Genetic Differentiation along a Hybrid Zone in Aquilegia
Introduction
Questions we want to answer
Determine the genetic factors responsible for the separation of the two species A.formosa A.pubescens
population studies:
a) Determine the degree of population of structure
b)Genotype structure on the landscape
c)phenotype on the landscape
d)associating genotypes with phenotypic traits
Material and Methods
Collection of Samples.
Samples were collected from the area of South Sierra specifically from…. Each indivitual is show on figure 1. The elevation differs significally. Pure A.pubescens is collected from high elevations(),pure A.formosa is collected from low elevations and in between we have collected hybrids.
Isolation of DNA
For the isolation of the DNA the protocol used by with minor modifications was used where 96 samples were treated for DNA extraction at the same time. In this protocol 96-well plate from Fischer Scientific were used to wash the leaves from samples coming from the different habitats. A part from Cell Lysis buffer was added and samples were mechanically grinded for 1 min. Centrifucation followed for 1min, and the rest two parts of the buffer were added. Another mechanical grinding followed for 1 min. The rest steps are the same as in Y.Li 2007.
Genotyping
A software from SEQUENOM Assay Design version 3.1 was used to develop the massively the primers for the markers from the Tentative Concensus (TC) sequences we used in our analysis. In total 180 markers were used. These were filtered down to XXXSNPs that had a minimun minor allele count of 5? Those markers were divided into six groups of assays containing roughly 30 markers each. The protocol used for genotyping is described in Söderlund-Strand A. et al., 2008. In house perl scripts were used in order to checked the integrity of our data.
Genetic analysis
Structure was used on our populations in order to infere population structure. Based on statistics we have decided to use K=7 with Burnins 10000 and three repetitions for each K.
Results
In total 1107 individuals were used from different areas from the South Sierras. Figure 1 shows the locations of each of the areas from where samples were collected.
Figure 1. The areas from where the 1107 individuals were collected.
Yan Li.(2007). Purification of Arabidopsis DNA in 96-Well Plates Using the PUREGENE DNA Purification Kit. Genetic Variation a laboratory manual. pp.87-89
Söderlund-Strand A, Dillner J, Carlson J.(2008). High-throughput genotyping of oncogenic human papilloma viruses with MALDI-TOF mass spectrometry. Clin Chem. Jan;54(1):86-92
Jonathan K. Pritchard, Matthew Stephens and Peter Donnelly(2000). Inference of Population Structure Using Multilocus Genotype Data. Genetics Jun;155(2):945-59
