GENETIC DIVERSITY AND PHENETIC RELATIONSHIPS OF FIVE TRIFOLIUM L. SPECIES (FABACEAE) BY INTER SIMPLE SEQUENCE REPEATS MARKERS

Five species of Trifolium L. (T. repens L., T. pretense L., T. hybridum L., T. campestre Schreb., and T. dubium Sibth.) were analyzed used to evaluate the genetic diversity and their phenetic relationships using inter-simple sequence repeats (ISSR) markers. Overall, T. pratense exhibited higher variation than other species. 114 amplicons were produced by ISSR markers, of which 77 (67.5%) bands were polymorphic. T. dubium showed the low genetic variation. Total genetic diversity values (HT) varied between 0.333 and 0.487, for an average over all polymorphic loci of 0.282. On a perlocus basis, the proportion of total genetic variation due to differences among species (GST) was 0.380. This indicated that about 38.0% of the total variation was among species. The estimate of gene flow, based on GST, was very low among species of genus Trifolium (Nm = 0.816). An assessment of the proportion of diversity present within species, HPOP/HSP, indicated that about 95.8% the total genetic diversity was within species. T. pratense and T. hybridum were grouped together and this clade was sister with T. repens. Two remainder species with yellow flowers were grouped together. Information on genetic diversity for Trifolium is valued for the management of germplasm and for evolving conservation strategies. Introduction Trifolium L., the clover genus, is one of the largest genera in Fabaceae family. This genus consists about 250-300 species with a wide distribution and adaption to different agro-ecological regions (Gillet et al., 2001; Ellison et al., 2006). Trifolium repens, also known as White or Dutch clover, originated in the Mediterranean region and quickly spread throughout Europe (Baker and Williams, 1987; Lane et al., 1997). White clover is adapted to a wide climate range from the Arctic to the subtropics and has a wide altitudinal ranges. It is found up as 6000 m in the Himalaya Range (Baker and Williams, 1987). It has also become naturalized in China, Mongolia, Korea, and Japan. The genus Trifolium includes more than 20 clover cultivated species as forages (Hirano, 2005). White clover (T. repens) in Korea has been introduced from Europe about two hundred years ago. Most species belonging to genus Trifolium can tolerate wide variations in temperature, sunlight, and pH of soil. With the recent development of organic farming, legumes have been considered candidates of fertilizer (Paplauskiene and Dabkeviciene, 2012). Many species of Trifolium are known to have been cultivated on a commercial scale including white and red clover (T. repens and T. pratense), the two most economically important pasture legumes in the UK (Taylor and Quesenberry, 1996). However, Trifolium is one of major weeds for lawns, farming fields, and golf courses in Korea. Especially many plants of Trifolium are also considered to cause 1 Corresponding author. E-mail: mkhuh@deu.ac.kr


Introduction
Trifolium L., the clover genus, is one of the largest genera in Fabaceae family.This genus consists about 250-300 species with a wide distribution and adaption to different agro-ecological regions (Gillet et al., 2001;Ellison et al., 2006).Trifolium repens, also known as White or Dutch clover, originated in the Mediterranean region and quickly spread throughout Europe (Baker and Williams, 1987;Lane et al., 1997).White clover is adapted to a wide climate range from the Arctic to the subtropics and has a wide altitudinal ranges.It is found up as 6000 m in the Himalaya Range (Baker and Williams, 1987).It has also become naturalized in China, Mongolia, Korea, and Japan.
The genus Trifolium includes more than 20 clover cultivated species as forages (Hirano, 2005).White clover (T.repens) in Korea has been introduced from Europe about two hundred years ago.Most species belonging to genus Trifolium can tolerate wide variations in temperature, sunlight, and pH of soil.With the recent development of organic farming, legumes have been considered candidates of fertilizer (Paplauskiene and Dabkeviciene, 2012).Many species of Trifolium are known to have been cultivated on a commercial scale including white and red clover (T.repens and T. pratense), the two most economically important pasture legumes in the UK (Taylor and Quesenberry, 1996).However, Trifolium is one of major weeds for lawns, farming fields, and golf courses in Korea.Especially many plants of Trifolium are also considered to cause damage to the environment and have gradually the superior competitive ability on golf courses to create fairways and teeing areas.
Alsike clover (T.hybridum), field clover (T.campestre), suckling clover (T.dubium), are European grassland legumes that have spread to many parts of the world.Recently they have been also introduced to Korea.These non-native clovers can rapidly invade and dominate vegetated and bare areas in Korea (Huh et al., 2005).
Many molecular marker techniques have been developed and they have been extensively used in plant systematic studies, measurement of variation to establish evolutionary relationships within or among species, and population genetic research (Hu and Vick, 2003;Gupta and Rustgi, 2004;Rizza et al., 2007).Inter simple sequence repeats (ISSR) markers have the advantage over randomly amplified polymorphic DNA (RAPD) in that the primers are longer, allowing for more stringent annealing temperatures (Wolfe and Liston, 1998).These higher temperatures apparently provide a higher reproducibility of bands than in RAPD (Nagaoka and Ogihara, 1997).Tsumura et al. (1996) found that most of their ISSR bands (96%) segregated according to Mendelian expectations.
The aim of this study was the estimation of population structure, genetic diversity, and genetic relationships of five clover species in Korea.

Plant materials
Five clover species, T. repens L., T. pratense L., T. hybridum L., T. campestre Schreb., and T. dubium Sibth.were used for ISSR analysis (Table 1).Thirty plants were collected for each species.Within populations, plants are genetically subdivided by micro-environmental heterogeneity (Sackville and Chorlton, 1995).Clover has a creeping growth habit and spreads with stolons or runners above the soil with adventitious roots forming at each node.The geographic distance between the selected individuals was about 1.0 m to avoid inclusion of individuals emanating from the same rhizome.Medicago sativa L. was used as an outgroup species in this study.

DNA extraction
Total genomic DNA was extracted from a fresh young leaves using the plant DNA Zol Kit (Life Technologies Inc., Grand Island, New York, U.S.A.) according to the manufacturer's protocol.Briefly, Approximately 1.2 g fresh leaves per individual was ground to fine powder in liquid nitrogen with a mortar and pestle.The pulverized material was transferred to a micro-tube and Plant DNA Zol solution was added.The sample was shaken gently at room temperature for 10 min.After adding 24:1 chloroform/isoamyl alcohol, the sample was centrifuged at 12,000 g.The DNA precipitate was recovered with 70% ethanol, dried, and dissolved in TE buffer.The extracted DNA concentrations were calculated with a fluorometer (DyNA Quant 200, Hoefler, Amersham Biosciences, USA) using bisbenzimide (Amersham Biosciences, USA) as the fluorescent dye.

ISSR analysis
The ISSR amplification assay developed by Zietkiewicz et al. (1994) using primers listed in Table 1.PCR was performed within a total volume of 25 using a PTC-100 DNA Engine Dyad Peltier thermal cycler (MJ Research, Watertown, MA, USA).Each PCR mixture contained PCR buffer (Promega; 20 mM This-HCl, 50 mM KCl), 1.5 mM MgCl 2 , 0.24 mM of each dNTP, 12.5 pmol of each primer, 0.25 units of BIOTAQ DNA polymerase (Bioline), and 25 ng of genomic DNA.An initial denaturation step of 5 min at 94ºC was followed by 30 cycles of amplification (1 min sec at 94ºC, 1 min at 50ºC, 1.5 min at 72ºC) and a final elongation step of 10 min at 72ºC.
The amplification products were separated by electrophoresis on 2.0% agarose gels in Tris-Borate buffer, and stained with ethidium bromide.A 100 bp ladder DNA marker (Pharmacia) was used in the end of for the estimation of fragment size.

Statistical analyses
PCR-amplified ISSR fragments detected on gels were scored absent (0) or present (1).Only unambiguously reproducible bands were scored and used in the analyses.The following genetic parameters were calculated using a POPGENE computer program (ver.1.31) developed by Yeh et al. (1999): the percentage of polymorphic loci (P p ), mean numbers of alleles per locus (A), effective number of alleles per locus (A e ), and gene diversity (H) (Nei, 1973) and Shannon's index (I) of phenotypic diversity.Shannon-Weaver index of diversity (Shannon and Weaver, 1963): the formula for calculating the Shannon diversity index (H') is: H' = -Σ pi ln pi pi is the proportion of important value of the ith species (pi = ni / N, ni is the important value index of ith species and N is the important value index of all the species).
Polymorphism information content (PIC) value was calculated using the formula PIC, PIC = 1 -p 2 -q 2 , where p = band frequency and q = no-band frequency (Rizza et al., 2007).
Nei's gene diversity formulae (H T , H S , and G ST ) were used to evaluate genetic diversity within and among cultivars (Nei, 1973).H T is the expected heterozygosity of an individual in an equivalent random mating total interspecies.H S is the expected heterozygosity of an individual in an equivalent random mating total intraspecies.The G ST coefficient corresponds to the relative amount of differentiation among cultivars.Furthermore, gene flow (Nm) between the pairs of species was calculated from G ST values by Nm = 0.5(1/G ST − 1) (McDermott and McDonald, 1993).
Shannon's index of genotypic diversity (H O ) for ISSR was estimated to quantity the degree of within species diversity following the formula (Bowman et al., 1971): H O = -∑pi log pi, where pi is the frequency of a particular phenotype i.
A phenetic relationship was constructed by the neighbor-joining (NJ) method in PHYLIP version 3.57 using MEGA5 program (Tamura et al., 2011).Parsimony analyses were conducted using PAUP * 4.0b3a (Swofford, 1999).Confidence values for individual branches were determined by a bootstrap analysis with 100 repeated sampling of the data.

Results and Discussion
From the 20 decamer primers used for a primary ISSR analysis, thirteen primers produced good amplification products both in quality and variability (Table 1).The remaining primers either did not amplify or showed unclear amplification across all genotypes.114 amplicons were produced by ISSR marker, of which 77 (67.5%) bands were polymorphic.Polymorphism information content (PIC) for ISSR primers ranged from 0.244 to 0.498 with an average of 0.287 per primer.
In a simple measure of inter-cultivars variability i.e. the percentage of polymorphic bands, T. pratense exhibited the highest variation (49.1%) among clovers and T. dubium the lowest (36.0%) (Table 2).The average number of alleles per locus (A) was 1.423 across species, varying from 1.360 to 1.491.The effective numbers of alleles per locus (A E ) was 1.311 across species, varying from 1.251 to 1.374.The mean genetic diversity within species was 0.175.Shannon's index of phenotypic diversity (I) of T. pratense (0.302) was highest of all taxa and T. hybridum was the second (0.286).Overall, T. pratense exhibited higher variation than other species.Two species (T.campestre and T. dubium) with yellow flowers were shown the low genetic variation.
The first fragment (ISSR-06-01) of primer ISSR-01 was specific band for T. repens which did not show at other species.The ISSR-01-04 fragment of primer ISSR-01 was also specific band for T. pratense.These specific fragments seemed to be useful markers to discriminate among species.Total genetic diversity values (H T ) for polymorphic loci varied between 0.333 (ISSR-04) and 0.487 (ISSR-12) (Table 3).An average (H T ) over all 114 loci for five species with 13 ISSR primers was 0.282.In interlocus variation in the within-species, mean genetic diversity (H S ) was low (0.175).On a per-locus basis, the proportion of total genetic variation due to differences among species (G ST ) ranged from 0.216 for ISSR-08 to 0.547 for ISSR-02, with a mean of 0.380.This indicated that about 38.0% of the total variation was among species.Thus, about genetic variation (62.0%) resided within species.The estimate of gene flow, based on G ST , was very low among species (N m = 0.816).Values of genetic distance (D) were ≤ 0.233 (Table 4).Genetic identity values among pairs of species ranged from 0.508 to 0.956.