Genetics

Carotenoids are 40 carbon isoprenoids pigment molecules, indispensable for plant life. They play essential roles in photosynthesis, and they serve as precursors to the plant hormones abscisic acid (ABA) and strigolactones. In addition, these compounds furnish many flowers and fruit with attractive yellow, orange and red colors. Carotenoids are important in human health since they serve as pro-vitamin A and are antioxidants with protective activity against various illnesses. Vegetables and fruits are a natural source of carotenoids, and apricot (Prunus armeniaca L.) has the potential to serve as a particularly rich source. Carotenoid biosynthesis in plants is well characterized thanks to extensive research in model plants such as tomato and Arabidopsis. However, little is known in regards to apricot. To better understand the factors that determine carotenoid profile in apricot fruit, we characterized the carotenoids composition and content of fruits from more than one hundred apricot cultivars and lines. We present the result of a HPLC analysis, showing a great variability within the different cultivars, in both carotenoid content and composition. Based on the carotenoid phenotype, we suggest candidate genes to explain the phenotypic diversity. The data obtained will be used to facilitate research on the genetic factors controlling carotenoid biosynthesis in apricot.

Genetic improvement of host resistance has been identified by the U.S. apple industry as a major way to mitigate production risks associated with economically important diseases such as fire blight and blue mold. Several fire blight resistance QTLs have been described in Malus, however most come from exotic germplasm of poor fruit quality. Two bi-parental populations derived from ‘Splendour’ apple, which has excellent flavor and is resistant to fire blight, were found to segregate for resistance to fire blight (1R:3S) when challenged with Erwinia amylovora. These populations are being genotyped to identify the genetic basis of this resistance. Another approach to identify marker trait associations for fire blight resistance within domesticated Malus cultivars is to utilize the pedigree-linked germplasm of the RosBREED project. The Malus Crop Reference and Washington State University (WSU) Breeder Pedigree Sets were propagated and a replicated research orchard will be established at WSU, Wenatchee, WA in spring 2015 and used to determine the resistance of this germplasm to fire blight. Although there is no known resistance to blue mold in M. ×domestica, M. sieversii PI613981 collected from the wild in Kazakhstan is resistant. Fruit collected from a ‘Royal Gala’ X PI613981 mapping population (GMAL4593) were inoculated with P. expansum and evaluated for decay over a three year period. A genetic framework map has been developed for the GMAL4593 population and was used to identify QTLs for blue mold resistance on linkage groups 4 and 10.

Japanese flowering-cherries called “Sakura” show various floral morphogenesis in such as the number of flowers in a cluster, flower size, shapes of petals, the number of petals, flower color, and so on. In this study, we are focusing on the variation of inflorescence structure which is possibly one of the important traits affecting ornamental quality for flowering-cherries. We investigated the lengths of inflorescence axis and peduncles in a number of selected cultivars of flowering cherries at the time of anthesis. The inflorescence types diverged widely from corymb to umbel. The expressions of genes expected to be involved in the inflorescence development were analyzed by RT-PCR. The expression of TFL1/CEN/FT homologous genes (PruTFL1, PruCEN, and PruFT), which are related to the flowering and inflorescence structure, were investigated in inflorescence axis and peduncles. Expression of PruTFL1 was hardly detected in selected cultivars. PruCEN expression was observed only in several cultivars. PruFT was highly expressed in the inflorescence axis and peduncles and the expression level was relatively higher in inflorescence. However, these expression levels were different in cultivars, and the relationship to morphological variation couldn’t be found. The expression of genes related to the biosynthesis of gibberellic acids, which are often involved in organ elongation, were also analyzed, but the expression pattern was ambiguous like the result of TFL1/CEN/FT homologous genes. We are planning transcriptome approach to found the other genes involved in the development of various inflorescences in flowering-cherries, and testing the effects of phytohormones to the inflorescence development.

CBFs are cold-induced transcription factors (TFs) that regulate the expression of many cold-acclimation-related genes. To better understand and improve cold hardiness in apple, transgenic lines of ‘M26’ apple have been created that overexpress a peach CBF gene or various native apple CBF genes. Alternatively, silencing constructs have been used to generally or specifically inhibit native CBF genes. These transgenic lines have been studied in both laboratory and field studies over the past several years. Overexpression of a peach CBF gene in apple improved cold hardiness and unexpectedly also made trees enter dormancy earlier in the fall, delayed budbreak in the spring, and significantly decreased growth. These effects have been verified in 3 years of field studies. Transcriptomic studies indicate that CBF genes regulate approximately 8.5% of the total gene set identified in apple during normal cold acclimation, including a number of other transcription factors, including dormancy-associated-MADs box (DAM) genes. A bioinformatic analysis has revealed that several apple DAM genes possess one to several C-repeats (CBF protein binding motif) in their upstream promoter sequences. CBFs have also been proposed to regulate growth by inducing the accumulation of growth repressing DELLA proteins in the nucleus. Thus it appears that, in addition to cold acclimation, CBF TFs have the potential to integrate the regulation of both dormancy and growth. A bioinformatic analysis of CBF and Inducer of CBF Expression(ICE) genes in several fruit (grape, apple, peach, citrus, and blueberry) and forest tree (poplar, eucalyptus) species has also been conducted and will be discussed.

Strawberries are economically important fruits around the world. Fragaria x ananassa, the cultivated strawberry, is an allo-octoploid specie (2n=8x=56), their ploidy level difficult the application of marker assisted breeding. F. vesca has been considered as a diploid model organism. Berries, including strawberries, are known for their rich nutritional profile. Understanding the genetic bases controlling the production of phenolic and volatile compounds is extremely important for the selection of new varieties of cultivated strawberry with greater consumer acceptation.
The genetic map of a F2 population (CamarosaxDover) posses 192 loci distributed along the 28 expected linkage groups, representing each homeologous group with a high coverage (>70%) when compared to F. vesca genome. LC-MS analysis for polyphenol metabolites in full ripen fruits allowed the quantification of 22 metabolites and mapping 146 metabolic QTLs.
The IStraw90™ (International Strawberry 90K Axiom®) Affymetrix array developed by Rosbreed based on diploid and octoploide sequences was hybridized with 120 F2 individuals and parentals. The analysis of the results using GenotypeConsole and SNPolisher softwares shows a high quality of hybridization , with a >97% probe call rate for all individuals. For the 93937 valid SNPs : 75% were classified as MonoHighResolution, 9356 as NoMinorHomozygous, and 4939 as PolyHighResolution segregating in our population. The SNPs position in F.vesca and the SSR map allow to construct 4x7 homeologous groups with an increased resolution and higher coverage. Distribution of SNPs in homeologous groups will help the assembly of contigs and scaffolds of the genome draft sequence covering 154x of one population individual.

Many species in the Rosaceae, Solanaceae and Plantaginaceae exhibit the S-RNase-based self-incompatibility (SI). Their S loci contain the genes encoding S-ribonuclease (S-RNase) and F-box protein(s) for the pistil and the pollen specificities, respectively. The pollen S specificity in the Solanaceae is determined by multiple S locus F-box proteins (SLFs), while that in Prunus (Rosaceae) is by a single S haplotype-specific F-box protein (SFB). In addition to SFB, three other pollen-expressed F-box genes called S locus F-box protein like 1-3 (SLFL1-3) are located in the flanking regions of the Prunus S locus, although it is not clear whether they are involved in SI. An F-box protein generally forms the SCF complex with Skp1, Cul1 and Rbx1. The SCF complex is known to polyubiquitinate substrate proteins to be degraded by the ubiquitin proteasome system. Although solanaceous SLFs have been shown to form the SCF complex and to participate in the SI system, it remained to be elucidated whether Prunus SFB and SLFLs would form the SCF complex. We previously identified SFB-interacting Skp1-like protein 1 (SSK1), which showed the binary protein interactions with SFB, SLFLs and the pollen-expressed Cul1 homologs (Cul1A and Cul1B). Here, we report the ternary protein interactions of GST-fused SFB/SLFLs, SSK1 and Cul1A/Cul1B, in pull-down assay, indicating that SFB and SLFLs could form the SCF complex with either of the Cul1 homologs and polyubiquitinate unidentified substrate proteins in pollen tube.

Anthocyanin-rich fruit, because of their antioxidant properties and strong attractiveness to consumers, generates great interest in varietal innovation programs that increasingly integrate genomics and plant breeding. This is the case in peach in which the anthocyanin concentration in the mesocarp of the fruit is controlled by three different loci: i) the dominant Cs locus, mapped to LG3 and probably associated with MYB10, determines a red pigmentation of the mesocarp around the stone; ii) the dominant locus DBF, for Dominant Blood-Flesh, that we recently mapped to LG5, determines a fully red mesocarp appearing at the later stages of the fruit development. Using the peach reference sequence, we identified as good candidates a cluster of three predicted members of the dihydroflavonol-4-reductase gene family in the 505-kbp region containing DBF; iii) similarly, we narrowed the region (<80-kpb) of the recessive locus bf, for blood-flesh, that had been previously mapped to LG4. It determines a more or less substantial accumulation of anthocyanin in both immature and mature fruits, associated with the red pigmentation of midrib (rpm) on the underside of leaves and a reduced tree height (rth). However for bf, uncertainty still exists over whether these simply inherited traits are controlled by three linked loci (bf, rpm, and rth) or one pleiotropic locus. From biochemical, molecular and genomics analyses of one segregating population of peach (850 progenies), we bring new elements to answer this question and propose candidate genes involved in anthocyanin expression and the growth of plants.

Apple scab and powdery mildew are a major concern for apple breeders and producers. Control of these apple diseases by the development of varieties with durable disease resistance is the most efficient and environmentally friendly way. Therefore, the gene-pyramiding route for the development of apple varieties with durable disease resistances requires a supply of a diverse resistance gene pool. The identification and mapping of these genes is therefore of paramount importance. In this study genetic mapping of Quantitative Trait Loci (QTLs) for apple scab and powdery mildew was performed on progeny of three mapping populations (‘Mildew Resistant’ x ‘Golden Delicious’, ‘Russian Seedling’ x ‘Golden Delicious’ and Malus platycarpa x ‘Mildew Resistant’). Three marker systems were used and these were Simple Sequence Repeats (SSRs), Diversity Arrays Technology (DArTs) and Single Nucleotide Polymorphisms (SNPs) markers. A total of 17 putative QTLs were detected for the ‘Mildew Resistant’ x ‘Golden Delicious’ population, 10 for the Malus platycarpa x ‘Mildew Resistant’ population and nine for the ‘Russian Seedling’ x ‘Golden Delicious’ populations for the three diseases. Two putative QTLs for apple scab detected on LG 02 of the ‘Russian Seedling’ x ‘Golden Delicious’ coincided with loci previously identified as encoding two apple scab resistance genes Vh2 and Vh4 on ‘Russian apple’. SNP markers R_8936738_Lg2 and R_32981524_Lg2 were strongly linked to the Vh4 and Vh2 QTL regions respectively. The analysis of chromosome two led to the identification of two candidate genes for the Vh4 locus and a cluster of ABC (PDR) for Vh2.

Flesh softening, particularly mealiness, which occurs in apples during storage, is an unfavorable characteristic as it decreases the commercial value of the produce. Although many studies have been conducted on firmness and texture at harvest as well as changes in them during storage, the degree of mealiness (i.e. loss of cellular adhesion) during storage has rarely been investigated. In the present study, we quantified flesh mealiness under 20°C with 85% relative humidity by measuring the degree of cell separation in flesh discs after 7 hours shaking in a sucrose solution. Samples were taken over a period of 2 years from 137 F1 individuals from a cross between ‘Orin’ x ‘Akane.’ QTL analysis revealed two QTLs for flesh mealiness on linkage group (LG) 10, and the genetic distance of these QTLs was approximately 35 cM. Md-PG1SSR10kd marker was the nearest to one of the QTLs with a LOD score of 5.13. This marker is located on 10 kbp downstream of the stop codon of MdPG1, known to be involved in firmness and texture of flesh. AF057134-SSR marker was the nearest to the other QTL with a LOD score of 5.30. The location of this marker is almost same as a QTL reported for flesh firmness at harvest and after cold storage for 2 months in ‘Mondial Gala’ apples. These results indicate that LG 10 has the significant importance in controlling not only fruit texture at harvest but also loss of texture during post-harvest storage.

The demand for blackberries (Rubus spp.) has increased in Costa Rica, which represents an important income for small farmers in the country. The material grown in Costa Rica consists of four species and nine genotypes, mostly landraces of R. adenotrichos and is grown from southern Mexico, to northern South America. Costa Rica possesses diversity of Rubus with desirable economic traits, such as high ORAC values, but little is known about their genetics and genomics. In this study, genotypes were analyzed using 9 RAPD, and 13 SSR markers as well as using preliminary flow cytometry work. Nei’s genetic distances were calculated, and the UPGMA procedure was used. ‘Qualicum’, ‘Tulameen’ and ‘Navaho’ were comparative controls. Both RAPD and SSR analyses concurred with morphological data except for R. urticifolius ld. ‘Caballo’, which showed a closer relationship to R. adenotrichos landraces than R. urticifolius ld. ‘Ratón’. Although, R. urticifolius was reported as a diploid, allelic patterns were similar to that of a tetraploid. R. glaucus was reported as a blackberry-raspberry hybrid; both RAPD and SSR analyses support this as the genotype showed the closest relationship to R. idaeus. Flow cytometry analyses showed that R. adenotrichos landraces were diploid. Ploidy measurements for all other genotypes are underway to determine whether different ploidy levels can be found in R. urticifolius, or that ‘Caballo’ is a different species from ‘Ratón’. Genetic relationships and ploidy information is crucial to breeders for determining crossing compatibly among the genotypes, and initializing a competent breeding program in the future.