Insights into the effect of human civilization on Malus evolution and domestication.

The evolutionary history of the Malus genus has not been well studied. In the current study, we presented genetic evidence on the origin of the Malus genus based on genome sequencing of 297 Malus accessions, revealing the genetic relationship between wild species and cultivated apples. Our results demonstrated that North American and East Asian wild species are closer to the outgroup (pear) than Central Asian species, and hybrid species including natural (separated before the Pleistocene, about 2.5 Mya) and artificial hybrids (including ornamental trees and rootstocks) are between East and Central Asian wild species. Introgressions from M. sylvestris in cultivated apples appeared to be more extensive than those from M. sieversii, whose genetic background flowed westward across Eurasia and eastward to wild species including M. prunifolia, M. × asiatica, M. × micromalus, and M. × robust. Our results suggested that the loss of ancestral gene flow from M. sieversii in cultivated apples accompanied the movement of European traders around the world since the Age of Discovery. Natural SNP variations showed that cultivated apples had higher nucleotide diversity than wild species and more unique SNPs than other apple groups. An apple ERECTA-like gene that underwent selection during domestication on 15th chromosome was identified as a likely major determinant of fruit length and diameter, and an NB-ARC domain-containing gene was found to strongly affect anthocyanin accumulation using a genome-wide association approach. Our results provide new insights into the origin and domestication of apples and will be useful in new breeding programmes and efforts to increase fruit crop productivity.

Development and validation of the Axiom(®) Apple480K SNP genotyping array.

Cultivated apple (Malus × domestica Borkh.) is one of the most important fruit crops in temperate regions, and has great economic and cultural value. The apple genome is highly heterozygous and has undergone a recent duplication which, combined with a rapid linkage disequilibrium decay, makes it difficult to perform genome-wide association (GWA) studies. Single nucleotide polymorphism arrays offer highly multiplexed assays at a relatively low cost per data point and can be a valid tool for the identification of the markers associated with traits of interest. Here, we describe the development and validation of a 487K SNP Affymetrix Axiom(®) genotyping array for apple and discuss its potential applications. The array has been built from the high-depth resequencing of 63 different cultivars covering most of the genetic diversity in cultivated apple. The SNPs were chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non-genic regions. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom(®) Apple480K to assess the effectiveness of the array. A large majority of SNPs (359 994 or 74%) fell in the stringent class of poly high resolution polymorphisms. We also devised a filtering procedure to identify a subset of 275K very robust markers that can be safely used for germplasm surveys in apple. The Axiom(®) Apple480K has now been commercially released both for public and proprietary use and will likely be a reference tool for GWA studies in apple.

Integrated QTL detection for key breeding traits in multiple peach progenies.

BACKGROUND: Peach (Prunus persica (L.) Batsch) is a major temperate fruit crop with an intense breeding activity. Breeding is facilitated by knowledge of the inheritance of the key traits that are often of a quantitative nature. QTLs have traditionally been studied using the phenotype of a single progeny (usually a full-sib progeny) and the correlation with a set of markers covering its genome. This approach has allowed the identification of various genes and QTLs but is limited by the small numbers of individuals used and by the narrow transect of the variability analyzed. In this article we propose the use of a multi-progeny mapping strategy that used pedigree information and Bayesian approaches that supports a more precise and complete survey of the available genetic variability. RESULTS: Seven key agronomic characters (data from 1 to 3 years) were analyzed in 18 progenies from crosses between occidental commercial genotypes and various exotic lines including accessions of other Prunus species. A total of 1467 plants from these progenies were genotyped with a 9 k SNP array. Forty-seven QTLs were identified, 22 coinciding with major genes and QTLs that have been consistently found in the same populations when studied individually and 25 were new. A substantial part of the QTLs observed (47%) would not have been detected in crosses between only commercial materials, showing the high value of exotic lines as a source of novel alleles for the commercial gene pool. Our strategy also provided estimations on the narrow sense heritability of each character, and the estimation of the QTL genotypes of each parent for the different QTLs and their breeding value. CONCLUSIONS: The integrated strategy used provides a broader and more accurate picture of the variability available for peach breeding with the identification of many new QTLs, information on the sources of the alleles of interest and the breeding values of the potential donors of such valuable alleles. These results are first-hand information for breeders and a step forward towards the implementation of DNA-informed strategies to facilitate selection of new cultivars with improved productivity and quality.

ASSIsT: an automatic SNP scoring tool for in- and outbreeding species.

UNLABELLED: ASSIsT (Automatic SNP ScorIng Tool) is a user-friendly customized pipeline for efficient calling and filtering of SNPs from Illumina Infinium arrays, specifically devised for custom genotyping arrays. Illumina has developed an integrated software for SNP data visualization and inspection called GenomeStudio (GS). ASSIsT builds on GS-derived data and identifies those markers that follow a bi-allelic genetic model and show reliable genotype calls. Moreover, ASSIsT re-edits SNP calls with null alleles or additional SNPs in the probe annealing site. ASSIsT can be employed in the analysis of different population types such as full-sib families and mating schemes used in the plant kingdom (backcross, F1, F2), and unrelated individuals. The final result can be directly exported in the format required by the most common software for genetic mapping and marker-trait association analysis. ASSIsT is developed in Python and runs in Windows and Linux. AVAILABILITY AND IMPLEMENTATION: The software, example data sets and tutorials are freely available at http://compbiotoolbox.fmach.it/assist/. CONTACT: eric.vandeweg@wur.nl.

Recombination rates and genomic shuffling in human and chimpanzee--a new twist in the chromosomal speciation theory.

A long-standing question in evolutionary biology concerns the effect of recombination in shaping the genomic architecture of organisms and, in particular, how this impacts the speciation process. Despite efforts employed in the last decade, the role of chromosomal reorganizations in the human-chimpanzee speciation process remains unresolved. Through whole-genome comparisons, we have analyzed the genome-wide impact of genomic shuffling in the distribution of human recombination rates during the human-chimpanzee speciation process. We have constructed a highly refined map of the reorganizations and evolutionary breakpoint regions in the human and chimpanzee genomes based on orthologous genes and genome sequence alignments. The analysis of the most recent human and chimpanzee recombination maps inferred from genome-wide single-nucleotide polymorphism data revealed that the standardized recombination rate was significantly lower in rearranged than in collinear chromosomes. In fact, rearranged chromosomes presented significantly lower recombination rates than chromosomes that have been maintained since the ancestor of great apes, and this was related with the lineage in which they become fixed. Importantly, inverted regions had lower recombination rates than collinear and noninverted regions, independently of the effect of centromeres. Our observations have implications for the chromosomal speciation theory, providing new evidences for the contribution of inversions in suppressing recombination in mammals.

An ancient duplication of apple MYB transcription factors is responsible for novel red fruit-flesh phenotypes.

Anthocyanin accumulation is coordinated in plants by a number of conserved transcription factors. In apple (Malus × domestica), an R2R3 MYB transcription factor has been shown to control fruit flesh and foliage anthocyanin pigmentation (MYB10) and fruit skin color (MYB1). However, the pattern of expression and allelic variation at these loci does not explain all anthocyanin-related apple phenotypes. One such example is an open-pollinated seedling of cv Sangrado that has green foliage and develops red flesh in the fruit cortex late in maturity. We used methods that combine plant breeding, molecular biology, and genomics to identify duplicated MYB transcription factors that could control this phenotype. We then demonstrated that the red-flesh cortex phenotype is associated with enhanced expression of MYB110a, a paralog of MYB10. Functional characterization of MYB110a showed that it was able to up-regulate anthocyanin biosynthesis in tobacco (Nicotiana tabacum). The chromosomal location of MYB110a is consistent with a whole-genome duplication event that occurred during the evolution of apple within the Maloideae family. Both MYB10 and MYB110a have conserved function in some cultivars, but they differ in their expression pattern and response to fruit maturity.

The de novo, chromosome-level genome assembly of the sweet chestnut (Castanea sativa Mill.) Cv. Marrone Di Chiusa Pesio.

OBJECTIVES: The sweet chestnut Castanea sativa Mill. is the only native Castanea species in Europe, and it is a tree of high economic value that provides appreciated fruits and valuable wood. In this study, we assembled a high-quality nuclear genome of the ancient Italian chestnut variety 'Marrone di Chiusa Pesio' using a combination of Oxford Nanopore Technologies long reads, whole-genome and Omni-C Illumina short reads. DATA DESCRIPTION: The genome was assembled into 238 scaffolds with an N50 size of 21.8 Mb and an N80 size of 7.1 Mb for a total assembled sequence of 750 Mb. The BUSCO assessment revealed that 98.6% of the genome matched the embryophyte dataset, highlighting good completeness of the genetic space. After chromosome-level scaffolding, 12 chromosomes with a total length of 715.8 and 713.0 Mb were constructed for haplotype 1 and haplotype 2, respectively. The repetitive elements represented 37.3% and 37.4% of the total assembled genome in haplotype 1 and haplotype 2, respectively. A total of 57,653 and 58,146 genes were predicted in the two haplotypes, and approximately 73% of the genes were functionally annotated using the EggNOG-mapper. The assembled genome will be a valuable resource and reference for future chestnut breeding and genetic improvement.

A validated protocol for eDNA-based monitoring of within-species genetic diversity in a pond-breeding amphibian.

In light of the dramatic decline in amphibian biodiversity, new cost-efficient tools to rapidly monitor species abundance and population genetic diversity in space and time are urgently needed. It has been amply demonstrated that the use of environmental DNA (eDNA) for single-species detection and characterization of community composition can increase the precision of amphibian monitoring compared to traditional (observational) approaches. However, it has been suggested that the efficiency and accuracy of the eDNA approach could be further improved by more timely sampling; in addition, the quality of genetic diversity data derived from the same DNA has been confirmed in other vertebrate taxa, but not amphibians. Given the availability of previous tissue-based genetic data, here we use the common frog Rana temporaria Linnaeus, 1758 as our target species and an improved eDNA protocol to: (i) investigate differences in species detection between three developmental stages in various freshwater environments; and (ii) study the diversity of mitochondrial DNA (mtDNA) haplotypes detected in eDNA (water) samples, by amplifying a specific fragment of the COI gene (331 base pairs, bp) commonly used as a barcode. Our protocol proved to be a reliable tool for monitoring population genetic diversity of this species, and could be a valuable addition to amphibian conservation and wetland management.

Susceptibility Evaluation to Fire Blight and Genome-Wide Associations within a Collection of Asturian Apple Accessions.

Fire blight, caused by Erwinia amylovora, is one of the most devastating apple diseases. The selection of cultivars of low susceptibility and the study of the genetic mechanisms of the disease play important roles in fire blight management. The susceptibility level to fire blight was evaluated in 102 accessions originating from Asturias, a cider-producing region located in the north of Spain with a wide apple germplasm. Evaluations took place under quarantine conditions using artificial inoculations of grafted plants. The results revealed wide variation in susceptibility responses and low-susceptible cultivars were identified. In addition, 91 cultivars were genotyped using the Affymetrix Axiom® Apple 480 K SNP array to conduct genome-wide association studies (GWAS). A statistically significant signal was detected on chromosome 10 using the multi-locus mixed model (MLMM). Two genes were identified as major putative candidate genes: a TIR-NBS-LRR class disease protein and a protein containing a development and cell death (DCD) domain. The outcomes of this study provide a promising source of information, particularly in the context of cider apples, and set a starting point for future genetic and breeding approaches.

Absence of increased genomic variants in the cyanobacterium Chroococcidiopsis exposed to Mars-like conditions outside the space station.

Despite the increasing interest in using microbial-based technologies to support human space exploration, many unknowns remain not only on bioprocesses but also on microbial survivability and genetic stability under non-Earth conditions. Here the desert cyanobacterium Chroococcidiopsis sp. CCMEE 029 was investigated for robustness of the repair capability of DNA lesions accumulated under Mars-like conditions (UV radiation and atmosphere) simulated in low Earth orbit using the EXPOSE-R2 facility installed outside the International Space Station. Genomic alterations were determined in a space-derivate of Chroococcidiopsis sp. CCMEE 029 obtained upon reactivation on Earth of the space-exposed cells. Comparative analysis of whole-genome sequences showed no increased variant numbers in the space-derivate compared to triplicates of the reference strain maintained on the ground. This result advanced cyanobacteria-based technologies to support human space exploration.

On the Origin and Propagation of the COVID-19 Outbreak in the Italian Province of Trento, a Tourist Region of Northern Italy.

BACKGROUND: Trentino is an Italian province with a tourism-based economy, bordering the regions of Lombardy and Veneto, where the two earliest and largest outbreaks of COVID-19 occurred in Italy. The earliest cases in Trentino were reported in the first week of March 2020, with most of the cases occurring in the winter sport areas in the Dolomites mountain range. The number of reported cases decreased over the summer months and was followed by a second wave in the autumn and winter of 2020. METHODS: we performed high-coverage Oxford Nanopore sequencing of 253 positive SARS-CoV-2 swabs collected in Trentino between March and December 2020. RESULTS: in this work, we analyzed genome sequences to trace the routes through which the virus entered the area, and assessed whether the autumnal resurgence could be attributed to lineages persisting undetected during summer, or as a consequence of new introductions. CONCLUSIONS: Comparing the draft genomes analyzed with a large selection of European sequences retrieved from GISAID we found that multiple introductions of the virus occurred at the early stage of the epidemics; the two epidemic waves were unrelated; the second wave was due to reintroductions of the virus in summer when traveling restrictions were uplifted.

High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex.

The biosynthesis of anthocyanin in many plants is affected by environmental conditions. In apple (Malus × domestica Borkh.), concentrations of fruit anthocyanins are lower under hot climatic conditions. We examined the anthocyanin accumulation in the peel of maturing 'Mondial Gala' and 'Royal Gala' apples, grown in both temperate and hot climates, and using artificial heating of on-tree fruit. Heat caused a dramatic reduction of both peel anthocyanin concentration and transcripts of the genes of the anthocyanin biosynthetic pathway. Heating fruit rapidly reduced expression of the R2R3 MYB transcription factor (MYB10) responsible for coordinative regulation for red skin colour, as well as expression of other genes in the transcriptional activation complex. A single night of low temperatures is sufficient to elicit a large increase in transcription of MYB10 and consequently the biosynthetic pathway. Candidate genes that can repress anthocyanin biosynthesis did not appear to be responsible for reductions in anthocyanin content. We propose that temperature-induced regulation of anthocyanin biosynthesis is primarily caused by altered transcript levels of the activating anthocyanin regulatory complex.

Candidate gene mapping identifies genomic variations in the fire blight susceptibility genes HIPM and DIPM across the Malus germplasm.

Development of apple (Malus domestica) cultivars resistant to fire blight, a devastating bacterial disease caused by Erwinia amylovora, is a priority for apple breeding programs. Towards this goal, the inactivation of members of the HIPM and DIPM gene families with a role in fire blight susceptibility (S genes) can help achieve sustainable tolerance. We have investigated the genomic diversity of HIPM and DIPM genes in Malus germplasm collections and used a candidate gene-based association mapping approach to identify SNPs (single nucleotide polymorphisms) with significant associations to fire blight susceptibility. A total of 87 unique SNP variants were identified in HIPM and DIPM genes across 93 Malus accessions. Thirty SNPs showed significant associations (p < 0.05) with fire blight susceptibility traits, while two of these SNPs showed highly significant (p < 0.001) associations across two different years. This research has provided knowledge about genetic diversity in fire blight S genes in diverse apple accessions and identified candidate HIPM and DIPM alleles that could be used to develop apple cultivars with decreased fire blight susceptibility via marker-assisted breeding or biotechnological approaches.

A SNP transferability survey within the genus Vitis.

BACKGROUND: 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 Vitis vinifera 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. RESULTS: 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 V. vinifera, of 18.8% across the wild forms of V. vinifera, of 2.3% among non-vinifera Vitis species, and of 0% with Muscadinia rotundifolia was found. In addition, mean allele frequencies were used to evaluate SNP informativeness and develop useful subsets of markers. CONCLUSION: Using SNPlex application and corroboration from the sequencing analysis, the informativeness of SNP markers from the heterozygous grape cultivar Pinot Noir was validated in V. vinifera (including cultivars and wild forms), but had a limited application for non-vinifera Vitis 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 V. vinifera.

Genomic signatures of different adaptations to environmental stimuli between wild and cultivated Vitis vinifera L.

The application of population genetic methods in combination with gene mapping strategies can help to identify genes and mutations selected during the evolution from wild plants to crops and to explore the considerable genetic variation still maintained in natural populations. We genotyped a grapevine germplasm collection of 44 wild (Vitis vinifera subsp. sylvestris) and 48 cultivated (V. vinifera subsp. sativa) accessions at 54 K single-nucleotide polymorphisms (SNPs) to perform a whole-genome comparison of the main population genetic statistics. The analysis of Wright Fixation Index (FST) along the whole genome allowed us to identify several putative "signatures of selection" spanning over two thousand SNPs significantly differentiated between sativa and sylvestris. Many of these genomic regions included genes involved in the adaptation to environmental changes. An overall reduction of nucleotide diversity was observed across the whole genome within sylvestris, supporting a small effective population size of the wild grapevine. Tajima's D resulted positive in both wild and cultivated subgroups, which may indicate an ongoing balancing selection. Association mapping for six domestication-related traits was performed in combination with population genetics, providing further evidence of different perception and response to environmental stresses between sativa and sylvestris.

PhytoTypeDB: a database of plant protein inter-cultivar variability and function.

Despite a fast-growing number of available plant genomes, available computational resources are poorly integrated and provide only limited access to the underlying data. Most existing databases focus on DNA/RNA data or specific gene families, with less emphasis on protein structure, function and variability. In particular, despite the economic importance of many plant accessions, there are no straightforward ways to retrieve or visualize information on their differences. To fill this gap, we developed PhytoTypeDB (http://phytotypedb.bio.unipd.it/), a scalable database containing plant protein annotations and genetic variants from resequencing of different accessions. The database content is generated by an integrated pipeline, exploiting state-of-the-art methods for protein characterization requiring only the proteome reference sequence and variant calling files. Protein names for unknown proteins are inferred by homology for over 95% of the entries. Single-nucleotide variants are visualized along with protein annotation in a user-friendly web interface. The server offers an effective querying system, which allows to compare variability among different species and accessions, to generate custom data sets based on shared functional features or to perform sequence searches. A documented set of exposed RESTful endpoints make the data accessible programmatically by third-party clients.

Towards map-based cloning of FB_Mfu10: identification of a receptor-like kinase candidate gene underlying the Malus fusca fire blight resistance locus on linkage group 10.

Breeding for resistance against the destructive fire blight disease of apples is the most sustainable strategy to control the menace of this disease, and has become increasingly important in European apple breeding programs. Since most cultivars are susceptible, wild accessions have been explored for resistance with quantitative trait loci detected in a few wild species. Fire blight resistance of Malus fusca was described following phenotypic evaluations with a C-type strain of Erwinia amylovora, Ea222_JKI, and the detection of a major QTL on chromosome 10 (Mfu10) of this crabapple. The stability of the resistance of M. fusca and Mfu10 has been evaluated using two other strains, the highly aggressive Canadian S-type strain-Ea3049, and the avrRpt2EA mutant-ZYRKD3-1, both of which overcome the resistance of Malus ×robusta 5, a wild species accession with an already described fire blight resistance gene. To pave the way for positional cloning of the underlying fire blight resistance gene of M. fusca, we have fine mapped the QTL region on linkage group 10 using 1888 individuals and 23 newly developed molecular markers, thus delimiting the interval of interest to 0.33 cM between markers FR39G5T7xT7y/FR24N24RP and FRMf7358424/FR46H22. Tightly linked SSR markers are suitable for marker-assisted selection in breeding programs. Furthermore, a bacterial artificial chromosome (BAC) clone spanning FB_Mfu10 region was isolated and sequenced. One putative fire blight resistance candidate gene of M. fusca was predicted on the sequence of BAC 46H22 within the resistance region that encodes B-lectin and serine/threonine kinase domains.

Relationship between hydroxycinnamic acids and the resistance of apple cultivars to rosy apple aphid.

The phenolic profiles of apple cultivars from the SERIDA Asturian cider apple breeding program, including parents and progenies, were determined by ultrahigh-performance liquid chromatography-diode array detector-electrospray ionization-quadrupole time of flight/mass spectrometer in order to study the relationship between phenols and the resistance of apple tree cultivars to rosy apple aphid (RAA). A pattern recognition technique named partial least square discriminant analysis (PLS-DA) was used to classify apple cultivars based on resistance to RAA, resistant and susceptible, reaching scores with accuracy higher than 97% and 91% respectively. Hydroxycinnamic acids, particularly 4-caffeoylquinic acid (4-CQA) and 4-p-coumaroylquinic acid (4-pCoQA), were identified as the major player in RAA resistance by the PLS-DA model. Indeed, the isomerisation 5-CQA → 4-CQA is favoured in resistant cultivars, whereas the isomerisation 5-pCoQA → 4-pCoQA is favoured in susceptible cultivars. As a result, resistant cultivars accumulate higher amounts of 4-CQA than susceptible ones, and the opposite occurs for 4-pCoQA. Also, minor isomerisations of 5-CQA to 1-CQA or 3-CQA show opposite behaviour for resistant and susceptible cultivars. Cultivar resistance to RAA is concluded to be related with the phenylpropanoid pathway, the isomerisation reactions being the key metabolic reaction for a cultivar to be resistant or susceptible to RAA.

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.