Genomics to Genetics

The development of cherry fruit follows a well-defined pattern that ends in a desirable, healthy and valuable product. Associated with development is a large scale change in gene expression pattern with differential regulation. Next-generation sequencing provides the tools for examining this process at a level of detail never before possible. This study constitutes three parts: sequence discovery to provide a reference transcriptome ; RNA-Seq analysis across fruit development; and validation of the RNA-Seq data as a measure of gene expression changes during development. A sweet cherry transcriptome was constructed from RNA libraries made from multiple stages of leaf, buds and fruit tissues. Assembly was done using SOAPdenovoTrans, producing more than 30k scaffolds over 500 bp and an N50 over 1500. Predicted orfs were compared to the peach model and over 12k cherry transcripts matched to peach in sequence and peptide size. RNA-Seq was conducted from 20 libraries spanning the entire fruit developmental timeframe. Alignment of these reads to the cherry transcriptome produced between five and seven million matches for each library. Read counts were roughly normalized by dividing by total counts and expression changes graphed for genes identified by homology to the peach model. Gene expression was further characterized by quantitative PCR to identify: “house-keeping” genes useful for normalizing expression levels across tissues; stage-specific changes flagging transitions in fruit development; and cell wall modifying gene products associated with fruit firmness. These quantitative PCR results confirmed the gene expression patterns observed by RNA-Seq.

Rose, as many other ornamental, vegetable and field crops, is tetraploid. This poses constraints in genetic analyses, due to the occurrence of multiple alleles at marker and trait loci and the existence of multiple allele dosages. Developments in marker discovery (next generation sequencing), genotyping (SNP arrays) and analysis (software for dosage scoring) now make it feasible to develop high-density molecular marker maps for the homologous chromosomes in tetraploids separately, and perform QTL analysis. We developed the WagRhSNP Axiom array based on sequences from tetraploid garden roses and cut roses, to be used for inheritance studies and genetic mapping. The array design can be used by all researchers working in rose. We have also developed a general strategy for mapping in polyploid crops including stringent error filtering, which we will also apply to other ornamentals, including tetraploid Alstroemeria and hexaploid Chrysanthemum. Using this strategy we have build genetics maps for cut and garden roses. They are based on the simplex:nulliplex, simplex:simplex and duplex:nulliplex segregating SNPs. The cut rose map contains a total of 2515 (parent P514) and 2762 (P867) SNP markers. Each linkage group has between 127 and 705 SNP markers. Twenty six of the 28 haplogroups (homologous chromosomes) are represented with 30-246 SNPs per haplogroup. Both maps enabled establishing that the predominant mode of inheritance is tetrasomic with a possible disomic inheritance in one chromosome. Furthermore, occurrence and distribution of double reduction is calculated, and a detailed overview of the synteny with the Fragaria vesca genome sequence is produced.

The large size and spreading growth habit of trees requires excessive labor, land space, and pesticides. Genetically improving tree shapes so that they can be planted at higher density and/or more readily adapted to mechanization would increase productivity and be more environmentally friendly. Currently, very little is known about the genes and signaling pathways that regulate tree growth and development. Using peach as a model system, we have mapped and identified several genes that control branch growth and tree size via a new, Next-Gen sequencing enabled method. The peach pillar trait associated with fastigiated growth was found to be controlled by a gene called PpeTAC1 (Tiller Angle Control) homologues of which were previously described in rice and maize. Knock outs of PpeTAC1 in Arabidopsis showed a similar fastigiated growth habit as did transgenic plums which were silenced for PpeTAC1. PpeTAC1 was found to be a member of a broader gene family that includes the previously described LAZY1. Knock outs of LAZY1 in rice and Arabidopsis displayed prostrate growth as well as a loss of gravitropism associated with impaired polar auxin transport. RNAseq studies of apical shoots from TAC1 and LAZY1 mutants in peach and Arabidopsis did not show changes in auxin biosynthesis, transport, or signaling but instead revealed coordinated changes in stress, defense, and secondary metabolic pathways. The knowledge gained from this work has important implications for improving agricultural productivity and sustainability in not only fruit trees but potentially a wide variety of different crops.

In sweet cherry, flowering process is controlled by specific chill and heat requirements. In the context of global climate change, the knowledge of the genetic determinism of flowering is necessary to develop new cultivars that are able to face the challenges associated with an increase of temperature. Two intraspecific F1 mapping progenies, ‘Regina’ × ‘Lapins’ (R×L, 124 individuals) and ‘Regina’ × ‘Garnet’ (R×G, 117 individuals), were genotyped and used in the detection of quantitative trait locus (QTL) involved in flowering process. Using a candidate gene approach, a list of one hundred functional candidate genes was established according to those found in other plant species. Cherry gene homologues were identified using the sweet cherry ‘Regina’ transcriptome database developed in our lab. Polymorphism between parents was analysed and candidate genes were mapped in the two progenies. The expression of the most promising candidate genes, located in QTL regions for flowering date, were analysed using qRT-PCR on reproductive buds at different stages during dormancy progression.

The phytohormone cytokinin plays a major role in plant development. Recently we have reported the identification and comparative analyses of genes associated with cytokinin signaling and homeostasis pathways in two hardwood tree species: Populus trichocarpa and Prunus persica (Immanen et al., 2013). To better understand the role that cytokinin responsive genes play in stone fruit development, we have performed RNA-seq and qPCR analyses of peach fruits treated with exogenously applied t-zeatin at different developmental stages. These analyses reveal a developmental stage specific expression of the cytokinin signaling and homeostasis pathway genes in peach fruits. We have also identified cytokinin responsive genes during pre-lignification, lignification and post-lignification stages of stone-fruit development in Prunus persica. Furthermore, transient overexpression of a putative type-B response regulator (PpRR1) in peach fruits increased the expression of putative downstream genes, PpShy2 and PpRR6. These results suggest that the cytokinin response pathway that has been described in Arabidopsis is conserved in Peach, and that this response pathway is differentially expressed during peach stone-fruit development. This work was funded by CONICYT Fondecyt /Regular N°1121021

Temperate sub tropical countries for instance South Africa receive very little chill measured in Richardson Units (RU) during winter months, which causes great concern for farmers producing high chill requirement fruit crops like apples. Failure to fulfil chill requirement in apples causes prolonged dormancy symptoms for instance reduced branching and extended fruiting period in trees. The genetic mechanism of chill accumulation during endodormancy is not well understood and the aim of this study is to investigate this phenomenon. The two apple varieties, ‘Anna’ and ‘Lady Williams’ were used to compare the differentially expressed genes during chill accumulation. ‘Anna’ is a low chill apple variety that requires less than 300 hours of chill. ‘Lady Williams’ is a very high chill variety that requires more than 1500 hours of chill. QTL analysis was performed for a Lady Williams’ x ‘Anna’ mapping population using the apple 8K Infinium SNP-array. One major QTL on linkage group 9 and several minor QTLs on linkage groups, 2, 8 and 15 were detected. RNA was extracted from Lady Williams and Anna at 100h time intervals and analysed by RNA-Seq. Transcripts were investigated between time intervals to identify genes that were differentially expressed and then mapped against the QTL’s to locate the genes within the loci. A number of transcripts were highlighted as being differentially expressed between time intervals. The differentially expressed genes from Anna were compared to those from Lady Williams. Differences between the patterns of expression were identified for further analysis.

In sweet cherry, fruit weight, firmness, and organoleptic quality are very important traits for both producers and consumers. Their genetic determinism has been analysed in three F1 sweet cherry progenies. ‘Regina’ × ‘Lapins’ (RxL), ‘Regina’ × ‘Garnet’ (R×G) and ‘Fercer’ × ‘Burlat’ (F×B), were analysed for fruit weight and firmness, during four to seven years. R×L was also analysed for pH and titratable acidity for three years. Major soluble carbohydrates (glucose, fructose, sucrose and sorbitol) and major organic acids (citrate and malate) were quantified during one year on R×L and F×B progenies. Genotyping was performed with the RosBREED’s cherry 6K SNP array v1 and linkage maps constructed with JoinMap 4.0. Many QTLs were detected for both fruit weight and firmness and in most cases in the same position, the most significant being detected on LG2, 5 and 6. Major QTLs for pH, titratable acidity and malic acid were identified on LG6, in the same region. On LG2 the QTL for fruit weight co-localised with a CELL NUMBER REGULATOR gene (CNR 12). For fruit weight and firmness, 14 candidate genes (CGs) were identified on the QTL on LG5 and 29 on the QTL on LG6. On LG5, three were selected for their potential involvement in fruit weight control: cytochrome p450 78A3-like, CNR1 and plac8 family protein. The 11 other CGs were selected for their potential involvement in fruit firmness. On LG6, three and 26 CGs were selected for their potential involvement in the control of fruit weight and firmness, respectively.

'Golden Delicious' (Malus x domestica Borkh.) (GD) is one of the most widely available pome fruit. External CO2-injury, a postharvest controlled-atmosphere storage related peel disorder, significantly impacts long-term storability and fruit quality for fresh apple and pear fruit cultivars, although annual incidence varies greatly. The experiment included 5%CO2-controlled atmosphere (CA), 5%CO2-CA plus 1-methylcyclopropene (CA+1-MCP), and regular air (RA) treatments and peel tissues were sampled at 14, 28, 56, 84 days storage over a three year period. RNA-Seq based transcriptome profiling (2011) and Lipdome profiling by LCMS (2011-2013) were carried out for sampled apple peel tissues. KEGG pathway analysis indicated oxidation/reduction, glycolysis, sodium transporters and glutamate biosynthesis enriched in CA but not RA and CA+1-MCP. Accordingly, gene ontology for CA emphasized the importance of the endoplasmic reticulum (ER), ER stress and the Unfolded Protein Response while CA+1-MCP down regulated these ER stress response and protein folding genes. Multivariate and time course analysis of RNA-seq and LCMS/APCI data provided genes and lipids of interest. SKP2A, an F-box protein known to be involved in severe ER stress, was detected only in CA stored fruit after 28 days storage. Results for the lipidome show (C18:3/18:2) diglyceride, β-SitoGluclinolenate, two monogalactose-diacylglycerol and two triacylglycerols species having a significant differences between treatments. Highly relevant network gene models selected by sparse Partial Least Squares is being validated by RTqPCR using all three years GD peel tissue. These results will determine the key molecular responses of apple peel tissue to high-CO2 controlled-atmosphere storage.

The meristems of many perennial plants in temperate and boreal climates remain in a cyclic quiescent state within buds during the cold period of autumn and winter, which ensures protection against the effects of cold and water stress. Photoperiod and temperature control growth cessation and bud dormancy induction, whereas dormancy release requires the quantitative perception of chilling. We performed two transcriptomic approaches for the identification of differently expressed transcripts in reproductive buds of peach (Prunus persica [L.] Batsch). Among different genes involved in transcriptional regulation of dormancy, pollen development and stress tolerance, we identified a gene coding for a protein similar to Stress Associated Proteins (SAP) containing two specific Zn-finger domains named A20 and AN1 (PpSAP). SAPs have been described as regulators of the abiotic stress response in plant species, emerging as potential candidates for improvement of stress tolerance in plants. We have studied the developmental and stress dependent expression of PpSAP in reproductive buds and vegetative tissues. PpSAP was highly expressed in leaves and dormant buds, being down-regulated after the release of bud dormancy and before bud break. PpSAP strongly interacted with ubiquitin proteins in the yeast two-hybrid system, in accordance with previous works supporting an E3 ubiquitin ligase activity for SAP proteins. PpSAP was constitutively expressed in transgenic plum plants under the control of CaMV 35S promoter. PpSAP over-expression led to alterations in leaf shape and an increased tolerance to leaf desiccation, which conferred to this gene a high interest for the manipulation of abiotic stress tolerance in plants.

The improvement of fruit aroma is currently one of the most desirable objectives in peach breeding programs. To better characterize and asses the genetic potential for increasing the aroma by breeding, a quantitative trait locus (QTL) analysis approach was carried out in a F1 population segregating largely for fruit traits. Linkage maps were constructed using the IPSC peach 9 K Infinium ® II array, rendering dense genetic maps except of some chromosomes, probably due to identity-by-descent of those chromosomes in the parental genotypes. Aroma variability was analyzed by a metabolomic approach based on GC-MS to profile 81 volatile compounds across the population in two locations. Additionally, quality-related traits were also studied to assess possible pleiotropic effects. Correlation-based analysis of the volatile dataset revealed that the peach volatilome is organized in modules formed by compounds from the same biosynthetic origin or sharing similar chemical structures. QTL mapping showed clustering of volatile QTL included in the same volatile modules, indicating that some of them are subjected to joint genetic control. The monoterpene module is controlled by a unique locus at the top of LG4, a locus previously showed to affect the levels of two terpenoid compounds. At the bottom of LG4 a locus controlling several volatiles but also melting/nonmelting and maturity-related traits was found, suggesting putative pleiotropic effects. In addition, two novel locus controlling lactones and esters at linkage group 5 and 6 were discovered. The results presented here confirmed previously locus controlling the aroma of peach but also identified novel ones.