Citrus Tristeza Virus Resistance Gene Research Articles

Construction of genetic linkage map and graphical genotyping of pseudo-backcrossed F2 (BC’2) progeny to introduce a CTV resistance from Poncirus trifoliata (L.) Raf. into Citrus by introgression breeding

Most citrus tristeza virus (CTV) strains infect almost all Citrus species and have caused serious damage on citrus production in many countries. Poncirus trifoliata (trifoliate orange) has a single dominant resistance gene effective against a broad range of CTV strains. P. trifoliata is sexually compatible with Citrus species, and introgression breeding programs have been initiated to introduce the CTV resistance into Citrus. “Kankitsu Chukanbohon Nou 8 Gou” (Nou-8) was developed by crossing Citrus sp. “Kiyomi” with an F1 hybrid derived from Citrus hassaku × P. trifoliata, but it still had undesirable eating traits. In aim to progress marker-assisted introgression breeding for CTV resistance efficiently, we selected 189 DNA markers which could be applied to genotyping for Citrus and P. trifoliata and constructed a genetic linkage map of Nou-8. The map included 189 DNA markers on 10 linkage groups and extended about 722.8 cM in total length. CTV resistance locus was located at 2.4 cM position in linkage group 02_(1), with the same location found when progeny were scored for resistance using an ELISA assay and for DNA marker for CTV resistant (CTV103). Graphical genotypes of Nou-8 and its progeny suggested that (1) 36 % of the chromosome in the BC’2 population were inherited from Nou-8 without any recombination sites, (2) Nou-8 had been already completely substituted by Citrus genotype in two linkage groups, and (3) four of 93 progeny of Nou-8 had recombination sites in the flanking region of the CTV resistance gene locus.

Identification of Genes Expressed Differentially in Grapefruit Infected with Candidatus Liberibacter asiaticus in the Late Stage of Disease

Citrus greening or Huanglongbing (HLB), a destructive disease of citrus worldwide, was reported from south of Iran in 2007. The molecular basis of compatibility and disease development in this system is poorly understood. We have carried out a cDNA-AFLP analysis to analyse gene expression of grapefruit infected by Candidatus Leiberibacter asiaticus in the late infection stage. We have applied a cDNA-AFLP approach on grafted infected grapefruit trees at the representing symptoms stage in susceptible host. Selective amplifications with 10 primer combinations allowed the visualization of approximately 24 transcript-derived fragments (TDFs) in the leaves of graft-inoculated trees, which were differentially expressed. We sequenced 14 fragments, which were identified as grapefruit transcripts after homology searching, whereas 12 were not homologous to sequences in NCBI databases. Many grapefruit genes spanning almost all functional categories were upregulated during infection, especially genes involved in ATP synthesize, cytochrome P450 synthesize, isoflavone 2'-hydroxylase, zeaxanthin epoxidase, cellulose synthase, DNA repair protein, aconitate hydratase 2 and citrus tristeza virus resistance gene. This study provides the first global catalogue of grapefruit genes expressed during inoculation, together with their functional annotations. This will help to elucidate the molecular basis of the resistance process and identify genes and chemicals that could help to inhibit the pathogen.

PCR Primers for Marker Assisted Backcrossing to Introduce a CTV Resistance Gene from Poncirus trifoliata (L.) Raf. into Citrus

Citrus tristeza virus (CTV) is an acute pathogen that causes serious damage to the citrus industry. Poncirus trifoliata (L.) Raf., a sexually compatible species with Citrus, has resistance against a broad range of CTV strains. Breeding programs have been conducted to introduce the CTV resistance gene from P. trifoliata to Citrus, but no commercial cultivar has yet been developed. In this study, we developed four selection markers linked to CTV resistance to enable marker assisted selection to efficiently introduce CTV resistance into Citrus. The four CTV resistance-linked markers were composed of a co-dominant single nucleotide polymorphism (SNP) marker and three dominant sequence tagged site (STS) markers. All four markers were fitted to the progeny and were linked to CTV resistance, with only 2.8% exceptions. We also developed 46 P. trifoliata allele identification markers from alleles of 35 Citrus species. Among the 46 markers, nine were located in linkage group 2, on which the CTV resistance locus is located. We located the other 31 markers on the rest of the linkage groups so that these markers could be used to distinguish P. trifoliata genome regions remaining in the hybrid progenies. The set of PCR primers developed in this study will be useful for marker assisted backcrossing to introduce the P. trifoliata CTV resistance gene into Citrus.

Refinement of the Citrus Tristeza Virus Resistance Gene (Ctv) Positional Map in Poncirus trifoliata and Generation of Transgenic Grapefruit (Citrus paradisi) Plant Lines with Candidate Resistance Genes in this Region

Citrus tristeza virus (CTV) is a major pathogen of Citrus. A single dominant gene Ctv present in the trifoliate relative of Citrus, Poncirus trifoliata confers broad spectrum resistance against CTV. Refinement of genetic maps has delimited this gene to a 121 kb region, comprising of ten candidate Ctv resistance genes. The ten candidate genes were individually cloned in Agrobacterium based binary vector and transformed into three CTV susceptible grapefruit varieties. Two of the candidate R-genes, R-2 and R-3 are exclusively expressed in transgenic plants and in Poncirus trifoliata, while five other genes are also expressed in non-transformed Citrus controls. Northern blotting with a CTV derived probe for assessment of infection in virus inoculated plants over a span of three growth periods, each comprising of six to eight weeks, indicates either an absence of initiation of infection or it's slow spread in R-2 plant lines or an initial appearance of infection and it's subsequent obliteration in some R-1 and R-4 plant lines. Limited genome walk up- and downstream form R-1 gene, based on it's 100% sequence identity between Poncirus and Citrus, indicates promoter identity of 92% between the two varieties. Further upstream and downstream sequencing indicates the presence of an O-methyl transferase and a Copia like gene respectively in Citrus instead of the amino acid transporter like gene upstream and a sugar transporter like gene downstream in Poncirus. The possibility of recombinations in the resistance locus of Citrus and the need for consistent monitoring for virus infection and gene expression in the transgenic Citrus trees is discussed.

Construction of a 1.2-Mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library ofPoncirus trifoliata(L.) Raf.

The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45 696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.Key words: Poncirus, citrus tristeza virus, chromosome walk, resistance gene.

Construction of a 1.2-Mb contig including the citrus tristeza virus resistance gene locus using a bacterial artificial chromosome library of <i>Poncirus trifoliata</i> (L.) Raf.

The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45,696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes.

Cloning and characterization of NBS-LRR class resistance-gene candidate sequences in citrus

Numerous disease resistance gene-like DNA sequences were cloned from an intergeneric hybrid of Poncirus and Citrus, using a PCR approach with degenerate primers designed from conserved NBS (nucleotide-binding site) motifs found in a number of plant resistance genes. Most of the cloned genomic sequences could be translated into polypeptides without stop codons, and the sequences contained the characteristic motifs found in the NBS-LRR class of plant disease resistance genes. Pairwise comparisons of these polypeptide sequences indicated that they shared various degrees of amino-acid identity and could be grouped into ten classes (RGC1–RGC10). When the sequences of each class were compared with known resistance-gene sequences, the percentage of amino-acid identity ranged from 18.6% to 48%. To facilitate genetic mapping of these sequences and to assess their potential linkage relationship with disease resistance genes in Poncirus, we developed CAPS markers by designing specific primers based on the cloned DNA sequences and subsequently identifying restriction enzymes that revealed genetic polymorphisms. Three of the amplified DNA fragment markers (designated as 18P33a, Pt9a, and Pt8a) were associated with the citrus tristeza virus resistance gene (Ctv), and one fragment (Pt8a) was associated with the major gene responsible for the citrus nematode resistance (Tyr1); both genes are from Poncirus and of importance to citrus survival and production. These polymorphic fragments were located on two local genetic linkage maps of the chromosome region from Ctv to Tyr1. These results indicate that resistance-gene candidate sequences amplified with the NBS-derived degenerate primers are valuable sources for developing markers in disease resistance-gene tagging, mapping, and cloning.

Inheritance of citrus nematode resistance and its linkage with molecular markers

Eleven RAPD markers linked to a gene region conferring resistance to citrus nematodes in an intergen-eric backcross family were identified. Two sequence- characterized amplified region markers linked to a citrus tristeza virus resistance gene and one selected resistance gene candidate marker were evaluated for their association with citrus nematode resistance. A nematode-susceptible citrus hybrid, LB6-2 [Clementine mandarin (Citrus reticulata)×Hamlin orange (C. sinensis)], was crossed with the citrus nematode-resistant hybrid Swingle citrumelo (C. paradisi×Poncirus trifoliata) to produce 62 hybrids that were reproduced by rooted cuttings. The plants were grown in a greenhouse and inoculated with nematodes isolated from infected field trees. The hybrids segregated widely for this trait in a continuous distribution, suggesting possible polygenic control of the resistance. Bulked segregant analysis was used to identify markers associated with resistance by bulking DNA samples from individuals at the phenotypic distribution extremes. Linkage relationships were established by the inheritance of the markers in the entire population. A single major gene region that contributes to nematode resistance was identified. The resistance was inherited in this backcross family from the grandparent Poncirus trifoliata as a single dominant gene. QTL analysis revealed that 53.6% of the phenotypic variance was explained by this major gene region. The existence of other resistance-associated loci was suggested by the continuous phenotypic distribution and the fact that some moderately susceptible hybrids possessed the resistance-linked markers. The markers may be useful in citrus rootstock breeding programs if it can be demonstrated that they are valid in other genetic backgrounds.

Development and characterization of SCAR markers linked to the citrus tristeza virus resistance gene from Poncirus trifoliata

Twelve new dominant randomly amplified polymorphic DNA (RAPD) fragments associated with a single dominant gene for resistance to citrus tristeza virus (CTV) were identified using bulked segregant analysis of an intergeneric backcross family. These and eight previously reported RAPDs were mapped in the resistance gene (Ctv) region; the resulting localized linkage map spans about 32 cM, with nine close flanking markers within 2.5 cM of Ctv. Seven of 20 RAPD fragments linked with the resistance gene were cloned and sequenced, and their sequences were used to design longer primers to develop sequence characterized amplified region (SCAR) markers that can be utilized reliably in marker-assisted selection, high-resolution mapping, and map-based cloning of the resistance gene. All seven cloned RAPDs were converted successfully into SCARs by redesigning primers, optimizing PCR parameters (especially the annealing temperature), or digesting amplification products with restriction enzymes. Four of the seven remained dominant markers, displaying presence-absence polymorphism patterns; the other three detected restriction site changes or length variations and thus were transformed into codominant markers. Two genomic regions rich in variability were also detected by two codominant SCAR markers.

Molecular markers flanking citrus tristeza virus resistance gene from Poncirus trifoliata (L.) Raf.

Two segregating populations for citrus tristeza virus (CTV) resistance derived from Poncirus trifoliata var ‘Flying Dragon’ by self-pollination and pollination to Citrus medica L. var ethrog ‘Arizona’ were inoculated with a common CTV isolate. The presence of virus was checked by the Double Antibody Sandwich Enzyme-Linked Assay and Direct Tissue Blot Inmunoassay at 3, 6, and 12 months after inoculation. Seven RAPDs were found linked to the CTV resistance gene by bulked segregant analysis. The closest linked RAPDs were cloned to obtain linked codominant RFLPs and to increase the precision of the genetic distance estimation. The CTV resistance gene seems to be located between cW18 and cK16. Differences in genetic distances among progenies are large and can be explained by genome-wide reduction in the recombination of progeny derived from male versus female gametes.

A localized linkage map of the citrus tristeza virus resistance gene region

A localized genetic linkage map was developed of the region surrounding the citrus tristeza virus (CTV) resistance gene (designated Ctv) from Poncirus trifoliate L., a sexually compatible Citrus relative. Bulked segregant analysis (BSA) was used to identify potential resistance-associated RAPD fragment markers in four intergeneric backcross families that were segregating for CTV resistance. Eight RAPD fragments were found that were consistently linked to Ctv in the four families. Map distances and locus order were determined with MAPMAKER 3.0, using the results obtained from 59 individuals in the largest family. Also, a consensus map was constructed with JOINMAP 1.3, using pooled results from the four backcross families. Marker orders were identical, except for 1 marker, on these independently developed maps. Family-specific resistance-associated markers were also identified, as were numerous susceptibility-associated markers. The identification of markers tightly linked to Ctv will enable citrus breeders to identify plants likely to be CTV-resistant by indirect, marker-assisted selection, rather than by labor-intensive direct challenge with the pathogen. These markers also provide a basis for future efforts to isolate Ctv for subsequent genetic manipulation.

A localized linkage map of the citrus tristeza virus resistance gene region

A localized linkage map of the citrus tristeza virus resistance gene region