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.

Reprogramming of a defense signaling pathway in rough lemon and sweet orange is a critical element of the early response to‘Candidatus Liberibacter asiaticus’

Huanglongbing(HLB)in citrus infected by Candidatus Liberibacter asiaticus(CLas)has caused tremendous losses to the citrus industry.No resistant genotypes have been identified in citrus species or close relatives.Among citrus varieties,rough lemon(Citrus jambhiri)has been considered tolerant due to its ability to produce a healthy flush of new growth after infection.The difference between tolerance and susceptibility is often defined by the speed and intensity of a plant’s response to a pathogen,especially early defense responses.RNA-seq data were collected from three biological replicates of CLas-and mock-inoculated rough lemon and sweet orange at week 0 and 7 following infection.Functional analysis of the differentially expressed genes(DEGs)indicated that genes involved in the mitogen activated protein kinase(MAPK)signaling pathway were highly upregulated in rough lemon.MAPK induces the transcription of WRKY and other transcription factors which potentially turn on multiple defense-related genes.A Subnetwork Enrichment Analysis further revealed different patterns of regulation of several functional categories,suggesting DEGs with different functions were subjected to reprogramming.In general,the amplitude of the expression of defense-related genes is much greater in rough lemon than in sweet orange.A quantitative disease resistance response may contribute to the durable tolerance level to HLB observed in rough lemon.

Expression of BpPIN is associated with IAA levels and the formation of lobed leaves in Betula pendula ’Dalecartica’

Auxin polar transport genes PIN(PINFORMED)determine the concentration gradient of auxin in plants.To understand the relationship between the development of different tissues in Betula pendula‘Dalecartica’,BpPIN gene expression and indole-3-acetic acid(IAA)content were analyzed using qRT-PCR,ELISA,and GUS staining.Gene expression of BpPIN genes and IAA levels in the leaves,buds,stems,xylem,and roots of B.pendula‘Dalecartica’and B.pendula as a control were measured.BpPIN1,BpPIN5 and BpPIN6 were upregulated during development in both species,suggesting a dominant role in the development of B.pendula‘Dalecartica’leaves.Moreover,BpPIN1 gene expression was positively associated with IAA levels during leaf,vein and petiole development in B.pendula‘Dalecartica’only.The correlation coefficient of the first three leaves was 0.69(P=0.04),while that of the first three petioles was 0.85(P=0.001).In addition,GUS staining of the pro-DR5::GUS transgenic line of cultivar was correlated with the results of BpPIN1 expression.Overall,these findings suggest that BpPIN1 is associated with the formation of lobed leaves in B.pendula‘Dalecartica’.

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.

Proteomic and metabolomic analyses provide insight into the off-flavour of fruits from citrus trees infected with‘Candidatus Liberibacter asiaticus’

Orange fruit from trees infected by‘Candidatus Liberibacter asiaticus’(CaLas)often do not look fully mature and exhibit off-flavours described as bitter,harsh,and metallic rather than juicy and fruity.Although previous studies have been carried out to understand the effect of CaLas on the flavour of orange juice using metabolomic methods,the mechanisms leading to the off-flavour that occurs in Huanglongbing(HLB)-symptomatic fruit are not well understood.In this study,fruits were collected from symptomatic and healthy Valencia sweet orange(Citrus sinensis)trees grafted on Swingle(C.paradisi X Poncirus trifoliata)rootstock.Isobaric tags for relative and absolute quantification(iTRAQ)and gas chromatography-mass spectrometry(GC-MS)were used to measure the proteins,sugars,organic acids,amino acids,and volatile terpenoids.The results showed that most of the differentially expressed proteins involved in glycolysis,the tricarboxylic acid(TCA)cycle and amino-acid biosynthesis were degraded,and terpenoid metabolism was significantly downregulated in the symptomatic fruit.Valencene,limonene,3-carene,linalool,myrcene,andα-terpineol levels were significantly lower in fruit from CaLas-infected trees than from healthy trees.Similar phenomena were observed for sucrose and glucose.Our study indicated that off-flavour of symptomatic fruit was associated with a reduction in the levels of terpenoid products and the downregulation of proteins in glycolysis,the TCA cycle,and the terpenoid biosynthesis pathway.

Selection of elite lines of BpGH3.5-transgenic Betula platyphylla using growth adaptability analysis

White birch(Betula platyphylla)is precious material for pulpwood and widely distributed in 14 provinces of China.Previous study indicated that inhibited expression of a gene encoding an auxin amide synthase,BpGH3.5,in transgenic plants reduced the level of IAA–amino acid conjugation,resulting in more free IAA,thereby better growth of birch.Utilizing transgenic-B pGH3.5 lines to increase wood production in a wide range of environments is the goal for breeders.In three field trials here,we measured tree height,diameter at breast height,and volume of 16 BpGH3.5-transgenic 7-year-old white birch lines(including 12 antisense strand lines and 4 overexpression lines)and a wild-type white birch line from three sites that varied greatly in their environmental conditions.To select elite BpGH3.5-transgenic lines for each target environment,we used an additive main effects and multiplicative interaction model to analyze genotype by environment interaction,growth adaptability and stability.The selection criteria for elite transgenic lines were set as the average volume plus 0.75 times the standard deviation for the tested lines at each test site.Results showed that the effect of line and site for height was highly significant(P<0.01),and the effect of line×site was significant(P<0.05);selected as the elite lines were FG12,FG13 and FG27 at the Maoershan Experimental Forest Farm,FG13 and FG32 at the Shidaohe Forest Farm,and FG3 and FG31 at the Ecological Experiment Forest Farm.These seven high-yield,stable lines can now be tested in production trials or adjacent trial areas with similar environmental conditions,while the high-yield,unstable lines should be tested in production trials in areas deemed suitable for their growth.These results provide guidance on which released transgenic elite lines will grow best in a wide range of conditions.