Genetics to Breeding

The Spanish Program of Plant Genetic Resources integrates, among others, the collections located at Public University of Navarre, Centro de Investigaciones Agrarias de Mabegondo, Cabildos (Tenerife, La Palma and Gran Canaria), University of Lleida, Estación Experimental de Aula Dei-CSIC and CITA of Aragon. Those collections include mainly local cultivars from their respective regions, covering most of the Spanish apple-growing areas. Though some previous studies about the genetic variability of apple genetics resources from Spain were already performed, a complete analysis is needed in order to evaluate the complete diversity of Malus spp. in Spain. For doing that, the Spanish Government funded the project “Harmonization of the methodology of characterization, assessment of genetic diversity and definition of the core collection of the apple germplasm conserved in Spanish genebanks”. In total, we have evaluated 1206 accessions using standardized methodologies, with SSR markers and morphological descriptors. SSR fingerprinting was performed with 13 SSR markers. SSR profiles were obtained independently and allele sizes were compared using a common set of cultivars selected as references. Results showed 601 genotypes for 1206 accessions. Most of the genotypes (438) were identified only in one accession. The other 163 genotypes were repeated in two to 81 accessions (involving 767 accessions in total). The harmonization of morphological descriptors will allow us to determine if the accessions with the same genotype are synonymies or closely related individuals. Results of this study highlight the interest of coordinated actions in order to optimize the management of germplasm collections and to evaluate the complete genetic diversity of Malus spp. in Spain.

Fruit colour it is a key trait in sweet cherry as it is a main determinant of fruit quality. Fruit and flower color in other rosaceous species is caused by anthocyanin accumulation, which is regulated by transcriptional factors of the anthocyanin biosynthetic pathway. In sweet cherry a transcription factor, MYB10, has been cloned. This transcription factor correlates with anthocyanin production during sweet cherry development and co-localizes with a major QTL for cherry fruit colour. In this work, we studied MYB10 transcription, structure and function in a sweet cherry cultivar that produces white fruits, and in three cultivars with different red fruits. Results revealed a lack of MYB10 transcription in white sweet cherries and a large insertion in one allele of the MYB10 genomic sequence. We postulate that this mutation and slight differences in promoter sequence of MYB10 result in low expression of this gene in the white cultivar, confirming that MYB10 is a major determinant of fruit color in sweet cherry.

Soft scald is a postharvest disorder of apple (Malus domestica) characterized by distinct brown and often longitudinal lesions on the fruit that often extend into the flesh and can lead to secondary infections in the fruit. Fruit that develop soft scald are unmarketable for fresh eating. The disorder is economically important because it usually develops in storage multiple weeks after producers invest considerable resources into harvest and cold storage. Incidence of soft scald varies among cultivars and can vary depending on orchard and storage conditions. Many cultivars are susceptible to the development of soft scald, including cultivars commonly used extensively as parents in US breeding programs. Observations by breeders suggest the trait is heritable. The development of new cultivars with reduced potential for soft scald development would benefit the apple industry and this could be made more efficient through the use of marker assisted breeding.
A quantitative trait locus (QTL) detection study was conducted using FlexQTL software with data collected on soft scald incidence in 2011 and 2012 from the RosBREED apple germplasm set and additional data collected in 2013 at the University of Minnesota comprised of 4 half-sib families with ‘Honeycrisp’ as a common parent. Putative QTL were detected, though they were not consistently detected in all years and locations.

Harvest date in apple (Malus x domestica) is an important trait for breeders making crossing and selection decisions. Early harvest date has been associated with lower hedonic ratings for fruit quality traits and decreased storability due to softening compared to apples that mature later in the season. An objective for breeders and producers is to have genotypes with early season ripening and improved texture and storage traits that could replace less desirable cultivars. Conversely, in production regions with short growing seasons, late season cultivars may never ripen fully before freezing and cannot be harvested. The development of molecular markers that inform plant breeders on harvest date would be useful in selecting parents for cultivar development and for screening seedlings as part of marker assisted breeding (MAB). Selected seedlings and advanced breeding lines could also be targeted to testing locations with appropriate season lengths. A pedigree-linked population was investigated for harvest date quantitative trait loci (QTL) for 2011 and 2012. Growing degree day accumulation (base 4.4 °C) at harvest was used as a proxy for calendar date in order to compare years and to normalize data for ancestors grown at other locations as part of the RosBREED project. The analyses evidenced QTL on linkage groups 3, 4, 7, 8, 11, 14, and 15. The QTL on LG 3 co-localized with a QTL for sensory firmness and harvest date from previous reports in apple. This study will provide marker-trait-loci associations through the identification of functional SNP haplotypes which span the QTLs and can be utilized in MAB.

In temperate fruit trees, most key phenological stages are highly dependent on environmental conditions. In particular, correct timing for dormancy and flowering is essential to ensure good fruit production and quality. As a result, in a swiftly-changing environment, temperate fruit crop adaptation in many areas will be at risk in the coming decades. Global changes in environmental conditions include warmer winters and higher risks of frosts in the early spring, leading to a wide range of problems: flower and fruit set, sun-scald, cross-pollination or novel host-pest interaction.
With the final aim of better understanding the response of flowering to climatic conditions, we present a large-scale analysis of flowering data for various sweet cherry cultivars from numerous sites in Europe, characterized by a wide range of climatic conditions. These phenology data were provided through a national network of experimentation (Ctifl) and a European COST action on sweet cherry that INRA-Bordeaux is leading. This approach allowed extracting the main trends in flowering behaviour under different temperature conditions and selecting the best phenology models.
These results represent the first step towards developing a predictive model for flowering in sweet cherry based on both genomic and phenology data.

Dormancy is the ability of some species, i.e. perennial woody plants in the Prunus genus, to suspend and resume growth in response to seasonal changes. In order to re-start growth flower buds need a certain amount of chill units in winter and subsequent heat hours in spring. Especially in warmer production areas and now increasingly due to global warming, the chilling requirements cannot be completely fulfilled. Therefore, different cyanide-based chemicals have been used to fully achieve the required chill units, and also to synchronize flowering time, which increases the fruit yield and facilitates fruit harvesting. The best chemical used to date is Dormex® (AlzChem), which can bring forward flowering time around one week compared to untreated controls. However, the EU has banned this chemical in 2008 due to concerns towards environmental effects and operator exposure. The molecular mechanisms by which cyanamide, the principal component of Dormex®, brings forward flowering time is still unknown. Some studies suggest that cyanamide acts through the release of hydrogen cyanide. Hydrogen cyanide can also be produced by the hydrolysis of cyanogenic glucosides present in Prunus. Furthermore, it is known to inhibit the antioxidant enzymes catalase and superoxide dismutase. We propose that the consequently increased ROS levels activate certain ‘breaking dormancy’ genes. To prove this, we have treated dormant cherry flower buds with Dormex® to analyze the effects on the transcript, protein and metabolite level. Finally, we will also detect the cyanogenic glucosides in cherry flower buds and in different parts of the fully developed flower.

The original taste of almond kernel is bitter. Due to human domestication, nowadays the majority of cultivated almond varieties are sweet. Since most of the cultivated almonds are heterozygous for bitterness, new bitter almond seedlings are usually obtained in the cross breeding programs. Therefore, it would be interesting to develop a molecular marker that enables the breeder to eliminate early in the nursery the bitter seedlings. Although its biochemical function remains unknown, the Sweet kernel (Sk) locus, was localized in linkage group five (G5) in an almond genetic linkage map. Fine mapping between the molecular markers flanking the Sk/sk gene, has been performed to obtain a shorter interval in which Sk locus should be localized. We will show new single nucleotide polymorphisms found in a 3 Mb region in which Sk locus is included within G5. In bitter almonds, hydrogen cyanide is produced upon hydrolysis of amygdalin following tissue disruption. Amygdalin and its precursor prunasin are cyanogenic glucosides. Amygdalin is mainly present in the kernels, whereas prunasin can be detected in the vegetative parts as well as in the fruit mother tissues. Parallel studies with candidate genes analysis based on the amygdalin pathway are also under study. The elucidation of the polymorphisms defining bitterness will be decisive to identify the Sk locus and the development of a molecular marker for bitterness in almond.

Self-incompatibility (SI) is one of the most important genetic systems maintaining genetic diversity in flowering plants. The gametophytic self-incompatibility (GSI) system of the Solanaceae, Rosaceae, and Plantaginaceae are called the S-RNase-based GSI system. In this GSI system, the self and cross incompatibility reaction specificities of pistil and pollen are determined by ribonuclease (RNase) and F-box protein, respectively. Although the three plant families use the same molecule as the pistil S and pollen S determinants, molecular and genetic analyses of Prunus SC S haplotypes and polyploid sour cherry reveal the possible existence of a distinct self and cross recognition mechanism in the S-RNase-based GSI system of Prunus. In Prunus, the specificity determinants of pistil and pollen are called S-RNase and S haplotype-specific F-box protein (SFB), respectively. We previously proposed a working hypothesis in which Prunus SFB is supposed to have a distinct function from the pollen S determinant F-box proteins (SLF) of Solanaceae and Plantaginaceae. Here, we performed a detailed phylogenetic analysis of Prunus SFB and its orthologs in the Prunus genome and other angiosperm genomes. Our results indicated that Prunus SFB was generated from recent Prunus-specific gene duplications. Prunus F-box protein genes that are located flanking regions the S locus (SLFLs) are classified to the same clade as the pollen S F-box protein genes of Solanaceae, Plantaginaceae, and the subtribe Pyrinae (Rosaceae), indicating that duplication and sub-functionalization of the original pollen S genes generated SFB. We discuss the possible mechanisms of (in)compatibility reaction in Prunus.

Next-generation sequencing technologies have dramatically accelerated the use of genetic information for genome diversity analysis and genetic mapping in plants. Genotyping-by-sequencing (GBS) is a next-generation DNA sequencing (NGS) technology that can analyzed hundreds of thousands of short reads from a genome, allowing a direct and inexpensive single nucleotide polymorphism (SNP) detection from germplasm collections. Our objective was to identify and characterize SNPs through commercial cultivars and segregant populations with known paternity relationships. We used the GBS technology to characterize a collection of 29 commercially important Japanese plum (Prunus salicina L.) cultivars plus 116 offspring derivated from controlled cross among Japanese plum cultivars. We analyzed 68 offspring from Angeleno (♀) by Aurora (♂) and 48 offspring from Flavor Rich (♀) by Larry Ann (♂). Previously, a paternity analysis was conducted in order to verify the filiations for individuals of each segregant population. We calculate the paternity index (PI), combinated paternity index (CPI) and probability of paternity (W) using 17 simple sequence repeat (SSR). The sequencing and filtering process gave 1,784,547 tags. The tags were aligned to the peach reference genome, where 50% aligned to unique position, 5% aligned to multiple position and 45% could not be aligned. Finally after filtering, 57,542 SNP were identified. We discussed the useful of high-throughput GBS to characterize the genetic diversity of commercial cultivars and segregant populations of Japanese plum. Acknowledgements: This research was supported by CONICYT, FONDECYT/Regular Nº1120261.

Fruit color in sour cherry (Prunus cerasus) is an important market-driven trait in the U.S. where the dominate cultivar ‘Montmorency’ has a brilliant red color. This unique red color allows for a distinction between sour cherries grown in the U.S. and those in Europe which have predominantly dark-purple flesh. The anthocyanin transcription factor, MYB10, has previously been shown to control flesh color in cherry and other rosaceous species. Thirteen allelic variants for the sour cherry MYB10 region were distinguished based on the linkage phase of 47 polymorphic SNPs determined using the 6K Infinium® II SNP array developed by the RosBREED project. Of these 13 haplotypes, four behaved as dominant alleles conferring dark-flesh color. No SNPs, however, were found in this region which would distinguish haplotypes conferring dark-flesh. Marker-assisted seedling selection (MASS) is a goal of the Michigan State University cherry breeding program to cull undesired seedlings before they are planted in the field. Those found to have dark-fleshed alleles could be culled if a simple DNA diagnostic tool were available. Due to the high synteny in Prunus, the peach genome sequence was used to design 36 SSRs that were screened for their ability to uniquely identify dark-fleshed haplotypes. One SSR primer pair was found to amplify fragments that successfully differentiated the two darkest-flesh haplotypes. This marker can now be used for MASS in any crosses with either of these two haplotypes to cull those individuals which are predicted to have dark-purple flesh.