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Citrus, one of the most economically important fruit crops in the world, is commercially propagated through grafting. Varying scion-rootstock combinations cause substantial effects on trees that often influence yield and fruit quality traits. Presently, the explanation for these differences has not been extensively studied at the molecular level. One potential reason for rootstock effects on fruit quality is the presence of small RNAs, molecules known to affect gene expression and plant development.It was hypothesized that grafting diverse rootstocks influences small RNA populations in citrus, which can greatly impact fruit quality. By using mRNA-seq and small RNA-seq, an integrative analysis of microRNA and mRNA expression profiles was performed on grafted citrus. In this study, fruit and root samples were collected from sweet orange scions grafted onto four genetically differing rootstocks (trifoliate orange, Carrizo citrange, rough lemon, and sweet orange). Differentially expressed microRNA (DEM) and mRNA profiles were identified according to fold change analysis and the relationships between these molecules were identified.A total of 1,633 unique genes in the fruit tissues and 11,368 in the root tissues were identified as differentially expressed between genotypes in at least one collection date during fruit development. The majority of the differences came from the comparisons between trifoliate and rough lemon rootstocks. GO and KEGG functional annotation analyses revealed that genes differentially expressed between trifoliate and rough lemon rootstocks were related to defense response, cell wall organization and biosynthesis, and carbohydrate metabolism. DEGs in fruit tissues grown on these rootstocks were related to transcription regulation and plant hormone signal transduction.The small RNA sequencing data identified 603 known, conserved miRNAs. Of these, 188 were differentially expressed between trifoliate and rough lemon rootstocks. Reads that were not homologous to any know plant miRNAs were assessed as potentially novel small RNAs. 17 potential novel microRNAs were identified in citrus, ten of which were differentially expressed between genotypes. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis confirmed the results from the sequencing data and revealed a negative correlation between several DEMs and their respective target genes, whose function may play a crucial role in citrus fruit development.

Additional file 5: Supplementary Fig. S1. Phylogenetic analysis of Spiroplasma strains. Maximum-likelihood phylogeny of Spiroplasma based on core orthologous genes. In total, 601 orthologous genes were concatenated, and a maximum-likelihood approach was used to generate the phylogeny with 1000 bootstrap replicates. Bootstrap values are indicated at each node. The resulting phylogeny was visualized using FigTree v.1.4.3 [70]. S. citri strains analyzed in this report are underlined.

Additional file 7: Supplementary Fig. S3. Predicted prophage sequences in Spiroplasma citri genomes. Prophage annotation was performed by PHASTER (PHAge Search Tool – Enhanced Release). The program predicts the completeness of the predicted prophages, based on the proportion of phage genes in the identified region. Green bars are scored as intact (score > 90); blue bars are questionable (score 70–90); red bars are incomplete (score

Additional file 6: Supplementary Fig. S2. Dot-Matrix representation of a BLASTn comparison of complete Spiroplasma citri genome sequences. The chromosome of each newly assembled strain (X-axis) was compared to the reference sequence, strain R8-A2 (Y-axis). A) C189; B) LB 319; C) BR12; D) CC-2; E) C5; F) BLH-13; G) BLH-MB.