Gennaro Fazio

Comparative analysis of response to phenotypic and marker-assisted selection for multiple lateral branching in cucumber (Cucumis sativus L.)

Abstract Yield increase in processing cucumber (Cucumis sativus L.) is positively correlated with an increase in number of fruit-bearing branches. Multiple lateral branching (MLB) is a metric trait controlled by at least five effective factors. Breeding efficacy might be improved through marker-assisted selection (MAS) for MLB. Experiments were designed to independently confirm previously determined linkage of molecular markers (L18-2-H19A SNP, CSWTAAA01 SSR, CSWCT13 SSR, W7-2 RAPD and BC-551 RAPD) to MLB, and to determine their utility in MAS. These markers were present in significantly higher frequency than expected (1, presence:3, absence; p < 0.001) in BC2 plants selected based on a high MLB phenotype (BC2PHE). However, markers that were considered selectively neutral fit the expected segregation of donor parent DNA in BC2 progeny. Markers linked to MLB were used in MAS of BC1 and BC2 plants to produce BC2MAS, and BC3MAS progeny. Means for MLB in MAS populations were compared with backcross populations developed through phenotypic selection (BC2PHE, BC3PHE) and by random mating where no selection had been applied (BC2RND, BC3RND). Statistical analysis showed no significant differences (p < 0.001) between means of phenotypic (BC2PHE = 3.02, BC3PHE = 3.29) and marker-aided selection (BC2MAS = 3.12, BC3MAS = 3.11) for MLB. However, both phenotypic and MAS population means were significantly higher than the random control (BC2RND = 2.27, BC3RND = 2.41) for MLB. Thus, given the observed response to selection and the rapid life-cycle of cucumber (4 months), markers linked to MLB when used in MAS will most likely be effective tools in cucumber improvement.

Rootstock-regulated gene expression patterns in apple tree scions

Apple trees (Malus x domestica) do not reproduce true-to-type from seed. Therefore, desirable cultivars are clonally propagated by grafting vegetative material onto rootstocks. Although cloned cultivars are genetically identical, rootstocks influence horticulturally important cultivar traits, including tree size, disease resistance, and abiotic stress tolerance. Here, ‘Gala’ scions were grafted to seven different rootstocks that produce a range of tree sizes and grown in a greenhouse. Global gene expression patterns in the scions were compared using a DNA microarray representing 55,230 apple transcripts. Each rootstock triggered a distinct, reproducible scion gene expression pattern. Two thousand nine hundred thirty-four scion transcripts were differentially regulated, by a factor of two or greater, by one or more rootstocks. Transcripts from genes predicted to be involved in responses to stress and biotic and abiotic stimuli were disproportionately represented among the rootstock-regulated transcripts. Microarray data analysis based on tree size identified 116 transcripts whose expression levels were correlated with tree size. The correlation of transcript level with tree size was tested for 14 of these transcripts using quantitative polymerase chain reaction in a population of orchard-grown ‘Mutsu’ cultivar trees grafted onto rootstocks from a breeding population of multiple crosses. Of those tested, transcripts encoding predicted sorbitol dehydrogenase, homeobox-leucine zipper, and hevein-like proteins were confirmed as being expressed at higher levels in larger trees, while a transcript predicted to encode an extensin-like protein was confirmed as being expressed at higher levels in smaller trees. This study illustrates the utility of using rootstock-regulated phenotypes to identify genes potentially associated with horticulturally important traits.

Interaction of Brassicaceous Seed Meal and Apple Rootstock on Recovery of Pythium spp. and Pratylenchus penetrans from Roots Grown in Replant Soils

Pythium spp. and Pratylenchus penetrans are significant components of the diverse pathogen complex that incites apple replant disease in Washington State. The structure of the Pythium population differs among orchard soils but is composed of multiple pathogenic species. Studies were conducted to determine the effect of brassicaceous seed meals and apple rootstock on the activity and composition of these pathogen populations. Brassicaceous seed meals differed in capacity to suppress Pythium numbers and apple root infection, as well as differentially transformed composition of the population recovered from apple roots. Brassica juncea seed meal (SM) was the sole seed meal examined to suppress Pythium numbers and root infection; however, a persisting population was always detected in which Pythium irregulare existed as the dominant or co-dominant species. In general, the Geneva series rootstocks were less susceptible to root infection by native populations of Pythium, whereas M26, MM106, and MM111 were highly susceptible. Apple rootstocks from the Geneva series consistently supported lower populations of P. penetrans than did Malling or Malling-Merton rootstocks. B. juncea SM was superior to Brassica napus SM or Sinapis alba SM in suppressing lesion nematode populations. Significant rootstock × seed meal interaction was detected, and nematode suppression in response to B. napus or S. alba SM was only observed when used in concert with a tolerant rootstock, while B. juncea SM suppressed lesion nematode root populations irrespective of rootstock. These findings demonstrate that utilization of brassicaceous seed meal amendments for replant disease suppression must employ an appropriate rootstock in order to achieve optimal disease control.

Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus ‘Robusta 5’ accessions

BackgroundBreeding of fire blight resistant scions and rootstocks is a goal of several international apple breeding programs, as options are limited for management of this destructive disease caused by the bacterial pathogen Erwinia amylovora. A broad, large-effect quantitative trait locus (QTL) for fire blight resistance has been reported on linkage group 3 of Malus ‘Robusta 5’. In this study we identified markers derived from putative fire blight resistance genes associated with the QTL by integrating further genetic mapping studies with bioinformatics analysis of transcript profiling data and genome sequence databases.ResultsWhen several defined E.amylovora strains were used to inoculate three progenies from international breeding programs, all with ‘Robusta 5’ as a common parent, two distinct QTLs were detected on linkage group 3, where only one had previously been mapped. In the New Zealand ‘Malling 9’ X ‘Robusta 5’ population inoculated with E. amylovora ICMP11176, the proximal QTL co-located with SNP markers derived from a leucine-rich repeat, receptor-like protein ( MxdRLP1) and a closely linked class 3 peroxidase gene. While the QTL detected in the German ‘Idared’ X ‘Robusta 5’ population inoculated with E. amylovora strains Ea222_JKI or ICMP11176 was approximately 6 cM distal to this, directly below a SNP marker derived from a heat shock 90 family protein gene ( HSP90). In the US ‘Otawa3’ X ‘Robusta5’ population inoculated with E. amylovora strains Ea273 or E2002a, the position of the LOD score peak on linkage group 3 was dependent upon the pathogen strains used for inoculation. One of the five MxdRLP1 alleles identified in fire blight resistant and susceptible cultivars was genetically associated with resistance and used to develop a high resolution melting PCR marker. A resistance QTL detected on linkage group 7 of the US population co-located with another HSP90 gene-family member and a WRKY transcription factor previously associated with fire blight resistance. However, this QTL was not observed in the New Zealand or German populations.ConclusionsThe results suggest that the upper region of ‘Robusta 5’ linkage group 3 contains multiple genes contributing to fire blight resistance and that their contributions to resistance can vary depending upon pathogen virulence and other factors. Mapping markers derived from putative fire blight resistance genes has proved a useful aid in defining these QTLs and developing markers for marker-assisted breeding of fire blight resistance.

Comparative analysis and functional annotation of a large expressed sequence tag collection of apple.

A total of 34 apple (Malus × domestica Borkh.) cDNA libraries were constructed from root, leaf, bud, shoot, flower, and fruit tissues, at various developmental stages and/or under biotic or abiotic stress conditions, and of several genotypes. From these libraries, 190,425 clones were partially sequenced from the 5′ end and 42,619 clones were sequenced from the 3′ end, and a total of 182,241 high‐quality expressed sequence tags (ESTs) were obtained. These coalesced into 23,442 tentative contigs and 9843 singletons, for a total of 33,825 apple unigenes. Functional annotation of this unigene set revealed an even distribution of apple sequences among the three main gene ontology categories. Of ∼33,000 apple unigenes, 8437 (25%) had no detectable homologs (E >0.1) in the Arabidopsis genome. When the entire apple unigene set was compared with the entire citrus [Citrus sinensis (L.) Osbeck] unigene set and the poplar (Populus trichocarpa Torr. & Gray) predicted proteome, both members of the core eudicot and rosids clade, 13,521 of apple unigenes matched one or more sequences in citrus, while 25,817 had counterparts in the poplar protein database. Apple–Arabidopsis–citrus–poplar comparisons revealed closer evolutionary relationships between apple and poplar than with the other two species. Genes involved in basic metabolic pathways appear to be largely conserved among apple, citrus, poplar, and Arabidopsis.

Genetic diversity in Malus ×domestica (Rosaceae) through time in response to domestication

UNLABELLED • PREMISE OF THE STUDY Patterns of genetic diversity in domesticated plants are affected by geographic region of origin and cultivation, intentional artificial selection, and unintentional genetic bottlenecks. While bottlenecks are mainly associated with the initial domestication process, they can also affect diversity during crop improvement. Here, we investigate the impact of the improvement process on the genetic diversity of domesticated apple in comparison with other perennial and annual fruit crops.• METHODS Apple cultivars that were developed at various times (ranging from the 13th through the 20th century) and 11 of the 15 apple cultivars that are used for 90% of the apple production in the United States were surveyed for genetic diversity based on either 9 or 19 simple sequence repeats (SSRs). Diversity was compared using standard metrics and model-based approaches based on expected heterozygosity (He) at equilibrium. Improvement bottleneck data for fruit crops were also collected from the literature.• KEY RESULTS Domesticated apples showed no significant reduction in genetic diversity through time across the last eight centuries. Diversity was generally high, with an average He > 0.7 for apples from all centuries. However, diversity of the apples currently used for the bulk of commercial production was lower.• CONCLUSIONS The improvement bottleneck in domesticated apples appears to be mild or nonexistent, in contrast to improvement bottlenecks in many annual and perennial fruit crops, as documented from the literature survey. The low diversity of the subset of cultivars used for commercial production, however, indicates that an improvement bottleneck may be in progress for this perennial crop.

Genome re-sequencing reveals the history of apple and supports a two-stage model for fruit enlargement

Human selection has reshaped crop genomes. Here we report an apple genome variation map generated through genome sequencing of 117 diverse accessions. A comprehensive model of apple speciation and domestication along the Silk Road is proposed based on evidence from diverse genomic analyses. Cultivated apples likely originate from Malus sieversii in Kazakhstan, followed by intensive introgressions from M. sylvestris. M. sieversii in Xinjiang of China turns out to be an “ancient” isolated ecotype not directly contributing to apple domestication. We have identified selective sweeps underlying quantitative trait loci/genes of important fruit quality traits including fruit texture and flavor, and provide evidences supporting a model of apple fruit size evolution comprising two major events with one occurring prior to domestication and the other during domestication. This study outlines the genetic basis of apple domestication and evolution, and provides valuable information for facilitating marker-assisted breeding and apple improvement.Apple is one of the most important fruit crops. Here, the authors perform deep genome resequencing of 117 diverse accessions and reveal comprehensive models of apple origin, speciation, domestication, and fruit size evolution as well as candidate genes associated with important agronomic traits.

Genome to Phenome Mapping in Apple Using Historical Data

Apple (Malus X. domestica Borkh.) is one of the worlds most valuable fruit crops. Its large size and long juvenile phase make it a particularly promising candidate for marker‐assisted selection (MAS). However, advances in MAS in apple have been limited by a lack of phenotype and genotype data from sufficiently large samples. To establish genotype‐phenotype relationships and advance MAS in apple, we extracted over 24,000 phenotype scores from the USDA‐Germplasm Resources Information Network (GRIN) database and linked them with over 8000 single nucleotide polymorphisms (SNPs) from 689 apple accessions from the USDA apple germplasm collection clonally preserved in Geneva, NY. We find significant genetic differentiation between Old World and New World cultivars and demonstrate that the genetic structure of the domesticated apple also reflects the time required for ripening. A genome‐wide association study (GWAS) of 36 phenotypes confirms the association between fruit color and the MYB1 locus, and we also report a novel association between the transcription factor, NAC18.1, and harvest date and fruit firmness. We demonstrate that harvest time and fruit size can be predicted with relatively high accuracies (r > 0.46) using genomic prediction. Rapid decay of linkage disequilibrium (LD) in apples means millions of SNPs may be required for well‐powered GWAS. However, rapid LD decay also promises to enable extremely high resolution mapping of causal variants, which holds great potential for advancing MAS.

Transcriptional regulation of ethylene and jasmonate mediated defense response in apple ( Malus domestica ) root during Pythium ultimum infection

Apple replant disease (ARD) is a significant economic restraint to the successful re-establishment of new apple orchards on sites previously planted to the same crop. Pythium ultimum, an oomycete, is a significant component of the ARD pathogen complex. Although ethylene (ET)- and jasmonic acid (JA)-mediated defense responses are intensively studied in the foliar pathosystem, the transferability of this knowledge to the interaction between a perennial root system and soilborne pathogens is unknown. The aim of this study was to test the hypothesis that the ET/JA-mediated defense response is conserved in roots of tree crops in response to infection by P. ultimum. Apple genes with the annotated function of ET/JA biosynthesis, MdERF (ethylene response factor) for signaling transduction and a gene encoding a pathogenesis-related (PR) protein (β-chitinase, the target of ERF) were identified from the apple genome sequences. The transcriptional profiles of these genes during P. ultimum infection and after exogenous ET and/or JA treatment were characterized using qRT-PCR. Several genes showed a 10- to 60-fold upregulation in apple root tissue 24-48 h post inoculation (hpi). Exogenous ET and JA treatment exhibited either a positive or negative influence on expression of ET or JA biosynthesis genes, depending upon gene isoforms and the tissue types, while the expression of MdERF and the PR protein encoding gene was upregulated by both ET and JA treatment. Our data are consistent with the hypothesis that ET/JA-mediated defense pathways are functional in the root system of perennial tree species defending soilborne pathogens.

Nomenclature and genetic relationships of apples and pears from Terceira Island

Heritage apple (Malus domestica Borkh. hybrids) and pear (Pyrus communis L. hybrid) trees grow in villages throughout Terceira Island, Azores, Portugal. Some of these pears have different names but similar morphology. The objective of this study was to determine synonymy, homology, and phylogeny of apples and pears from Terceira and to examine potential relationships of the island pears with standard apples and pears of Portuguese or American descent. Nine apple microsatellite markers were used to determine genetic relationships. Distance- and parsimony-based cluster analysis grouped these genotypes into separate apple and pear clades. The Terceira apples were divided into two clades: the maçā and the reineta-reinette. Among the 17 heritage apple genotypes, seven unique accessions were identified and four groups of synonyms, or possibly clones, were detected including: ‘Reineta Agosto’ and ‘Reineta Verde’ from Altares; ‘Reineta Castanha’ and ‘Reineta Verde Miuda’; ‘Maçā Pêra,’ ‘Maçā Calhau’, ‘Pêro Branco’ from Salga and from Terra-Chā and ‘Maçā Marmelo’; and the five genotypes ‘Maçā Sao Joao’, ‘Malápio Rosa’, ‘Maçā Gaspar’, ‘Maçā Branca’ and ‘Maçā Pato’. In addition, two homonyms were detected. ‘Pêro Vermelho’ from Terra Chā was a separate genotype from a tree from Doze Ribeiras of the same name, but Pêro Branco from Terra Chā appears to be a clone that can be distinguished by an additional allele at CH1F07a from a tree with that name from Salga. One pair of apple clones, ‘Reineta Agosto’ and ‘Reineta Verde’ from Altares appear to be derived from an unreduced gamete of ‘Golden Delicious.’ Another apple genotype ‘Maçā Acida’ could be a sibling of the ‘Maçā Pêra’ clonal group. Other tested standard apples from the US genebank were unrelated to Terceira genotypes. Of the seven heritage pears, five unique genotypes and one pair of synonyms were detected. ‘Pêra Papo Pintassilgo’ from Raminho and ‘Pêra Vermelha’ from the nursery of Serviço de Desenvolvimento Agario da Terceira (SDAT) were synonyms. ‘Passans du Portugal’ was related to ‘Pêra Cabaca’ but other standard pears from the US genebank were unrelated to Terceira genotypes. Future studies will include additional apple and pear cultivars from other Islands of the Azores and continental Portugal, and wild Asian species to further explore genetic relationships.