A DNA test for high incidence of soft scald and soggy breakdown postharvest disorders in Malus domestica Borkh.

The 'Honeycrisp' apple, an economically important cultivar and breeding parent, is prone to soft scald and soggy breakdown postharvest physiological disorders. Phenotypic evaluation of soft scald is time consuming and costly, making it an excellent target for DNA-informed breeding. The objective of this study was to develop a DNA test for a soft scald and soggy breakdown quantitative trait locus (QTL) on linkage group two (LG2) that was characterized in a previous study. 'Honeycrisp' is homozygous for the undesirable high disorder incidence haplotype (HDI) at this QTL. In this study, sixteen single nucleotide polymorphism markers were evaluated for their associations with the HDI haplotype in a set of 132 unique cultivars and important breeding parents. A DNA test was successfully developed utilizing KASP™ (Kompetitive Allele Specific PCR) chemistry to identify the number of HDI haplotypes in individuals. This test had a 100% accuracy for detecting homozygous unfavorable HDI individuals and has an expected 88% accuracy over all three haplotype copy groups across the evaluated germplasm. This DNA test is a promising tool for minimizing the chances of selecting individuals that exhibit high incidence of soft scald postharvest disorder in 'Honeycrisp'-related germplasm. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01245-w.

Genomic ancestry estimation quantifies use of wild species in grape breeding.

BACKGROUND: Grapes are one of the world's most valuable crops and most are made into wine. Grapes belong to the genus Vitis, which includes over 60 inter-fertile species. The most common grape cultivars derive their entire ancestry from the species Vitis vinifera, but wild relatives have also been exploited to create hybrid cultivars, often with increased disease resistance. RESULTS: We evaluate the genetic ancestry of some of the most widely grown commercial hybrids from North America and Europe. Using genotyping-by-sequencing (GBS), we generated 2482 SNPs and 56 indels from 7 wild Vitis, 7 V. vinifera, and 64 hybrid cultivars. We used a principal component analysis (PCA) based ancestry estimation procedure and verified its accuracy with both empirical and simulated data. V. vinifera ancestry ranged from 11 % to 76 % across hybrids studied. Approximately one third (22/64) of the hybrids have ancestry estimates consistent with F1 hybridization: they derive half of their ancestry from wild Vitis and half from V. vinifera. CONCLUSIONS: Our results suggest that hybrid grape breeding is in its infancy. The distribution of V. vinifera ancestry across hybrids also suggests that backcrosses to wild Vitis species have been more frequent than backcrosses to V. vinifera during hybrid grape breeding. This pattern is unusual in crop breeding, as it is most common to repeatedly backcross to elite, or domesticated, germplasm. We anticipate our method can be extended to facilitate marker-assisted selection in order to introgress beneficial wild Vitis traits, while allowing for offspring with the highest V. vinifera content to be selected at the seedling stage.

The 2012 West Nile encephalitis epidemic in Dallas, Texas.

IMPORTANCE: After progressive declines over recent years, in 2012 West Nile virus epidemics resurged nationwide, with the greatest number of cases centered in Dallas County, Texas. OBJECTIVE: To analyze the epidemiologic, meteorologic, and geospatial features of the 2012 Dallas West Nile virus epidemic to guide future prevention efforts. DESIGN, SETTING, AND PATIENTS: Public health surveillance of Dallas County, an area of 2257 km2 and population of 2.4 million. Surveillance data included numbers of residents diagnosed with West Nile virus infection between May 30, 2012, and December 3, 2012; mosquito trap results; weather data; and syndromic surveillance from area emergency departments. MAIN OUTCOMES AND MEASURES: Incidence and age-adjusted incidence rates of West Nile neuroinvasive disease (WNND), daily prevalence of emergency department visits for asthma and skin rash, and Culex quinquefasciatus species-specific vector index (an estimate of the average number of West Nile virus-infected mosquitoes per trap-night). RESULTS: The investigation identified 173 cases of WNND, 225 of West Nile fever, 17 West Nile virus-positive blood donors, and 19 deaths in 2012. The incidence rate for WNND was 7.30 per 100,000 residents in 2012, compared with 2.91 per 100,000 in 2006, the largest previous Dallas County outbreak. An unusually rapid and early escalation of large numbers of human cases closely followed increasing infection trends in mosquitoes. The Cx quinquefasciatus species-specific vector index predicted the onset of symptoms among WNND cases 1 to 2 weeks later (count regression ß = 2.97 [95% CI, 2.34 to 3.60]; P < .001). Although initially widely distributed, WNND cases soon clustered in neighborhoods with high housing density in the north central area of the county, reflecting higher vector indices and following geospatial patterns of West Nile virus in prior years. During the 11 years since West Nile virus was first identified in Dallas, the log-transformed annual prevalence of WNND was inversely associated with the number of days with low temperatures below 28°F (-2.2°C) in December through February (ß = -0.29 [95% CI, -0.36 to -0.21]; P < .001). Aerial insecticide spraying was not associated with increases in emergency department visits for respiratory symptoms (ß = -4.03 [95% CI, -13.76 to 5.70]; P = .42) or skin rash (ß = -1.00 [95% CI, -6.92 to 4.92]; P = .74). CONCLUSIONS AND RELEVANCE: Large West Nile virus epidemics in Dallas County begin early after unusually warm winters, revisit similar geographical distributions, and are strongly predicted by the mosquito vector index. Consideration of weather patterns and historical geographical hot spots and acting on the vector index may help prevent West Nile virus-associated illness.

Genomewide selection for fruit quality traits in apple: breeding insights gained from prediction and postdiction.

Many fruit quality traits in apple (Malus domestica Borkh.) are controlled by multiple small-effect quantitative trait loci (QTLs). Genomewide selection (genomic selection) might be an effective breeding approach for highly quantitative traits in woody perennial crops with long generation times like apple. The goal of this study was to determine if genomewide prediction is an effective breeding approach for fruit quality traits in an apple scion breeding program. Representative apple scion breeding germplasm (nindividuals = 955), high-quality single nucleotide polymorphism (SNP) data (nSNPs = 977), and breeding program fruit quality trait data at harvest were analyzed. Breeding parents `Honeycrisp' and `Minneiska' were highly represented. Moderate to high predictive abilities were observed for most fruit quality traits at harvest. For example, when 25% random subsets of the germplasm set were used as training sets, mean predictive abilities ranged from 0.35 to 0.54 across traits. Trait, training and test sets, family size for within family prediction, and number of SNPs per chromosome affected model predictive ability. Inclusion of large-effect QTLs as fixed effects resulted in higher predictive abilities for some traits (e.g. percent red overcolor). Postdiction (i.e. retrospective) analyses demonstrated the impact of culling threshold on selection decisions. The results of this study demonstrate that genomewide selection is a useful breeding approach for certain fruit quality traits in apple.

A new method to reconstruct the direction of parent-offspring duo relationships using SNP array data and its demonstration on ancient and modern cultivars in the outcrossing species malus × domestica.

Unordered parent-offspring (PO) relationships are an outstanding issue in pedigree reconstruction studies. Resolution of the order of these relationships would expand the results, conclusions, and usefulness of such studies; however, no such PO order resolution (POR) tests currently exist. This study describes two such tests, demonstrated using SNP array data in the outcrossing species apple (Malus × domestica) on a PO relationship of known order ("Keepsake" as a parent of "Honeycrisp") and two PO relationships previously ordered only via provenance information. The first test, POR-1, tests whether some of the extended haplotypes deduced from homozygous SNP calls from one individual in an unordered PO duo are composed of recombinant haplotypes from accurately phased SNP genotypes from the second individual. If so, the first individual would be the offspring of the second individual, otherwise the opposite relationship would be present. The second test, POR-2, does not require phased SNP genotypes and uses similar logic as the POR-1 test, albeit in a different approach. The POR-1 and POR-2 tests determined the correct relationship between "Keepsake" and "Honeycrisp". The POR-2 test confirmed "Reinette Franche" as a parent of "Nonpareil" and "Brabant Bellefleur" as a parent of "Court Pendu Plat". The latter finding conflicted with the recorded provenance information, demonstrating the need for these tests. The successful demonstration of these tests suggests they can add insights to future pedigree reconstruction studies, though caveats, like extreme inbreeding or selfing, would need to be considered where relevant.

The use of shared haplotype length information for pedigree reconstruction in asexually propagated outbreeding crops, demonstrated for apple and sweet cherry.

Pedigree information is of fundamental importance in breeding programs and related genetics efforts. However, many individuals have unknown pedigrees. While methods to identify and confirm direct parent-offspring relationships are routine, those for other types of close relationships have yet to be effectively and widely implemented with plants, due to complications such as asexual propagation and extensive inbreeding. The objective of this study was to develop and demonstrate methods that support complex pedigree reconstruction via the total length of identical by state haplotypes (referred to in this study as "summed potential lengths of shared haplotypes", SPLoSH). A custom Python script, HapShared, was developed to generate SPLoSH data in apple and sweet cherry. HapShared was used to establish empirical distributions of SPLoSH data for known relationships in these crops. These distributions were then used to estimate previously unknown relationships. Case studies in each crop demonstrated various pedigree reconstruction scenarios using SPLoSH data. For cherry, a full-sib relationship was deduced for 'Emperor Francis, and 'Schmidt', a half-sib relationship for 'Van' and 'Windsor', and the paternal grandparents of 'Stella' were confirmed. For apple, 29 cultivars were found to share an unknown parent, the pedigree of the unknown parent of 'Cox's Pomona' was reconstructed, and 'Fameuse' was deduced to be a likely grandparent of 'McIntosh'. Key genetic resources that enabled this empirical study were large genome-wide SNP array datasets, integrated genetic maps, and previously identified pedigree relationships. Crops with similar resources are also expected to benefit from using HapShared for empowering pedigree reconstruction.

RosBREED: bridging the chasm between discovery and application to enable DNA-informed breeding in rosaceous crops.

The Rosaceae crop family (including almond, apple, apricot, blackberry, peach, pear, plum, raspberry, rose, strawberry, sweet cherry, and sour cherry) provides vital contributions to human well-being and is economically significant across the U.S. In 2003, industry stakeholder initiatives prioritized the utilization of genomics, genetics, and breeding to develop new cultivars exhibiting both disease resistance and superior horticultural quality. However, rosaceous crop breeders lacked certain knowledge and tools to fully implement DNA-informed breeding-a "chasm" existed between existing genomics and genetic information and the application of this knowledge in breeding. The RosBREED project ("Ros" signifying a Rosaceae genomics, genetics, and breeding community initiative, and "BREED", indicating the core focus on breeding programs), addressed this challenge through a comprehensive and coordinated 10-year effort funded by the USDA-NIFA Specialty Crop Research Initiative. RosBREED was designed to enable the routine application of modern genomics and genetics technologies in U.S. rosaceous crop breeding programs, thereby enhancing their efficiency and effectiveness in delivering cultivars with producer-required disease resistances and market-essential horticultural quality. This review presents a synopsis of the approach, deliverables, and impacts of RosBREED, highlighting synergistic global collaborations and future needs. Enabling technologies and tools developed are described, including genome-wide scanning platforms and DNA diagnostic tests. Examples of DNA-informed breeding use by project participants are presented for all breeding stages, including pre-breeding for disease resistance, parental and seedling selection, and elite selection advancement. The chasm is now bridged, accelerating rosaceous crop genetic improvement.

Genetics of zonal leaf chlorosis and genetic linkage to a major gene regulating skin anthocyanin production (MdMYB1) in the apple (Malus × domestica) cultivar Honeycrisp.

'Honeycrisp' is a widely grown and acclaimed apple cultivar that is commonly used in breeding programs. It also has a well-documented tendency to develop the physiological disorder, zonal leaf chlorosis (ZLC). This disorder causes reduced photosynthetic capacity and is thought to be due to a problem with phloem loading, although the underlying genetics of the disorder have not previously been discerned. In order to understand the breeding implications of the disorder, six families with 'Honeycrisp' as a parent and one family with 'Honeycrisp' as both a maternal and paternal grandparent were evaluated for ZLC incidence over two years. One major quantitative trait locus (QTL) for ZLC incidence was identified on linkage group (LG) 9. A haplotype in 'Honeycrisp' that originated from grandparent 'Duchess of Oldenburg' was associated with increased ZLC incidence in offspring in both years and all families evaluated. The LG9 QTL was 5 to 10 cM from MdMYB1, which is a major gene regulating fruit skin anthocyanin production. 'Honeycrisp' is heterozygous for red fruit skin overcolor at MdMYB1. The 'Honeycrisp' haplotype at the LG9 QTL associated with increased ZLC is in linkage phase with the allele at MdMYB1 associated with red color. Selection for the red allele from 'Honeycrisp' at MdMYB1 will result in most offspring also inheriting the haplotype at the LG9 QTL associated with high ZLC. The occurrence of two copies of this haplotype was sub-lethal in seedlings of a family where both parents inherited both the red overcolor allele at MdMYB1 and the haplotype at the LG9 QTL associated with high ZLC. This is the first study to have identified a genetic component of ZLC with clear breeding implications.

Correction: Genetics of zonal leaf chlorosis and genetic linkage to a major gene regulating skin anthocyanin production (MdMYB1) in the apple (Malus × domestica) cultivar Honeycrisp.

[This corrects the article DOI: 10.1371/journal.pone.0210611.].

Genetic analysis of stilbenoid profiles in grapevine stems reveals a major mQTL hotspot on chromosome 18 associated with disease-resistance motifs.

Grapevine (Vitis spp.) contains a wealth of phytochemicals that have received considerable attention due to health-promoting properties and biological activities as phytoalexins. To date, the genetic basis of the quantitative variations for these potentially beneficial compounds has been limited. Here, metabolic quantitative trait locus (mQTL) mapping was conducted using grapevine stems of a segregating F2 population. Metabolic profiling of grapevine stems was performed using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), resulting in the detection of 1317 ions/features. In total, 19 of these features matched with literature-reported stilbenoid masses and were genetically mapped using a 1449-SNP linkage map and R/qtl software, resulting in the identification of four mQTLs. Two large-effect mQTLs that corresponded to a stilbenoid dimer and a trimer were mapped on chromosome 18, accounting for phenotypic variances of 29.0% and 38.4%. Functional annotations of these large-effect mQTLs on the VitisNet network database revealed a major hotspot of disease-resistance motifs on chromosome 18. This 2.8-Mbp region contains 48 genes with R-gene motifs, including variants of TIR, NBS, and LRR, that might potentially confer resistance to powdery mildew, downy mildew, or other pathogens. The locus also encompasses genes associated with flavonoid and biosynthetic pathways that are likely involved in the production of secondary metabolites, including phytoalexins. In addition, haplotype dosage effects of the five mQTLs further characterized the genomic regions for differential production of stilbenoids that can be applied in resistance breeding through manipulation of stilbenoid production in planta.

Detection and diagnosis of herpes simplex virus infection in adults with acute liver failure.

Disseminated herpes simplex virus (HSV) infection may lead to acute liver failure (ALF) and the need for emergency liver transplantation (LT). The primary aim of this study was to determine the utility of HSV serological testing and HSV DNA testing by polymerase chain reaction (PCR) in the diagnosis and management of indeterminate, pregnancy-related, and known HSV-related ALF. Stored sera obtained on study day 1 or 2 from patients enrolled in the United States ALF Study Group with indeterminate (n = 51), pregnancy-related (n = 12), and HSV-related (n = 4) ALF were screened for HSV DNA by PCR and serology. While 7 of the indeterminate and pregnant patients had positive anti-HSV immunoglobulin M, none had detectable HSV DNA. The 4 known HSV cases all had high-titer HSV DNA on presentation (range: 3.5 to 36 x 10(8) copies/mL). Two HSV patients underwent LT but developed posttransplant extrahepatic HSV infection despite suppression of HSV DNA with acyclovir treatment, and one of them eventually died. The 2 other fulminant HSV patients died within 48 hours of presentation. In conclusion, serum HSV DNA indicative of occult HSV infection was not detected in 51 indeterminate and 12 pregnancy-related ALF patients. The 4 patients with known HSV-related ALF all had high HSV DNA levels at presentation, and despite the rapid use of antiviral therapy and emergency LT, substantial morbidity and mortality were encountered, highlighting the poor prognosis with severe disseminated HSV infection.

Pandemic influenza: a primer.

Influenza maintains a special position in human medicine as the cause of the greatest pandemic of disease in all of human history as well as a continuous and significant source of worldwide morbidity and mortality every year. The very nature of the influenza virus allows it to evade and adapt to the human population. We currently have useful tools such as vaccination and antiviral medications to limit this burden; however, appropriate vaccines take time to prepare, and antiviral drug resistance has emerged as a significant problem. Continued vigilance and pandemic planning are essential, given the continued threat of novel strains that can arise and rapidly cause disease across the world.

Genetic dissection of powdery mildew resistance in interspecific half-sib grapevine families using SNP-based maps.

Quantitative trait locus (QTL) identification in perennial fruit crops is impeded largely by their lengthy generation time, resulting in costly and labor-intensive maintenance of breeding programs. In a grapevine (genus Vitis) breeding program, although experimental families are typically unreplicated, the genetic backgrounds may contain similar progenitors previously selected due to their contribution of favorable alleles. In this study, we investigated the utility of joint QTL identification provided by analyzing half-sib families. The genetic control of powdery mildew was studied using two half-sib F1 families, namely GE0711/1009 (MN1264 × MN1214; N = 147) and GE1025 (MN1264 × MN1246; N = 125) with multiple species in their ancestry. Maternal genetic maps consisting of 1077 and 1641 single nucleotide polymorphism (SNP) markers, respectively, were constructed using a pseudo-testcross strategy. Ratings of field resistance to powdery mildew were obtained based on whole-plant evaluation of disease severity. This 2-year analysis uncovered two QTLs that were validated on a consensus map in these half-sib families with improved precision relative to the parental maps. Examination of haplotype combinations based on the two QTL regions identified strong association of haplotypes inherited from 'Seyval blanc', through MN1264, with powdery mildew resistance. This investigation also encompassed the use of microsatellite markers to establish a correlation between 206-bp (UDV-015b) and 357-bp (VViv67) fragment sizes with resistance-carrying haplotypes. Our work is one of the first reports in grapevine demonstrating the use of SNP-based maps and haplotypes for QTL identification and tagging of powdery mildew resistance in half-sib families.

Elucidation of the 'Honeycrisp' pedigree through haplotype analysis with a multi-family integrated SNP linkage map and a large apple (Malus×domestica) pedigree-connected SNP data set.

The apple (Malus×domestica) cultivar Honeycrisp has become important economically and as a breeding parent. An earlier study with SSR markers indicated the original recorded pedigree of 'Honeycrisp' was incorrect and 'Keepsake' was identified as one putative parent, the other being unknown. The objective of this study was to verify 'Keepsake' as a parent and identify and genetically describe the unknown parent and its grandparents. A multi-family based dense and high-quality integrated SNP map was created using the apple 8 K Illumina Infinium SNP array. This map was used alongside a large pedigree-connected data set from the RosBREED project to build extended SNP haplotypes and to identify pedigree relationships. 'Keepsake' was verified as one parent of 'Honeycrisp' and 'Duchess of Oldenburg' and 'Golden Delicious' were identified as grandparents through the unknown parent. Following this finding, siblings of 'Honeycrisp' were identified using the SNP data. Breeding records from several of these siblings suggested that the previously unreported parent is a University of Minnesota selection, MN1627. This selection is no longer available, but now is genetically described through imputed SNP haplotypes. We also present the mosaic grandparental composition of 'Honeycrisp' for each of its 17 chromosome pairs. This new pedigree and genetic information will be useful in future pedigree-based genetic studies to connect 'Honeycrisp' with other cultivars used widely in apple breeding programs. The created SNP linkage map will benefit future research using the data from the Illumina apple 8 and 20 K and Affymetrix 480 K SNP arrays.

Risk factors for deep sternal wound infection after cardiac surgery: Influence of red blood cell transfusions and chronic infection.

BACKGROUND: Deep sternal wound infection (DSWI) following cardiac surgery is a serious complication, but risk factors associated with DSWI have not been fully elucidated. METHODS: We analyzed all DSWI cases at our institution from 2010-2013 in adult cardiac median sternotomy cases, based on Society of Thoracic Surgeons or National Healthcare Safety Network definitions, but with 1-year surveillance postsurgery. Controls were matched 3:1 per case for procedure, age, and year of surgery. Demographic and operative data were pulled from Society of Thoracic Surgeons database and chart review. Potential variables were evaluated using univariate and multivariate conditional logistic regression. RESULTS: Out of 1,894 surgeries performed, 39 DSWI cases (2%) and 117 controls were identified. In univariate analyses, patients with red blood cell (RBC) transfusion ≥ 4 units, any platelet transfusion, previous infections, and chronic infections were associated with higher DSWI. RBC transfusion ≥ 4 units (P = .037) and chronic infections (P = .029) remained significant risk factors for DSWI in multivariate analysis. Preoperative anemia alone was not associated with more DSWI, but its interaction with RBC transfusion ≥ 4 units was significant. CONCLUSIONS: High-volume RBC transfusions and chronic infections were strongly associated with DSWI in our population and represent potentially modifiable areas for improvement.

Catheter-Related Bloodstream Infections in Patients on Emergent Hemodialysis.

OBJECTIVE: This study had 2 objectives: (1) to describe the epidemiology of catheter-related bloodstream infections (CRBSI) in patients with end-stage renal disease (ESRD) who have no access to scheduled dialysis and (2) to evaluate whether a positive culture of the heparin-lock solution is associated with subsequent development of bacteremia. DESIGN: Retrospective observational cohort design for objective 1; and prospective cohort design for objective 2. SETTING AND PARTICIPANTS: The study was conducted in a 770-bed public academic tertiary hospital in Dallas, Texas. The participants were patients with ESRD undergoing scheduled or emergent hemodialysis. METHODS: We reviewed the records of 147 patients who received hemodialysis between January 2011 and May 2011 and evaluated the rate of CRBSI in the previous 5 years. For the prospective study, we cultured the catheter heparin-lock solution in 62 consecutive patients between June 2012 and August 2012 and evaluated the incidence of CRBSI at 6 months. RESULTS: Of the 147 patients on emergent hemodialysis, 125 had a tunneled catheter, with a CRBSI rate of 2.61 per 1,000 catheter days. The predominant organisms were Gram-negative rods (GNR). In the prospective study, we found that the dialysis catheter was colonized more frequently in patients on emergent hemodialysis than in those on scheduled hemodialysis. Colonization with GNR or Staphylococcus aureus was associated with subsequent CRBSI at 6 months follow-up. CONCLUSIONS: Patients undergoing emergent hemodialysis via tunneled catheter are predisposed to Gram-negative CRBSI. Culturing the heparin-lock solution may predict subsequent episodes of CRBSI if it shows colonization with GNR or Staphylococcus aureus. Prevention approaches in this population need to be studied further.

Next Generation Mapping of Enological Traits in an F2 Interspecific Grapevine Hybrid Family.

In winegrapes (Vitis spp.), fruit quality traits such as berry color, total soluble solids content (SS), malic acid content (MA), and yeast assimilable nitrogen (YAN) affect fermentation or wine quality, and are important traits in selecting new hybrid winegrape cultivars. Given the high genetic diversity and heterozygosity of Vitis species and their tendency to exhibit inbreeding depression, linkage map construction and quantitative trait locus (QTL) mapping has relied on F1 families with the use of simple sequence repeat (SSR) and other markers. This study presents the construction of a genetic map by single nucleotide polymorphisms identified through genotyping-by-sequencing (GBS) technology in an F2 mapping family of 424 progeny derived from a cross between the wild species V. riparia Michx. and the interspecific hybrid winegrape cultivar, 'Seyval'. The resulting map has 1449 markers spanning 2424 cM in genetic length across 19 linkage groups, covering 95% of the genome with an average distance between markers of 1.67 cM. Compared to an SSR map previously developed for this F2 family, these results represent an improved map covering a greater portion of the genome with higher marker density. The accuracy of the map was validated using the well-studied trait berry color. QTL affecting YAN, MA and SS related traits were detected. A joint MA and SS QTL spans a region with candidate genes involved in the malate metabolism pathway. We present an analytical pipeline for calling intercross GBS markers and a high-density linkage map for a large F2 family of the highly heterozygous Vitis genus. This study serves as a model for further genetic investigations of the molecular basis of additional unique characters of North American hybrid wine cultivars and to enhance the breeding process by marker-assisted selection. The GBS protocols for identifying intercross markers developed in this study can be adapted for other heterozygous species.

A next-generation marker genotyping platform (AmpSeq) in heterozygous crops: a case study for marker-assisted selection in grapevine.

Marker-assisted selection (MAS) is often employed in crop breeding programs to accelerate and enhance cultivar development, via selection during the juvenile phase and parental selection prior to crossing. Next-generation sequencing and its derivative technologies have been used for genome-wide molecular marker discovery. To bridge the gap between marker development and MAS implementation, this study developed a novel practical strategy with a semi-automated pipeline that incorporates trait-associated single nucleotide polymorphism marker discovery, low-cost genotyping through amplicon sequencing (AmpSeq) and decision making. The results document the development of a MAS package derived from genotyping-by-sequencing using three traits (flower sex, disease resistance and acylated anthocyanins) in grapevine breeding. The vast majority of sequence reads (⩾99%) were from the targeted regions. Across 380 individuals and up to 31 amplicons sequenced in each lane of MiSeq data, most amplicons (83 to 87%) had <10% missing data, and read depth had a median of 220-244×. Several strengths of the AmpSeq platform that make this approach of broad interest in diverse crop species include accuracy, flexibility, speed, high-throughput, low-cost and easily automated analysis.

Recurrent exposure to Histoplasma capsulatum in modern air-conditioned buildings.

BACKGROUND: Between 1989 and 1996, an epidemic of histoplasmosis occurred on a medical school campus. There had been numerous construction projects on the campus that involved previously wooded land and were adjacent to a large bird sanctuary. METHODS: We investigated the epidemic with active surveillance to detect cases, a histoplasmin skin-test survey, inspection of the air-filtration systems of the involved buildings, and cultures of soil samples. The investigation also included a simulation of entry into air-intakes of the buildings from spore sources by means of a wind-tunnel analysis of a model of the campus that used inert gas. After control procedures were instituted, sentinel population groups had follow-up with yearly serological tests. RESULTS: From 1989 through 1996, there were 29 cases of histoplasmosis that occurred among school employees. All cases with a defined onset began during periods of ongoing campus construction. Positivity rates for histoplasmin skin testing were higher among on-campus personnel (47%) than among off-campus employee control subjects (28%) (P<.001); the rates were highest in employees who worked on the upper floors of 2 research buildings. The air-handling units on the roofs of these buildings were not designed to exclude Histoplasma spores. The wind-tunnel experiment indicated that spores aerosolized in the bird sanctuary were not taken into campus buildings. CONCLUSIONS: The major sources of employee exposure to H. capsulatum spores were the construction sites. Low-level, recurrent exposures occurred over several years inside modern research buildings. This phenomenon, which has not been previously described, may play a role in the epidemiology of spore-transmitted diseases in urban settings.