Tracking seasonal changes in diversity of pollen allergen exposure: Targeted metabarcoding of a subtropical aerobiome.

The importance of grass pollen to the global burden of allergic respiratory disease is well established but exposure to subtropical and temperate pollens is difficult to discern. Current monitoring of airborne pollen relies on light microscopy, limiting identification of taxa to family level. This informs seasonal fluctuations in pollen aerobiology but restricts analysis of aerobiological composition. We aimed to test the utility of DNA metabarcoding to identify specific taxa contributing to the aerobiome of environmental air samples, using routine pollen and spore monitoring equipment, as well as assess temporal variation of Poaceae pollen across an entire season. Airborne pollen concentrations were determined by light microscopy over two pollen seasons in the subtropical city of Brisbane (27°32'S, 153°00E), Australia. Thirty daily pollen samples were subjected to high throughput sequencing of the plastid rbcL amplicon. Amplicons corresponded to plants observed in the local biogeographical region with up to 3238 different operational taxonomic units (OTU) detected. The aerobiome sequencing data frequently identified pollen to genus levels with significant quantitative differences in aerobiome diversity between the months and seasons detected. Moreover, multiple peaks of Chloridoideae and Panicoideae pollen were evident over the collection period confirming these grasses as the dominant Poaceae pollen source across the season. Targeted high throughput sequencing of routinely collected airborne pollen samples appears to offer utility to track temporal changes in the aerobiome and shifts in pollen exposure. Precise identification of the composition and temporal distributions of airborne pollen is important for tracking biodiversity and for management of allergic respiratory disease.

Phylogenetic analysis reveals multiple introductions of Cynodon species in Australia.

The distinction between native and introduced flora within isolated land masses presents unique challenges. The geological and colonisation history of Australia, the world's largest island, makes it a valuable system for studying species endemism, introduction, and phylogeny. Using this strategy we investigated Australian cosmopolitan grasses belonging to the genus Cynodon. While it is believed that seven species of Cynodon are present in Australia, no genetic analyses have investigated the origin, diversity and phylogenetic history of Cynodon within Australia. To address this gap, 147 samples (92 from across Australia and 55 representing global distribution) were sequenced for a total of 3336bp of chloroplast DNA spanning six genes. Data showed the presence of at least six putatively introduced Cynodon species (C. transvaalensis, C. incompletus, C. hirsutus, C. radiatus, C. plectostachyus and C. dactylon) in Australia and suggested multiple recent introductions. C. plectostachyus, a species often confused with C. nlemfuensis, was not previously considered to be present in Australia. Most significantly, we identified two common haplotypes that formed a monophyletic clade diverging from previously identified Cynodon species. We hypothesise that these two haplotypes may represent a previously undescribed species of Cynodon. We provide further evidence that two Australian native species, Brachyachne tenella and B. convergens belong in the genus Cynodon and, therefore, argue for the taxonomic revision of the genus Cynodon.

Allelic variation of the ß-, γ- and δ-kafirin genes in diverse Sorghum genotypes.

The ß-, γ- and δ-kafirin genes were sequenced from 35 Sorghum genotypes to investigate the allelic diversity of seed storage proteins. A range of grain sorghums, including inbred parents from internationally diverse breeding programs and landraces, and three wild Sorghum relatives were selected to encompass an extensive array of improved and unimproved germplasm in the Eusorghum. A single locus exists for each of the expressed kafirin-encoding genes, unlike the multigenic α-kafirins. Significant diversity was found for each locus, with the cysteine-rich ß-kafirin having four alleles, including the first natural null mutant reported for this prolamin subfamily. This allele contains a frame shift insertion at +206 resulting in a premature stop codon. SDS-PAGE revealed that lines with this allele do not produce ß-kafirin. An analysis of flour viscosity reveals that these ß-kafirin null lines have a difference in grain quality, with significantly lower viscosity observed over the entire Rapid ViscoAnalyser time course. There was less diversity at the protein level within the cysteine-rich γ-kafirin, with only two alleles in the cultivated sorghums. There were only two alleles for the δ-kafirin locus among the S. bicolor germplasm, with one allele encoding ten extra amino acids, of which five were methionine residues, with an additional methionine resulting from a nucleotide substitution. This longer allele encodes a protein with 19.1% methionine. The Asian species, S. propinquum, had distinct alleles for all three kafirin genes. We found no evidence for selection on the three kafirin genes during sorghum domestication even though the δ-kafirin locus displayed comparatively low genetic variation. This study has identified genetic diversity in all single copy seed storage protein genes, including a null mutant for ß-kafirin in Sorghum.

Development and characterization of microsatellite loci for Khaya senegalensis (Meliaceae)1.

PREMISE OF THE STUDY: Microsatellite loci were developed to characterize genetic variation and population subdivision in Khaya senegalensis (Desr.) A. Juss. (Meliaceae). • METHODS AND RESULTS: Microsatellite loci were identified from genomic DNA sequences generated using the 454 GS-FLX titanium platform. Primers were designed for 67 tri- and tetranucleotide repeats, of which 20 were selected for 2 multiplexes based on amplification success and band size. Eleven of these loci showed polymorphism in two populations of Khaya senegalensis and amplified in individuals from across the species range. • CONCLUSIONS: These new microsatellite loci will be useful for investigation of the evolutionary and conservation genetics of Khaya senegalensis.

QTL analysis of ergot resistance in sorghum.

Sorghum ergot, caused predominantly by Claviceps africana Frederickson, Mantle, de Milliano, is a significant threat to the sorghum industry worldwide. The objectives of this study were firstly, to identify molecular markers linked to ergot resistance and to two pollen traits, pollen quantity (PQ) and pollen viability (PV), and secondly, to assess the relationship between the two pollen traits and ergot resistance in sorghum. A genetic linkage map of sorghum RIL population R931945-2-2 x IS 8525 (resistance source) was constructed using 303 markers including 36 SSR, 117 AFLP , 148 DArT and two morphological trait loci. Composite interval mapping identified nine, five, and four QTL linked to molecular markers for percentage ergot infection (PCERGOT), PQ and PV, respectively, at a LOD >2.0. Co-location/linkage of QTL were identified on four chromosomes while other QTL for the three traits mapped independently, indicating that both pollen and non pollen-based mechanisms of ergot resistance were operating in this sorghum population. Of the nine QTL identified for PCERGOT, five were identified using the overall data set while four were specific to the group data sets defined by temperature and humidity. QTL identified on SBI-02 and SBI-06 were further validated in additional populations. This is the first report of QTL associated with ergot resistance in sorghum. The markers reported herein could be used for marker-assisted selection for this important disease of sorghum.

Segregation distortion for seed testa color in Mungbean (Vigna radiata L. Wilcek).

Genetic segregation experiments with plant species are commonly used for understanding the inheritance of traits. A basic assumption in these experiments is that each gamete developed from megasporogenesis has an equal chance of fusing with a gamete developed from microsporogenesis, and every zygote formed has an equal chance of survival. If gametic and/or zygotic selection occurs whereby certain gametes or zygotic combinations have a reduced chance of survival, progeny distributions are skewed and are said to exhibit segregation distortion. In this study, inheritance data are presented for the trait seed testa color segregating in large populations (more than 200 individuals) derived from closely related mungbean (Vigna radiata L. Wilcek) taxa. Segregation ratios suggested complex inheritance, including dominant and recessive epistasis. However, this genetic model was rejected in favor of a single-gene model based on evidence of segregation distortion provided by molecular marker data. The segregation distortion occurred after each generation of self-pollination from F1 thru F7 resulting in F7 phenotypic frequencies of 151:56 instead of the expected 103.5:103.5. This study highlights the value of molecular markers for understanding the inheritance of a simply inherited trait influenced by segregation distortion.

Sequence diversity in the coat protein coding region of the genome RNA of Johnsongrass mosaic virus in Australia.

Sequence diversity in the coat protein coding region of Australian strains of Johnsongrass mosaic virus (JGMV) was investigated. Field isolates were sampled during a seven year period from Johnsongrass, sorghum and corn across the northern grain growing region. The 23 isolates were found to have greater than 94% nucleotide and amino acid sequence identity. The Australian isolates and two strains from the U.S.A. had about 90% nucleotide sequence identity and were between 19 and 30% different in the N-terminus of the coat protein. Two amino acid residues were found in the core region of the coat protein in isolates obtained from sorghum having the Krish gene for JGMV resistance that differed from those found in isolates from other hosts which did not have this single dominant resistance gene. These amino acid changes may have been responsible for overcoming the resistance conferred by the Krish gene for JGMV resistance in sorghum. The identification of these variable regions was essential for the development of durable pathogen-derived resistance to JGMV in sorghum.

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum.

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class ( Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue ( Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.

Prediction of hybrid performance in grain sorghum using RFLP markers.

Heterosis is an important component of hybrid yield performance. Identifying high yielding hybrids is expensive and involves testing large numbers of hybrid combinations in multi-environment trials. Molecular marker diversity has been proposed as a more efficient method of selecting superior combinations. The aim of this study was to investigate the value of molecular marker-based distance information to identify high yielding grain sorghum hybrids in Australia. Data from 48 trials were used to produce hybrid performance-estimates for four traits (yield, height, maturity and stay green) for 162 hybrid combinations derived from 70 inbred parent lines. Each line was screened with 113 mapped RFLP markers. The Rogers distances between the parents of each hybrid were calculated from the marker information on a genome basis and individually for each of the ten linkage groups of sorghum. Some of the inbred parents were related so the hybrids were classified into 75 groups with each group containing individual hybrids that showed similar patterns of Rogers distances across linkage groups. Correlations between hybrid-group performance and hybrid-group Rogers distances were calculated. A significant correlation was observed between whole genome-based Rogers distance and yield ( r = 0.42). This association is too weak to be of value for identifying superior hybrid combinations. One reason for the generally poor association between parental genetic diversity and yield may be that important QTLs influencing heterosis are located in particular chromosome regions and not distributed evenly over the genome. Variation in the sign and magnitude of correlations between Rogers distance and hybrid-group performance for particular linkage groups observed in this study support this hypothesis. The concept of using diversity on individual linkage groups to predict performance was explored. Using data from just two linkage groups 38% of the variation in hybrid performance for grain yield could be explained. A model combining phenotypic trait data and parental diversity on particular linkage groups explained 71% of the variation in grain yield and has potential for use in the selection of heterotic hybrids.

Application of inter simple sequence repeat (ISSR) markers to plant genetics.

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)n, can be made with a degenerate 3'-anchor, such as (CA)8RG or (AGC)6TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with 32P or 33P via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.

RAPD polymorphisms among variant and phenotypically normal rice (Oryza sativa var.indica) somaclonal progenies.

RAPD analysis was performed among eight rice somaclonal families known to vary for specific characters and four somaclonal families which were phenotypically normal. The parental cultivar,indica rice cv. FR13A, was found to be homogeneous and homozygous at all but one of the 45 RAPD loci. Polymorphisms were found at 28 of the 45 bands among the somaclonal families, including both loss of parental bands, and the appearance of novel non-parental bands. Segregation data revealed both heterozygous and homozygous mutation events, with recessive mutations more prevalent than dominant. All somaclonal families differed significantly from the parental material, indicating that genomic alterations occurred in all families regardless of phenotype. None of the variant families could be regarded as isogenic lines of FR13A at the DNA level. However, some of the DNA level variation may be in highly repeated sequences with no phenotypic effects. The implications for somaclonal breeding and genetic engineering programs are discussed.

Random amplified polymorphic DNA (RAPD) detection of dwarf off-types in micropropagated Cavendish (Musa spp. AAA) bananas.

A RAPD marker specific to the dwarf off-type (hereafter known as dwarf) from micropropagation of Cavendish banana (Musa spp. AAA) cultivars New Guinea Cavendish and Williams was identified following an analysis of 57 normal (true-to-type) and 59 dwarf plants generated from several different micropropagation events. Sixty-six random decamer primers were used in the initial screen, of which 19 (28.8%) revealed polymorphisms between normal and dwarf plants. Primer OPJ-04 (5'-CCGAACACGG-3') was found to amplify an approx. 1.5 kb band which was consistently present in all normal but absent in all dwarf plants of both cultivars. Reliable detection of dwarf plants was achieved using this marker, providing the only available means ofin vitro detection of dwarfs. The use of this marker could facilitate early detection and elimination of dwarfs from batches of micropropagated bananas, and may be a useful tool in determining what factors in the tissue culture process lead to this off type production.Other micropropagation-induced RAPD polymorphisms were observed but were not associated with the dwarf trait.

Assessment of genome origins and genetic diversity in the genus Eleusine with DNA markers.

Finger millet (Eleusine coracana), an allotetraploid cereal, is widely cultivated in the arid and semiarid regions of the world. Three DNA marker techniques, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), and inter simple sequence repeat amplification (ISSR), were employed to analyze 22 accessions belonging to 5 species of Eleusine. An 8 probe--3 enzyme RFLP combination, 18 RAPD primers, and 6 ISSR primers, respectively, revealed 14, 10, and 26% polymorphism in 17 accessions of E. coracana from Africa and Asia. These results indicated a very low level of DNA sequence variability in the finger millets but did allow each line to be distinguished. The different Eleusine species could be easily identified by DNA marker technology and the 16% intraspecific polymorphism exhibited by the two analyzed accessions of E. floccifolia suggested a much higher level of diversity in this species than in E. coracana. Between species, E. coracana and E. indica shared the most markers, while E. indica and E. tristachya shared a considerable number of markers, indicating that these three species form a close genetic assemblage within the Eleusine. Eleusine floccifolia and E. compressa were found to be the most divergent among the species examined. Comparison of RFLP, RAPD, and ISSR technologies, in terms of the quantity and quality of data output, indicated that ISSRs are particularly promising for the analysis of plant genome diversity.

Interpretation of randomly amplified polymorphic DNA marker data for fingerprinting sweet potato (Ipomoea batatas L.) genotypes.

In this paper we present a method for the generation of randomly amplified polymorphic DNA (RAPD) markers for sweet potato. These were applied to produce genetic fingerprints of six clonal cultivars and to estimate genetic distances between these cultivars. The level of polymorphism within the species was extremely high. From the 36-decamer random primers used, 170 fragments were amplified, of which 132 (77.6%) were polymorphic. Ten primers resulted in no detected amplification. Of the remaining 26 primers for which amplification was achieved, only one did not reveal polymorphism. Six primers used alone enabled the discrimination of all six genotypes. Pattern analysis, which employed both a classification and ordination method, enabled the grouping of cultivars and the identification of primers which gave greatest discrimination among the cultivars.

Vitamin B12 and folate. Pediatric reference ranges.

Pediatric reference ranges were obtained for vitamin B12 and folate (Quantaphase radioimmunoassay, BioRad, Hercules, Calif) using a large hospital population. Data were analyzed employing the Hoffman approach, which was computer adapted. For folic acid, the range for subjects aged 1 to 12 years averaged 5.7 to 31.3 nmol/L for female subjects and 4.5 to 27.0 nmol/L for male subjects. There was a significant decrease in folic acid concentrations after 13 years, the upper limit being 16.5 nmol/L in female subjects and 19.9 nmol/L in male subjects. For vitamin B12, the values in the 0- to 1-year age group were nearly double those in the 13- to 18-year age group. For female subjects, the values in the 0- to 1-year age group were 168 to 1116 pmol/L and in male subjects were 216 to 891 pmol/L. At 13 to 18 years, for female subjects the values were 158 to 637 pmol/L and for male subjects, 134 to 605 pmol/L.

Demographics map route to MSV membership growth.

The Medical Society of Virginia's new database revealed 1) an aging membership with a high percentage of dues exemptions; 2) an apparent reluctance on the part of many young physicians to consider MSV dues a worthwhile investment; and 3) a marked underrepresentation in the MSV membership of women, who now comprise nearly half of most medical school classes. In order to maintain a viable organization, the Medical Society of Virginia must do more to attract those who are underrepresented in the membership. This is not a choice. It is an imperative.

Within-person fluctuations of serum cholesterol and lipoproteins.

The National Cholesterol Education Program has begun a National Campaign to screen millions of adult Americans for serum cholesterol. To determine whether such random samples represent an individual's true lipoprotein status, we measured fasting total serum cholesterol and lipoproteins, on a weekly basis for 4 weeks, in 20 subjects ages 22 to 63 years. Duplicate samples were tested by two standardized laboratories, each on five consecutive days. Variations of more than +/- 20% in the serum levels of total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were seen in 75%, 95%, and 65% of the subjects, respectively. On retesting, 40% of the subjects moved in or out of one "risk category"; and in 10% two categories, from "desirable" to "high risk," or vice versa. These data demonstrate that random testing may fall to detect wide fluctuations in the levels of serum lipoproteins, and therefore result in erroneous risk assignment or therapeutic intervention.

Normal hematologic values obtained with a Coulter counter, model S.

A survey was made of hematologic values obtained with the Coulter Counter, Model S, at American Medical Laboratories, Inc. Blood specimens from 6,887 outpatient men, women, and children were analyzed according to age and sex. Patients being seen by nephrologists, hematologists, and oncologists were excluded from the study. The mean values for white blood cell counts, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration correlated well with values frequently quoted as normal. However, the mean values for hemoglobin, hematocrit, and red blood cell counts were slightly lower than the commonly accepted normal mean values. This may be a result of newer and more accurate methods of hematologic analysis.