A total of 28 polymorphic loci were tested for suitability in parentage analysis in whooping crane. 67 Table 4.2 Success of mapping Grus microsatellite loci in the chicken genome 68 Table 4.3 Location of centromeres according to passerines (Table a) and Grus (Table b).
INTRODUCTION
THE GRUIDAE
In addition to the blue crane (G. paradisea also known as Anthropoides paradisea) whose general biology will be discussed in more detail (section 1.2), other species included in this study (Figure 1.2) are: the whooping crane (0. americana) , which occurs in North America; red-crowned crane (0. japonensis), found in East Asia; the crane (0. carunculatus), found in Ethiopia, south-central and southern Africa; and gray-crowned crane (Balearica regulorum) which occurs in east-central and southern Africa (Meineet al. 1996a). The five crane species mentioned in this study. a) whooping crane (Grus americana); (b) red-crowned crane (G. japonensis);.
STUDY SPECIES
- Nomenclature
- Distribution
- Status
- Anthropogenic threats Habitat alteration
Ultimately, future predictions of blue crane population sizes may be less stable than figures currently show (McCann et al. 2002). The blue crane is listed under CITES Appendix 11 (CITES 2006), which defines species as having a low current threat of extinction but may become endangered unless their trade is strictly regulated (Hemley 1994).
ILLEGAL TRADE IN WILDLIFE
- Its financial worth on an international scale
- CITES
- Control of the wildlife trade in South Africa
However, the DNA parentage test would not be able to identify the chicken as being wild-caught, but that the alleged parents of the chicken are not its parents, suggesting that the chicken was either wild-caught or obtained through illegal sources. either from another existing captive population. Ultimately, this method of detecting illicit trade could provide the evidence required for use in enforcing existing trade laws. i) to hunt, capture, trap or kill any living specimen of a listed threatened or protected species by any means, method or device, including searching, pursuing, driving, lying in wait, baiting, luring, launching a missile or wounding with intend to hunt, catch, trap or kill such specimen. ii) collecting, gathering or picking any specimen of a listed endangered or protected species. iii) picking off parts of, or cutting, chipping, uprooting, damaging or destroying any specimen of a listed threatened or protected species. iv) importation into the Republic, including importation from the sea, of any specimen of a listed endangered or protected species. v) export from the Republic, including re-export from the Republic, of any specimen of a listed endangered or protected species. vi) being in possession of or exercising physical control over any specimen of a listed threatened or protected species. vii) to cultivate, breed or otherwise propagate any specimen of a listed threatened or protected species or cause it to reproduce. viii) transporting, moving or otherwise translocating any specimen of a listed endangered or protected species. ix) sell or otherwise deal in, buy, receive, give, donate or accept as a gift or in any way acquire or dispose of any specimen of a listed endangered or protected species; or. x) any other prescribed activity involving a specimen of a listed endangered or protected species. This does not apply to a specimen of a listed threatened or protected species transported outside the Republic in transit through the Republic to a destination outside the Republic, provided that such transit through the Republic occurs under the control of an Environmental Management Inspector.
MOLECULAR MARKERS
- Minisatellites
- Microsatellites
Microsatellites are also known as simple sequence repeats (SSRs), variable number repeats (VNTRs) and short tandem repeats (STRs) (Selkoeet al.2006). Typically, tandem repeats span 5–40 units ( Selkoe et al. 2006 ), and different sequences of each microsatellite locus (called alleles) are subject to many variations in sequence length (polymorphism).
STUDY OBJECTIVES
- Thesis outline
The aim of this chapter is twofold: first, to identify the most suitable specimens for sexing the whooping crane, as well as the whooping crane and whooping crane; and secondly to identify sex-linked loci among those microsatellite loci tested for polymorphism in these three species. Using the information obtained from Chapters 3 and 4, as well as the information for the package analyzes described here, this chapter details the selection of an appropriate set of microsatellite loci to be used for parentage analysis in whooping crane.
METHODS
INTRODUCTION
GENERAL EXPERIMENT PROTOCOLS
- Collection and storage of DNA samples
Sampling kits were assembled to be provided to members of the SACWG involved in ringing crane chicks. One instruction leaflet (see below) with details of the kit components, as well as guidance on how to avoid contamination when collecting tissue, blood and feather samples.
Tubes for collection of tissue and/or blood samples
One envelope for each bird. Please make sure that the feathers of different specimens are not mixed. Please read 'Important Considerations' below for further Eppendorfs instructions for collecting blood samples for DNA analysis.
Important considerations
DNA visualisation
Concentrations of extracted DNA samples were estimated by comparing the extracted DNA with lambda DNA of known concentrations. Extracted DNA (2 III) was added to a 96-well BMG black plate (BMG LABTECH); reserve the last seven wells for six calf thymus DNA standards and one negative control (H20).
DNA standardisation: preparing 10 ng/1l1 (pCR-ready) samples
The band intensity of each DNA sample was compared by eye to seven lambda standards to obtain an estimate of concentration. DNA concentrations obtained by fluorimetry and lambda standards were averaged to generate a final concentration estimate for each crane sample.
Polymerase Chain Reaction
PCR amplification was performed using either a DNA Engine Tetrad 2 Thermal Cyeler (MJ Research, Bio-Rad), DNA Engine Tetrad PTC-225 Peltier Thermal Cyeler (Bio-Rad), or MyCyleer Thermal Cyeler (Bio-Rad). The PCR program used was 94 QC for 3 min; 35 cycles of 94 QC for 30 s, annealing temperature (Ta) for 30 s, 72 QC for 30 s, followed by a cycle of 72 QC for 10 mm. For optimization of MgCh concentration, a 2 mM MgCh PCR reaction was first used for all primer sets over an appropriate temperature gradient (see next paragraph).
SOURCING AND DEVELOPING MICROSATELLITE LOCI
- Unpublished whooping crane sequences
- Existing Grus microsatellite loci
- Blue crane microsatellite libraries
- DNA Sequencing
- Primer design
- Individual Genotyping
GC clamp: 1. A'GC' clamp refers to the composition of two base pairs at the 5' end of the forward primer. Pigtailing involves adding nucleotides with the sequence 'GTTTCTT' to the 5' end of the reverse primer (Brownstein et al. 1996).
SEX DETERMINATION IN THREE AFRICAN CRANE SPECIES
- INTRODUCTION
- Molecular sexing
- A review of three sexing primers to be used the three crane species
- Sex ratio analysis
- METHODS
- Samples
- Primer testing
- Sex-linked loci
- Sex ratio analysis
- RESULTS
- Primer testing
- DISCUSSION
A preliminary investigation into the sex ratio of the wild South African blue crane population was briefly carried out here. PCR products for blue crane generated from Griffiths P2 and P8 sexing primers (Griffiths et al. 1998) could not be separated on an agarose gel and are not recommended for sexing using this technique.
A PREDICTED MAP OF GRUS
MICROSATELLITE LOCI BASED ON THE CHICKEN GENOME
INTRODUCTION
- Can predictive mapping be successful?
- The predicted passerine map
- Predicted Grus map
The results showed high homology between two distantly related birds: the chicken and the emu (Shetty et al. 1999). Passerine species represent a family of birds separated by short genetic distances (Sibley et al. 1990). To test the correct prediction of the chromosomal locations of microsatellite loci in sparrows, Dawson et al. (2006) made a comparison between the predicted sparrow map and the connectivity map developed by Hansson et al.
METHODS
1995). Therefore, it may be possible that the synteny between these two species is not as conserved as between chickens and passerines. 2006) were able to test the accuracy of the predicted transient map by comparing it to a linkage map generated for a transient species. For predictive mapping purposes, the information collected from the Ensembl BLASTn chicken genome search included a) chromosome number and b) the start and end position of the locus sequence on the respective chicken chromosomes. Where possible, centromere locations were determined using the centromere position provided by the transient predictive map relative to the position of loci on the Grus map.
RESULTS
- Description of the chromosome map
Centromere locations were determined for five of the nine mapped chromosomes based on the location of sparrow loci relative to each chicken centromere. However, the Gamu022 locus on the Grus map has an initial position of 9755662, which lies between the two canine values indicating the location of the centromere (Table 4.3), so the position of the centromere relative to this location on the Grus predicted map could not be determined. The chromosomal locations of loci mapped using sequences from the whooping crane were shown to be in close agreement with the location of the same loci mapped using sequences from the native species (Table 4.4).
DISCUSSION
- Future directions
Work in the latter direction is underway with large-scale genome analysis proposed for several phylogenetically important bird taxa (as reviewed in Edwardset al. 2004). Importantly for this study in the blue crane, the accuracy of the predicted map in assigning loci genetic locations would first have to be tested using comparisons with a linkage map assessed for a Gruiformes species, as was done for Passeriformes in the predicted passerine map (Dawson et al. 2006). If proven accurate, the Grus map would provide a rapid method for mapping microsatellite loci in the blue crane.
CHARACTERISATION OF GRUS MICROSATELLITE LOCI
- INTRODUCTION
- Pre-characterisation procedures
- General locus characteristics
- Cross-species amplification of Grus loci in other species
- METHODS
- Pre-characterisation procedures
- Samples used
- General locus characteristics
- Further cross-species amplification of Grus loci
- RESULTS
- General locus characteristics
- DISCUSSION
- Pre-characterisation procedures
- General characterisation
- Further cross-species amplification of Grus loci
- SUMMARY
A selection of microsatellite loci originally developed in whooping crane (Glenn 1997), whooping crane (Hasegawaet al. 2000) and whooping crane (developed for this study) were tested for amplification in whooping crane. Seven unique red crane loci were previously submitted to EMBL (Hasegawa et al. 2000). All seven loci originally developed in red crane (Hasegawa et al. 2000) were able to amplify a pure PCR product in blue crane (Table 5.6b).
In their study, Glenn et al. 1997) show all seven loci amplifying successfully in the blue crane. However, cross-species amplification is more likely to occur between closely related species (Primmer et al. 1996).
Characterization of 14 blue crane Grus paradisea (Gruidae, AVES) microsatellite loci for use in detecting illegal trade
Characterization of microsatellite loci in 14 blue cranes Grus paradisea (Gruidae, AVES) for use in detecting illegal trade. Cross-species utilization of 14 blue crane (Grus paradisea) microsatellites in nine species and covering seven taxonomic orders. Meares KM (2006) Characterization of Grus microsatellite loci in the blue crane Grus paradisea MSc thesis, University of KwaZulu-Natal, South Africa.
PRIMER NOTE
CONCLUSION & FUTURE DIRECTIONS
- GRUS MICROSATELLITE MARKERS
- Characterisation
- Cross-species analysis
- FUTURE DIRECTIONS
- Validation of markers for forensic use
- Research on population structure of the blue crane
- Studbook management
- CONCLUDING REMARKS
The whooping crane (Grus paradisea) is listed on CITES Appendix II and the IUCN Red List of Threatened Species (OVCN 2006). A whooping crane locus, Gamu007, previously identified as sex-linked (Jones et al. 1999), was also Z-linked in whooping crane. Microsatellite markers used for herdbook management have proven useful for the conservation of the endangered whooping crane (G. americana) (lones et al. 2002).
Brohede J, Primmer CR, Moller AP, Ellegren H (2002) Heterogeneity in the rate and pattern of germline mutation at individual microsatellite loci. DEFRA (2005) Wildlife crime: a guide to the use of forensic and specialist techniques in the investigation of wildlife crime, p. Hasegawa0, Ishibashi Y, Abe S (2000) Isolation and characterization of microsatellite loci in the whooping crane Grus japonensis.
APPENDICES
Sample database
Recipes for laboratory reagents
- M EDTA 200 ml pH 8.0 Dissolve 37.2 gin 200 ml H 20
Blue crane sequences
Genotype database
Specimens not genotyped for any of the sexing primers (eg, crane sample 3212) were sexed using primer products 2550F/2718R visualized on a 3% agarose gel.
