Literature DB >> 27843723

Development of single-nucleotide polymorphism markers for Bromus tectorum (Poaceae) from a partially sequenced transcriptome.

Keith R Merrill1, Craig E Coleman2, Susan E Meyer3, Elizabeth A Leger4, Katherine A Collins2.   

Abstract

PREMISE OF THE STUDY: Bromus tectorum (Poaceae) is an annual grass species that is invasive in many areas of the world but most especially in the U.S. Intermountain West. Single-nucleotide polymorphism (SNP) markers were developed for use in investigating the geospatial and ecological diversity of B. tectorum in the Intermountain West to better understand the mechanisms behind its successful invasion. METHODS AND
RESULTS: Normalized cDNA libraries from six diverse B. tectorum individuals were pooled and sequenced using 454 sequencing. Ninety-five SNP assays were developed for use on 96.96 arrays with the Fluidigm EP1 genotyping platform. Verification of the 95 SNPs by genotyping 251 individuals from 12 populations is reported, along with amplification data from four related Bromus species.
CONCLUSIONS: These SNP markers are polymorphic across populations of B. tectorum, are optimized for high-throughput applications, and may be applicable to other, related Bromus species.

Entities:  

Keywords:  Bromus tectorum; Poaceae; cheatgrass; invasive; single-nucleotide polymorphism (SNP) development

Year:  2016        PMID： 27843723      PMCID： PMC5104524          DOI： 10.3732/apps.1600068

Source DB:  PubMed          Journal:  Appl Plant Sci        ISSN： 2168-0450            Impact factor:   1.936


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. 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.
Table 1.

Bromus tectorum SNP primers used in the KASP SNP genotyping assays.

SNP IDGenBank accession no.SNP positionaAllele-specific primers (5′–3′)bCommon primer (5′–3′)c
BTEC.0001GELF01054254231ATCCTTTCGAATTCAGATTTATCTGC(A/G)ACGTTCCTCTGGTTTTTTGAATTTTCCATT
BTEC.0004GELF0100516871CCCTTTGGTAAGAAATAAAGACGC(G/A)AGTGTCAAACGCCAATCAAAATACTGGTA
BTEC.0007GELF01054606264GTCGATGAAGCGCAAGCTGT(T/C)GCCTTCCAAGTTACCAGGTCCTTT
BTEC.0009GELF01000627159CACTCCTTGATCCATGAGATAAC(G/A)CCCTCGTGCCATTGTTTATTCTGCAA
BTEC.0013GELF010188461035CTGCAGCTGCTCATATGAAATTG(C/T)TGGCTTCTATGTTCCCTGCCTCAAT
BTEC.0014GELF01051811434GTGTTGCGTGGCGGCCT(G/A)AACATGAGGAATCATCGCCTGAAACAAA
BTEC.0019GELF01010834307GATCACTGCACACCAACCTCAA(T/C)CAAGCTTCAGAGCGTCACACTTGAA
BTEC.0082GELF01018777284GTAAAAGTTAGGTTGTTTTTGTTGGAG(T/C)GCTAAATGAAAATAGGTTGAGAACAACCTT
BTEC.0129GELF010077071215GGCGCTCTTCCATGGTGC(C/T)CAAGGTACTTCTTCGTTCTTCAAGACAT
BTEC.0175GELF010100571079AATGGGTTTTGGATAATGCCCTG(G/A)GTCCTGAGGATGCTGTAGGCTCAT
BTEC.0229GELF01028688297AATGCAGTGTCAATGTAGAGTCAAT(C/A)GAGTTCGACAGCAGCAATCCCATAT
BTEC.0232GELF0105042759AATAGACCATCTCGAGATGAACC(A/G)TGCGAGGGACAACATTTACATTGTGAAAA
BTEC.0402GELF010170571337GCCTTCTTCTTGCTGCCAGG(T/A)TGCTGGCAGCTCCCACACCATT
BTEC.0433GELF01002345817GTGACCGGTCTCACGTGAGT(A/G)GGTCTCTGAGTGCACAACGATACTT
BTEC.0448GELF010141611080GGATGTCTGATATTCCGCTGG(T/C)GCTGTTTCTCTACCCCTCTGTTGTA
BTEC.0449GELF0101458465GAGATTGCTGTAATGCAGTATCG(T/C)CGCAAACCCTTCTCCTCAAGAGATT
BTEC.0468GELF01052618538CTATCCCCATCACGATGAAATTTC(T/C)CGCTACACTGATGTATTCAACGAGTATAT
BTEC.0505GELF010131891094GAAGTTCAAGAACAACAGATTTCGC(C/T)ATCCAGTTCTGGAATAAGAACAAGTCCAT
BTEC.0583GELF010153101024ACCAGGGCGCCCTTATTACTG(A/T)GAGATCTTCCTCTTGCCATCTCCTT
BTEC.0601GELF01023207395GACAGTGACACCGAGATTGAG(G/T)ATCTTCTCGTCCAAGTCCTCAGCAA
BTEC.0605GELF01029244874AGCGTCTTCTTTGTTATCGTCA(G/A)TCATACAGTATTTTGTAGCTTCGCCTCAA
BTEC.0637GELF01004375952GTTGGATAAGCAAGGCTGCATG(T/G)ATTAAGGGAGCATACATAAGCCAAAAACAA
BTEC.0657GELF01009986233CAATGATGCACTCATGGGAATTCT(T/C)GTAAGGCGGACTGCACAAATTACGAA
BTEC.0663GELF01012084401GAAAACTGATGAAGCTACATGCCAT(G/C)CCGTGGGGGAAGCTGGCAATAT
BTEC.0696GELF01021947714GTACATCATCCATTATTCCTCCTTG(A/G)GAAGCCTATTCGTACCTGATCTGACAA
BTEC.0697GELF01022416433CTGCAGATCTCCCGTATCCT(T/C)CCCTGGATCTTTCTACCTACTCCTT
BTEC.0718GELF010501601124AAGGTCCCAGGGAAGGCG(C/T)CAACCTCCCCAGTCTCCAAAGAAAA
BTEC.0751GELF010058301123AAGTCAAGTTCTGTTAATTTTCCTCC(A/G)CGACTCGATTGGCTCCAACATTTGAA
BTEC.0775GELF01017138777GGACAAGCTCTAAATTTTTGGTTCTG(T/A)GCTACTCTAAACAACGGGAGCAAGTA
BTEC.0790GELF01026632341CCTGGATATATTATGTTCTGTAATATTCTA(G/A)GGTTTACAATGTGAGGTAAAGGAAGGAAA
BTEC.0818GELF01001943125GTAGTTGGCTCACCTGTACCTA(T/G)CCATTCATAGTGAAGGAGTATGCTACAAA
BTEC.0853GELF01009140810CTGACTGTTCTCCTGAGGTGT(A/C)CGGACATTGCTGAATACTTTTCTCGTT
BTEC.0854GELF01009368538ATGCAACAGCAATCTTCAGACC(C/T)GGGGGGCTCTGGAACTCATCAT
BTEC.0874GELF0101519395CACCCACGTACCAGTGGAC(G/A)CACAAGTTTGACCTCATGTACGCCAA
BTEC.0882GELF01017525452CGCCATCGAGAACTTCAGCTT(C/T)CCAGGGCGATTTTGTTTCGTCGAA
BTEC.0887GELF01018801791CAGTACTAGCCCCCAGATGA(T/C)GGTGGCAGATGGTTCCTGTGGAA
BTEC.0904GELF01024980474GCAGCCACTGAGCAATGTTAC(G/A)TTAAGCAGAGTCGGATCCACAGGAA
BTEC.0973GELF01004564197AGAACAATTGTAGTATGGTTATTGTCTA(A/C)GATGTGACAAAGTGATATTTTCCGGTGTA
BTEC.0992GELF01009440193AGTACTAGAGGTATGCCTGGC(G/T)GCAGCTAATAACAGTGTAGTATCTTGGTA
BTEC.0997GELF01010059331CAGAAAGAACCGACTTGCCG(C/T)CAGAAGATACTGCATGTTCCAGAGGTT
BTEC.1013GELF01014723978CATCACTGAAGCTTCTCAAGG(C/T)CAAGAGCAAATCCTCAACAGAAGGATATA
BTEC.1058GELF01024676911AGGCCTCGATTGATTGATTTCAG(C/T)GATAACAGTCCTTCTAGGGTTCAAGAAT
BTEC.1064GELF01026664300GAGGCGTCGGCCATCTC(C/T)CTTCGTCTTGTGGACCACGGGTT
BTEC.1125GELF01004063529GTAGTGAAAATTATCCATAGCCTGAT(T/C)CACAAAAAAACATTAAGAGGGGATAGCAAA
BTEC.1203GELF010177131086ATACCCCCAGCAAGCTTATATACA(G/A)CTTGGATTTATGATTCATGGTACCACTATT
BTEC.1204GELF01017791682GTCATGACTTCAGGATCCCTTAA(C/T)CATAGTTAATGTGCTGCGTCGGCAA
BTEC.1211GELF01019599169ATAGAAACATGAACCTTCTGCGTG(G/A)AACGTAAACAGGAGGGCGTAAATAATCTT
BTEC.1270GELF01051217739ATGTCCTGACTGAGGTGCCTT(A/G)CAAGGTAAGACATCTCAGGCAGGTA
BTEC.1352GELF010076341909ACAGGTTCAACGTTCCATGGAA(T/C)TTATCTTAACGGGGCACACCTCACT
BTEC.1383GELF01011814596CCTCAGTACCATCACAAAGATC(G/A)GAATACTATGCACAAGTCGGTAACTGTAT
BTEC.1388GELF010122461343GGAGAGCAAACAGCTGCGCA(A/G)TGCTGTGGCCTACATGTACCCAAT
BTEC.1398GELF01012763530AAATGGCAGCAGCTCTGGTGT(T/C)GTTCAGTGCTGCTCCCGTTTCTTTT
BTEC.1400GELF010128571753CGAGGTAGAGCTTTATCCAAC(A/G)TTATCGCTCAATCCGTAACTTCTTGCTTT
BTEC.1407GELF01014240790AAAAGAGATCCGGGCCATCG(G/A)GTACACCTTTTGGGGATATGGGTCAT
BTEC.1413GELF01015084869CCATACGCGTCTCCACTGGA(C/T)GAAGGAGATGAGAGGCGCGCTA
BTEC.1438GELF010186651266AATTGTGTTTCTCAAATCAGTGGAG(A/G)TTTTTTTACGAGCAAAAAAGAATCATCCAT
BTEC.1450GELF01020563958CATCAGGTCATCAGAAGCTAATGC(A/T)TGTAAATCTGATCTTGAGACCGCAGTA
BTEC.1489GELF01027878561GCATCATCGGAGCACTGGC(C/G)GTGCTGCTACTTTTGTTGCTGTCCTT
BTEC.1507GELF01049408106ATTTTAAGCGGTACAAAATTTAAGACCA(A/G)GAGGTGGGAGAGGGAGACTGAA
BTEC.1586GELF01002187770CCCTGCACGCGTCGGTTT(A/G)ATGGAGCGGCAGCAAGGATAACAAA
BTEC.1635GELF01007503274GTGTTGAGAAGTTCATCGTGCAA(G/A)TGAGCGTCCGTGGGTGTCTGTT
BTEC.1640GELF01008175568ACCGACTAGCGACTCTGAAGA(A/G)CCATTTCACCAGGTAGAGGTTCCAT
BTEC.1647GELF01008776986CATCAATGGGCTCAGTGTGAG(A/G)CGTCTACTGGCAGTTTCCGCCAT
BTEC.1652GELF010089271096AAATCGGGATTCTGGAGCTGAT(A/C)TCCAATTTCATGAGACCAATACGGTTCAA
BTEC.1654GELF01009235888CCAATATCAAAGGAGTTTGCTTGTAT(A/G)CAAACTCCTACTGTGAACACCTATACTTT
BTEC.1724GELF01016487282GGGAAGCATCGGTGCATTCT(T/G)ATTACGCTACTGGTTCCTCGCCTTT
BTEC.1794GELF01027724449GTTTCCTTGTGTCACTAGTGTATAG(C/T)CAACATAGAGCAACCGGAAGCCAAA
BTEC.1818GELF01049436173AGGGTGTTGGAACAATATCCCTT(G/C)ATTCCATCAGAAGAGAGACTAGGAACTAA
BTEC.1832GELF0105041898GCGCGAGAACATGGTCTGA(C/A)GGTCCTAGGTGTCTGTCGGACTA
BTEC.1873GELF01053648678GAGGTGTATCCTTAATGTCATGTG(C/T)GAAGAACATTATCTCAAAGCTTGGACCAT
BTEC.1907GELF01000259337GACAGCCTCAACTATTCCATTATC(G/A)GGAAGGCAGTGTTCAACTATGATTTTGAT
BTEC.1930GELF01003039619CGTACAACCCTTCCATCGTCT(C/T)TAGGGAGAATCCCAATCCGCATCAA
BTEC.2120GELF01021228171TTGGCCGAGGCATGGATGA(C/T)AAACCTCTCCTTAATTCACACGCAGAAAT
BTEC.2141GELF010248981271CATCTGGAATTTTTCATGTACATTCTC(G/A)GTGTTTGAACTCAGCCTTATATCTGGAAT
BTEC.2142GELF0102539335AGGGTCAATCGTGATAAGGCATT(G/A)GCAGAGTGGTTGTGGTTTCAGGATA
BTEC.2148GELF0102636644GGAGCCACTGTATGAAGATTTCA(C/T)TTTCAAGAAATAGCAAACAGGCGAGCAT
BTEC.2166GELF01030989710GTAGCTCTAGTTTGACAGCGCA(A/G)GGTCACTGAACAAAAGAATACAAAGTATAA
BTEC.2399GELF01003355403AGCATTCCAAGAGCAGCCAC(A/G)GCCTTGCCGCGATGTATGGTGTT
BTEC.2409GELF01003846146CACTACACTGCTAGAGCAGGT(C/T)ACATACGCCGGATCTGAACTCTCTT
BTEC.2436GELF01005070247AACCAATTGATTTTCTTCTCCTTATAAG(T/G)GACATTGTGTAGTTGTGTGACTAGTCAAA
BTEC.2521GELF010082151041CATTGAATTTCATAGACACATTACCTCT(A/G)CCGATAAATAGCCTGAGTGGATCCAA
BTEC.2704GELF0101496949AAATCGCTGTCAGAAGGCCCA(A/G)TTCTATCTATCGCTCCCCTGGCTT
BTEC.2773GELF01018572131CAGAAAATGATTCATTATCCTCTACATC(A/G)TAACTTCACTTCTGCAGTTTCCACCAT
BTEC.2795GELF010193661033GTTTCCCAATACCACTAGGCAC(T/C)TGGTTCTGGAAGACCTCGCCATATA
BTEC.2807GELF01019660178GCATATGACACATGTGCCATCATTT(G/A)CAAGACAGTACAAGACTAACACGAAGTAT
BTEC.2834GELF01020921158AATCAGGTTCGCTCCTGACAG(C/T)CAAGATAGGACCCGATGTAGGTCAT
BTEC.2850GELF01021363198AACATGCTGTATACTAGTGGCCA(T/G)TTGTGTCTCTGTGGTACATAATGGGAAT
BTEC.2869GELF01022307405CCCTAACGAGTTTACGAGACTC(G/A)GGCCTATGTGCCGCCGGAGAT
BTEC.2877GELF01022686436GGAGACCTACAGCTTTGGATTC(C/A)CCATTGATGTGTGAATATCAGAAGTTCATA
BTEC.2918GELF01024948398GAATTGCAATCCAATTAAAGTCGTC(G/A)AGGGCGACCAAGTAGATGACCTTTA
BTEC.2919GELF01024958395CGTACACCGGAGATGAAGAC(T/C)CCCCACTCGCGGCGGAAGAA
BTEC.3025GELF01035468119CAACATCGAGACCAGGCATTC(G/T)GGCAGAAGAGCAGGCACAGAGAA
BTEC.3049GELF01042054144AATCATGTCCCTTTCTTGTTTCTATC(A/G)GTTAAGCATGATGGGAAGGACTGCAA
BTEC.3142GELF01051573612CCCCCTAGTAACTCAAACGGC(T/A)CGCCTGAGGCACTTGAGGTACTT
BTEC.3285GELF01054303450GGATATGTTTTTTATTCTTGCTCATGTT(G/A)GGGCGCCATACAAAGATAATTTTGTTGTA

The SNP position is the number of the variable nucleotide within the contig counting from the 5′ end of the sequence as deposited in GenBank.

Allele-specific primers differ at the 3′ terminal nucleotide and each includes a distinctive sequence of nucleotides at the 5′ end that are complementary to separate universal FRET cassettes contained in the KASP reagent.

The common primer is used during amplification of products containing both SNPs.

Bromus tectorum SNP primers used in the KASP SNP genotyping assays. The SNP position is the number of the variable nucleotide within the contig counting from the 5′ end of the sequence as deposited in GenBank. Allele-specific primers differ at the 3′ terminal nucleotide and each includes a distinctive sequence of nucleotides at the 5′ end that are complementary to separate universal FRET cassettes contained in the KASP reagent. The common primer is used during amplification of products containing both SNPs. 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).
Table 2.

Information for the 95 Bromus tectorum SNPs used to genotype 251 individuals from 12 populations.

Population-level major allele frequenciesc,d
SNP IDMajor alleleaMinor alleleMajor freq.bALBBN1BN2BERGA1GA2GRALOSMILPR1PR2SAN
BTEC.0001GA0.8760.6790.9571.0000.8460.7600.9480.8850.9500.9810.9551.0000.613
BTEC.0004CT0.7540.8080.9091.0000.1920.1600.8970.8460.5500.9630.8180.6670.935
BTEC.0007GA0.5120.6790.9520.0000.7860.4400.8970.3330.4740.5931.0000.2500.000
BTEC.0009TC0.9481.0001.0001.0001.0000.7500.8970.9231.0000.9261.0000.9171.000
BTEC.0013CT0.9110.7861.0001.0000.8570.6400.8970.8851.0000.8891.0000.9171.000
BTEC.0014TC0.6630.8210.9320.9470.2140.3800.7590.7880.3250.4440.5000.5830.935
BTEC.0019GA0.9321.0001.0001.0000.9290.8000.8970.9621.0000.8151.0000.8331.000
BTEC.0082GA0.8900.7690.9571.0000.8460.6600.8790.7400.9000.9261.0000.9170.964
BTEC.0129CT0.7570.6430.8641.0000.2140.6000.8620.6920.7250.9260.8180.9170.517
BTEC.0175TC0.8200.3930.9090.9470.0710.6670.8970.8460.8000.9261.0000.9171.000
BTEC.0229GT0.5640.2860.0431.0000.1070.5600.2410.8270.5500.8890.0910.9170.871
BTEC.0232GA0.8650.7860.9571.0000.2860.6800.8790.9040.8500.8891.0000.9171.000
BTEC.0402AT0.9060.7861.0001.0000.9290.6600.8790.9041.0000.9261.0000.9170.935
BTEC.0433AG0.6540.7501.0000.0000.7500.8400.8280.3080.8000.6301.0000.5000.419
BTEC.0448GA0.8590.8571.0000.9470.2860.6400.8620.9230.7500.9261.0000.9170.968
BTEC.0449GA0.9421.0001.0001.0000.8570.8400.8971.0001.0000.0001.0000.5001.000
BTEC.0468AG0.9090.7691.0001.0000.8570.6400.8620.9041.0000.8891.0000.9171.000
BTEC.0505TC0.8430.6920.9091.0000.2500.6400.8790.9230.8000.9261.0000.9170.968
BTEC.0583AT0.8821.0000.9551.0000.2140.8000.8280.9230.8500.9261.0000.9170.967
BTEC.0601TG0.9500.7861.0001.0000.9290.8000.9830.8851.0001.0001.0001.0001.000
BTEC.0605TC0.8610.8570.8891.0000.3570.7780.4001.0001.0000.9261.0000.9170.613
BTEC.0637AC0.9741.0001.0001.0000.9290.9200.9811.0000.9501.0001.0001.0000.935
BTEC.0657AG0.8510.7860.9571.0000.2860.6400.8620.8850.8000.9261.0000.9170.935
BTEC.0663CG0.8080.8931.0000.9440.2500.9470.8520.7600.9000.4440.4550.9171.000
BTEC.0696GA0.8620.7860.9570.9470.2500.6800.8970.9230.8000.9261.0000.9171.000
BTEC.0697AG0.8160.9640.9521.0000.9290.9570.8280.6191.0000.4810.1000.9170.857
BTEC.0718TG0.9501.0000.9571.0000.8850.8200.8970.9811.0000.9261.0000.9171.000
BTEC.0751AG0.5420.1790.0430.9470.2140.5600.3620.6150.7000.9260.5450.9170.452
BTEC.0775AG0.9100.7861.0001.0000.8930.6600.8620.9041.0000.9261.0000.9171.000
BTEC.0790TC0.8040.7140.8701.0000.1790.6400.8790.8000.6500.9260.9000.9170.821
BTEC0.818GA0.9120.7861.0001.0000.9290.6600.8790.9231.0000.9261.0000.9170.968
BTEC.0853CA0.5220.7860.9350.0000.9290.6400.7760.2001.0000.0740.0910.4170.419
BTEC.0854TC0.6430.643——0.643————————
BTEC.0874CT0.7800.5710.9131.0000.2860.6600.8100.8850.7500.9070.9090.9170.581
BTEC.0882TC0.8451.0001.0000.9470.9290.7600.8790.9421.0000.3890.0910.9171.000
BTEC.0887GA0.8080.9290.6001.0000.2861.0001.0001.0001.0001.0000.6671.0000.800
BTEC.0904CT0.9070.7860.9321.0000.9290.6400.8970.8181.0000.9261.0000.9170.967
BTEC.0973CA0.5840.2860.1301.0000.8570.4200.2930.8460.2500.9630.9090.5830.629
BTEC.0992TC0.8560.9641.0001.0000.7500.8800.8280.8961.0000.4810.3000.9171.000
BTEC.0997TC0.7661.0000.7371.0000.8930.8800.8280.6670.7780.4810.0000.9170.667
BTEC.1013TC0.9200.7860.9571.0001.0000.6800.8970.8851.0000.9261.0000.9171.000
BTEC.1058TC0.8941.0001.0001.0001.0001.0000.8620.8851.0000.5000.5450.9171.000
BTEC.1064TC0.9781.0001.0001.0000.8210.9600.9310.9621.0001.0001.0000.9171.000
BTEC.1125GA0.9000.7500.9570.9470.9230.6600.8450.9041.0000.9441.0000.9171.000
BTEC.1203TC0.9320.7140.9571.0000.7860.8400.9660.9231.0000.9630.9091.0000.968
BTEC.1204TC0.8630.7860.9571.0000.2500.6600.8970.9040.8500.9261.0000.9171.000
BTEC.1211TC0.9120.7861.0001.0000.8570.6600.8620.9041.0000.8891.0000.9171.000
BTEC.1270GA0.7360.5000.9350.9740.2140.6600.7240.8270.7750.9260.6360.8330.548
BTEC.1352GA0.9050.5360.9570.8950.7500.7600.9310.8850.9001.0000.2730.9170.774
BTEC.1383GT0.5020.2310.6190.4740.7500.6000.9310.4580.5500.0740.6360.0830.400
BTEC.1388AG0.9100.7861.0001.0000.8570.6400.8790.9231.0000.9261.0000.9171.000
BTEC.1398GA0.9310.9621.0000.9470.8210.8750.7930.9521.0000.9630.2731.0001.000
BTEC.1400GA0.8660.7860.7221.0000.9290.7500.7860.7080.8571.0000.4501.0000.952
BTEC.1407TC0.5210.8850.3640.5260.6430.6090.1720.3910.6000.4810.0910.9170.393
BTEC.1413CT0.9180.7861.0001.0000.9640.6800.8970.8851.0000.9261.0000.9171.000
BTEC.1438GA0.5540.2860.0431.0000.6790.4000.1381.0000.2000.9631.0000.5830.548
BTEC.1450TA0.8571.0000.9571.0000.8211.0000.8280.9201.0000.4810.1000.8330.968
BTEC.1489GC0.953—0.8891.000—0.9440.8001.0001.0001.0001.0001.0000.947
BTEC.1507AG0.8410.7140.9570.9470.8930.9600.8280.9620.8000.4440.4550.9170.935
BTEC.1586AG0.7680.7861.0001.0000.2310.5000.2000.8240.8890.8890.7500.9170.895
BTEC.1635TC0.8570.7860.9570.9440.3210.6400.8790.9230.8500.9070.9090.9171.000
BTEC.1640GA0.7150.6920.8701.0000.2500.6400.7590.6400.5000.9260.0000.9170.806
BTEC.1647GA0.9281.0001.0001.0000.8930.8200.8790.9811.0000.0001.0000.5000.935
BTEC.1652CA0.8330.7860.9571.0000.8210.3750.9480.8270.5500.9631.0000.6670.968
BTEC.1654AG0.8430.0001.0001.0000.1070.8571.0001.0001.000—1.0000.0001.000
BTEC.1724CA0.7840.4290.9781.0000.2860.8400.6720.9230.5791.0000.9550.9170.629
BTEC.1794GA0.9380.9231.0001.0000.7140.8200.8791.0001.0000.9441.0000.9170.968
BTEC.1818CG0.8330.7860.9381.0000.3570.6400.8750.7060.7500.8891.0000.9170.964
BTEC.1832TG0.7060.5000.0870.9470.9640.9600.2410.6150.6501.0001.0000.8330.903
BTEC.1873CT0.7730.9290.8260.6390.8570.5000.8450.7690.5000.9420.9090.7270.935
BTEC.1907TC0.9340.8931.0001.0000.8210.8200.8790.9420.9500.9261.0000.9171.000
BTEC.1930TC0.5790.4621.0000.9000.2861.0001.0000.5380.2500.4440.7500.9170.176
BTEC.2120TC0.5710.6431.0001.0000.1430.5000.2000.4410.7780.0000.2500.5000.895
BTEC.2141CT0.5620.2860.6670.5000.2141.0001.0000.7330.3330.4070.5000.6670.615
BTEC.2142CT0.8191.0001.0000.9470.2860.9200.8620.8850.9000.4810.2730.9170.968
BTEC.2148TC0.8820.6431.0001.0000.8210.6400.7930.9230.9500.9261.0000.9170.935
BTEC.2166GA0.8370.9291.0001.0000.8210.9600.9310.7311.0000.4810.0000.9170.935
BTEC.2399GA0.9320.5711.0001.0000.7140.8330.9660.9231.0001.0001.0001.0001.000
BTEC.2409CT0.7040.7690.7390.9470.2140.6400.8620.4230.7000.9260.8180.9170.548
BTEC.2436CA0.7290.9230.6820.9470.8750.9200.9660.5000.6500.5190.6360.8330.484
BTEC.2521GA0.6400.5000.0871.0000.4641.0000.3620.6150.9000.4810.3000.9170.871
BTEC.2704CT0.8200.2310.9571.0000.1430.6400.8620.8851.0000.9261.0000.9170.903
BTEC.2773AG0.8850.9290.9570.9470.8570.9200.9660.9231.0000.4810.5451.0001.000
BTEC.2795AG0.7140.5710.1300.9470.9291.0000.3450.7310.8000.4621.0000.9171.000
BTEC.2807CT0.5000.3930.0430.9470.2860.8800.1720.8200.4500.4440.1360.8330.484
BTEC.2834TC0.9521.0001.0001.0001.0000.8000.8970.9620.9500.9261.0000.9171.000
BTEC.2850AC0.9461.0001.0001.0000.8930.8200.8790.9811.0000.9071.0000.9171.000
BTEC.2869TC0.9020.6791.0001.0000.8570.6400.8620.9231.0000.9261.0000.9171.000
BTEC.2877GT0.8930.8570.9571.0000.8210.8200.7241.0000.8000.9071.0000.9170.871
BTEC.2918TC0.7660.5360.9131.0000.7690.1600.8280.8460.5500.8891.0000.5831.000
BTEC.2919GA0.6950.5000.2860.8420.8930.9600.3100.7880.8500.4630.3000.9170.935
BTEC.3025TG0.9070.7861.0001.0000.8930.6600.8750.8650.9470.9231.0000.9171.000
BTEC.3049GA0.9021.0001.0001.0000.2500.8400.8971.0000.8000.9261.0000.9171.000
BTEC.3142TA0.6530.9290.9520.0000.7140.9200.7930.2730.8950.6301.0000.4170.423
BTEC.3285TC0.8960.8571.0000.9470.7140.6400.8620.8851.0000.8891.0000.9171.000

The major allele is the nucleotide that occurs at the highest frequency among the 251 individuals sampled.

Frequency of the major allele across all 12 populations.

Dash marks within a population indicate that no lines amplified successfully at that locus.

Population abbreviations are defined in Appendix 1.

Table 3.

Cross-amplification for the 95 Bromus tectorum SNPs within related Bromus species.

SNP IDB. rubens (N = 5)B. diandrus (N = 5)B. sterilis (N = 5)B. arvensis (N = 5)
BTEC.0001+++—
BTEC.0004++++
BTEC.0007++++
BTEC.0009+———
BTEC.0013++++
BTEC.0014++++
BTEC.0019++++
BTEC.0082++++
BTEC.0129++++
BTEC.0175++++
BTEC.0229+++—
BTEC.0232++++
BTEC.0402++++
BTEC.0433+++—
BTEC.0448++++
BTEC.0449++++
BTEC.0468++++
BTEC.0505++++
BTEC.0583+++—
BTEC.0601+++—
BTEC.0605++++
BTEC.0637++++
BTEC.0657++++
BTEC.0663++++
BTEC.0696++++
BTEC.0697++++
BTEC.0718+—+—
BTEC.0751++++
BTEC.0775++++
BTEC.0790+++—
BTEC.0818+++—
BTEC.0853++++
BTEC.0854————
BTEC.0874++++
BTEC.0882++++
BTEC.0887++++
BTEC.0904++++
BTEC.0973+++—
BTEC.0992+++—
BTEC.0997++++
BTEC.1013++++
BTEC.1058++—+
BTEC.1064++++
BTEC.1125++—+
BTEC.1203++++
BTEC.1204++++
BTEC.1211+++—
BTEC.1270++++
BTEC.1352————
BTEC.1383++++
BTEC.1388++++
BTEC.1398++++
BTEC.1400++++
BTEC.1407++++
BTEC.1413++++
BTEC.1438++——
BTEC.1450++++
BTEC.1489++++
BTEC.1507+++—
BTEC.1586++++
BTEC.1635++++
BTEC.1640++++
BTEC.1647++++
BTEC.1652++++
BTEC.1654++++
BTEC.1724++++
BTEC.1794++++
BTEC.1818+++—
BTEC.1832++++
BTEC.1873++++
BTEC.1907++++
BTEC.1930++++
BTEC.2120++++
BTEC.2141++++
BTEC.2142++——
BTEC.2148++++
BTEC.2166++++
BTEC.2399++++
BTEC.2409++++
BTEC.2436+++—
BTEC.2521+++—
BTEC.2704++++
BTEC.2773++++
BTEC.2795++++
BTEC.2807+++—
BTEC.2834+++—
BTEC.2850++++
BTEC.2869+++—
BTEC.2877++++
BTEC.2918++++
BTEC.2919++++
BTEC.3025++——
BTEC.3049++—+
BTEC.3142++++
BTEC.3285++——

Note: + = successful amplification; — = unsuccessful amplification; N = number of individuals sampled.

Information for the 95 Bromus tectorum SNPs used to genotype 251 individuals from 12 populations. The major allele is the nucleotide that occurs at the highest frequency among the 251 individuals sampled. Frequency of the major allele across all 12 populations. Dash marks within a population indicate that no lines amplified successfully at that locus. Population abbreviations are defined in Appendix 1. Cross-amplification for the 95 Bromus tectorum SNPs within related Bromus species. Note: + = successful amplification; — = unsuccessful amplification; N = number of individuals sampled.

CONCLUSIONS

Using B. tectorum from the IMW, we successfully developed 95 polymorphic SNP assays for studying its population and ecological genetics and found they have potential use in related Bromus species. SNPs were optimized for use with both the KASP and Fluidigm EP1 SNP genotyping platforms.
Appendix 1.

Information for 12 New Mexico (USA) populations of Bromus tectorum included in the survey of SNP polymorphisms.

Population nameaNo. sampledLatitudeLongitudeElevation (m)
Albuquerque (ALB)1434.906900−106.6717001500
Belen 1 (BN1)2334.675814−106.7716921465
Belen 2 (BN2)1934.654545−106.7784181466
Bernalillo (BER)1435.301900−106.5366001566
Gallup 1 (GA1)2535.528598−108.6672432017
Gallup 2 (GA2)2935.527506−108.7222112004
Grants (GRA)2635.143402−107.8386661972
Los Lunas (LOS)2034.811533−106.7538981480
Milan (MIL)2735.189228−107.9003391999
Prewitt 1 (PR1)1135.363436−108.0467642088
Prewitt 2 (PR2)1235.365321−108.0538252093
San Fidel (SAN)3135.076028−107.5551272003

Seeds collected from individual plants are deposited in the laboratory of S. E. Meyer.

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