Genetic diversity in red clover (Trifolium pratense L.) revealed by morphological and microsatellite (SSR) markers

Abstract

The NPGS-USDA core collection with 85 accessions of red clover, an important forage species, is little described. The goal of the present study was to evaluate the diversity of a set of accessions from the core collection at the morphological and molecular level in order to extract some valuable accessions for Brazilian red clover breeding programs. Twentyone morphological traits, collected in field and greenhouse in South Brazil, and seven SSR markers were used to describe 57 accessions from the U.S. core collection and one population cultivated in Southern Brazil. Variation between accessions was large for most of the 21 morphological traits. A cluster analysis based on the morphological traits revealed five distinct clusters that separated the populations according to flowering earliness, as already described, but also according to persistency, growth habit and dry matter productivity. Over seven SSR loci, the number of alleles averaged 11.1 alleles per locus. Genetic diversity measured with SSR markers was high, with a mean expected heterozygosity of 0.86. An analysis of molecular variance revealed that the largest proportion of variation (83.6%) resided at the within population level. Although the molecular markers also separated accessions into five clusters, there was no coincidence between the composition of groups found with morphological and molecular data. Use of genetic diversity in breeding programs requires to use the most promising populations, to combine positive traits such as persistency and forage yield, and probably to use within population variation to detect valuable genotypes that could be used as parents of synthetic varieties.

Microsatellite diversity and chromosome number in natural populations of Trifolium riograndense Burkart

“…No clearcut relation between genetic diversity assessed by SSR and geographical origin could be established in the present study, same as reported by Dias et al (2008) for T. pratense. In Trifolium repens, Zhang et al (2010) observed some relationship between genetic diversity and geographical origin and in Lotus corniculatus genetic distances and place of collection were highly correlated (Sardaro et al, 2008).…”

“…A similar result, with an average of 4.8 alleles per locus was reported by Kölliker et al (2001) in T. repens. In T. pratense, Dias et al (2008), also using SSR found high intrapopulational variability in a core collection of red clover (T. pratense), number of alleles per locus ranging from seven to 12. Null alleles, those alleles that are consistently not amplified (Martins et al, 2008) were observed in SSR ats 070, TM0133 and TM0256 (Table 2).…”

“…PCR reactions were prepared in a final volume of 7 µL per reaction, containing 1 µL of the work DNA solution (20 ng µL -1 ), 1 µL of PCR 10X buffer, 0.6 µL of MgCl 2 (50 nM), 0.8 µL of DNTP mix containing 2.5 mM of each of the four nucleotides, 0.6 µL of the forward primer (10 µM), 0.6 µL of the reverse primer (10 µM), 0.2 µL of Taq polimerase Quiagen (5 U µL -1 ) and sterilized water to complete the volume. PCR conditions followed Dias et al (2008) tions: initial denaturation at 94 ºC for 5 min, followed by seven cycles of 1 min at 94 ºC, 1 min at 61 ºC, 1 min at 72 ºC, and annealing temperature reduction of 1 ºC per cycle, followed by 25 cycles of 1 min at 94 ºC, 1 min at 55 ºC and 1 min at 72 ºC and six cycles of 45 seconds at 94 ºC, 45 seconds at 54 ºC and 45 seconds at 72 ºC and a final extension for 8 min at 72 ºC and storage at 7 ºC. The amplification products were separated in a 4 % high resolution agarose gel (Agarose 1000) stained with 0.08 µL mL -1 ethidium bromide (10 mg mL -1 ) and immersed in TBE 1X buffer under an electrophoretic current of 96 V for approximately 1 h and 15 min.…”

Morphoagronomic variation in natural populations of Trifolium riograndense (Burkart)

“…Bortolini et al (2006) reported a positive correlation between plant height and leaf area in white clover suggesting that plants with larger leaf area also had higher petioles. In red clover, Dias et al (2008) verified that plants with larger leaf area were higher. Other works with white clover also showed correlation between leaf area and petiole length (Gustine et al, 2002) and between leaf length and plant heigth (Mather et al, 1995).…”

Genetic variability of isoflavones in the USDA red clover core collection

“…Many works using morphologic (Christie & Choo, 1991;Kouamé & Quesenberry, 1993), molecular (Greene et al, 2004;Sato et al, 2005;Dias et al, 2008a) and biochemical traits (Yu et al, 2001;Mosjidis & Klinger, 2006) have showed the high genetic diversity present in this species. All red clovers may be grouped into three divisions corresponding to early, medium and late maturity types (Bird, 1948;Taylor & Smith, 1995).…”