important group of ornamental plants in the world.

佛罗里达热带观赏植物产业厚积薄发

佛罗里达州是美国重要的观赏植物生产州，其生产数量和销售额在美国仅次于加利福尼亚州，位居第二。其中，热带观叶植物和盆栽植物是佛罗里达最有优势和特色的产业。佛罗里达热带观叶植物生产开始于1912年，以波士顿蕨为代表的观叶植物生产从北部的波士顿和纽约迁移到了气候条件优越的美国南方。经过近一个世纪特别是近40年来的快速发展，佛罗里达成为当今美国热带观叶植物、吊篮、兰花、凤梨等热带植物生产的主要基地，产品销售额占全美同类产品销售额的70％左右，居全美第一位。