Genetic analysis of breeding stocks Of LWFG (Anser erythropus)

Transcription

1 Genetic analysis of breeding stocks Of LWFG (Anser erythropus) Michael Wink Institut für Pharmazie & Molekulare Biotechnologie Universität Heidelberg;

2 Aims & Scope: to determine: samples Genetic composition and variability of LWFG breeding stocks kept in Germany, Sweden and Finnland 270 LWFG Germany (n=84), Sweden and Finnland; 27 wild birds from Russia

3 DNA A C T G 1-10 million nucleotide differences between 2 individuals of a species Differences are inherited DNA is a blueprint of evolution, phylogeny & phylogeography A T G C A T G C T A C G T A C G 1x10 9 Base pairs

4 Methods of molecular systematics and evolution DNA Sequencing of marker genes Microsatellite-PCR AFLP-PCR; ISSR-PCR PCR SNP-analysis DNA-Fingerprinting Systematics, phylogeny, phylogeography Population genetics; Paternity determination Population genetics; Gene mapping molecular sexing paternity analysis, forensic Paternity determination Forensics

5 ncdna Genetic Marker -DNA Sequences -Length polymorphisms (z.b. Microsatellites) mtdna Maternal inheritance ND2

6 From Sample to Sequence Feathers Blood Muscle tissue DNA buffer ethanol Isolation of total DNA Amplification of marker genes by Polymerase Chain Reaction (PCR) with specific primers Cycle Sequencing PCR and Automatic DNA Sequencing From Sequences to Phylogeny and Taxonomy Alignment taxon 1 taxon 2 taxon 3 taxon 4 taxon 5 DNA Sequence ATG CAT GGG CTT TAA GGC CT ATG CAT GGT CTA GAA GGC CT ATG CAA GGT CTA GAA GGT CT ATG CAT GGT CTA TAA GGT CT ATG CAA GGG CTT TAA GGT CT Phylogeny reconstruction Programs PAUP*, PHYLIP, MEGA, MrBayes Maximum Parsimony MP Neighbor Joining NJ Maximum likelihood (ML) networks

7 Anser is a young group! cytochrome b (1,043 bp); nd2 (1,041 bp) ML

8 269Ae 270Ae 268Ae 267Ae 266Ae 265Ae 264Ae 263Ae 262Ae 261Ae 260Ae 259Ae 258Ae 257Ae 242Ae 241Ae 240Ae 239Ae 238Ae 237Ae 236Ae 235Ae 232Ae 231Ae 228Ae 227Ae 226Ae 225Ae 224Ae 222Ae 221Ae 220Ae 219Ae 218Ae 217Ae 216Ae 214Ae 213Ae 212Ae 211Ae 210Ae 182AeR 181AeR 180AeR 179AeR 178AeR 177AeR 176AeR 091Ae 090Ae 089Ae 088Ae 087Ae 086Ae 085Ae 084Ae 083Ae 082Ae 081Ae 080Ae 079Ae 078Ae 077Ae 076Ae 075Ae 074Ae 073Ae 072Ae 071Ae 070Ae 069Ae 048Aa 047Aa 046Aa 045Aa 044Aa 043Aa 042Aa 041Aa 040Aa 039Aa 038Aa 037Aa 036Aa 035Aa 034Aa 033Aa 032Aa 031Aa 030Aa 029Aa 028Aa 027Aa 026Aa 025Aa 024Aa 023Aa 022Aa 021Aa 020Aa 019Aa 018Aa 017Aa 016Aa 015Aa 014Aa 013Aa 012Aa 011Aa 010Aa 009Aa 008Aa 007Aa 006Aa 005Aa 001Aa 002Aa 272Ae 168AeR 173AeR 174AeR 183AeR 169AeR 184AeR 223Ae 170AeR 172AeR 185AeR 187AeR 175AeR 229Ae 209Ae 233Ae 234Ae 344Aan 341Aan 342Aan 343Aan 345Aan 346Aan 256Ae 334Af 055Aa 335Af 336Af 337Af 338Af 339Af 340Af186AeR 049Aa 050Aa 051Aa 052Aa 053Aa 054Aa 056Aa 057Aa 058Aa 060Aa 061Aa 062Aa German LWFG + GLG/GWFG/BG Lineage I lineage II GLG GWFG-II BG LWFG I/ GWFG I mtdna Russian LWFG Russian LWFG

9 Ia Network analysis mtdna Ib IId Id Ic IIb Ie IIa IIc Lineage I Western 9 Haplotypes Lineage II Eastern

10 Results of mtdna-analysis LWFG show 2 main genetic lineages with 9 haplotypes German LWFG: no evidence for maternal hybridisation with greyleg, barbacle, bean, brent, or Canada geese 20% of Swedish and Finnish birds are hybrids with greylegs In both lineages LWFG 2 haplotypes are present that are shared with White fronts

11 STR (microsatellite analysis) CACACACACANNNNNNNCACACACACACANNNN father Homologe Chromosomes mother V M Homologe Chromosomes Allel A B C D foreign

12 From Sample to STR analysis Feathers Blood Muscle tissue DNA buffer ethanol Isolation of total DNA Amplification of STR marker by Polymerase Chain Reaction (PCR) with specific primers High resolution PAGE or Automatic DNA Sequencing mother father sib 1 sib 2 sib 3 Bird Locus 1 Locus 2 A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6 1a b

13 Microsatellite-Analysis 8 polymorphic Gene-Loci GWFG GLG LWFG

14 #outgroup2 #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery.R33079 #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery.R33073 #Ans.ery.R33080 #Ans.ery #Ans.ery #Ans.ery.R33074 #Ans.ery.R33075 #Ans.ery.R33076 #Ans.ery.R33078 #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery.R32752 #Ans.ery.R32753 #Ans.ery.R33064 #Ans.ery.R32754 #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery.R33066 #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery.R33062 #Ans.ery #Ans.ery.R33063 #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery.R33069 #Ans.ery.R33071 #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery.R32756 #Ans.ery #Ans.ery.R33072 #Ans.ery #Ans.ery #Ans.Alb #Ans.ery #Ans.ery #Ans.ery.R32757 #Ans.ery.R33070 #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery #Ans.ery.R32755 #Ans.ery.R33065 #Ans.ery.R33067 #Ans.ery.R33068 #Ans.ery #Ans.ery #Ans.Alb #Ans.Alb #Ans.ery #Ans.ery #Ans.Alb #Ans.ery #Ans.Alb #Ans.Alb #Ans.Alb #Ans.ery #Ans.ery #Ans.Alb #Ans.Alb #Ans.ery #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.Alb #Ans.ery #Ans.Alb #Ans.Alb #Ans.Ans.6965 #Ans.Ans.6966 #Ans.Ans.6984 #Ans.Ans.6963 #Ans.Ans.6964 #Ans.Ans.6985 #Ans.Ans.6987 UPGMA #Ans.ery.R32758 GLG GWFG Microsatellite analysis; 8 Loci LWFG Cluster analysis

15 Results of microsatellite analysis LWFG, GWFG and GLG alleles cluster in distinct clades No general hybridisation! GWFG and LWFG are separated But some birds are obvious hybrids with GWFG or GLG

16 From Sample to ISSR analysis Feathers Blood Muscle tissue DNA buffer ethanol Isolation of total DNA Amplification by Polymerase Chain Reaction (PCR) with a specific single ISSR primer 1a 1b High resolution PAGE or Automatic DNA Sequencing Bird A1 A2 A3 A4 A5 A6 A7 A8 A9 B1 B2 B3 B4 1a b

17 ISSR-Analysis GLG WFG LWFG GLG WFG

18 Results of ISSR-fingerprinting Similar as STR-analysis

19 Conclusion Genetic methods provide evidence for the genetic structure and evolutionary history of LWFG They identify most hybridisation events Breeding stocks can be optimised according to the genetic data (i.e. hybrids and birds with douptful genetic composition will be excluded from breeding & reintroduction programs) Genetic diversity is relatively high. Genetically, breeding stocks of LWFG are suitable for the reintroduction project after reorganisation

20 Thanks for your attention Acknowledgements Hedi Sauer-Gürth Javier Gonzalez Wolfgang Scholze Johan Mooij Torsten Larsson Ake Andersson Lauri Kahanpää Deutsche Bundesstiftung Umwelt (DBU) Aktion Zwerggans Friends of LWFG Swedish Environmental Protection Agency

21