In A. squamosa, for example, the beginning of fowering of adult plants, originated from seeds, under specific cultivation conditions, would take at least three years until the characterization of the fruits, limiting the number of genotypes that can be studied, which makes the breeding process slow and expensive. The PCR assay using a combination of the primers detecting the wild-type and mutant alleles allows plants to be genotyped in the seedling stage, accelerating the breeding program. Given the generality of the INO gene detecting primers for use in multiple species of Annona , and the fertility of interspecific crosses in this genus, our codominant genotyping is applicable to introgression of the seedless trait into elite sugar apple varieties and into other cultivated Annona species.Understanding host responses to pathogen infection is essential to clarify the mechanisms of plant-microbe interactions and to develop novel strategies for therapy. Studies have been conducted to identify key genes and proteins induced by HLB in leaf tissues. These studies showed that key pathways and processes such as cell defense, transport, photosynthesis, carbohydrate metabolism, and hormone metabolism are affected by the disease. Clarifying host responses and disease development in fruit peel and leaves is critical for disease detection at the earliest stages.

CaLas is typically found in leaves, container size for blueberries but can be present in bark, root, flower , and fruit of infected trees. Although pathogen detection is typically conducted on leaf tissues, fruit peel can also be employed for the analysis of host responses. The analysis of host responses in both leaves and fruits is important to understand the mechanisms of the fruit disorder and tree decline induced by the disease.CaLas-infected peel tissues often show a characteristic color inversion as the fruit changes from green to yellow/orange. HLB also results in fruits that are small, asymmetric, and lopsided, with a bent fruit axis, small or aborted seeds, and a strong yellow to brown stain in vascular bundles within the axis at the peduncular end. Microarray technology has been used in numerous studies of host response to infection by pathogens including bacteria, viruses, and fungi. However, this technology can reveal the expression of only those genes represented on the array. Possible misleading interpretations of microarray results can occur due to non-specific hybridization. Next-generation DNA sequencing technology can reveal very rare and unknown transcripts, offering a more precise and accurate picture of the transcriptome. These tools, already applied to plants, assume extensive prior knowledge of the organism under investigation. For plant species that lack whole-genome sequence information, an extensive EST database is required.

Indeed, data obtained are usually confirmed with qRT-PCR analysis or integrated with proteomic and metabolomic analyses. In addition, analysis of the deep transcriptome profile using biological network theory can help define gene regulatory networks. Protein networks are increasingly used to describe the molecular basis of disease related subnetworks and to define protein-protein interaction networks that regulate disease resistance in plants and plant pathogen interactions. Bioinformatic tools are now available for visualizing and characterizing statistical properties of these networks . At present, no therapeutic treatments are available for HLB, and removal of infected trees and insect control are the main management strategies to limit or prevent its spread. Traditional diagnostic approaches rely on symptom recognition in the field, confirmed by PCR based on primers developed for individual Candidatus Liberibacter species. These practices were very useful to speed up pathogen detection and accelerate management procedures, although the pathogen may elude detection at asymptomatic stages. This is probably due to the fact that the pathogen is phloem-limited and not uniformly distributed within the tissues of infected trees. This study examines global changes in host gene expression due to CaLas infection in fruit peel. It aims to elucidate metabolic changes induced by the disease in the fruit. Using next generation sequencing technology, mRNA transcripts from fruit peel sample types representing various stages of disease were compared.

Fruits displaying symptoms were compared to asymptomatic fruits from the same tree and to apparently healthy fruits taken from trees free of HLB symptoms in the same orchard. Fruits were categorized into one of the three stages based on qRT-PCR in addition to commonly observed symptoms.Between 24 and 41 million 85-nt paired-end reads were obtained from each of four cDNA libraries derived from mature fruit peel at different disease stages: fruit peel from uninfected trees in an orchard with no HLB present; fruit peel from apparently healthy trees in an orchard with HLB; symptomatic and asymptomatic fruit peel from the same trees infected with CaLas . These reads were aligned to the NCBI citrus unigene set, with 42 to 46% of reads per sample mapping to a unigene. Six pairwise comparisons were made between symptomatic, asymptomatic, apparently healthy, and healthy control fruit peel to calculate changes in expression of individual genes . The comparison between asymptomatic and symptomatic stages of the disease identifies genes related to the appearance of symptoms. Interestingly, the overall expression profiles of apparently healthy and asymptomatic fruit peel were very similar, indicated by fewer differentially expressed transcripts in this comparison. The four fruit types were also tested for the presence of Citrus Tristeza Virus using CTV CP reference sequence T36 to show cross-responses to multiple pathogens. CTV was detected in all four sample types. No significant differences were observed among the three fruit categories from the infected orchard .Linear combination of transcriptomic data generated vectors or groups to best explain overall variance in the data set without prior assumptions about whether and how clusters might form. It was clearly evident that apparently healthy and asymptomatic fruits showed close similarities and were separated from the other two fruit types. Healthy fruits and symptomatic fruits were clearly distinguished from each other. The 21 target genes most specific to each category are listed in Table S7. Three complementary methods were used for functional analysis of the transcriptomic data: Fisher’s Exact Test and Gene Ontology descriptions, PageMan gene set enrichment analysis, and pathway enrichment analysis using Pathexpress. The Fischer Exact Test as provided in Blast2GO is useful to determine the specific GO terms affected by the disease. Some GO terms were significantly over-represented among differentially expressed genes obtained from pairwise comparisons . Among these, several GO terms associated with cell wall bio-genesis, modification, and organization and related metabolic processes were over-represented among more abundant transcripts in healthy control fruit than in apparently healthy, asymptomatic, or symptomatic fruit. Interestingly, GO terms related to photosynthetic reactions were over-represented in fruits showing the typical symptoms of HLB infection. Over-represented GO terms in four pairwise comparisons were analyzed to determine which GO terms correlated significantly with HLB disease . It is interesting to note that when symptoms are clearly evident, GO terms of small carbohydrate metabolism and other important pathways of primary metabolism such as the pentose-phosphate cycle are over-represented. As expected from visual analysis of fruit symptoms, many gene functions related to photosynthesis were over-represented at the symptomatic stage compared with all other conditions. GO terms for ion transport were also over-represented at the symptomatic stage. The high number of GO terms for oxidoreductase activity is consistent with the hypothesis that the disease induces oxidative stress. Several categories of GO terms involved in vesicle transportand cell wall bio-genesis, metabolism, raspberry grow in pots and organization were over represented among genes expressed at the symptomatic stage. Interesting defense-related and lipid transport GO terms were over-represented at the asymptomatic stage. PageMan software was used to visualize functional classes that were significantly affected by HLB disease . This method pinpoints which subcategory of genes were upregulated and downregulated in each main gene category based not only on metabolic pathways but also on cell functions. Increased expression in diseased samples is seen in photosynthesis, N-metabolism, amino acid synthesis, isoprenoids, jasmonate and salicylic acid, and several transcription factors.

Functional classes with decreased expression include sucrose and starch biosynthesis, glycolysis, gibberellins, DNA and protein synthesis, and flavonoids metabolism. Additional changes in expression can be seen in the comparison between samples from the disease-free location and apparently healthy fruits . These changes, eventually affected by environmental variability, probably were induced in early stages of HLB disease. Differential expression of transcripts comparing different stages of HLB infection and their functions were visualized using MapMan software. This provided more specific information on pathways and functions identified by Fisher’s Exact Test and PageMan. For MapMan data analyses, a mapping file composed of NCBI Citrus sinensis unigenes was used. The third enrichment method was conducted using the Pathexpress web-tool to determine which metabolic pathways were significantly affected by the disease by comparing symptomatic and asymptomatic fruits . The MapMan graphical metabolic overview identifies transcripts that are differentially expressed in symptomatic and apparently healthy fruit, with each colored square representing a single annotated gene in a particular pathway . Several genes involved in light reactions of photosynthesis, mitochondrial electron transport, sucrose metabolism, glycolysis, and fermentation were up-regulated. In contrast, gene transcripts for cell wall modification and degradation, pectin esterase activity, and cellulose synthesis were mostly downregulated. We also identified several differentially expressed genes involved in secondary metabolic pathways, including terpenes, flavonoids, and phenylpropanoids.The expression of several genes involved in photosynthesis and carbohydrate metabolism increased significantly in symptomatic fruits, when compared to asymptomatic fruit from the same tree or different trees. Transcripts for oxygen-evolving enhancer 3 , photosystem II subunit Q-2, photosystem II reaction center protein J, and other genes encoding different subunits of photosystem II were highly abundant in symptomatic fruit. In addition, genes encoding subunits of cytochrome b6/f and genes encoding ATP synthase subunits were induced . Several transcripts encoding subunits of photosystem I increased, including chlorophyll A apoprotein subunit G, photosystem I reaction center subunit , and D1 subunit. Several genes for enzymes involved in the first steps of glycolysis were differentially expressed. Up-regulation was observed for genes encoding protein serine/threonine kinase and fructose-2,6- biphosphatase . There were significant changes in transcripts related to carbohydrate metabolism in symptomatic and asymptomatic fruit. The citrus orthologs of two Arabidopsis genes encoding different isoforms of invertase were induced in symptomatic fruit. In starch metabolism, transcription of glucose-1-phosphate adenylyltransferase was diminished, while several genes involved in starch degradation were significantly differentially expressed . Expression of genes involved in raffinose synthesis were differentially regulated, while transcripts involved in galactinol metabolism were abundant in symptomatic fruits .Significant transcriptional changes in response to CaLas infection were observed for a group of genes involved in hormone biosynthesis, mobilization, and signal transduction. Log fold ratios for differentially expressed genes in symptomatic fruits compared to apparently healthy and asymptomatic fruits are shown . Transcripts related to GH3-like proteins involved in auxin synthesis were more abundant in symptomatic fruit. Transcripts for GRAM-domain containing protein, involved in the abscisic acid pathway, were more abundant. Several genes involved in ethylene biosynthesis and signal transduction were upregulated in HLB-affected fruit including ACO4, ethylene receptor 1 , ethylene response element binding factors , ethylene forming enzyme , while ACO1 and an ethylene-responsive protein were downregulated. Interestingly, genes for ATHK1 and Snakin-1, involved in the cytokinin and gibberellin pathways, were repressed in infected fruit .Real-time PCR analyses were conducted using all four fruit types to validate the expression patterns of a subset of differentially expressed genes identified by next generation sequencing . Among hormone-related transcripts, indole-3-acetic acid amido synthetase was expressed more at all disease stages. Ethylene responsive factor 1 expression was higher at the symptomatic stage. Transcripts for ent-kaurenoic acid hydroxylase 2 and gibberellin-responsive protein GASA1 were less abundant in infected fruits. The jasmonic acid and salicylic acid pathways were affected by HLB disease. Transcript abundance of lipoxigenase2 , which is involved in jasmonate biosynthesis, was lower in fruit from infected trees at asymptomatic and symptomatic stages than in healthy trees from the disease-free location. Salicylic acid methyltransferase was strongly up-regulated in infected fruits. Transcription factors are key players in transducing signals generated in response to pathogen infection. Among them, the WRKY family was highly involved in HLB response. WRKY70 was highly up-regulated in symptomatic fruits and its transcript abundance was also significantly higher in asymptomatic, apparently healthy fruit. Transcripts of RD26, NAC-1, and Myb-related transcription factors were more abundant at asymptomatic stages. Heat shock protein 82 , the hub with the highest degree in the protein-protein interaction network, was down-regulated in all infected fruits, with likely consequences for overall fruit metabolism.