Plants of ‘Alamo’, ‘Kanlow’, ‘Nebraska 28’, ‘Cave-in-Rock’, ‘Summer’, ‘Docotah’, ‘Shelter’, and ‘Blackwell’ , were grown in an Oklahoma State University (OSU) greenhouse, Stillwater, OK. These cultivars had been widely used in the USA and represented eco- and cyto-type diversity within the species . For initial SSR marker screening, equimolar DNAs from the eight cultivars were mixed to form a pooled DNA sample. For each cultivar, DNA sample was a mix from four to six plants.
In an OSU switchgrass nursery, ‘Alamo’, ‘Kanlow’, and ‘Cimarron’ plants were space planted on 3.5 feet × 3.5 feet centers in 2008. Four individuals from each cultivar were randomly selected. These 12 individual plants with additional four ‘Summer’ plants grown in the greenhouse constituted a panel (totally 16 plants), which was used for marker polymorphism analysis.
Open-pollinated seeds from a randomly selected ‘Kanlow’ genotype (encoded K4) in the nursery were harvested in 2010, and then they were germinated on filter paper in petri dishes after pre-chilling treatment for two weeks . The obtained seedlings were transplanted into conetainers and grown in the greenhouse for leaf collection. The obtained half-sib progeny population (Population 1) of 46 individuals (encoded as K4-1 to 46) was used to examine selfing and outcrossing rates of a plant grown in the open-pollinating, natural field condition.
In 2010, 22 first-generation selfed (S1) plants of two genotypes NL94 and SL93  were selected according to spring growth vigor, plant height, and crown size. And then two inflorescences from each plant were bagged with pillow cages  in the field before inflorescences fully emerged out. The obtained seeds were germinated respectively in a growth chamber in the spring of 2011. Survived plants were transplanted in a field plot on August 1, 2011 and constituted 22 families of totally 99 progeny plants (Population 2). The family size ranged from 1 to 13 in Population 2. Population 3 included 44 progeny collected from SL93 in a growth chamber, in which NL94 served as the pollen donor . Genomic DNA was isolated from healthy leaf tissues using the CTAB method . The DNA concentration was measured using an ND1000 spectrophotometer (NanoDrop Products, Wilmington, DE). The working solutions were adjusted to10 ng/μl as PCR templates.
SSR primer screening
PCR PPs were obtained principally from two sources: those on two-sister linkage maps (totally 585 PPs) , and the others from a recent linkage map (totally 473 PPs) . Primer sequence information was collected from previous studies [15–19]. After excluding SSR redundancy, unique PPs were used in this study. All forward primers were appended with a M13 sequence (5’-CACGACGTTGTAAAACGAC-3’) at the 5’ end to allow indirect labeling in PCR reactions. The initial screening for polymorphism was performed using the pooled DNA sample to determine the number of alleles at each locus. Candidate SSR PPs were selected based on the previous data generated in the mapping experiment  and screening results of amplifying clear bands, displaying four or more alleles per locus and avoiding tight linkages (i.e., >10 cM between two neighboring loci). And then selected PPs were tested on the panel with 16 individual DNA samples to determine polymorphism. Monoplex PCR with 10 μl volume mixtures each reaction and a ‘Touchdown’ thermal cycling program was used [68, 69].
Development and optimization of duplex PCR
Based on amplified allele size, map position and heterozygosity for each of the candidate SSR markers, a smaller set of SSRs was selected for testing in duplex PCR. The criteria used to combine SSR markers into duplex PCR were as the following: (1) Non-overlapping allele size for each pair of two markers; (2) Primer compatibility and genotyping quality in duplex PCR; (3) High polymorphism estimated by PIC value ; (4) Two high-quality bands in each genotype due to disomic inheritance identified in tetraploid switchgrass [9, 18]; (5) Genetic distance between selected SSRs ≥ 10 cM; (6) SSRs with tri-, tetra-, or higher nucleotide repeats were preferred to lessen slippage during PCR .
An optimization procedure was carried out before the final PP combinations for duplex PCR were assembled. Eight switchgrass genotypes from Alamo (A) and Kanlow (K), i.e., A2, A4, A5, A10, K1, K3, K4 and K5, were used as amplification templates. The SSR PPs used here to optimize duplexes were PVCAG-2397/8 and 2517/8. The adjustment of duplex PCR parameters on the amplification effect followed: increasing Taq polymerase (BioLabs®, Catalog #M0273X, NEW ENGLAND Inc., USA) from 0.25 to 0.5 units, dNTPs from 0.2 to 0.4 mM, template DNAs from 15 to 30 ng, PCR buffer from 1 × to 1.6 ×, Mg 2+ concentration from 1.5 to 2.4 mM, IR-M13 forward primer (labeled with either 700 nm or 800 nm florescence) concentration from 0.02 to 0.04 μM and PP quantity from 1.0 to 2.0 pmoles. Subsequently, the compatibilities of different SSR primer combinations were tested on the same eight genotypes.
Duplex PCRs were performed in 10 μl of reaction mixture containing 1 × PCR buffer, 2.4 mM of Mg 2+, 0.2 mM each of dNTPs, 0.125 to 4.0 pmoles of each primer (Table 2), 0.5 units of Taq polymerase (BioLabs®, USA), 0.02 μM IR-M13 forward primer, and 15 ng of genomic DNA. The cycling parameters were the same as monoplex PCR mentioned above. PCR products labeled with 700 and 800-nm dye were pooled, and mixed thoroughly. After denaturation, they were separated using 6.5% KB plus polyacrylamide gels with a LI-COR 4300 DNA Analyzer (LI-COR Biosciences, Lincoln, NE, USA) .
Genotyping and data analysis
The gel bands were visually scored and band sizes were determined using Saga Generation 2 software, version 3.3 (LI-COR Biosciences, Lincoln, NE, USA). For Population 3, the scoring of PCR bands and identification of selfed progeny were the same as our former study . For Populations 1 and 2, PCR bands were recorded as “ab” if two bands (“a” indicated upper band and “b” for lower band) or “aa” if only one band for the parent. In the progeny, if bands were from parents, they were scored in the same way as described above. If different bands were scored in the progeny, letters from “c” to “g” (or “h”, “i” and so on, if more alleles appeared) were assigned to each band based on the molecular sizes (“c” for the largest and “d” for a smaller band than “c” but larger than other non-parental bands) . If the alleles of a tested individual were all derived from its corresponding maternal parent, it was identified as a selfed progeny. Apart from maternal allele(s), if non-parental alleles of a progeny individual appeared, it was identified as cross-fertilized (i.e., hybrid). If none of the alleles of a tested progeny was from the seed parents on more than two marker loci (≥2), it was determined as contaminants and excluded in further study.
T-test was carried out using Microsoft® Excel 2007. The allele frequency, heterozygosity, PIC, and average non-exclusion probability for one known parent (NE-1P) were estimated using Cervus 3.0 . The output options were set as the following: Header row = yes, Read locus names = yes, First allele in column =3, Number of loci=166. For comparisons of expected heterozygosity among 18 LGs, the Scheffe’s method for general linear model (GLM) procedure was used in SAS 9.3 (SAS Institute Inc.) with a significance level of 0.05.