Comparison of different methods for SNP detection in grapevine

Single nucleotide polymorphisms (SNPs) are the most abundant of all markers, both in animal and plant genomes. In crops and tree species considerable investment has been recently made on this genomic technology. While large-scale characterisation of SNPs by high-throughput techniques is possible, such highthroughput platforms are not available to all plant breeding laboratories. This report compares alternative multi-purpose and affordable methods for SNP assay in grapevine (Vitis spp.). In particular, the efficiency, sensitivity and reliability of single-strand conformation polymorphism (SSCP) on both non-denaturant gels and fluorescence-based capillary electrophoresis are compared with minisequencing (single nucleotide extension reaction). The results indicate that when multiplexing in combination with minisequencing is a mid-throughput, reliable and flexible technique for the detection of SNPs and can therefore be used effectively to improve marker assisted breeding in grapevine.

A SNP transferability survey within the genus Vitis

<p>Abstract</p> <p>Background</p> <p>Efforts to sequence the genomes of different organisms continue to increase. The DNA sequence is usually decoded for one individual and its application is for the whole species. The recent sequencing of the highly heterozygous <it>Vitis vinifera </it>L. cultivar Pinot Noir (clone ENTAV 115) genome gave rise to several thousand polymorphisms and offers a good model to study the transferability of its degree of polymorphism to other individuals of the same species and within the genus.</p> <p>Results</p> <p>This study was performed by genotyping 137 SNPs through the SNPlex™ Genotyping System (Applied Biosystems Inc.) and by comparing the SNPlex sequencing results across 35 (of the 137) regions from 69 grape accessions. A heterozygous state transferability of 31.5% across the unrelated cultivars of <it>V. vinifera</it>, of 18.8% across the wild forms of <it>V. vinifera</it>, of 2.3% among non-<it>vinifera Vitis </it>species, and of 0% with <it>Muscadinia rotundifolia </it>was found. In addition, mean allele frequencies were used to evaluate SNP informativeness and develop useful subsets of markers.</p> <p>Conclusion</p> <p>Using SNPlex application and corroboration from the sequencing analysis, the informativeness of SNP markers from the heterozygous grape cultivar Pinot Noir was validated in <it>V. vinifera </it>(including cultivars and wild forms), but had a limited application for non-<it>vinifera Vitis </it>species where a resequencing strategy may be preferred, knowing that homology at priming sites is sufficient. This work will allow future applications such as mapping and diversity studies, accession identification and genomic-research assisted breeding within <it>V. vinifera</it>.</p

Development and Evaluation of a 9K SNP Addition to the Peach Ipsc 9K SNP Array v1

The IPSC 9K peach SNP array released by the international community has been a valuable tool in research and application. Even though majority of SNPs (84%) were polymorphic in the evaluation panels there were many genomic regions with low coverage, including those important for breeding. The existing peach array has been updated with 9K additional SNPs covering previously identified gaps and including recently identified SNPs important for breeding. SNPs (1,808,996) identified by sequencing 49 genomes of additional peach accessions were used as the main source of additional SNPs. Focal point strategy was used to select 8,971 SNPs within 40kb window from the 2,821 focal points distributed across the genome. Additional 129 SNPs were chosen to saturate either regions important for breeding or close the gaps larger than 100kb. The array was validated with 1,770 peach and 26 Prunus accessions (almond, plum, apricot, wild relatives). The add-on contained 7,862 SNPs evenly spread across 8 peach pseudo-molecules with only one SNP positioned on scaffold 13 covering 224.99Mbp of peach genome. The 9K add-on improved the 9K peach array by increasing the total number of usable SNPs by 7,206. The number of SNPs per chromosome increased on average by 50% with only on average 0.18% increase in total physical coverage. Number of gaps larger than 0.3 Mbp was reduced to 2 one on each chromosome 3 and 8. Overall genotyping efficiency in all material was &gt;90% except in almond, 82%. Number of informative markers, assessed by ASSIsT software, were highest in peach 64% and lowest in almond 10%, with 61% of markers being informative in wild Prunus (12) and 35% in apricot (4) and 2 - 33% in Japanese and European plum, respectively. Among 36.2% discarded markers 33% were monomorphic and 30% shifted homozygous in material used. Those markers could be informative in different background raising total number of informative markers. Ann addition of new SNPs to array improved the density and usefulness of the array in Prunus species. The practical applications of new 16K Illumina SNP peach array will be discussed. Specified Source(s) of Funding: USDA-NIFA-SCRI-Ros- BREED (2014-51181-22378

Genetic Variability Study in a Wide Germplasm of Domesticated Peach Through High Throughput

Peach (Prunus persica (L.) Batsch) is one of the most economically important fruit crops in temperate areas. Classical fruit tree breeding is generally slow and inefficient. Molecular markers could improve its efficiency but, although nowadays many Mendelian traits are mapped in peach and SSR markers have been found to be linked to some of the key major genes, its use in breeding programs is still limited. Main reasons for that are insufficient linkage between the markers and the genes and the lack of markers suitable for medium-high degree of multiplexing. To address this limitation, about 1,300 peach cultivars were genotyped with the 9K peach SNP chip (Verde et al. 2012) in the frame of FruitBreedomics project. This germplasm was chosen to be representative of the genetic diversity present in five germplasm collection in Europe and in China. Out of the 8144SNPs present in the chip, about 4300 were positively genotyped and used for the further analysis. The average number of heterozygous loci in the genotyped accessions was 1186 (spanning from 13 to 2775). The preliminary results of the population structure reveal three main subpopulations and the presence of high number of admixed individuals. LD seems to decay at distance longer than ca. 1 Mb. These results will be instrumental for implementing LD-based mapping of QTLs and genes in peach

Reconstruction of multi-generation pedigrees involving numerous old apple cultivars thanks to whole-genome SNP data

A number of European apple cultivars are old, some of them dating back to the Renaissance, Middle Ages or even earlier. Many other cultivars have been developed during subsequent times. In order to decipher the relationships that link some of these old cultivars, whole-genome SNP data (~ 250K) for over 1400 genotypes were analyzed to infer first-degree relationships and reconstruct pedigrees. We used simple exclusion tests based on a count of Mendelian error to identify up to a thousand potential parent-offspring duos, including 295 complete parent-offspring trios and a hundred duos that could be oriented. grand-parents for some missing parents could also be inferred. Combining all this information allowed us to reconstruct pedigrees (up to 6 generations) highlighting the central role of major founders such as ‘Reinette Franche’, ‘Margil’, and ‘Alexander’. Haplotypes were deduced from genotypic data and pedigrees, and used to measure haplotype sharing between supposedly unrelated cultivars, allowing investigating further links between them.To our knowledge, such a large analysis to reconstruct multigeneration pedigrees involving (very) old cultivars selected over such time has never before been performed in perennial fruit species

Collaborative project to identify direct and distant pedigree relationships in apple

Pedigree information is fundamentally important in breeding programs, enabling breeders to know the source of valuable attributes and underlying alleles and to enlarge genetic diversity in a directed way. Many apple cultivars are related to each other through both recent and distant common ancestors. As apple trees are clonally propagated, long-lived, and widely adapted, many of the ancestors of modern cultivars are still present in global germplasm collections. Use of apple SNP arrays enables identification of direct and distant pedigree relationships with precision. An example is the elucidation of the \u27Honeycrisp\u27 pedigree using the 8K SNP array, which enabled further findings regarding the inheritance of important alleles for traits including scab resistance and soft scald susceptibility. To facilitate more discoveries across apple germplasm, a large-scale collaborative apple pedigree reconstruction project has been initiated. This project utilizes output from the Illumina Infinium 20K and Affymetrix Axiom 480K apple SNP arrays, a high quality genetic linkage map for the 20K array SNPs, and a data curation pipeline developed through the FruitBreedomics and RosBREED projects. Techniques using shared haplotype length statistics will be used alongside historical information to deduce distant pedigree relationships. The project involves various experts, germplasm collections, and academic institutions around the world and is open for further extension. It will provide findings useful for breeding programs, germplasm collections, geneticists, and historians

An integrated approach for increasing breeding efficiency in apple and peach in Europe

Despite the availability of whole genome sequences of apple and peach, there has been a considerable gap between genomics and breeding. To bridge the gap, the European Union funded the FruitBreedomics project (March 2011 to August 2015) involving 28 research institutes and private companies. Three complementary approaches were pursued: (i) tool and software development, (ii) deciphering genetic control of main horticultural traits taking into account allelic diversity and (iii) developing plant materials, tools and methodologies for breeders. Decisive breakthroughs were made including the making available of ready-to-go DNA diagnostic tests for Marker Assisted Breeding, development of new, dense SNP arrays in apple and peach, new phenotypic methods for some complex traits, software for gene/QTL discovery on breeding germplasm via Pedigree Based Analysis (PBA). This resulted in the discovery of highly predictive molecular markers for traits of horticultural interest via PBA and via Genome Wide Association Studies (GWAS) on several European genebank collections. FruitBreedomics also developed pre-breeding plant materials in which multiple sources of resistance were pyramided and software that can support breeders in their selection activities. Through FruitBreedomics, significant progresses were made in the field of apple and peach breeding, genetics, genomics and bioinformatics of which advantage will be made by breeders, germplasm curators and scientists. A major part of the data collected during the project has been stored in the FruitBreedomics database and has been made available to the public. This review covers the scientific discoveries made in this major endeavour, and perspective in the apple and peach breeding and genomics in Europe and beyond

High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development

Using the latest sequencing and optical mapping technologies, we have produced a high-quality de novo assembly of the apple (Malus domestica Borkh.) genome. Repeat sequences, which represented over half of the assembly, provided an unprecedented opportunity to investigate the uncharacterized regions of a tree genome; we identified a new hyper-repetitive retrotransposon sequence that was over-represented in heterochromatic regions and estimated that a major burst of different transposable elements (TEs) occurred 21 million years ago. Notably, the timing of this TE burst coincided with the uplift of the Tian Shan mountains, which is thought to be the center of the location where the apple originated, suggesting that TEs and associated processes may have contributed to the diversification of the apple ancestor and possibly to its divergence from pear. Finally, genome-wide DNA methylation data suggest that epigenetic marks may contribute to agronomically relevant aspects, such as apple fruit development

QTL mapping for phenolic compounds in apple fruit and apple juice from a cider apple progeny

Polyphenols have favorable antioxidant potential on human health suggesting that their high content in apple is responsible for the beneficial effects of apple consumption. tehy are also related to the quality of ciders as they predominantly account for astringency, bitterness, color and aroma. Five groups of phenolic compounds are described in the apple fruit: flavanols, hydroxynnamic acids, dihydrochalcones, flavonols and anthocyanins. So far, only two studies have been published on the genetic basis of the phenolic content of dessert apples. As cider apples are commonly described to be much more concentrated in phenolic compounds than dessert varieties, the present study focuses on a cider apple progeny. 32 compounds belonging to the five groups were identified and quantified by HPLC-UV and UHPLC-UV-MS/MS in fruit extracts and juices. 53 QTL controlling phenolic compounds concentration were detected on nine linkage groups (LG) on the integrated linkage map, for all phenolic groups except anthocyanins. QTL clusters located on LG1, 12, 14, 15 and 17 were stable across the year or the studied material. QTL detected on LG1, 14 and 17 for quercitrin, p-coumaroylquinic acid, rutin and chlorogenic acid confirmed results of previous studies. However, no significant QTL was obtained on the LG16 where a major locus for flavanols was previously located. With the two previous studies, this study shows the diversity of genomic regions traits of interest in apple

Identification of Pyrus Single Nucleotide Polymorphisms (SNPs) and Evaluation for Genetic Mapping in European Pear and Interspecific Pyrus Hybrids

We have used new generation sequencing (NGS) technologies to identify single nucleotide polymorphism (SNP) markers from three European pear (Pyrus communis L.) cultivars and subsequently developed a subset of 1096 pear SNPs into high throughput markers by combining them with the set of 7692 apple SNPs on the IRSC apple Infinium® II 8K array. We then evaluated this apple and pear Infinium® II 9K SNP array for large-scale genotyping in pear across several species, using both pear and apple SNPs. The segregating populations employed for array validation included a segregating population of European pear (‘Old Home’בLouise Bon Jersey’) and four interspecific breeding families derived from Asian (P. pyrifolia Nakai and P. bretschneideri Rehd.) and European pear pedigrees. In total, we mapped 857 polymorphic pear markers to construct the first SNP-based genetic maps for pear, comprising 78% of the total pear SNPs included in the array. In addition, 1031 SNP markers derived from apple (13% of the total apple SNPs included in the array) were polymorphic and were mapped in one or more of the pear populations. These results are the first to demonstrate SNP transferability across the genera Malus and Pyrus. Our construction of high density SNP-based and gene-based genetic maps in pear represents an important step towards the identification of chromosomal regions associated with a range of horticultural characters, such as pest and disease resistance, orchard yield and fruit quality