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).