Bromus tectorum L. (Poaceae) is an annual grass species that is extremely successful at invading shrubland habitats in the U.S. Intermountain West (IMW). Over the past 40 years, its range has expanded into both desert and montane habitats previously considered resistant to invasion pressure. Population and ecological genetic research on B. tectorum in the North American invaded range has relied on either six allozyme loci, with between two and four alleles per locus (Novak et al., 1991; Valliant et al., 2007; Schachner et al., 2008), or seven microsatellite (simple sequence repeat [SSR]) markers (Ramakrishnan et al., 2002, 2004), although some studies used only four of the seven markers (Leger et al., 2009; Merrill et al., 2012). Use of these marker systems has revealed populations throughout the invaded region that are largely homogeneous, dominated by one or a few common genotypes, with very few heterozygous individuals. Debate exists over the relative role of outcrossing in the success of B. tectorum, especially in adapting to novel or stringent habitats (Ramakrishnan et al., 2006; Ashley and Longland, 2007; Valliant et al., 2007; Leger et al., 2009). It is possible that outcrossing rates in B. tectorum have been underestimated in homogeneous populations because the small number of markers and the low level of polymorphism may not provide enough resolution to observe recombinant genotypes (Meyer et al., 2013). To provide a larger genetic marker set, we report here the development of single-nucleotide polymorphism (SNP) marker assays for the population genetic study of B. tectorum. SNPs are ideal for examining the role of outcrossing in the B. tectorum invasion and recent range expansion, as well as quantifying variation in these invasive populations, because of the ease of assaying numerous polymorphic loci simultaneously.
METHODS AND RESULTS
For cDNA library construction, we used inflorescences and whole seedlings of six individuals with diverse SSR genotypes commonly found in multiple habitats within the IMW. Inflorescence tissue was collected and combined from three individuals with SSR genotypes IEBB, DCBB, and FEDD at SSR loci BT05, BT26, BT30, and BT33 and, likewise, for whole seedlings collected from three individuals with SSR genotypes EZBY, DABB, and KCBB (Merrill et al., 2012). RNA was extracted from each tissue sample using the ZR Plant RNA MiniPrep Kit (Zymo Research, Irvine, California, USA). A SMART approach cDNA synthesis was performed separately with RNA from the two tissue types (Zhu et al., 2001). After synthesis, cDNA was combined and normalized by treatment with a duplex-specific nuclease (Zhulidov et al., 2004).
The normalized cDNA was sequenced on a single run using a Roche 454 GS FLX instrument and Titanium reagents (454 Life Sciences, a Roche Company, Branford, Connecticut, USA) without DNA fragmentation at the Brigham Young University DNA Sequencing Center (Provo, Utah, USA). Newbler (version 2.0.01; 454 Life Sciences, a Roche Company) was used to assemble, de novo, 1,258,041 DNA reads into 65,486 contigs. For assembly, the minimum overlap length was set to 50 bp and the minimum overlap identity to 95%. This Transcriptome Shotgun Assembly project has been deposited at the DDBJ/ENA/ GenBank International Nucleotide Sequence Database (INSD; a collaboration between the DNA Data Bank of Japan [DDBJ], the European Nucleotide Archive [ENA], and GenBank) under the accession GELF00000000. The version described in this paper is the first version, GELF01000000. A total of 3333 putative SNPs were identified using a SNP finder tool within the BamBam genome sequence analysis package (Page et al., 2014), employing the following criteria: (1) sequence coverage depth at the SNP must be ≥10, (2) the minor allele must represent at least 30% of the alleles observed, and (3) only SNPs that did not have another SNP within 50 bp of either side were considered for possible assay development.
Information for the 95 Bromus tectorum SNPs used to genotype 251 individuals from 12 populations.
Cross-amplification for the 95 Bromus tectorum SNPs within related Bromus species.
A diverse panel of 23 individuals was created for validating SNP assays by selecting a wide range of SSR genotypes using the four B. tectorum SSR loci BT05, BT26, BT30, and BT33 (Merrill et al., 2012). These 23 individuals were full siblings of previously genotyped individuals. Due to the inbred nature of B. tectorum, high levels of homozygosity are commonly observed and seeds from the same maternal plant are expected to be near-isogenic. Seeds collected from individual plants in the field were sown in the greenhouse, and leaf tissue DNA was extracted using a DNeasy Plant Mini Kit (QIAGEN, Germantown, Maryland, USA) or a modified cetyltrimethylammonium bromide (CTAB) extraction protocol (Fulton et al., 1995). A set of 101 SNPs were validated for KASP genotyping (LGC Genomics, Beverly, Massachusetts, USA) following the KASP Genotyping Manual (version 3.0) using a PHERAstar Plus Microplate Reader (BMG Labtech, Ortenberg, Germany), and the data were analyzed using KlusterCaller (version 2.15, LGC Genomics). Primers for each assay were designed using PrimerPicker Lite for KASPar (version 0.26, LGC Genomics). This software designs two forward allele–specific primers (one for each allele) and two potential common reverse primers, only one of which is used in the actual assay (Table 1). The two allele-specific primers differ at the terminal, 3′ nucleotide, which defines the SNP. Each assay was tested on the panel of 23 individuals, with one nontemplate control per assay, in a 384-well plate. Data displays from successful assays had two (or three, in the case of heterozygotes) distinct clusters with good separation. If initial separation was poor, samples were amplified for an additional five, 10, or 15 cycles, as needed. For assays that failed using the first common reverse primer, the second common reverse primer was substituted and validated.
KASP assays for 95 polymorphic SNPs were converted for use on the Fluidigm EP1 SNP Genotyping System (Fluidigm Corporation, San Francisco, California, USA) with the 96.96 Dynamic Array IFC. The assays were tested on 95 individuals according to the Fluidigm SNP Genotyping Advanced Development Protocol. As positive controls, four individuals were included that had already been genotyped using all 95 SNPs on the PHERAStar during the marker development process. These four individuals collectively represented both alleles for each SNP assayed. Genotypes of these individuals as determined on the Fluidigm platform were 100% identical to the genotypes generated by the PHERAStar method. At least one nontemplate control was used for each 96.96 array. Data were analyzed using the Fluidigm SNP Genotyping Analysis Software (version 3.0.2).
Verification of the 95 SNPs is demonstrated for 251 individuals collected from 12 populations located in New Mexico, USA (Table 2, Appendix 1). Genotyping of 10 of the 12 populations is described in Lara (2013). All of the 95 SNP markers are polymorphic across the 12 populations but not necessarily within populations. It is assumed that none of the populations are in Hardy–Weinberg equilibrium, with respect to these markers, because B. tectorum is cleistogamous and heterozygosity is very low within any given population (Meyer et al., 2013). The 95 SNP assays were performed on five individuals each from four related species—B. rubens L., B. diandrus Roth, B. sterilis L., and B. arvensis L.—with 93, 91, 85, and 70 loci amplifying, respectively (Table 3).
The authors thank S. Harrison for his help with tissue collection and preparation. This research was funded in part through grants from the Joint Fire Sciences Program (2007-1-3-10) and the Cooperative State Research, Education, and Extension Service (CSREES) National Resource Initiative Biology of Weedy and Invasive Species Program (2008-35320-18677), and through support from the University of Nevada, Reno.