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Genetic Variation Within and Among Lowland Switchgrass Cultivars as Revealed with AFLP Polymorphisms

Switchgrass (Panicum virgatum L.) has gained wider attention due to its recognition and use as a model herbaceous crop species for bioenergy production. Genetic diversity information in lowland switchgrass cultivars can help to specify cultivars to be used in the breeding programs aiming for hybrid vigor. The objective of this research was to analyze genetic variation within and among five lowland switchgrass cultivars using amplified fragment length polymorphism (AFLP) markers. AFLP polymorphisms indicated the presence of high genetic variation within lowland switchgrass cultivars with ‘Alamo’ exhibiting the highest genetic variation and ‘Performer’ the lowest. The Nei’s genetic diversity parameters revealed the lowest genetic distance between cultivars ‘Alamo’ and ‘Cimarron’ and the highest value between cultivars ‘Alamo’ and ‘Kanlow’. ‘Alamo’ and ‘Cimarron’ were clustered together while ‘BoMaster’, ‘Kanlow’, and ‘Performer’ were grouped into the other cluster. In addition, there were clusters with mixed genotypes. The findings of this study can be used to select diverse lines as parents for heterosis and inbreeding studies. © 2020 NAPA. All rights reserved. Citation: Makaju, S. O., Wu, Y. Q., Anderson, M. P., Kakani, V. G., Smith, M, Todd, J. & Adhikari, L. (2020). Genetic Variation Within and Among Lowland Switchgrass Cultivars as Revealed with AFLP Polymorphisms. Global Journal of Agricultural and Allied Sciences, 2(1), 11-17.

Genetic diversity is the result of selection, mutation, migration, genetic drift and/or recombination (de Vicente & Fulton, 2003). Variation can be evaluated on phenotypic and/or genotypic levels. Genotypic variation is evaluated at the level of DNA molecules responsible for transmitting genetic information (de Vicente & Fulton, 2003). DNA markers simply refer to a DNA sequence in the genome that can be used to genotype individuals or a population. The markers are selected based on the nucleic acid hybridization (RFLP), PCR (RAPD, AFLP, SSR), single base-pair change [single nucleotide polymorphisms (SNP)], array hybridization [diversity arrays technology (DArT)], and restriction site associated DNA (RAD). Different molecular markers have been used in the switchgrass diversity studies. They include random amplified polymorphic DNA (RAPD) (Casler, Stendal, Kapich & VOgel, 2007;Gunter, Tuskan & Wullschleger, 1996;Nageswara-Rao, Soneji Kwit & Stewart, 2013), restriction fragment length polymorphism (RFLP) (Missaoui, Paterson & Bouton, 2006), expressed sequence tag-simple sequence repeat markers (EST-SSRs) (Cortese, Honig, Miller & Bonos, 2010;Huang, Bughrara, Zhang, Bales-Arcelo & Bin, 2011;Narasimhamoorthy et al., 2008), amplified fragment length polymorphism (AFLP) (Todd et al., 2011), simple sequence repeats (SSR) (Zalapa et al., 2011), sequence-related amplified polymorphism (SRAP) (Huang et al., 2011) and a network-based single nucleotide polymorphism (SNP) (Lu et al., 2013). SNP markers were used in the switchgrass genetic diversity analysis to identify seven population groups that corresponded to ecotype, ploidy, and geographic distribution (Evans et al., 2017). AFLP markers can delineate between upland and lowland ecotypes and related plants according to broad geographic regions (Todd, Wu, Wang & Samuels, 2011). SSR markers were used to delineate diversity between ecotypes and between ploidy levels (Zhang et al., 2011). RFLP markers were used to determine extensive diversity between lowland tetraploid cultivar 'Alamo' (AP13) and upland tetraploid cultivar 'Summer' (VS16) and to develop linkage maps (Missaoui, Paterson & Bouton, 2005, 2006. The information on the extent of diversity in lowland cultivars will help determine the specific cultivars to be used in future crop improvement programs to develop potentially high yielding switchgrass cultivars. The immediate benefit of such diversity information will be in the development of advanced inbreds [selfing generations 5 to 6 (S5 to S6)] which can be used to produce hybrids for harnessing hybrid vigor. Bhandari, Nayak, Dalid & Sykes (2017) found up to 23% high parent heterosis from a cross between selections of divergent populations of switchgrass. Inbred populations can be utilized in the development of linkage maps (Liu, Wu, Wang & Samuels, 2012) and identification of quantitative trait loci (QTL) associated with agronomic, quality and disease traits. The QTL information can then be used in the marker assisted selection in switchgrass breeding.
Polymerase chain reaction (PCR) is a simple, automated technique for repeated copying of a short DNA molecule (Conner & Hartl, 2004). AFLP is a PCR based dominant marker and used in genetic research, DNA fingerprinting, and genetic engineering (Vos et al., 1995). It is a highly sensitive method for detecting polymorphisms in DNA. Unlike SNP genotyping, AFLP genotyping lack the information regarding the DNA sequence, which hinders in comparative genome analysis such as coregulation of markers or orthologous gene studies using common techniques such as nucleotide BLAST. However, AFLP is still a proven method that at low cost without specialized equipment can generate many bands. It has been successful at accessing genetic diversity and linkage mapping in many crops around the world (Meudt & Clarke, 2007). Because information on genetic diversity with outcrossing lowland switchgrass populations is generally lacking, this study aimed to analyze genetic diversity among and within five lowland switchgrass cultivars using AFLP. The cultivars included 'Alamo', 'BoMaster', 'Cimarron', 'Kanlow', and 'Performer'.

Plant Materials and Genomic DNA Extraction
Plant materials consisted of 384 plants from five lowland tetraploid cultivars 'Alamo', 'BoMaster', 'Cimarron', 'Performer', and 'Kanlow'. Seventy-six plants from the cultivar 'Performer' and 77 plants from each of the remaining four cultivars were seed-propagated and transplanted in individual 10-cm plastic pots with SUN-GRO Metro-Mix 200 series soil (Sun Gro Horticulture, WA) in a greenhouse at the Agronomy Research Station, Oklahoma State University, Stillwater, OK. The study began in July 2011 and completed in December 2013. Genomic DNA samples were extracted from leaf tissues using Zymo Research ZR Plant/Seed DNA Kit TM (Zymo Research Corporation, CA). DNA quality was checked with 1% agarose gel electrophoresis. The DNA samples were diluted to a final concentration of 100 ng μL -1 before enzyme digestion.
In the study for genetic variation among five cultivars, a total of 64 plants were used including 12 plants from cultivar 'Performer' and 13 plants from each of the remaining four cultivars (Fig. 1). The within genetic variation was studied separately for each of the five cultivars with 64 plants in each of them. The decision to use the above-mentioned numbers were based on capacity of polyacrylamide gel which can accommodate 64 sample lanes and two additional size marker lanes in a LI-COR 4300 DNA Analyzer.

AFLP Analysis
AFLP analysis was performed following Vos et al. (1995), with minor modifications (Todd et al., 2011;Wu, Taliaferro, Bai & Anderson, 2005). In the first step, the genomic DNA was double-digested with EcoRI and MSeI restriction enzymes and the DNA fragments were ligated to oligonucleotide AFLP adapters. The ligated DNA fragments were preamplified by PCR using a primer combination based on adapter sequences. In the second step, 12 AFLP selective primer combinations (Table 1) were used for selective amplification. The EcoRI primers were labeled with either IRD-700 or IRD-800 infrared fluorescence dye. The number of polymorphic bands (loci) considered appropriate for genetic variation in switchgrass is >400 (Todd et al., 2011). Accordingly, 12 selective primer pairs were used to generate >400 amplification products (polymorphic loci). All PCRs were conducted in an Applied Biosystems 2720 thermocycler (Applied Biosystems Inc., IL). In the third step, approximately one microliter of selectively amplified PCR products were loaded on a 0.25 mM thick 6.5% (w/v) polyacrylamide gel with 66 wells in a LI-COR 4300 DNA Analyzer (LI-COR Inc., Lincoln, NE) and run in 1x TBE buffer at 1500 V for 2.5 h. Standard DNA size markers (50-700 bp) (LI-COR Inc., Lincoln, NE) were loaded on first and last lanes to determine the size of the selectively amplified fragments in the final gel image. A total of 36 gels including 6 gels for among cultivar genetic variation and 30 gels (6 gels for each of the five cultivars) for within cultivar genetic variation were run.

Data Analysis
AFLP bands throughout the gel profile were visually scored as present (1), absent (0), and ambiguous (9). The scoring is repeated at least twice for all gel profiles to accurately collect data. The bands were scored between ~75 and 500 bp. The binary data matrix was recorded in a Microsoft Excel data sheet. Numerical Taxonomy System version 2.0 (NTSYSpc 2) program (Rohlf, 1998) was used to analyze the data. Each gel gave two images based on IRD-700 or IRD-800 infrared fluorescence dye. Data from six gels (12 images) were used for among-cultivar variation study. Data from six gels (12 images) for each of the five cultivars were separately analyzed for the within-cultivar variation analysis. A total of 72 gel images, including 12 gel images for among genetic variation and 60 gel images for within genetic variation, were scored and analyzed. In NTSYSpc 2 program, SIMQUAL module was used to compute genetic similarity coefficients (SC). The cluster analysis was based on unweighted pair-group method with arithmetic mean (UPGMA) within the SAHN module. DCENTER module was used for the principle coordinate analysis.   Shannon's information index (I), expected heterozygosity (He), and unbiased expected heterozygosity (uHe) were computed separately for each of the five cultivars; analysis of molecular variance (AMOVA) and Nei's genetic distance (Nei 1972) calculation were performed in among-variation data. GenAlEx 6 (Peakall & Smouse, 2012, Peakall & Smouse, 2006 software was used for these computations. AFLP bands initially scored as present (1), absent (0), and ambiguous (9) for NTSYSpc 2 were converted into present (1), absent (0), and ambiguous/missing (-1) for calculations in GenAlEx 6. AMOVA was performed to partition variation between cultivars. Pairwise genetic distance in different cultivars was computed using Nei's distance (Nei, 1972). Table 1 shows polymorphic band data with 12 different selective amplification primer pairs used in the experiment. In the analysis among five cultivars together, 85.5% of bands were polymorphic (Table 1). In the analysis within each of the five cultivars separately, polymorphic band percentages ranged from a minimum of 70.9% in 'Performer' to a maximum of 91.8% in 'Kanlow'. Similarity coefficients from analysis among five cultivars were given in Table S1 and the summary of similarity coefficients, for among cultivars and for each of the five cultivars, was provided in Table 2.

Results
'Alamo' exhibited the highest within-cultivar genetic variation (coefficient of variation=9.53) and 'Performer' exhibited the lowest withincultivar genetic variation (coefficient of variation=4.21) ( Table 2). Analysis using five cultivars together showed 'A4' from 'Alamo' and 'P4' from 'Performer' were the most divergent (similarity coefficient=0.60) ( Table 2 and Table S1). The average similarity coefficient ranged from 0.76 to 0.82 indicating the presence of high genetic variation among switchgrass genotypes.
The cluster analysis on AFLP variation among five cultivars generated a dendrogram with big cluster (m) which included 61 genotypes from different cultivars and a small cluster (n) that included genotypes P4, P6, and P8 from cultivar 'Performer' (Fig. 2). A genotype C4 from cultivar 'Cimarron' was observed separate from the rest of the individuals in the big cluster. The cluster m produced a cluster (m-1) of mixed genotypes from 'Alamo', 'BoMaster', and 'Cimarron' and a cluster (m-2) with two subclusters (a and b). The sub-cluster 'a' included cultivars 'Alamo' (a-1) and 'Cimarron' (a-2) while the sub-cluster 'b' included 'BoMaster' (b-1), 'Kanlow' (b-2), and 'Performer' (b-3). In the sub-cluster 'b', 'BoMaster' and 'Kanlow' were genetically more similar. The two-dimensional plot from principal coordinates analysis produced groupings (Fig. S1) mostly consistent with the clusters generated from the cluster analysis. The principal coordinates analysis revealed that the first principal coordinate explained 10.34% variation and the second principal coordinate explained 7.85% variation. The dendrograms from cluster analysis and twodimensional plots from principal coordinates analysis are mostly congruent for AFLP variation within each of the five cultivars 'Alamo', 'BoMaster', 'Cimarron', 'Kanlow', and 'Performer' (Figs. S2, S3, S4, S5, S6, S7, S8, S9, S10, S11). In these five cultivars, the first principal coordinate explained 11. 80, 9.38, 8.09, 7.98, and 11.40% variations, respectively, while the second principal coordinate explained 5.90, 6.27, 5.61, 5.72, and 4.33% variations, respectively. Mantel test results are shown in Table S2. The goodness of fit of the dendrograms to the original dissimilarity matrices (i.e., similarity coefficient table) was poor for among-cultivars (analysis of five cultivars together) and for 'Kanlow', however, the dendrograms were not significantly different from dissimilarity matrices (P = 1 > 0.05 in both cases). The dendrograms were a good or a very good fit to the dissimilarity matrices for each of 'Alamo', 'BoMaster', 'Cimarron', and 'Performer'.
AMOVA analysis carried out in the data from AFLP variation among five lowland switchgrass cultivars partitioned variation between cultivars at 15% as estimated variances and degrees of freedom (df) between cultivars, within cultivar, and total were 13.34 and 4, 74.39 and 59, and 87.73 and 63, respectively. Nei's genetic diversity revealed the lowest genetic distance between cultivars 'Alamo' and 'Cimarron' and the highest value between cultivars 'Alamo' and 'Kanlow' (Table S3). Shannon's information index (I), expected heterozygosity (He), and unbiased heterozygosity (uHe) calculated separately for each of the five cultivars revealed higher values for 'Kanlow' and 'Alamo' compared to the other three cultivars (Table 3).

Discussion
The morphological and physiological variation in switchgrass is closely associated with climatic factors and the adaptation along north-south range is dependent on photoperiod (Casler, 2012). Switchgrass is a native crop from North America with a large morphological diversity and wider adaptation (Parrish & Fike, 2005). The cultivars 'Alamo' and 'Kanlow' were developed from wild germplasm sources. The original ancestor of cultivar 'Cimarron' was primarily from 'Alamo'. 'Cimarron' was developed as a synthetic cultivar by polycrossing seven elite clonal parents in 2001 at Oklahoma State University (Wu, 2014). The selection of parent plants for 'Cimarron' was based on the evaluation of biomass yield of their half-sib families (Wu, 2014). The dendrogram and two-dimensional plot showed 'Alamo' and 'Cimarron' in the same group exhibiting the genetic relatedness consistent with the pedigree information. It appears that lowland switchgrass can be divided into two broad populations, including those from the south related to 'Alamo' like 'Cimarron' and the northern populations related to 'Kanlow' such as 'BoMaster' and 'Performer'. It was between these populations, 'Alamo' and 'Kanlow', that midparent heterosis was found and one of the crosses showed 23% high parent heterosis (Bhandari et al., 2017).
'BoMaster' and 'Performer' switchgrass cultivars were developed by North Carolina Agricultural Research Service, NC. Both 'BoMaster' (Reg. No. CV-248, PI 645256) (Burns, Godshalk & Timothy, 2008a) and 'Performer' (Reg. No. CV-247) (Burns, Godshalk & Timothy, 2008b) switchgrass cultivars were developed through three cycles of selection from a selected group of 161 lowland switchgrass plants that represented 11 different germplasm sources which included 'Kanlow'. The method in the development of these cultivars was recurrent half-sib selection. The selection for both cultivars was based on dry matter yield and in vitro dry matter digestibility. 'BoMaster' was selected for dry matter yield (Burns et al., 2008a) and 'Performer' was for in vitro dry matter digestion (Burns et al., 2008b) during the cultivar development. Similarly, the dendrogram and the two-dimensional plot showed 'Kanlow', 'BoMaster', and 'Performer' in the same group. The seven population groups identified in a genetic diversity analysis include Upland West, Upland East, Upland North, Upland Montane, Lowland North, Lowland South, Lowland Central, and admixed (Evans et al., 2017), indicating the genetic diversity across North-South and across East-West. 'Alamo', 'Cimarron', and 'Kanlow' were originated in George West in Texas, Stillwater in Oklahoma, and Wetumka in Oklahoma, respectively. 'BoMaster' and 'Performer' both were originated in Raleigh in North Carolina. The variation among these cultivars can also be attributed to the switchgrass genetic diversity across North-South and East-West locational gradients. Self-incompatibility and inter-cultivar gene flow, the characteristics of switchgrass, can also be the possible attributing factors for the clusters of mixed genotypes. We have used genetic distance based on AFLP markers as the basis to separate cultivars as heterotic. However, a further study may be required to determine if AFLP genetic distance is correlated with heterosis.

Conclusions and Recommendations
Lowland tetraploid switchgrass cultivars showed a high level of genetic variation. 'Alamo' showed the highest genetic variation while 'Performer' showed the lowest. The plant 'A4' from 'Alamo' and the plant 'P4' from 'Performer' were the most divergent genotypes. 'Alamo' and 'Cimarron' were clustered together while 'BoMaster', 'Kanlow', and 'Performer' were grouped into the other cluster. In addition, there were clusters with mixed genotypes as well. The findings of this research would be useful for future plant breeding and genetic improvement programs in lowland switchgrass. The results can be used for the selection of diverse lines as parents for heterosis and inbreeding studies.    Table S1. Continue ….