CAPÍTULO V: PROPUESTA DE PLAN DE MARKETING
5.2 Ofertas del distrito
stranded sequence that was unreadable (See chromatogram below). Despite this, a 981 bp DNA sequence of the control region of the Western Yellow Robin was constructed from 2 separate fragments of DNA.
Figure 3.2a A Rufous Treecreeper DNA control region fragment produced by L436 and 12S primers. CGGGGCTTAAACCTCCGTTTTCCATGGAGATGAGTTCCAGTACACCTTGCGAATTTCACCGC GTCATAAGTTTCGCCCCACCTCCTAGGATATGTTATCTCCCTACAGCTTTCAAGTCCACCCAA GCCAGAGGACCAGGTCATCTATTAACGGTGCACCTCACGAGAACCGAGCTACTCAACGTCA GTTATACCCTCGTTATTGGCTTCAAGGCCATACTTTCCCCCTACACCCTAGCCCAACTTGCTC TTTTGCGCCTCTGGTTCCTATTTCAGGGCCATAAATCTCCTGATTCCTTCTCAATTGCTCTTCA CAGATACAAGTGGTTGGTCTGCATAAATCCTCCTTTTAACTCGTGATCGCGGCATCTGACCGT TTTTCCTCTTGTTTTCTTTCTGGGGTCTCTTCAATAAACCCTTCAAGTGCGTAGCAGGTGTTAT CTTCCTCTTGACATGTACATCATATGACATCCGAGCGGCCTCATCGCCCGCAGAGCTATCTA AGTGTAATGGTTTCGTTGGATAACCTGTCGCATACTTAGACTCTGATGCACTTTGCCCCCATT CATGAAACCCGCGCTGTTTACCTCTTGGGTCACAGATGGTGTTATGGTTGTGGGACATGACT ATTTTTTCATGCAGTTCTAGGGACTTATAGTAAAACCCCTATTTCACGCATTATTTGCGCAAT TTTTCTTTTTTGTTTGTCATTTTTTTGTTAACATAACAAAAAAATTAACCGAACCTACCCTACA TTGTCCAAACCATTAATAATTCATCAAACTGTTTATGCACTTTCCACCTAAACACACATTACC TTTCTTCATGACATTGGAACCAAACAAAAACACGGACACCACCTCACCCAACAAACCAGCA AACCCCTACCCCATGCCCTTGTAGCTTACAACAAAGCATGGCACTGAAGATGCCAAGATGGC CGTCATAAAACGCCCAAGGACAAAAGACTTAGTCCTAACCT
3.2.1 DNA Sequencing of the Mitochondrial Cytochrome b Gene
The sequencing of the cytochrome b gene was carried out in two parts, using 4 different primers.
Figure 3.2.1a Chromatogram for part of Rufous Treecreeper Cytochrome b region of the mitochondria. ACATCTGCCGAGACGTTCAATTCGGCTGATTAATCCGCAATCTCCATGCTAACGGAGCCTCT ATGTTCTTCATCTGCATCTACCTACACATCGGCCGAGGCTTCTACTATGGATCCTACGCAAAC AAGGAAACCTGAAACACCGGAGTCCTCCTACTTCTCACCTTAATAGCAACAGCCTTCGTAGG CTACGTACTCCCCTGAGGACAAATATCATTCTGAGGGGCTACAGTCATCACCAACCTATTCT CCGCTATCCCATACATCGGCCAAACCCTCGTAGAATGAGCTTGAGGAGGCTTCTCAGTAGAC AACCCGACCCTCACACGATTCTTTGCCCTCCACTTCCTACTGCCATTCGTAATCGCAGGACTC ACCCTAGTCCACCTAACCTTCCTACACGAAACAGGCTCCAACAACCCCTTAGGCATCCCCTC AGACTGCGACAAAATCCCATTCCACCCATACCACACCACAAAAGACATCCTAGGATTCGCAC TAATATTTGTCCTCCTTGCATCACTCGCTTTATTCTCCCCAAACCTGCTAGGAGACCCAGAAA ACTTTACCCCCGCTAACCCCCTAGCCACACCTCCCCACATCAAACCAGAATGATACTTCCTGT TTGCCTACGCCATCCTGCGTTCCATCCCCAACAAACTAGGAGGAGTC
Figure 3.2.1b Rufous Treecreeper cytochrome b region of the mitochondria, 671bp.
GGATGCGGCGAGGGCTAGGACTCCTCCTAGTTTGNGGGGAGGGAACGCAGGATGGCGTAGG CAAACAGGAAGTATCATTCTGGTTTGATGTGGGGAGGTGTGGCTAGGGGATTAGCGGGGGT AAAGTTTTCTGGGTCTCCTAGCAGGTTTGGGGAGAATAAAGCGAGTGATGCAAGGAGGACA AATATTAGTGCGAATCCTAGGATGTCTTTTGTGGTGTGGTATGGGTGGAATGGGATTTTGTC GCAGTCTGAGGGGATGCCTAGGGGGTTGTTGGAGCCTGTTTCGTGTAGGAAGGTTAGGTGGA CTAGGGTGAGTCCTGCGATTACGAATGGCAGTAGGAAGTGGAGGGCAAAGAATCGTGTGAG GGTCGGGTTGTCTACTGAGAAGCCTCCTCAAGCTCATTCTACGAGGGTTTGGCCGATGTATG GGATAGCGGAGAATAGGTTGGTGATGACTGTAGCCCCTCAGAATGATATTTGTCCTCAGGGG AGTACGTAGCCTACGAAGGCTGTTGCTATTAAGGTGAGAAGTAGGAGGACTCCGGTGTTTCA GGTTTCCTTGTTTGCGTAGGATCCATAGTAGAAGCCTCGGCCGATGTGTAGGTAGATGCAGA TGAAGAACATAGAGGCTCCGTTAGCATGGAGATTGCGGATTAATCAGCCGAATTGAACGTCT CGGCAGATGTGGGCAACGGAGGCGAAGGCTAGGGAAGTGTCTGCTGTGTAGTGTATAGCGA GAA
Figure 3.1.4c Yellow-plumed Honeyeater cytochrome b region of the mitochondria, 742bp.
3.2.2 Analysis of Mitochondrial DNA Sequences
The sequencing of the control region and cytochrome b segments of mitochondrial DNA was synthesised in two fragments and he overlapping sequences were used to join the segments together. The isolation of mitochondrial DNA from whole genomic DNA, could have avoided cellular components from contaminating the sequencing reactions, especially of the control region.
Mitochondrial Gene Order
The Mitochondrial gene order was established in the Rufous Treecreeper.
Consideration also had to be given to 2 different orientations and four different gene regions. After some experimentation with a selection of primers, the second orientation
of a cytochrome b - glutamic acid - ND6 – control region – phenylalanine - 12S order was found. Amplification of the entire 4 segmented gene region was completed and the amplified fragments were separated by gel electrophoresis to check fragment size and sequenced. The size of target gene regions was referenced by using published
sequences in GENBANK (NCBI). A final continuous DNA sequence was traced with multiple sequence alignments of each gene region, which contained regions of similarity in overlapping segments.
Control Region
Amplifying the control region was problematic, which resulted in what appeared to be sequences generated from a mixed template. Mitochondrial pseudogenes or numpts, were suspected to be the cause for the problems in sequencing the control region (Refer to section 1.5, p.45). Therefore, it is recommended to purify mitochondrial DNA from total DNA prior to sequencing to avoid the interference of pseudogene sequences being
produced in the reaction. White, et al., (1998) provides a Density-Gradient Ultra- centrifugation Method which isolates pure mitochondrial DNA, prior to any sequencing reactions. Also, the results for this study may have improved if feather samples had been first processed using this method. However, a 981bp sequence of the control region was found for the Western Yellow Robin. Part of the cytochrome b gene was sequenced for the Rufous Treecreeper (671bp) and Yellow-plumed Honeyeater (742bp).
Cytochrome b Gene
Primers for cytochrome b (cytb) were designed from a collection 50 mitochondrial gene sequences from highly conserved gene regions (from within the same family group of species). They were located in GENBANK (NCBI) and aligned by a computer program BioEDIT (Hall, 1999). This allows a multiple sequence alignment showing consensus sequences within coding regions of a transcribed gene. Specific primer sets were then designed from the areas of high consensus which also flanked a variable region. This variable part of the gene containing a higher rate of mutations (synonymous) was selected as the targeted area for analysis.
The intention of designing new primers was to increase specific binding to template DNA. Designing a new set of primers (de novo), allows the faithful replication and prediction of the expected length of the DNA product. The Rufous Treecreeper mitochondrial Cytochrome b Gene (partial cyb sequence 671bp), was translated into a 255 amino acid sequence code and identified with Blast at NCBI database. The
Conserved Domains data base produced a 100% alignment with cytochrome b segment of the Rufous Treecreeper with accession number AAB05474. Please see Appendix 6 for details. .
3.2.3 Detection of Natural Selection of Cytochrome b DNA
Detection of Natural Selection in mitochondrial DNA of partial cytochrome b (cytb) gene was conducted with the synonymous (silent) and non-synonymous basesubstitution rate method and the second method estimates any recombination events in the target gene to test for natural selection.
The rates of mutations in the Rufous Treecreeper cytb gene, were found to be under influence of natural selection. Since cytb is transcribed into a functional protein of the mitochondria, the specific amino acid combinations would have to be faithfully
translated, avoiding a faulty functioning protein. Under this evolutionary pressure, the mutations that do occur in the functioning cytb gene are silent (non-lethal) mutations. These mutations are a form of redundancy of the genetic code (base substitutions), whereby an amino acid is specified by more than one codon. The test for neutrality for mitochondrial DNA using synonymous (silent) and non- synonymous base substitution rates using DnaSP software (Rozas, et al., 2003), was conducted with ten different haplotypes. For each haplotype there were 223 codons analysed from 669 sites. The average number of synonymous sites was 165.78 and 503.22 for the number of non- synonymous sites. The relative levels for these rates (synonymous < non-synonymous) indicate diversifying selection as the mode of selection for the Rufous Treecreeper cytb
gene.
The second recombination event method was applied tothe Rufous Treecreeper partial cytochrome b gene, where 671 sites and 13 polymorphic sites were tested. The number of pair wise comparisons analysed was 78, the number of sites with four gametic types: was 0 and the minimum number of recombination events (Rm) was 0. This test