We tested the normality of the data with qq-plots and Kolmogorov–Smirnov tests of model residuals. We conducted all statistical analyses with SPSS .Our study represents one of the first field experiments showing that a broad survey of ants reduce colonization of coffee berries by the CBB. This is in contrast to previous studies that suggest ants may not have any effects on CBB, especially in field experiments . Our results are in accordance with other observational studies that show that specific ant species may limit CBB in coffee plantations, yet these studies have either focused on the most dominant or abundant species observed or investigated the broad community-wide impacts of ants on the CBB . Our experimental approach is limited to our understanding of how ants control CBB colonization of berries and not other life stages of the CBB. Our study suggests that ant occupation of coffee bushes is very important during a seasonal period when new coffee berries develop and the CBB begins to disperse from old infested berries to developing un-infested berries . It is surprising that Crematogaster spp. and S. picea did not limit the colonization of berries, considering that other studies have shown species within these two genera have important effects on herbivores . Low ant activity on coffee bushes with Crematogaster spp. or S. picea cannot explain these results because thesespecies had greater activity per branch than P. ejectus and P. simplex and equivalent activity to A. instabilis and P. synanthropica, species that did limit CBB damage.
One explanation could be that because we grouped five Crematogaster spp. together into a single treatment, round plastic planter effects of individual species may be masked. Solenopsis picea may have an effect on CBB colonization, but only with higher ant activity or when CBB are in closer proximity to nest entrances. This species also has a small body size and moves relatively slowly in comparison to the species that did have an effect, which might have limited it from removing or easily capturing CBBs. Wasmannia auropunctata is of similar size to S. picea and still had strong effects on CBB. However, W. auropunctata had significantly higher ant activity on branches as compared to S. picea. Perhaps the combination of low activity, small body size, and slower movement limited S. picea from affecting the CBB. While we found no effect of S. picea on CBB colonization of berries, it may be that S. picea, and other smaller ants, have important impacts on the CBB at other stages of the CBB life cycle because they can pass into entrance holes of the CBB . Experiments with both P. simplex and P. ejectus employed slightly different methodologies than the other ant species, which may have intensified the effect of these ants. For these two species, hollow twigs that contained ants were attached to a branch with berries and this branch was used as the control branch in the experiment. This likely elevated the number of ants per branch per minute. However, in the lab, P. simplex had similar effects on the CBB . Additionally these two species had the lowest densities on control branches of all other species, averaging 3.6 and 3.7 ants per branch for P. ejectus and P. simplex, respectively. Thus, these species have effects at very low numbers, and the results of this study should only pertain to branches for which the density of these species reaches this mark. Certain aggressive ants that limit CBB colonization of berries might also benefit CBB after colonization.
Larger ants cannot enter berries, but if they are aggressive competitors for space, they will prevent other ants from occupying the branches they patrol . These ants, likely A. instabilis and P. synanthropica, may provide CBB with enemy free space after the CBBs colonize berries in their territories. In conclusion, we find that six of eight ant species limited CBB colonization of coffee berries suggesting that ants, generally, provide important pest control services within coffee agroecosystems. This is the first field experiment to demonstrate general ant limitation of CBB colonization. This finding is important considering that chemical pesticides are thought to be ineffective at controlling the CBB . Nonetheless, ants do not completely control the CBB, other control agents like birds, parasitoids, and fungal pathogens also aid in the control of the CBB . Further work should look at larger scale impacts of ants on the CBB, such as farm scale impacts. Also, more theoretical work is needed to understand how ants impact the CBB at different stages of its life cycle and to reveal which stage of the life cycle is most important for population regulation. Nonetheless, this study provides strong evidence that ants defend coffee from CBB colonization.Grape berries undergo a series of complex physiological and biochemical changes during their development that determine their characteristics at harvest . Genome-wide expression studies using microarray and, more recently, RNA sequencing revealed that berry development involves the expression and modulation of approximately 23,000 genes and that the ripening transition is associated with a major transcriptome shift .
Transcriptomic studies characterized the ripening program across grapevine cultivars , identifying key ripening-related genes and determining the impact of stress and viticultural practices on ripening . This knowledge increases the possibility of exerting control over the ripening process, improving fruit composition under suboptimal or adverse conditions, and enhancing desirable traits in a crop with outstanding cultural and commercial significance . These genome-wide expression analyses were possible because a highly contiguous assembly for the species was produced ;this first effort used a grape line created by several rounds of back crossing to reduce heterozygosity, facilitating genome assembly . Though poor by current standards, this pioneering, chromosome-resolved assembly served as the basis for numerous publications. However, the structural diversity of grape genomes makes using a single one-size-fits-all reference genome inappropriate . There is substantial unshared gene content between cultivars, with 8–10% of the genes missing when two cultivars are compared . Although many of these genes are not essential for plant survival, they can account for 80% of the expression within their respective families and expand key gene families possibly associated with cultivar-specific traits . Assembling genome references for all interesting cultivars is impractical, in part because its cost remains prohibitive and because of genomic features that impede the development of high-quality genome assemblies for any grape cultivar. Although the V. vinifera genome is relatively small and as repetitive as other plant genomes of similar size , it is highly heterozygous . Most domesticated grape cultivars are crosses between distantly related parents; this and clonal propagation cause the high heterozygosity observed in the species . Earlier attempts using short reads struggled to resolve complex, highly heterozygous genomes . A limited ability to call consensus polymorphic regions yields highly fragmented assemblies where structural ambiguity occurs and alternative alleles at heterozygous sites are excluded altogether . Single Molecule Real Time DNA sequencing has emerged as the leading technology for reconstructing highly contiguous, diploid assemblies of long, repetitive genomes that include phased information about heterozygous sites . Recently, we used Vitis vinifera cv. Cabernet Sauvignon to test the ability of SMRT reads and the FALCON-Unzip assembly pipeline to resolve both alleles at heterozygous sites in the genome . The assembly produced was significantly more contiguous than the original PN40024 assembly and provided the first phased sequences of the diploid V. vinifera genome . Despite recent advances in genome reconstruction methodologies, round plastic plant pot assembling a complex plant genome is still costly. Transcriptome reconstruction is the only alternative strategy to depict known and unknown gene content information . De novo assembly of RNA-seq reads is widely used for this purpose . SMRT technology was recently deployed to investigate expressed gene isoforms in a variety of organisms, including a handful of plant species . Long reads delivered by this methodology report full-length transcripts sequenced from their 59-ends to polyadenylated tails , making Iso-Seq an ideal technology for reconstructing a transcriptome without a reference genome sequence and without assembling fragments to resolve the complete isoform sequence . Moreover, alternative transcripts that contribute to the gene space complexity and vary with cell type , developmental stage , and stress cannot be definitively characterized without full-length transcript information.
The objective of this study was to test whether full-length cDNA sequencingwith Iso-Seq technology is a suitable alternative to traditional genome sequencing, assembly, and annotation for reconstructing a grape transcriptome reference for transcriptional profiling. We compared how Cabernet Sauvignon’s Iso-Seq transcriptome fares as a reference for RNA-seq analysis vs. its annotated genome. We sequenced the full-length transcripts of ripening berries with Iso-Seq and Illumina RNA-seq reads. The high-coverage short-read data were used to profile gene expression and to error-correct low-expression isoforms that would have been otherwise lost by the standard Iso-Seq pipeline. The transcriptome reference built with Iso-Seq data represented most of the expressed genes in the grape berries and included cultivar specific or “private” genes. When used as the reference for RNAseq, Iso-Seq generated transcriptome profiles quantitatively similar to those obtained by mapping on a complete genome reference. These results support using Iso-Seq to capture the gene space of a plant and build a comprehensive reference for transcriptional pro- filing without a pre-defined reference genome.To obtain a comprehensive representation of the transcripts expressed during berry development, we isolated RNA from Cabernet Sauvignon berries before the onset of ripening , at véraison , after véraison , and at commercial ripeness . To avoid loading bias, cDNAs were fractionated based on their length to produce four libraries at each developmental stage in size ranges of 1-2 kbp, 2-3 kbp, 3-6 kbp, or 5-10 kbp . Libraries derived from different developmental stages were barcoded and libraries with similar cDNA size were pooled together. Each library pool was sequenced independently on two SMRT cells of a Pacific Biosciences Sequel system generating a total of 23.6 Gbp. In parallel, the same samples were sequenced using Illumina technology to provide high-coverage sequence information for error correction and for gene expression quantification . Demultiplexing, filtering and quality control of SMRT sequencing data were performed using SMRT Link as described in the Methods section. A total of 672,635 full-length non-chimeric reads with a maximum length of 14.6 kbp and a N50 of 3.5 kbp were generated . FLNC reads were further polished and clustered into 46,675 single representatives of expressed transcripts ranging from 400 bp to 8.8 kbp with a N50 of 3.6 kbp . The alignment of FLNC reads and PCIRs to the genomic DNA contigs of the same Cabernet Sauvignon clone confirmed that sequence clustering and polishing successfully increased sequence accuracy, whose median values were 95.4% in FLNC reads and 99.6% in the PCIRs. The increase in sequence accuracy was also reflected by the significantly longer detectable coding sequences in the PCIRs compared to the short and fragmented CDS found in the FLNC reads . The residual sequence discrepancy between PCIRs and the genomic contigs could be explained by heterozygosity and/or sequencing errors, but unexpectedly not by expression level . Over 18.5% of the FLNC reads did not cluster with any other reads and were discarded by the SMRT Link pipeline. When mapped on the genomic contigs, the uncorrected reads displayed a sequence accuracy that reflected the typical error rate of 10–20% of the technology . To recover the information carried by these 124,185 uncorrected FLNCs, which represented an important fraction of the transcriptome , we error-corrected their sequences with LSC using the short reads generated with Illumina technology. As for the PCIRs, error correction resulted in greater sequence accuracy and longer CDS . PCIRs and error-corrected FLNC reads were finally combined into a single dataset of 170,860 corrected Iso-Seq isoforms . As low as 1.7% of the CISIs showed significant homology with interspersed repeats. LTRs and LINEs were the most abundant orders with 778 and 729 representatives, respectively. Chloroplast and mitochondria genes represented a small fraction of the CISIs with only 89 isoforms having a significant match . CISIs were also searched for non-coding RNA using the covariance models of the Rfam database; only 182 isoforms were annotated as ncRNAs and were all ribosomal RNA . Excluding these transcribed isoforms, only 164 CISIs failed to align to the Cabernet Sauvignon genomic contigs, confirming the completeness of the genome assembly and the negligible biological contamination of the berry samples.