SSR genotyping
To develop polymorphic genetic markers for kochia, Roche 454 sequencing
technology (Keck Center, University of Illinois) was used to determine
partial genomic sequence from a single GR kochia individual.
Approximately 75.2 million aligned bases (from 357 million total bases
sequenced) with reads having an average length of 557 bases were
obtained. This dataset was screened for simple sequence repeats (SSRs)
with pentanucleotide repeat units to use as molecular markers for
genotyping. Out of a total of 30 SSR markers initially tested, 11 SSR
markers (Table 2) exhibiting polymorphisms were selected for genotyping
the 44 kochia populations.
Amplification of 100 to 200 bp sequence regions containing the selected
SSR markers was carried out using polymerase chain reaction with
specific primers (Table 2; together with expected fragment size of the
amplified loci). Amplification of 5 ng of genomic DNA was performed
using EconoTaq PLUS Master Mix (Lucigen) in a BioRad CFX96 Real-Time
System (C1000 Touch Thermal Cycler). After an initial denaturation
period of 2 minutes at 94°C, PCR was run for 37 cycles, consisting of
denaturation at 94°C for 30 seconds, annealing at either 57°C or 62°C
for 30 seconds (Table 2), and extension at 72°C for 45 seconds. A final
extension of 2 minutes at 72°C was included. Amplified fragment size
analysis was carried out by capillary electrophoresis using an Advanced
Analytical Fragment Analyzer, using the 35-500 bp dsDNA method.
Fragments were sized by ProSize 2.0 software. Alleles were binned using
Flexibin software (Amos, 2005; Amos et al., 2007).