Frankia nepalensis sp. nov., a non-infective non-nitrogen-fixing isolate from root nodules of Coriaria nepalensis Wall.

Strains CN4T, CN6, CN7 and CNm7 were isolated from root nodules of Coriaria nepalensis from Murree in Pakistan. They do not form root nodules on C. nepalensis nor on Alnus glutinosa although they deformed root hairs of Alnus. The colonies are bright red-pigmented, the strains form hyphae and sporangia but no N2-fixing vesicles and do not fix nitrogen in vitro. The peptidoglycan of strain CN4T contains meso-diaminopimelic acid; whole cell sugars consist of ribose, mannose, glucose, galactose and rhamnose. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two unknown lipids represent the major polar lipids; MK-9(H4) and MK-9(H6) are the predominant menaquinones (>15 %), and iso-C16 : 0 and C17 : 1ω8c are the major fatty acids (>15 %). The results of comparative 16S rRNA gene sequence analyses indicated that strain CN4T is most closely related to Frankia saprophytica CN 3T. An MLSA phylogeny using amino acids sequences of AtpD, DnaA, FtsZ, Pgk and RpoB, assigned the strain to cluster 4 non-nodulating species, close to F. saprophytica CN 3T , Frankia asymbiotica M16386T and Frankia inefficax EuI1cT with 0.04 substitutions per site, while that value was 0.075 with other strains. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between CN4T and all species of the genus Frankia with validly published names were below the defined threshold for prokaryotic species demarcation, with dDDH and ANI values at or below 27.8 and 83.7 %, respectively. The four strains CN4T, CN6, CN7 and CNm7 had dDDH (98.6-99.6 %) and ANI values that grouped them as representing a single species. CN4T has a 10.76 Mb genome. CN4T was different from its close phylogenetic neighbours with validly published names in being red-pigmented, in having several lantibiotic-coding clusters, a carbon monoxide dehydrogenase cluster and a clustered regularly interspaced short palindromic repeats (CRISPR) cluster. The results of phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain CN4T (=DSM 114740T = LMG 32595T) to a novel species, with CN4T as type strain, for which the name Frankia nepalensis sp. nov. is proposed.

Quantification of chlamydiae in the endangered Houston toad (Anaxyrus houstonensis).

Two primer set/probe combinations targeting variable regions on the 23S rRNA gene were designed to detect and quantify chlamydiae in DNA extracted from brain swabs of the endangered Houston toad (Anaxyrus houstonensis) using SYBRGreen- and Taqman-based quantitative polymerase chain reaction (qPCR). Prevalence and abundance values for samples were generally different between SYBRGreen- and Taqman-based detection methods, with higher specificity observed for Taqman-based detection. Of the 314 samples analyzed, initial screening with SYBRGreen-based qPCR retrieved 138 positive samples, of which 52 were confirmed by Taqman-based analyses as chlamydiae. All of these samples were subsequently identified as Chlamydia pneumoniae by specific qPCR and confirmed by comparative sequence analyses of 23S rRNA gene amplicons. These results demonstrate the usefulness of our developed qPCR methods to screen for and verify prevalence of chlamydiae in DNA of brain swabs, and ultimately specifically identify and quantify chlamydiae, specifically C. pneumoniae in these samples.

Frankia umida sp. nov., isolated from root nodules of Alnus glutinosa L.

Frankia strain Ag45/Mut15T was isolated from a root nodule of Alnus glutinosa growing in a swamp at lake Grossensee, Germany. The strain forms root nodules on A. glutinosa, in which it produces hyphae and clusters of N2-fixing vesicles. N2-fixing vesicles are also produced in nitrogen-free growth medium, in addition to hyphae and sporangia. The whole-cell hydrolysates of strain Ag45/Mut15T contained meso-diaminopimelic acid in the peptidoglycan and ribose, xylose, mannose, glucose, galactose and a trace of rhamnose as cell-wall sugars. The major polar lipids were phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol and glyco-phospholipid. The predominant (>20 %) menaquinones were MK-9(H6) and MK-9(H4). The major fatty acid profile (>10 %) consisted of iso-C16:0, C17 : 1 ω8c and C17 : 0. Pairwise 16S rRNA gene distances showed that strain Ag45/Mut15T was most closely related to Frankia torreyi CpI1T and Candidatus Frankia nodulisporulans with 16S rRNA gene similarity values of 0.001335 substitutions per site. An multilocus sequence analysis phylogeny based on atpD, dnaA, ftsZ, pgk and rpoB amino acid sequences positioned the strain within cluster 1 of Alnus- and Myrica-nodulating species, close to Candidatus F. nodulisporulans AgTrST and F. canadensis ARgP5T. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the studied strain Ag45/Mut15T and all validly named Frankia species were below the defined threshold for prokaryotic species demarcation. Candidatus F. nodulisporulans AgTrST, which cannot be cultivated in vitro, was found to be the closest phylogenetic neighbour to strain strain Ag45/Mut15T with dDDH and ANI values of 61.8 and 97 %, respectively. Strain Ag45/Mut15T was not able to sporulate in nodule tissues like strain AgTrST.Phenotypic, physiological and phylogenomic analyses confirmed the assignment of strain Ag45/Mut15T (=DSM 114737T=LMG 326O1T) to a novel species, with Ag45/Mut15T as type strain, for which the name Frankia umida sp. nov. is proposed.

Draft Genomes of Frankia strains AiPa1 and AiPs1 Retrieved from Soil with Monocultures of Picea abies or Pinus sylvestris using Alnus incana as Capture Plant.

The genomes of two nitrogen-fixing Frankia strains, AiPa1 and AiPs1, are described as representatives of two novel candidate species. Both strains were isolated from root nodules of Alnus incana, used as capture plants in bioassays on soils from a reforested site at Karttula, Finland, that was devoid of actinorhizal plants but contained 25 year-old monocultures of spruce (Picea abies (L.) Karsten) or pine (Pinus sylvestris L.), respectively. ANI analyses indicate that each strain represents a novel Frankia species, with genome sizes of 6.98 and 7.35 Mb for AiPa1 and AiPs1, respectively. Both genomes harbored genes typical for many other symbiotic frankiae, including genes essential for nitrogen-fixation, for synthesis of hopanoid lipids and iron-sulfur clusters, as well as clusters of orthologous genes, secondary metabolite determinants and transcriptional regulators. Genomes of AiPa1 and AiPs1 had lost 475 and 112 genes, respectively, compared to those of other cultivated Alnus-infective strains with large genomes. Lost genes included one hup cluster in AiPa1 and the gvp cluster in AiPs1, suggesting that some genome erosion has started to occur in a different manner in the two strains.

Draft Genome of Frankia sp. Strain R82, an Isolate from Root Nodules of Myrica gale.

The Frankia sp. strain R82 genome is described as representative of a novel candidate species within Frankia cluster 1, as indicated by average nucleotide identity (ANI) analyses, with its closest relatives being Frankia nodulisporulans AgTrs and strains Ag45/Mut15 and AgPM24 (86% identity).

Draft Genomes of Symbiotic Frankia Strains AgB32 and AgKG'84/4 from Root Nodules of Alnus Glutinosa growing under Contrasted Environmental Conditions.

The genomes of two nitrogen-fixing Frankia strains, AgB32 and AgKG'84/4, were isolated from spore-containing (spore+) and spore-free (spore-) root nodules of Alnus glutinosa, but they did not sporulate upon reinfection. The two strains are described as representatives of two novel candidate species. Phylogenomic and ANI analyses indicate that each strain represents a novel species within cluster 1, with genome sizes of 6.3 and 6.7 Mb smaller than or similar to those of other cultivated Alnus-infective cluster 1 strains. Genes essential for nitrogen-fixation, clusters of orthologous genes, secondary metabolite clusters and transcriptional regulators analyzed by comparative genomic analyses were typical of those from Alnus-infective cluster 1 cultivated strains in both genomes. Compared to other cultivated Alnus-infective strains with large genomes, those of AgB32 and AgKG'84/4 had lost 380 or 409 genes, among which one hup cluster, one shc gene and the gvp cluster, which indicates genome erosion is taking place in these two strains.

Draft Genomes of Nitrogen-fixing Frankia Strains Ag45/Mut15 and AgPM24 Isolated from Root Nodules of Alnus Glutinosa.

The genomes of two nitrogen-fixing Frankia strains, Ag45/Mut15 and AgPM24, isolated from root nodules of Alnus glutinosa are described as representatives of a novel candidate species. Phylogenomic and ANI analyses confirmed that both strains are related to cluster 1 frankiae, and that both strains belong to a novel species. At 6.4 - 6.7 Mb, their genomes were smaller than those of other cultivated Alnus-infective cluster 1 strains but larger than that of the non-cultivated Alnus-infective cluster 1 Sp+ strain AgTrS that was their closest neighbor as assessed by ANI. Comparative genomic analyses identified genes essential for nitrogen-fixation, gene composition as regards COGs, secondary metabolites clusters and transcriptional regulators typical of those from Alnus-infective cluster 1 cultivated strains in both genomes. There were 459 genes present in other cultivated Alnus-infective strains lost in the two genomes, spread over the whole of the genome, which indicates genome erosion is taking place in these two strains.

Quantification of members of the Mycobacterium chelonae-abscessus complex in lesions of the endangered houston toad (Anaxyrus houstonensis).

Illumina-based 16S rRNA V3 amplicon sequencing of total DNA obtained from soft tissue lesions (joint granulomas) of the endangered Houston toad (Anaxyrus houstonensis) demonstrated that many reads represented members of the actinobacterial Mycobacterium chelonae-abscessus complex. In order to quantify members of this complex in those lesions, we designed three complex-specific primer set/probe combinations (sets I, II and III) targeting variable regions on the 23S rRNA gene for SybrGreen- and Taqman-based quantitative polymerase chain reaction (qPCR). Both SybrGreen- and Taqman-based analyses specifically detected members of the M. chelonae-abscessus complex in lesion samples, with numbers between 104 and 107 cells per 100-mg sample. Values within individual samples were generally comparable between SybrGreen- and Taqman-based detection methods and between all primer set/probe combinations, except for SybrGreen-based analyses of a few samples analyzed with primer set I that used a less specific forward primer. The development of highly specific detection and quantification methods for members of the M. chelonae-abscessus complex in lesion samples can enable group specific tracking of these organisms, particularly in captive or stewardship settings where source and transmission monitoring are valuable tools to husbandry and species conservation.

Effect of different Alnus taxa on abundance and diversity of introduced and indigenous Frankia in soils and root nodules.

The effect of host plants on the abundance and distribution of introduced and indigenous Frankia populations was assessed in soils and root nodules of four alder species, Alnus glutinosa,Alnus cordata,Alnus rubra and Alnus viridis. Plants were grown in microcosms with either a sandy soil without detectable frankiae, with or without inoculation of a mixture of Frankia isolates, or a silty clay loam soil with indigenous Frankia. The presence of frankiae in soils increased plant height and root nodule formation, with significant increases in the presence of indigenous frankiae. Abundance in soils increased significantly for both introduced and indigenous Frankia populations independent of alder species, with generally largest increases in cluster 1b frankiae. Root nodules formed by introduced frankiae did not reflect the diversity of strains inoculated, with nodules generally only formed by strain ArI3 representing cluster 1a/d. All indigenous Frankia populations detected in soil were also found in A. glutinosa nodules, while A. cordata or A. rubra nodules contained different subsets of frankiae with unique abundances dependent on plant species. These results demonstrate the intrageneric differences of host plants in the selection of specific Frankia populations in soils for root nodule formation.

Draft genomes of non-nitrogen-fixing Frankia strains.

In this study, we describe the genomes of two novel candidate species of non-nitrogen fixing Frankia that were isolated from the root nodules of Coriaria nepalensis and Alnus glutinosa, genospecies CN and Ag, respectively. Comparative genomic analyses revealed that both genospecies lack genes essential for nitrogen-fixation and possess genes involved in the degradation of plant cell walls. Additionally, we found distinct biosynthetic gene clusters in each genospecies. The availability of these genomes will contribute to the study of the taxonomy and evolution of actinorhizal symbioses.

MYCOBACTERIA IN SKIN LESIONS AND THE HABITAT OF THE ENDANGERED HOUSTON TOAD (ANAXYRUS HOUSTONENSIS).

Head-starting of the federally endangered Houston toad (Anaxyrus houstonensis), that is, the release of egg strands, tadpoles, and metamorphic juveniles produced in captivity into the original breeding ponds, requires assessment of potential threats for the transmission of pathogens from captive to free-ranging toads. We used Illumina-based 16S rRNA V3 amplicon sequencing to investigate the community structure of bacteria from skin lesions of captive Houston toad and habitat (pond) samples. Proteobacteria, alone or together with Actinobacteria and, in some samples, Cyanobacteria represented virtually all reads in tissue lesion samples, whereas pond samples were much more diverse, with Acidobacteria, Actinobacteria, Bacteriodetes, Chloroflexi, Cyanobacteria, Firmicutes, Planctomycetes, Proteobacteria, and Verrucomicrobia present with little variation between samples. If present in lesions, Actinobacteria were largely represented by Mycobacteriaceae, and here mainly by one sequence identical to sequences of members of the Mycobacterium chelonae-abscessus complex. In pond samples, mycobacteria represented only a small portion of the actinobacteria, although at higher diversity with six distinct reads. Sequences for reads obtained from pond samples were identical to those representing the M. chelonae-abscessus complex, a group with Mycobacterium marinum, Mycobacterium kansasii, Mycobacterium avium, a group with Mycobacterium vaccae, Mycobacterium fortuitum, Mycobacterium poriferae, and a group with Mycobacterium elephantis and Mycobacterium celeriflavum, whereas sequences of high similarity were detected for reads related to those of Mycobacterium holsaticum, Mycobacterium pallens, and Mycobacterium obuense, and Mycobacterium goodii. Our results indicated that lesions observed on the Houston toad in captivity are not the result of mycobacteria in every case, and that the presence of mycobacteria in the captive colony does not represent a novel pathogen threat to the wild populations because such bacteria are also seen in the natural pond habitats for the Houston toad.

The prevalence of Leptospira among invasive small mammals on Puerto Rican cattle farms.

Leptospirosis, an emerging infectious disease caused by bacteria of the genus Leptospira, is thought to be the most widespread zoonotic disease in the world. A first step in preventing the spread of Leptospira is delineating the animal reservoirs that maintain and disperse the bacteria. Quantitative PCR (qPCR) methods targeting the LipL32 gene were used to analyze kidney samples from 124 House mice (Mus musculus), 94 Black rats (Rattus rattus), 5 Norway rats (R. norvegicus), and 89 small Indian mongooses (Herpestes auropunctatus) from five cattle farms in Puerto Rico. Renal carriage of Leptospira was found in 38% of the sampled individuals, with 59% of the sampled mice, 34% of Black rats, 20% of Norway rats, and 13% of the mongooses. A heterogeneous distribution of prevalence was also found among sites, with the highest prevalence of Leptospira-positive samples at 52% and the lowest at 30%. Comparative sequence analysis of the LipL32 gene from positive samples revealed the presence of two species of Leptospira, L. borgpetersenii and L. interrogans in mice, detected in similar percentages in samples from four farms, while samples from the fifth farm almost exclusively harbored L. interrogans. In rats, both Leptospira species were found, while mongooses only harbored L. interrogans. Numbers tested for both animals, however, were too small (n = 7 each) to relate prevalence of Leptospira species to location. Significant associations of Leptospira prevalence with anthropogenic landscape features were observed at farms in Naguabo and Sabana Grande, where infected individuals were closer to human dwellings, milking barns, and ponds than were uninfected individuals. These results show that rural areas of Puerto Rico are in need of management and longitudinal surveillance of Leptospira in order to prevent continued infection of focal susceptible species (i.e. humans and cattle).

Detection of Salmonella in the intestine of Hypostomus plecostomus from the upper San Marcos River, Texas.

The prevalence of salmonellae in the intestines of the invasive suckermouth catfish Hypostomus plecostomus was assessed in the San Marcos River, just down-stream of its spring-fed headwaters. In 2014, H. plecostomus, sediment, and water samples were collected during 15 sampling events. A combination of semi-selective enrichment and quantitative polymerase chain reaction (qPCR) revealed the presence of salmonellae in 45% of the fish intestines across the entire year, with a prevalence range of 13-100% per sampling event. Repetitive element sequence-based PCR (rep-PCR) and multi-locus sequence typing (MLST) revealed a high diversity of salmonellae from fish intestine samples at individual sampling times, single or multiple presence of rep-PCR patterns and serotypes within individual fish, and identical rep-PCR patterns and serotypes for different fish within and across sampling events. Overall, 15 serotypes were identified by MLST, with a diversity range between one and seven serotypes per sampling event. Some serotypes were retrieved only once, while others were detected more frequently. A few serotypes were retrieved at several sampling times, nearly evenly distributed over the entire sampling period. Prevalence and diversity were independent of precipitation events, indicating the potential presence of environmental strains that are capable of long-term persistence in the environment.

Frankia Diversity in Host Plant Root Nodules Is Independent of Abundance or Relative Diversity of Frankia Populations in Corresponding Rhizosphere Soils.

Actinorhizal plants form nitrogen-fixing root nodules in symbiosis with soil-dwelling actinobacteria within the genus Frankia, and specific Frankia taxonomic clusters nodulate plants in corresponding host infection groups. In same-soil microcosms, we observed that some host species were nodulated (Alnus glutinosa, Alnus cordata, Shepherdia argentea, Casuarina equisetifolia) while others were not (Alnus viridis, Hippophaë rhamnoides). Nodule populations were represented by eight different sequences of nifH gene fragments. Two of these sequences characterized frankiae in S. argentea nodules, and three others characterized frankiae in A. glutinosa nodules. Frankiae in A. cordata nodules were represented by five sequences, one of which was also found in nodules from A. glutinosa and C. equisetifolia, while another was detected in nodules from A. glutinosa Quantitative PCR assays showed that vegetation generally increased the abundance of frankiae in soil, independently of the target gene (i.e., nifH or the 23S rRNA gene). Targeted Illumina sequencing of Frankia-specific nifH gene fragments detected 24 unique sequences from rhizosphere soils, 4 of which were also found in nodules, while the remaining 4 sequences in nodules were not found in soils. Seven of the 24 sequences from soils represented >90% of the reads obtained in most samples; the 2 most abundant sequences from soils were not found in root nodules, and only 2 of the sequences from soils were detected in nodules. These results demonstrate large differences between detectable Frankia populations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specific Frankia populations in soils.IMPORTANCE The nitrogen-fixing actinobacterium Frankia forms root nodules on actinorhizal plants, with members of specific Frankia taxonomic clusters nodulating plants in corresponding host infection groups. We assessed Frankia diversity in root nodules of different host plant species, and we related specific populations to the abundance and relative distribution of indigenous frankiae in rhizosphere soils. Large differences were observed between detectable Frankia populations in soil and those in root nodules, suggesting that root nodule formation is not a function of the abundance or relative diversity of specific Frankia populations in soils but rather results from plants potentially selecting frankiae from the soil for root nodule formation. These data also highlight the necessity of using a combination of different assessment tools so as to adequately address methodological constraints that could produce contradictory data sets.

The Prevalence of Trypanosoma cruzi, the Causal Agent of Chagas Disease, in Texas Rodent Populations.

Rodent species were assessed as potential hosts of Trypanosoma cruzi, the etiologic agent of Chagas disease, from five sites throughout Texas in sylvan and disturbed habitats. A total of 592 rodents were captured, resulting in a wide taxonomic representation of 11 genera and 15 species. Heart samples of 543 individuals were successfully analyzed by SybrGreen-based quantitative PCR (qPCR) targeting a 166 bp fragment of satellite DNA of T. cruzi. Eight rodents representing six species from six genera and two families were infected with T. cruzi. This is the first report of T. cruzi in the pygmy mouse (Baiomys taylori) and the white-footed mouse (Peromyscus leucopus) for the USA. All infected rodents were from the southernmost site (Las Palomas Wildlife Management Area). No differences in pathogen prevalence existed between disturbed habitats (5 of 131 tested; 3.8%) and sylvan habitats (3 of 40 tested; 7.5%). Most positives (n = 6, 16% prevalence) were detected in late winter with single positives in both spring (3% prevalence) and fall (1% prevalence). Additionally, 30 Triatoma insects were collected opportunistically from sites in central Texas. Fifty percent of these insects, i.e., 13 T. gerstaeckeri (68%), and two T. lecticularia (100%) were positive for T. cruzi. Comparative sequence analyses of 18S rRNA of samples provided identical results with respect to detection of the presence or absence of T. cruzi and assigned T. cruzi from rodents collected in late winter to lineage TcI. T. cruzi from Triatoma sp. and rodents from subsequent collections in spring and fall were different, however, and could not be assigned to other lineages with certainty.

Sybr Green- and TaqMan-Based Quantitative PCR Approaches Allow Assessment of the Abundance and Relative Distribution of Frankia Clusters in Soils.

The nodule-forming actinobacterial genus Frankia can generally be divided into 4 taxonomic clusters, with clusters 1, 2, and 3 representing nitrogen-fixing strains of different host infection groups and cluster 4 representing atypical, generally non-nitrogen-fixing strains. Recently, quantitative PCR (qPCR)-based quantification methods have been developed for frankiae of clusters 1 and 3; however, similar approaches for clusters 2 and 4 were missing. We amended a database of partial 23S rRNA gene sequences of Frankia strains belonging to clusters 1 and 3 with sequences of frankiae representing clusters 2 and 4. The alignment allowed us to design primers and probes for the specific detection and quantification of these Frankia clusters by either Sybr Green- or TaqMan-based qPCR. Analyses of frankiae in different soils, all obtained from the same region in Illinois, USA, provided similar results, independent of the qPCR method applied, with abundance estimates of 10 × 105 to 15 × 105 cells (g soil)-1 depending on the soil. Diversity was higher in prairie soils (native, restored, and cultivated), with frankiae of all 4 clusters detected and those of cluster 4 dominating, while diversity in soils under Alnus glutinosa, a host plant for cluster 1 frankiae, or Betula nigra, a related nonhost plant, was restricted to cluster 1 and 3 frankiae and generally members of subgroup 1b were dominating. These results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.IMPORTANCE Root nodule formation by the actinobacterium Frankia is host plant specific and largely, but not exclusively, correlates with assignments of strains to specific clusters within the genus. Due to the lack of adequate detection and quantification tools, studies on Frankia have been limited to clusters 1 and 3 and generally excluded clusters 2 and 4. We have developed tools for the detection and quantification of clusters 2 and 4, which can now be used in combination with those developed for clusters 1 and 3 to retrieve information on the ecology of all clusters delineated within the genus Frankia Our initial results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.

Diversity of Frankia in soil assessed by Illumina sequencing of nifH gene fragments.

Targeted Illumina sequencing of nitrogenase reductase (nifH) gene fragments and analyses of pair-end reads through a modified QIIME pipeline were used to assess the diversity of the actinomyceteous genus Frankia in three soils. Soils were vegetated with host or non-host plants, and included locations in Illinois (ABA, host), Colorado (CoMt, non-host), and Wisconsin (FMWI, non-host). After filtering, seven unique sequences were recovered for soil ABA, six for CoMt, and four sequences for FMWI. These sequences were included in a Bayesian topology anchored by published sequence data from pure cultures of Frankia. Sequences from all three soils showed affinities to Frankia strains from both the Alnus and Elaeagnus host infection groups. Reads representing Casuarina-infective strains were not detected. Four sequences from soil CoMt and five sequences from soil ABA did not cluster, at 97% similarity, into a shared OTU that contained a cultured relative. These results demonstrate that targeted Illumina sequencing provides an efficient and economical method for assessing haplotype diversity of ecofunctional genes (e.g. nifH) at the genus level in microorganisms that perform important ecosystem functions.

Amino acid profiles in first trimester amniotic fluids of healthy bovine cloned pregnancies are similar to those of IVF pregnancies, but not nonviable cloned pregnancies.

Somatic cell nuclear transfer (SCNT), or cloning, is one of the assisted reproductive technologies currently used in agriculture. Commercial applications of SCNT are presently limited to the production of animals of high genetic merit or the production of the most elite show cattle owing to its relatively low efficiency. In current practice, 20% to 40% of SCNT pregnancies do not result in viable offspring. In an effort to better understand some of the anomalies associated with SCNT pregnancies, we investigated amino acid compositions of first trimester amniotic fluid. In this retrospective study, amniotic fluids were collected from SCNT and control IVF pregnancies at Day 75 of gestation and grouped according to the pregnancy results: control IVF (IVF), viable SCNT pregnancies that resulted in live healthy calves (SCNT-HL), nonviable SCNT pregnancies that were aborted before Day 150 (SCNT-ED), and nonviable SCNT pregnancies that were aborted after Day 150 or produced deceased calves (SCNT-LD). High-performance liquid chromatography (HPLC) was used to analyze the concentrations of 22 amino acids (AAs) in the amniotic fluid samples. There were no differences in average AA concentrations between IVF and SCNT-HL groups, whereas SCNT-LD and SCNT-ED had higher levels of total AA concentrations. Concentrations of asparagine, citruline, arginine, and valine were significantly higher in the SCNT-LD group. Both SCNT-LD and SCNT-ED groups had relatively large intragroup variances in AA concentrations. Urea concentration was also measured in the SCNT amniotic fluid samples. No correlations between urea concentrations and arginine concentrations or pregnancy outcomes were found. The findings in this study not only deepen the understanding on SCNT pregnancy anomalies, but also provide a potentially useful screening tool for assessing viable and nonviable SCNT pregnancies.

Chromosome-specific microsatellite multiplex sets for linkage studies in the domestic dog.

To expedite linkage studies and positional cloning efforts in the dog, Minimal Screening Set 2 (MSS-2) of 327 canine microsatellite markers has been multiplexed into chromosome-specific panels. MSS-2 provides 9 Mb coverage of the canine genome with no gaps larger than 17.1 Mb and is the most recent and comprehensive set of microsatellites available for whole-genome scans. Markers were labeled with fluorescent dyes based on locations and expected product sizes to facilitate the multiplexing of a maximum number of markers for each chromosome. All markers are amplified using a single thermal cycling program and PCR mix and are optimized for resolution on an ABI 3100 genetic analyzer. Sixty-nine chromosome-specific panels were created by coamplification of a maximum number of markers and subsequent coloading of the remaining markers.