BackgroundCitrus breeding programs have many limitations associated with the species biology and physiology, requiring the incorporation of new biotechnological tools to provide new breeding possibilities. Diversity Arrays Technology (DArT) markers, combined with next-generation sequencing, have wide applicability in the construction of high-resolution genetic maps and in quantitative trait locus (QTL) mapping. This study aimed to construct an integrated genetic map using full-sib progeny derived from Murcott tangor and Pera sweet orange and DArTseq™ molecular markers and to perform QTL mapping of twelve fruit quality traits. A controlled Murcott x Pera crossing was conducted at the Citrus Germplasm Repository at the Sylvio Moreira Citrus Centre of the Agronomic Institute (IAC) located in Cordeirópolis, SP, in 1997. In 2012, 278 F1 individuals out of a family of 312 confirmed hybrid individuals were analyzed for fruit traits and genotyped using the DArTseq markers. Using OneMap software to obtain the integrated genetic map, we considered only the DArT loci that showed no segregation deviation. The likelihood ratio and the genomic information from the available Citrus sinensis L. Osbeck genome were used to determine the linkage groups (LGs).ResultsThe resulting integrated map contained 661 markers in 13 LGs, with a genomic coverage of 2,774 cM and a mean density of 0.23 markers/cM. The groups were assigned to the nine Citrus haploid chromosomes; however, some of the chromosomes were represented by two LGs due the lack of information for a single integration, as in cases where markers segregated in a 3:1 fashion. A total of 19 QTLs were identified through composite interval mapping (CIM) of the 12 analyzed fruit characteristics: fruit diameter (cm), height (cm), height/diameter ratio, weight (g), rind thickness (cm), segments per fruit, total soluble solids (TSS, %), total titratable acidity (TTA, %), juice content (%), number of seeds, TSS/TTA ratio and number of fruits per box. The genomic sequence (pseudochromosomes) of C. sinensis was compared to the genetic map, and synteny was clearly identified. Further analysis of the map regions with the highest LOD scores enabled the identification of putative genes that could be associated with the fruit quality characteristics.ConclusionAn integrated linkage map of Murcott tangor and Pera sweet orange using DArTseq™ molecular markers was established and it was useful to perform QTL mapping of twelve fruit quality traits. The next generation sequences data allowed the comparison between the linkage map and the genomic sequence (pseudochromosomes) of C. sinensis and the identification of genes that may be responsible for phenotypic traits in Citrus. The obtained linkage map was used to assign sequences that had not been previously assigned to a position in the reference genome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3629-2) contains supplementary material, which is available to authorized users.

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BackgroundPhytophthora nicotianae Breda de Haan (Phytophthora parasitica Dastur) causes severe damage to citrus crops worldwide. A population of citrandarins was created from the cross between the susceptible parent Citrus sunki Hort. Ex Tan. and the resistant parent Poncirus trifoliata (L.) Raf. cv. Rubidoux, both parents and two reference rootstocks (Rangpur lime and Swingle citrumelo) were grafted in a greenhouse on Rangpur lime. Inoculations were performed at 10 cm and 15 cm above the grafting region and the resulting lesions were evaluated by measuring the lesion length 60 days after inoculation. As control, non-inoculated plants of each genotype were used. In addition, we evaluated the expression of 19 candidate genes involved in citrus defense response 48 h after pathogen infection by quantitative Real-Time PCR (qPCR). We mapped genomic regions of Quantitative Trait Loci (QTLs) and Expression Quantitative Trait Loci (eQTLs) associated with resistance to P. parasitica in the linkage groups (LGs) of the previously constructed maps of C. sunki and P. trifoliata.ResultsWe found disease severity differences among the generated hybrids, with lesion lengths varying from 1.15 to 11.13 mm. The heritability of the character was 65%. These results indicate that there is a great possibility of success in the selection of resistant hybrids within this experiment. The analysis of gene expression profile demonstrated a great variation of responses regarding the activation of plant defense pathways, indicating that citrandarins have several defense strategies to control oomycete infection. The information of the phenotypic and gene expression data made possible to detect genomic regions associated with resistance. Three QTLs and 84 eQTLs were detected in the linkage map of P. trifoliata, while one QTL and 110 eQTLs were detected in C. sunki.ConclusionsThis is the first study to use eQTLs mapping in the Phytophthora-citrus interaction. Our results from the QTLs and eQTLs mapping allow us to conclude that the resistance of some citrandarins to the infection by P. parasitica is due to a favorable combination of QTLs and eQTLs transmitted by both parents.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-4888-2) contains supplementary material, which is available to authorized users.

Huanglongbing (HLB), caused mainly by ‘Candidatus Liberibacter asiaticus’ (CLas), is the most devastating citrus disease because all commercial species are susceptible. HLB tolerance has been observed in Poncirus trifoliata and their hybrids. A wide-ranging transcriptomic analysis using contrasting genotypes regarding HLB severity was performed to identify the genetic mechanism associated with tolerance to HLB. The genotypes included Citrus sinensis, Citrus sunki, Poncirus trifoliata and three distinct groups of hybrids obtained from crosses between C. sunki and P. trifoliata. According to bacterial titer and symptomatology studies, the hybrids were clustered as susceptible, tolerant and resistant to HLB. In P. trifoliata and resistant hybrids, genes related to specific pathways were differentially expressed, in contrast to C. sinensis, C. sunki and susceptible hybrids, where several pathways were reprogrammed in response to CLas. Notably, a genetic tolerance mechanism was associated with the downregulation of gibberellin (GA) synthesis and the induction of cell wall strengthening. These defense mechanisms were triggered by a class of receptor-related genes and the induction of WRKY transcription factors. These results led us to build a hypothetical model to understand the genetic mechanisms involved in HLB tolerance that can be used as target guidance to develop citrus varieties or rootstocks with potential resistance to HLB.

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Huanglongbing (HLB) is a severe disease of citrus caused by the bacterium Candidatus Liberibacter. In America, the most common species is Candidatus Liberibacter asiaticus (CLas). In a previous study of the Citrus-HLB pathosystem, our group found differences in CLas multiplication in a population of hybrids obtained by hybridization between Citrus sunki Hort. Ex Tan. and Poncirus trifoliata (L.) Raf. cv. Rubidoux. The bacterial concentration was higher in C. sunki than in P. trifoliata. Thus, this study aims to map phenotypic (QTL) and gene expression (eQTL) data associated with host response to CLas in the linkage groups (LGs) of the previously constructed parental maps of C. sunki and P. trifoliata cv. Rubidoux. For the phenotypic analysis, we used a population of 79 F1 hybrids (termed citrandarins) between C. sunki and P. trifoliata. CLas and starch were quantified in the leaves of the plants two years after pathogen inoculation, allowing the classification of hybrids as resistant, tolerant, and susceptible. The expression of 14 candidate genes was measured in 72 hybrids of the population and used as expression data for the eQTL mapping. We located nine QTL and 52 eQTL in the C. sunki map and 17 QTL and 40 eQTL in the P. trifoliata map. The overlap of eQTL of the majority of genes with QTL from the phenotypic data indicates that the genes are related to the phenotype and are probably related to pathogen infection.

Citrus plants have been extremely affected by Huanglongbing (HLB) worldwide, causing economic losses. HLB disease causes disorders in citrus plants, leading to callose deposition in the phloem vessel sieve plates. Callose is synthesized by callose synthases, which are encoded by 12 genes (calS1-calS12) in Arabidopsis thaliana. We evaluated the expression of eight callose synthase genes from Citrus in hybrids between Citrus sunki and Poncirus trifoliata infected with HLB. The objective of this work was to identify possible tolerance loci combining the expression quantitative trait loci (eQTL) of different callose synthases and genetic Single-Nucleotide Polymorphism (SNP) maps of C. sunki and P. trifoliata. The expression data from all CscalS ranged widely among the hybrids. Furthermore, the data allowed the detection of 18 eQTL in the C. sunki map and 34 eQTL in the P. trifoliata map. In both maps, some eQTL for different CscalS were overlapped; thus, a single region could be associated with the regulation of more than one CscalS. The regions identified in this work can be interesting targets for future studies of Citrus breeding programs to manipulate callose synthesis during HLB infection.

Citrus orchards have some limitations, such as the occurrence of phytosanitary problems. Alternaria brown spot (ABS) is caused by fungus Alternaria alternata, which affects several parts of the plant by producing a host-specific toxin, known as ACT. ABS is a limiting factor in orchards due to the susceptibility of most planted cultivars: ‘Murcott’ tangor and ‘Ponkan’ tangerine. The selection of varieties resistant/tolerant to the disease has economic importance. Therefore, the aim of this experiment was to evaluate the response to A. alternata inoculation in a population of ‘Murcott’ tangor vs ‘Pera’ sweet orange hybrids. Leaves of 2-3 centimeters in length of ‘Murcott’ tangor, ‘Pera’ sweet orange, ‘Ponkan’, ‘Dancy’, ‘Fremont’ tangerine and 198 hybrids were collected. For in vitro inoculation, monosporic A. alternata culture at concentration of 105 conidia mL-1 was used. Inoculated leaves were stored in humid chamber. After 24, 48 and 72 hours of inoculation, leaf lesions were evaluated following a diagrammatic scale. The results obtained showed that most hybrids from the crossing of ‘Murcott’ tangor vs ‘Pera’ sweet orange are susceptible to ABS. However, 44 are resistant and ten are tolerant. Among ABS-tolerant hybrids, some have phenotype similar to that of cultivated and commercialized hybrids.