Response of citrus hybrids to Alternaria alternata inoculation

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.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

QTL mapping for fruit quality in Citrus using DArT-seq markers

A incorporação de novas ferramentas biotecnológicas aos programas de melhoramento de citros oferece inúmeras possibilidades. Os marcadores DArTTM (Diversity Arrays Technology), combinados à técnica de sequenciamento de última geração, apresentam boa aplicabilidade na construção de mapas genéticos de alta resolução e no mapeamento de QTL (Quantitative Trai Loci). Assim, este estudo teve como objetivo construir um mapa genético integrado de tangor \'Murcott\' e laranja \'Pera\' usando os marcadores moleculares do tipo DArT_seqTM e localizar QTLs para doze caracteres de qualidade de frutos. A partir de um cruzamento controlado entre tangor \'Murcott\' e laranja \'Pera\', realizado no banco de germoplasma de Citros do Centro de Citricultura \"Sylvio Moreira\", Instituto Agronômico de Campinas, localizado em Cordeirópolis-SP, em 1997. Foi obtida uma família de 350 indivíduos híbridos, dos quais 278 foram selecionados para avaliação das características de fruto em 2012. No presente trabalho, esses 278 indivíduos foram genotipados usando os marcadores DArTseqTM. Para construir o mapa integrado foi utilizado o programa OneMap e foram considerados apenas os marcadores que não apresentaram desvio de segregação mendeliana. A razão de verossimilhança foi utilizada para a formação de grupos de ligação, além da informação genômica obtida a partir do genoma sequenciado de Citrus sinensis L. Osbeck disponível em (http://citrus.hzau.edu.cn/orange/index.php). O mapa parcialmente integrado foi composto de 661 marcadores, ligados em 13 grupos, que correspondem ao número haplóide de cromossomos da espécie, com cobertura genômica de 2.774 cM. De acordo com as análises de mapeamento por intervalo composto e os resultados do teste de permutação, um total 19 QTLs foram identificados, tendo em conta as características de fruto analisadas: peso (g), diâmetro (cm), altura (cm), relação diâmetro / altura, espessura da casca (cm), número de gomos, teor de sólidos solúveis (°Brix), acidez titulável (%), rendimento de suco (%), número de sementes, valor do índice tecnológico (IT) e número estimado de frutos por caixa. O mapa genético integrado foi comparado com o genoma (pseudochromosomes) de Citrus sinensis e sintenias foram claramente identificadas. A análise mais aprofundada das regiões genômicas (QTLs) apresentando os maiores valores de LOD score permitiu identificar a presença de genes candidatos que podem estar associados com as características analisadasThe incorporation of new biotechnological tools to citrus breeding programs provides many new possibilities. The DArT markers (Diversity Arrays Technology), combined with Next-Generation Sequencing presents good applicability in the construction of high resolution genetic maps and QTL mapping. This study aimed to construct an integrated genetic map of \'Murcott\' tangor and \'Pera\' sweet orange using the DArTseqTM molecular markers, and localize QTL for twelve fruit quality traits. A controlled cross between \'Murcott\' tangor and \'Pera\' sweet orange was conducted at the Citrus Germplasm Bank at the \"Sylvio Moreira\" Citrus Centre of Agronomic Institute, located in Cordeirópolis-SP in 1997.A family with 350 hybrid individuals was obtained, from which 278 were selected for evaluation of fruit traits in 2012. In this study, the 278 F1 individuals were genotyped using the DArTseqTM markers. To build the integrated map, we used the OneMap program and considered all DArT loci that showed no segregation deviation. The likelihood ratio was used for formation of linkage groups, besides the genomic information obtained from the available Citrus sinensis genome sequence (http://citrus.hzau.edu.cn/orange/index.php). The partially integrated map contained 661 markers linked in 13 linkage groups, with genomic coverage of 2,774 cM, the map is saturated and represent the species haploid chromosome number. According to the analyses using \"Composite Interval Mapping\" (CIM) and the results of the permutation test, a total of 19 QTL were identified for the 12 fruits characteristics analyzed: diameter (cm), height (cm), ratio of H/D, weight (g), rind thickness (cm), segments per fruit, total soluble solids ((°Brix), Acidity (%), Juice content (%), number of seeds, ratio of total soluble solids /acidity and number of fruits per box. The genome sequence (pseudochromosomes) of Citrus sinensis L. Osbeck was compared to the genetic map, and sinteny was clearly identified. Further analysis of the map regions with the highest LOD score enabled the identification of the presence of genes that could be associated with the characteristics. The genome sequences allowed identification of genes that may respond for phenotypic traits in Citru

QTLs and eQTLs mapping related to citrandarins’ resistance to citrus gummosis disease

Abstract Background Phytophthora 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. Results We 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. Conclusions This 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

Additional file 2: of QTLs and eQTLs mapping related to citrandarins’ resistance to citrus gummosis disease

Gene expression data with mean Ct and SD for 19 candidate genes. (XLS 2436 kb

Additional file 5: of QTLs and eQTLs mapping related to citrandarins’ resistance to citrus gummosis disease

Gene, linkage group (LG), flanking markers, position of the eQTL in centiMorgans (cM position), LOD score, proportion of phenotypic variation (R2) explained by eQTL in % and additive effect by the eQTLs mapping in the Citrus sunki map. (XLSX 18 kb

Additional file 3: of QTLs and eQTLs mapping related to citrandarins’ resistance to citrus gummosis disease

Real lesion length (RLL) caused from the inoculation with P. parasitica in the individuals of the experiment. (XLSX 12 kb

Additional file 1: of QTLs and eQTLs mapping related to citrandarins’ resistance to citrus gummosis disease

Selected genes for RT-qPCR assays. (XLSX 11 kb

Additional file 4: of QTLs and eQTLs mapping related to citrandarins’ resistance to citrus gummosis disease

Gene, linkage group (LG), flanking markers, position of the eQTL in centiMorgans (cM position), LOD score, proportion of phenotypic variation (R2) explained by eQTL in % and additive effect by the eQTLs mapping in the Poncirus trifoliata map. (XLSX 16 kb

Additional file 1: Table S1. of QTL mapping for fruit quality in Citrus using DArTseq markers

DArTseq dataset used for BLASTN analysis to compare the integrated linkage map constructed with the Citrus sinensis reference genome. (XLSX 38 kb