Cross-Reactivity of 24 Cannabinoids and Metabolites in blood using the immunalysis cannabinoids direct enzyme-linked immunosorbent assay kit.

With a wider availability of synthetic and semi-synthetic cannabinoids in the consumer space, there is a growing impact on public health and safety. Forensic toxicology laboratories should keep these compounds in mind as they attempt to remain effective in screening for potential sources of human performance impairment. Enzyme-linked immunosorbent assay (ELISA) is a commonly utilized tool in forensic toxicology, as its efficiency and sensitivity make it useful for rapid and easy screening for a large number of drugs. This screening technique has lower specificity, which allows for broad cross-reactivity among structurally-similar compounds. In this study, the Cannabinoids Direct ELISA kit from Immunalysis was utilized to assess the cross-reactivities of 24 cannabinoids and metabolites in whole blood. The assay was calibrated with 5 ng/mL of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol and the analytes of interest were evaluated at concentrations ranging from 5 to 500 ng/mL. Most parent compounds demonstrated cross-reactivity ≥ 20 ng/mL, with increasing alkyl side chain length relative to Δ9-tetrahydrocannabinol resulting in decreased cross-reactivity. Of the 24 analytes, only the carboxylic acid metabolites, 11-nor-9-carboxy-Δ8-tetrahydrocannabinol, 11-nor-9(R)-carboxy-hexahydrocannabinol, and 11-nor-9(S)-carboxy-hexahydrocannabinol, were cross-reactive at levels ≤ 10 ng/mL. Interestingly, 11-nor-9(R)-carboxy-hexahydrocannabinol demonstrated cross-reactivity at 5 ng/mL, where its stereoisomer 11-nor-9(S)-carboxy-hexahydrocannabinol, did not. As more information emerges about the prevalence of these analytes in blood specimens, it is important to understand and characterize their impact on current testing paradigms.

A Past Genetic Bottleneck from Argentine Beans and a Selective Sweep Led to the Race Chile of the Common Bean (Phaseolus vulgaris L.).

The domestication process of the common bean gave rise to six different races which come from the two ancestral genetic pools, the Mesoamerican (Durango, Jalisco, and Mesoamerica races) and the Andean (New Granada, Peru, and Chile races). In this study, a collection of 281 common bean landraces from Chile was analyzed using a 12K-SNP microarray. Additionally, 401 accessions representing the rest of the five common bean races were analyzed. A total of 2543 SNPs allowed us to differentiate a genetic group of 165 accessions that corresponds to the race Chile, 90 of which were classified as pure accessions, such as the bean types 'Tórtola', 'Sapito', 'Coscorrón', and 'Frutilla'. Our genetic analysis indicates that the race Chile has a close relationship with accessions from Argentina, suggesting that nomadic ancestral peoples introduced the bean seed to Chile. Previous archaeological and genetic studies support this hypothesis. Additionally, the low genetic diversity (π = 0.053; uHe = 0.53) and the negative value of Tajima' D (D = -1.371) indicate that the race Chile suffered a bottleneck and a selective sweep after its introduction, supporting the hypothesis that a small group of Argentine bean genotypes led to the race Chile. A total of 235 genes were identified within haplotype blocks detected exclusively in the race Chile, most of them involved in signal transduction, supporting the hypothesis that intracellular signaling pathways play a fundamental role in the adaptation of organisms to changes in the environment. To date, our findings are the most complete investigation associated with the origin of the race Chile of common bean.

An LTR retrotransposon in the promoter of a PsMYB10.2 gene associated with the regulation of fruit flesh color in Japanese plum.

Japanese plums exhibit wide diversity of fruit coloration. The red to black hues are caused by the accumulation of anthocyanins, while their absence results in yellow, orange or green fruits. In Prunus, MYB10 genes are determinants for anthocyanin accumulation. In peach, QTLs for red plant organ traits map in an LG3 region with three MYB10 copies (PpMYB10.1, PpMYB10.2 and PpMYB10.3). In Japanese plum the gene copy number in this region differs with respect to peach: there are at least three copies of PsMYB10.1, with the expression of one of them (PsMYB10.1a) correlating with fruit skin color. The objective of this study was to determine a possible role of LG3-PsMYB10 genes in the natural variability of the flesh color trait and to develop a molecular marker for marker-assisted selection (MAS). We explored the variability within the LG3-PsMYB10 region using long-range sequences obtained in previous studies through CRISPR-Cas9 enrichment sequencing. We found that the PsMYB10.2 gene was only expressed in red flesh fruits. Its role in promoting anthocyanin biosynthesis was validated by transient overexpression in Japanese plum fruits. The analysis of long-range sequences identified an LTR retrotransposon in the promoter of the expressed PsMYB10.2 gene that explained the trait in 93.1% of the 145 individuals analyzed. We hypothesize that the LTR retrotransposon may promote the PsMYB10.2 expression and activate the anthocyanin biosynthesis pathway. We propose for the first time the PsMYB10.2 gene as candidate for the flesh color natural variation in Japanese plum and provide a molecular marker for MAS.

An Upgraded, Highly Saturated Linkage Map of Japanese Plum (Prunus salicina Lindl.), and Identification of a New Major Locus Controlling the Flavan-3-ol Composition in Fruits.

Japanese plum fruits are rich in phenolic compounds, such as anthocyanins and flavan-3-ols, whose contents vary significantly among cultivars. Catechin (C) and epicatechin (EC) are flavan-3-ol monomers described in the fruits of this species and are associated with bitterness, astringency, antioxidant capacity, and susceptibility to enzymatic mesocarp browning. In this study, we aimed to identify quantitative trait loci (QTL) associated with the content of flavan-3-ol in Japanese plum fruits. We evaluated the content of C and EC in the mesocarp and exocarp of samples from 79 and 64 seedlings of an F1 progeny (<'98-99' × 'Angeleno'>) in the first and second seasons, respectively. We also constructed improved versions of linkage maps from '98-99' and 'Angeleno,' presently called single-nucleotide polymorphisms (SNPs) after mapping the already available GBS reads to Prunus salicina Lindl. cv. 'Sanyueli' v2.0 reference genome. These data allowed for describing a cluster of QTLs in the cultivar, 'Angeleno,' associated with the flavan-3-ol composition of mesocarp and exocarp, which explain up to 100% of the C/EC ratio. Additionally, we developed a C/EC metabolic marker, which was mapped between the markers with the highest log of odds (LOD) scores detected by the QTL analysis. The C/EC locus was located in the LG1, at an interval spanning 0.70 cM at 108.30-108.90 cM. Our results suggest the presence of a novel major gene controlling the preferential synthesis of C or EC in the Japanese plum fruits. This study is a significant advance in understanding the regulation of synthesizing compounds associated with fruit quality, postharvest, and human health promotion.

Detection of Quantitative Trait Loci Controlling the Content of Phenolic Compounds in an Asian Plum (Prunus salicina L.) F1 Population.

Consumption of fresh fruit is known to protect against non-communicable diseases due to the fruit's content in compounds with an antioxidant capacity, among them is polyphenols. Asian plums (Prunus salicina L.) accumulate more than 40 phenolic compounds, with a remarkable diversity in their profiles, depending on the variety and environmental conditions. Although candidate genes have been indicated to control this trait, the loci controlling its phenotypic variation have not yet been defined in this species. The aim of this work was to identify the quantitative trait Loci (QTL) controlling the phenolic compounds content in the Asian plum skin and flesh. Using UHPLC-DAD-Orbitrap-MS, we determined that cyanidin-3-glucoside and cyanidin-3-rutinoside are the main anthocyanins in Asian plums. Other anthocyanins found to a lesser extent were tentatively identified as cyanidin bound to different sugar and procyanidin moieties. Then we phenotyped fruits of 92 and 80 F1 seedlings from the cross < "98.99" × "Angeleno"> (98 Ang) for two harvest seasons. We used HPLC-DAD to quantify single anthocyanin and spectrophotometric techniques to determine the total content of phenols, flavonoids, procyanidins, and antioxidant activity (DPPH and FRAP). To determine the phenotype-genotype association of phenolic compounds content, phenotypic values (adjusted by linear mixed-effects models), genotypic data and linkage maps were analyzed with the multiple QTL model (MQM) approach. We found a total of 21 significant trait-marker associations: 13 QTLs segregating from "98.99" and 8 QTLs from "Angeleno." From these associations, 8 corresponded to phenolic compound content in the flesh and 13 in the skin. Phenotype variance was explained by the detected loci, ranging from 12.4 to 27.1%. The identified loci are related to the content of cyanidin-3-glucoside (LG4), cyanidin-3-rutinoside (LG4), total flavonoids and procyanidins (LG5 and LG8), and minor anthocyanin compounds (LG3 and LG4). These results will help improve the efficiency of breeding programs for the generation of Asian plum varieties with high phenolic compound content.

Isolated myeloid sarcoma of lumbar spine without bone marrow involvement: a rare case report and treatment dilemma.

Granulocytic sarcoma, chloroma, myeloblastoma, or here referred as myeloid sarcoma (MS), is a rare extramedullary tumor composed of immature myeloid cells called myeloblasts. MS is seen most commonly in patients with acute myeloid leukemia and less frequently in chronic myeloid leukemia, myelodysplastic syndrome. In rarer instances, MS has been shown to precede the development of myeloid tumors by acute myeloblastic leukemia (AML). In particular, isolated MS involving spine is extremely rare. We herein present a rare case of isolated spinal MS in non-leukemic patient. This is a previously relatively healthy 47-year-old man who presented with signs of lumbar spinal cord compression, initially reported as schwannoma on imaging, later diagnosed with spinal MS on pathology. Further workup did not reveal any evidence of bone marrow or other hematological involvement. The patient successfully treated by L4/L5 laminectomy and debulking with subsequent radiation resulting in substantial decrease in size of tumor with significant improvement in symptoms during follow up. This case not only describes a rare case of isolated MS of lumbar spine, but also highlights the potential treatment challenges of such a rare diagnosis. We review the available literature, discuss available treatment options, and highlight the need for further investigations along with increased clinician awareness.

The Multisite PeachRefPop Collection: A True Cultural Heritage and International Scientific Tool for Fruit Trees.

Plants have evolved a range of adaptive mechanisms that adjust their development and physiology to variable external conditions, particularly in perennial species subjected to long-term interplay with the environment. Exploiting the allelic diversity within available germplasm and leveraging the knowledge of the mechanisms regulating genotype interaction with the environment are crucial to address climatic challenges and assist the breeding of novel cultivars with improved resilience. The development of multisite collections is of utmost importance for the conservation and utilization of genetic materials and will greatly facilitate the dissection of genotype-by-environment interaction. Such resources are still lacking for perennial trees, especially with the intrinsic difficulties of successful propagation, material exchange, and living collection maintenance. This work describes the concept, design, and realization of the first multisite peach (Prunus persica) reference collection (PeachRefPop) located across different European countries and sharing the same experimental design. Other than an invaluable tool for scientific studies in perennial species, PeachRefPop provides a milestone in an international collaborative project for the conservation and exploitation of European peach germplasm resources and, ultimately, as a true heritage for future generations.

Hydroethanolic Extract of Lampaya Medicinalis Phil. (Verbenaceae) Decreases Proinflammatory Marker Expression in Palmitic Acid-exposed Macrophages.

BACKGROUND: Obesity is a major health problem associated with increased comorbidities, which are partially triggered by inflammation. Proinflammatory macrophage infiltration in adipose tissue of individuals with obesity increases chronic inflammation. Obesity is associated with elevated plasma levels of saturated fatty acids, such as palmitic acid (PA), which promotes inflammation in vivo and in vitro. Infusions of Lampaya medicinalis Phil. (Verbenaceae) are used in the folk medicine of Northern Chile to counteract inflammation of rheumatic diseases. Hydroethanolic extract of lampaya (HEL) contains spectrophotometrically defined compounds that may contribute to the observed effect on inflammation. METHODS: We evaluated the phytochemical composition of HEL by high-performance liquid chromatography coupled to diode array detection (HPLC-DAD) and liquid chromatography-electrospray ionization- tandem mass spectrometry (LC-ESI-MS/MS). We assessed whether the exposure to HEL affects PA-induced expression of proinflammatory factors in THP-1 macrophages. RESULTS: HPLC-DAD and LC-ESI-MS/MS analyses showed the presence of considerable amounts of flavonoids in HEL. The PA-induced phosphorylation of the inflammatory pathway mediators IKK and NF-κB, as well as the elevated expression and secretion of proinflammatory cytokines (IL-6, TNF-α), were reduced in cells pre-exposed to HEL. CONCLUSION: These findings give new insights about the effect of HEL reducing IKK/NF-κB proinflammatory pathway, likely explained by the number of flavonoids contained in the extract. More studies would be needed to define the possible role of Lampaya as a preventive approach in subjects with obesity whose circulating PA might contribute to chronic inflammation.

Deletion of the miR172 target site in a TOE-type gene is a strong candidate variant for dominant double-flower trait in Rosaceae.

Double flowers with supernumerary petals have been selected by humans for their attractive appearance and commercial value in several ornamental plants, including Prunus persica (peach), a recognized model for Rosaceae genetics and genomics. Despite the relevance of this trait, knowledge of the underlying genes is limited. Of two distinct loci controlling the double-flower phenotype in peach, we focused on the dominant Di2 locus. High-resolution linkage mapping in five segregating progenies delimited Di2 to an interval spanning 150 858 bp and 22 genes, including Prupe.6G242400 encoding an euAP2 transcription factor. Analyzing genomic resequencing data from single- and double-flower accessions, we identified a deletion spanning the binding site for miR172 in Prupe.6G242400 as a candidate variant for the double-flower trait, and we showed transcript expression for both wild-type and deleted alleles. Consistent with the proposed role in controlling petal number, Prupe.6G242400 is expressed in buds at critical times for floral development. The indelDi2 molecular marker designed on this sequence variant co-segregated with the phenotype in 621 progenies, accounting for the dominant inheritance of the Di2 locus. Further corroborating the results in peach, we identified a distinct but similar mutation in the ortholog of Prupe.6G242400 in double-flower roses. Phylogenetic analysis showed that these two genes belong to a TARGET OF EAT (TOE)-type clade not represented in Arabidopsis, indicating a divergence of gene functions between AP2-type and TOE-type factors in Arabidopsis and other species. The identification of orthologous candidate genes for the double-flower phenotype in two important Rosaceae species provides valuable information to understand the genetic control of this trait in other major ornamental plants.

Genome-enabled predictions for fruit weight and quality from repeated records in European peach progenies.

BACKGROUND: Highly polygenic traits such as fruit weight, sugar content and acidity strongly influence the agroeconomic value of peach varieties. Genomic Selection (GS) can accelerate peach yield and quality gain if predictions show higher levels of accuracy compared to phenotypic selection. The available IPSC 9K SNP array V1 allows standardized and highly reliable genotyping, preparing the ground for GS in peach. RESULTS: A repeatability model (multiple records per individual plant) for genome-enabled predictions in eleven European peach populations is presented. The analysis included 1147 individuals derived from both commercial and non-commercial peach or peach-related accessions. Considered traits were average fruit weight (FW), sugar content (SC) and titratable acidity (TA). Plants were genotyped with the 9K IPSC array, grown in three countries (France, Italy, Spain) and phenotyped for 3-5 years. An analysis of imputation accuracy of missing genotypic data was conducted using the software Beagle, showing that two of the eleven populations were highly sensitive to increasing levels of missing data. The regression model produced, for each trait and each population, estimates of heritability (FW:0.35, SC:0.48, TA:0.53, on average) and repeatability (FW:0.56, SC:0.63, TA:0.62, on average). Predictive ability was estimated in a five-fold cross validation scheme within population as the correlation of true and predicted phenotypes. Results differed by populations and traits, but predictive abilities were in general high (FW:0.60, SC:0.72, TA:0.65, on average). CONCLUSIONS: This study assessed the feasibility of Genomic Selection in peach for highly polygenic traits linked to yield and fruit quality. The accuracy of imputing missing genotypes was as high as 96%, and the genomic predictive ability was on average 0.65, but could be as high as 0.84 for fruit weight or 0.83 for titratable acidity. The estimated repeatability may prove very useful in the management of the typical long cycles involved in peach productions. All together, these results are very promising for the application of genomic selection to peach breeding programmes.

Genotyping by Sequencing for SNP-Based Linkage Analysis and Identification of QTLs Linked to Fruit Quality Traits in Japanese Plum (Prunus salicina Lindl.).

Marker-assisted selection (MAS) in stone fruit (Prunus species) breeding is currently difficult to achieve due to the polygenic nature of the most relevant agronomic traits linked to fruit quality. Genotyping by sequencing (GBS), however, provides a large quantity of useful data suitable for fine mapping using Single Nucleotide Polymorphisms (SNPs) from a reference genome. In this study, GBS was used to genotype 272 seedlings of three F1 Japanese plum (Prunus salicina Lindl) progenies derived from crossing "98-99" (as a common female parent) with "Angeleno," "September King," and "September Queen" as male parents. Raw sequences were aligned to the Peach genome v1, and 42,909 filtered SNPs were obtained after sequence alignment. In addition, 153 seedlings from the "98-99" × "Angeleno" cross were used to develop a genetic map for each parent. A total of 981 SNPs were mapped (479 for "98-99" and 502 for "Angeleno"), covering a genetic distance of 688.8 and 647.03 cM, respectively. Fifty five seedlings from this progeny were phenotyped for different fruit quality traits including ripening time, fruit weight, fruit shape, chlorophyll index, skin color, flesh color, over color, firmness, and soluble solids content in the years 2015 and 2016. Linkage-based QTL analysis allowed the identification of genomic regions significantly associated with ripening time (LG4 of both parents and both phenotyping years), fruit skin color (LG3 and LG4 of both parents and both years), chlorophyll degradation index (LG3 of both parents in 2015) and fruit weight (LG7 of both parents in 2016). These results represent a promising situation for GBS in the identification of SNP variants associated to fruit quality traits, potentially applicable in breeding programs through MAS, in a highly heterozygous crop species such as Japanese plum.

Leaching and residual activity of imidazolinone herbicides in lowland soils / Lixiviação dos herbicidas imidazolinonas em solo de várzea

ABSTRACT: Herbicides used in the Clearfield® rice (Oryza sativa L.) production system have a potential for leaching. This can result in contamination of underground water resources and cause injury to not tolerant crops that are sown in a succession and/or crop rotation. The objective of this study was to determine the leaching potential and the residual activity of the herbicides used in the Clearfield® rice system. The experiment was conducted over a period of two years and consisted of conducting a field test to be followed by two bioassays with a year of difference between their implementation. Initially an experiment was conducted in lowland area where it was planted the cultivar of rice ‘PUITA INTA CL’. Approximately one and two years thereafter, soil samples from each plot were collected at intervals of 5cm to a depth of 30cm (B factor) for the bioassay to evaluate persistence of herbicides. Factor A was composed of mixtures formulated of imazethapyr + imazapic (75 + 25g a.i. L-1), imazapyr + imazapic (525 + 175g a.i. kg-1) in two doses, imazethapyr (100g a.i. L-1) and treatment control without application. Basing on results, it was concluded that the mixtures imazethapyr + imazapic, imazapyr + imazapic and imazethapyr leached into the soil, reaching depths of up to 25cm in lowland soil. Imidazolinone herbicides used today in the irrigated rice Clearfield® system are persistent in soil, and their phytotoxic activity can be observed up to two years after application.

RESUMO: Os herbicidas utilizados no sistema Clearfield® de produção de arroz irrigado (Oryza sativa L.) apresentam potencial de lixiviação. Isso pode causar contaminação de mananciais hídricos subterrâneos e fitotoxicidade as culturas não tolerantes semeadas em sucessão e/ou rotação. O objetivo deste trabalho foi determinar o potencial de lixiviação e o efeito residual dos herbicidas utilizados no sistema Clearfield® de arroz irrigado. O experimento foi conduzido durante dois anos e consistiu da realização de dois bioensaio com diferença de um ano entre a implantação dos mesmos. Inicialmente foi instalado o experimento em área de várzea onde foi semeada a cultivar de arroz ‘Puitá INTA CL’ em área de várzea. Aproximadamente um e dois anos após, foram coletadas amostras de solo de cada parcela, em intervalos de 5cm até a profundidade de 30cm (fator B) para a realização do bioensaio a fim de avaliar persistência dos herbicidas. O fator A foi constituído pelas misturas formuladas imazethapyr + imazapic (75 + 25g i.a. L-1), imazapyr + imazapic (525 + 175g i.a. kg-1) em duas doses, imazethapyr (100g i.a. L-1) e um tratamento testemunha sem aplicação. Com os resultados obtidos, foi possível concluir que as misturas imazethapyr + imazapic, imazapyr + imazapic e imazethapyr lixiviam no solo, atingindo profundidades de até 25cm em solo de várzea. Os herbicidas imidazolinonas, hoje utilizados no sistema Clearfiel® de arroz irrigado, são persistentes no solo podendo seus efeitos fitotóxicos serem observados até dois anos após sua aplicação.

Brown Rot Strikes Prunus Fruit: An Ancient Fight Almost Always Lost.

Brown rot (BR) caused by Monilinia spp., has been an economic problem for the stone fruit market due to dramatic losses, mainly during the postharvest period. There is much literature about basic aspects of Monilinia spp. infection, which indicates that environment significantly influences its occurrence in the orchard. However, progress is needed to sustainably limit this disease: the pathogen is able to develop resistance to pesticides, and most of BR resistance research programs in plant models perish. Solving this problem becomes important due to the need to decrease chemical treatments and reduce residues on fruit. Thus, research has recently increased, exploring a wide range of disease control strategies (e.g., genetic, chemical, physical). Summarizing this information is difficult, as studies evaluate different Monilinia and Prunus model species, with diverse strategies and protocols. Thus, the purpose of this review is to present the diversity and distribution of agents causing BR, focusing on the biochemical mechanisms of Monilinia spp. infection both of the fungi and of the fruit, and report on the resistance sources in Prunus germplasm. This review comprehensively compiles the information currently available to better understand mechanisms related to BR resistance.

Whole-Genome Analysis of Diversity and SNP-Major Gene Association in Peach Germplasm.

Peach was domesticated in China more than four millennia ago and from there it spread world-wide. Since the middle of the last century, peach breeding programs have been very dynamic generating hundreds of new commercial varieties, however, in most cases such varieties derive from a limited collection of parental lines (founders). This is one reason for the observed low levels of variability of the commercial gene pool, implying that knowledge of the extent and distribution of genetic variability in peach is critical to allow the choice of adequate parents to confer enhanced productivity, adaptation and quality to improved varieties. With this aim we genotyped 1,580 peach accessions (including a few closely related Prunus species) maintained and phenotyped in five germplasm collections (four European and one Chinese) with the International Peach SNP Consortium 9K SNP peach array. The study of population structure revealed the subdivision of the panel in three main populations, one mainly made up of Occidental varieties from breeding programs (POP1OCB), one of Occidental landraces (POP2OCT) and the third of Oriental accessions (POP3OR). Analysis of linkage disequilibrium (LD) identified differential patterns of genome-wide LD blocks in each of the populations. Phenotypic data for seven monogenic traits were integrated in a genome-wide association study (GWAS). The significantly associated SNPs were always in the regions predicted by linkage analysis, forming haplotypes of markers. These diagnostic haplotypes could be used for marker-assisted selection (MAS) in modern breeding programs.

A unique mutation in a MYB gene cosegregates with the nectarine phenotype in peach.

Nectarines play a key role in peach industry; the fuzzless skin has implications for consumer acceptance. The peach/nectarine (G/g) trait was described as monogenic and previously mapped on chromosome 5. Here, the position of the G locus was delimited within a 1.1 cM interval (635 kb) based on linkage analysis of an F2 progeny from the cross 'Contender' (C, peach) x 'Ambra' (A, nectarine). Careful inspection of the genes annotated in the corresponding genomic sequence (Peach v1.0), coupled with variant discovery, led to the identification of MYB gene PpeMYB25 as a candidate for trichome formation on fruit skin. Analysis of genomic re-sequencing data from five peach/nectarine accessions pointed to the insertion of a LTR retroelement in exon 3 of the PpeMYB25 gene as the cause of the recessive glabrous phenotype. A functional marker (indelG) developed on the LTR insertion cosegregated with the trait in the CxA F2 progeny and was validated on a broad panel of genotypes, including all known putative donors of the nectarine trait. This marker was shown to efficiently discriminate between peach and nectarine plants, indicating that a unique mutational event gave rise to the nectarine trait and providing a useful diagnostic tool for early seedling selection in peach breeding programs.

Fine mapping and identification of a candidate gene for a major locus controlling maturity date in peach.

BACKGROUND: Maturity date (MD) is a crucial factor for marketing of fresh fruit, especially those with limited shelf-life such as peach (Prunus persica L. Batsch): selection of several cultivars with differing MD would be advantageous to cover and extend the marketing season. Aims of this work were the fine mapping and identification of candidate genes for the major maturity date locus previously identified on peach linkage group 4. To improve genetic resolution of the target locus two F2 populations derived from the crosses Contender x Ambra (CxA, 306 individuals) and PI91459 (NJ Weeping) x Bounty (WxBy, 103 individuals) were genotyped with the Sequenom and 9K Illumina Peach Chip SNP platforms, respectively. RESULTS: Recombinant individuals from the WxBy F2 population allowed the localisation of maturity date locus to a 220 kb region of the peach genome. Among the 25 annotated genes within this interval, functional classification identified ppa007577m and ppa008301m as the most likely candidates, both encoding transcription factors of the NAC (NAM/ATAF1, 2/CUC2) family. Re-sequencing of the four parents and comparison with the reference genome sequence uncovered a deletion of 232 bp in the upstream region of ppa007577m that is homozygous in NJ Weeping and heterozygous in Ambra, Bounty and the WxBy F1 parent. However, this variation did not segregate in the CxA F2 population being the CxA F1 parent homozygous for the reference allele. The second gene was thus examined as a candidate for maturity date. Re-sequencing of ppa008301m, showed an in-frame insertion of 9 bp in the last exon that co-segregated with the maturity date locus in both CxA and WxBy F2 populations. CONCLUSIONS: Using two different segregating populations, the map position of the maturity date locus was refined from 3.56 Mb to 220 kb. A sequence variant in the NAC gene ppa008301m was shown to co-segregate with the maturity date locus, suggesting this gene as a candidate controlling ripening time in peach. If confirmed on other genetic materials, this variant may be used for marker-assisted breeding of new cultivars with differing maturity date.

Identification of woolliness response genes in peach fruit after post-harvest treatments.

Woolliness is a physiological disorder of peaches and nectarines that becomes apparent when fruit are ripened after prolonged periods of cold storage. This disorder is of commercial importance since shipping of peaches to distant markets and storage before selling require low temperature. However, knowledge about the molecular basis of peach woolliness is still incomplete. To address this issue, a nylon macroarray containing 847 non-redundant expressed sequence tags (ESTs) from a ripe peach fruit cDNA library was developed and used. Gene expression changes of peach fruit (Prunus persica cv. O'Henry) ripened for 7 d at 21 degrees C (juicy fruit) were compared with those of fruit stored for 15 d at 4 degrees C and then ripened for 7 d at 21 degrees C (woolly fruit). A total of 106 genes were found to be differentially expressed between juicy and woolly fruit. Data analysis indicated that the activity of most of these genes (>90%) was repressed in the woolly fruit. In cold-stored peaches (cv. O'Henry), the expression level of selected genes (cobra, endopolygalacturonase, cinnamoyl-CoA-reductase, and rab11) was lower than in the juicy fruit, and it remained low in woolly peaches after ripening, a pattern that was conserved in woolly fruit from two other commercial cultivars (cv. Flamekist and cv. Elegant Lady). In addition, the results of this study indicate that molecular changes during fruit woolliness involve changes in the expression of genes associated with cell wall metabolism and endomembrane trafficking. Overall, the results reported here provide an initial characterization of the transcriptome activity of peach fruit under different post-harvest treatments.