SSR individual identification system construction and population genetics analysis for Chamaecyparis formosensis.

Chamaecyparis formosensis is an endemic species of Taiwan, threatened from intensive use and illegal felling. An individual identification system for C. formosensis is required to provide scientific evidence for court use and deter illegal felling. In this study, 36 polymorphic simple sequence repeat markers were developed. By applying up to 28 non-linked of the developed markers, it is calculated that the cumulative random probability of identity (CPI) is as low as 1.652 × 10-12, and the identifiable population size is up to 60 million, which is greater than the known C. formosensis population size in Taiwan. Biogeographical analysis data show that C. formosensis from four geographic areas belong to the same genetic population, which can be further divided into three clusters: SY (Eastern Taiwan), HV and GW (Northwestern Taiwan), and MM (Southwestern Taiwan). The developed system was applied to assess the provenance of samples with 88.44% accuracy rate and therefore can serve as a prescreening tool to reduce the range required for comparison. The system developed in this study is a potential crime-fighting tool against illegal felling.

Development and technical application of SSR-based individual identification system for Chamaecyparis taiwanensis against illegal logging convictions.

Chamaecyparis taiwanensis is an endemic plant suffering illegal logging in Taiwan for its high economic value. Lack of direct evidence to correlate stump and timber remains a hurdle for law enforcement. In this report, 23 polymorphic Genomic Simple Sequence Repeat (gSSR) and 12 Expressed Sequence Tag (EST)-SSR markers were developed and their transferability was assessed. The individual identification system built from selected non-linkage 30 SSR markers has a combined probability of identity as 5.596 × 10-12 equivalents to identifying an individual in a population of up to 18 million C. taiwanensis with 99.99% confidence level. We also applied the system in an actual criminal case by selecting 19 of these markers to correlate illegally felled timbers and victim trees. Our data demonstrate that molecular signals from three timbers hit with three victim trees with confidence level more than 99.99%. This is the first example of successfully applying SSR in C. taiwanensis as a court evidence for law enforcement. The identification system adapted advanced molecular technology and exhibits its great potential for natural resource management on C. taiwanensis.

Genome skimming and exploration of DNA barcodes for Taiwan endemic cypresses.

Cypresses are characterized by their longevity and valuable timber. In Taiwan, two endemic cypress species, Chamaecyparis formosensis and C. obtusa var. formosana, are threatened by prevalent illegal logging. A DNA barcode system is urgently needed for reforestation and conservation of these two cypresses. In this study, both plastomes and 35S rDNAs from 16, 10, and 6 individuals of C. formosensis, C. obtusa var. formosana, and C. obtusa var. obtusa were sequenced, respectively. We show that the loss of plastid trnT-GGU readily distinguishes C. formosensis from its congeneric species. We demonstrate that entire sequences of plastomes or 35S rDNAs are capable of correctly identifying cypress species and varieties, suggesting that they are effective super-barcodes. We also discover three short hypervariable loci (i.e., 3'ETS, ITS1, and trnH-psbA) that are promising barcodes for identifying cypress species and varieties. Moreover, nine species-specific indels of > 100 bp were detected in the cypress plastomes. These indels, together with the three aforementioned short barcodes, constitute an alternative and powerful barcode system crucial for identifying specimens that are fragmentary or contain degraded/poor DNA. Our sequenced data and barcode systems not only enrich the genetic reference for cypresses, but also contribute to future reforestation, conservation, and forensic investigations.

Isolation and characterization of SSR and EST-SSR loci in Chamaecyparis formosensis (Cupressaceae).

PREMISE OF THE STUDY: Simple sequence repeat (SSR) and expressed sequence tag (EST)-SSR markers were developed as tools for marker-assisted selection of Chamaecyparis formosensis and for the molecular differentiation of cypress species. METHODS AND RESULTS: Based on the SSR-enriched genomic libraries and transcriptome data of C. formosensis, 300 primer pairs were selected for initial confirmation, of which 19 polymorphic SSR and eight polymorphic EST-SSR loci were chosen after testing in 92 individuals. The number of alleles observed for these 27 loci ranged from one to 17. The levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.000 to 0.903, respectively. Most markers also amplified in C. obtusa var. formosana. CONCLUSIONS: The developed SSR and EST-SSR sequences are the first reported markers specific to C. formosensis. These markers will be useful for individual identification of C. formosensis and to distinguish cypress species such as C. obtusa var. formosana.

Sex identification of owls (family Strigidae) using oligonucleotide microarrays.

Molecular sexing of the diversified avian family Strigidae is difficult. Sex identification using the intron length difference between W and Z chromosomal CHD1 genes, as visualized by agarose gel electrophoreses, often produces ambiguous results. Here we describe a simple method for sexing a variety of Strigidae species using oligonucleotide microarrays, on which several sex-specific probes operated complementarily or in concert. The sex of 8 owl species was identified clearly on the microarrays through sequence recognition. This sequence-directed method can be easily applied to a wider range of Strigidae species.

Simultaneous detection and differentiation of Newcastle disease and avian influenza viruses using oligonucleotide microarrays.

Newcastle disease (ND) and avian influenza (AI) are two of the most important zoonotic viral diseases of birds throughout the world. These two viruses often have a great impact upon the poultry industry. Both viruses are associated with transmission from wild to domestic birds, and often display similar signs that need to be differentiated. A rapid surveillance among wild and domestic birds is important for early disease detection and intervention, and is the basis for what measures should be taken. The surveillance, thus, should be able to differentiate the diseases and provide a detailed analysis of the virus strains. Here, we described a fast, simultaneous and inexpensive approach to the detection of Newcastle disease virus (NDV) and avian influenza virus (AIV) using oligonucleotide microarrays. The NDV pathotypes and the AIV haemagglutinin subtypes H5 and H7 were determined at the same time. Different probes on a microarray targeting the same gene were implemented in order to encompass the diversified virus strains or provide multiple confirmations of the genotype. This ensures good sensitivity and specificity among divergent viruses. Twenty-four virus isolates and twenty-four various combinations of the viruses were tested in this study. All viruses were successfully detected and typed. The hybridization results on microarrays were clearly identified with the naked eyes, with no further imaging equipment needed. The results demonstrate that the detection and typing of multiple viruses can be performed simultaneously and easily using oligonucleotide microarrays. The proposed method may provide potential for rapid surveillance and differential diagnosis of these two important zoonoses in both wild and domestic birds.

Sexing a wider range of avian species based on two CHD1 introns with a unified reaction condition.

Identifying the sex of a bird is important to ensure successful breeding strategies and effective conservation programs. Sex may be identified from the intron size of the CHD1 gene located on the avian sex chromosomes Z and W. However, because of the great nucleotide diversity across different avian species, no given intron is in widespread use without ambiguous results. Complicated modifications of the reaction condition are required to suit different species. Two CHD1 introns were used with a unified reaction condition in this study to simplify the procedure. Consequently, genders of 73 avian species covering 19 families were successfully identified based on this two-intron approach. This means the ability to sex a wider range of avian species using a simplified procedure, greatly assisting in population management at zoos. Zoo Biol 26:425-431, 2007. (c) 2007 Wiley-Liss, Inc.