Conservation of grass genomes has been documented in distantly related grass species (Ahn and Tanksley, 1993; Kurata et al., 1994; Gale and Devos, 1998). Comparative genetics revealed the conservation of gene and marker orders within the Poaceae family (Ahn and Tanksley, 1993; Van Deynze et al., 1995; Jones et al., 2002; Alm et al., 2003; Kuleung et al., 2004). Comparative genomics facilitates identification of putative orthologous loci (genes that encode proteins with the same function in different species) controlling agronomic traits within the Poaceae (Paterson et al., 1995; Devos and Gale, 1997). It also assists extending genetic information from model species to more complicated species (Gale and Devos, 1998). As the grass genomes are highly conserved, molecular markers developed in one species can be used for genetic analysis of other species.
In the past, comparative genomics efforts relied primarily on the hybridization-based RFLP technique. For example, three chromosome I specific maize clones were detected on the chromosomes of homeologous group 3 (3a, 3b, and 3c) in a tall fescue map (Xu et al., 1995). Attempts were undertaken to assess the evolutionary relationship of meadow fescue with tall fescue through comparative RFLP mapping (Chen et al., 1998). A group of 33 RFLP markers that was mapped in meadow fescue and tall fescue detected highly conserved linkage groups (LGs) between the two species. Eight of the nine markers that mapped to LG I of meadow fescue also were present in LG I of tall fescue. However, changes in marker sequences, map distances, and homeologous LGs were detected between the two species. This indicated that the P genome diverged substantially during evolution from the diploid meadow fescue to the hexaploid tall fescue (Chen et al., 1998).
A major drawback of RFLP-based maps was low resolution, which often failed to determine the preserved order of genes between related species at micro levels. The application of a PCR-based codominant marker system for comparative genomics would be highly desirable. These markers have been found to be efficient in transferring genetic information across species (Kantety et al., 2002; Thiel et al., 2003; Eujayl et al., 2004; Saha et al., 2004). Studies by Jones et al. (2002) and Alm et al. (2003) revealed that forage species (ryegrass and meadow fescue) were highly orthologous; that is, they had a common ancestry and were colinear with the ancestries of rice, oat (Avena sativa L.), maize, and sorghum. High rates of transferability of SSR loci across species (>50%) within a genus have been reported (Peakall et al., 1998; Gaitán-Solís et al., 2002; Dirlewanger et al., 2002; Thiel et al., 2003; Eujayl et al., 2004; Saha et al., 2004). However, the transferability of SSR loci across genera and beyond appears to be low (White and Powell, 1997; Peakall et al., 1998; Roa et al., 2000; Thiel et al., 2003). The EST-SSR markers are expected to be more conserved and have a higher rate of transferability across species than genomic SSR markers (Scott et al., 2000). Thanks to this transferability, the EST-SSR markers have good potential for application in cross species genetic studies (Kantety et al., 2002; Thiel et al., 2003; Eujayl et al., 2004; Saha et al., 2004).
Tall fescue EST-SSR markers are fairly polymorphic across species and can be used to discriminate genotypes with wider genetic bases (Mian et al., 2005). One genotype from each of 12 forage and cereal grass species was screened using tall fescue EST-SSR primer pairs. Nearly 43% of the primer pairs produced PCR bands in at least 10 species. Ten percent of primer pairs were species specific (Mian et al., 2005). From this initial screening, 71 primer pairs with clear banding patterns were selected for screening 54 genotypes. Forty-six of the 71 selected primer pairs worked in all 12 species. In a similar study, tall fescue EST-SSRs were found highly conserved (Saha et al., 2004). A set of tall fescue EST-SSR markers was identified which can amplify characteristic SSR type products across a wide range of species in the Poaceae family (Mian et al., 2005; Saha et al., 2004). In many forage grass species, molecular markers have yet to be developed. Thus, highly conserved tall fescue microsatellite markers are of great value for species in which molecular marker information is very limited or not developed at all. Tall fescue EST-SSRs were found most effective in closely related species, and the complexity of banding pattern increased with the increasing genetic distance from tall fescue. A total of 511 tall fescue genomic SSRs was tested for functionality in 10 genotypes representing six different grass species (Saha et al., 2006). The proportion of PPs that produced clean SSR products ranged from 66% (tall fescue) to 48% (rice). Polymorphism rates were tested in the parents of mapping populations, and rates varied from 68% (tall fescue) to 34% (rice).
Breeders often are interested in incorporating ryegrass genes into tall fescue, or vice versa. Molecular markers have proven useful in monitoring introgression between species. A repetitive DNA sequence specific to the Festuca genome was used as a probe to monitor chromatin introgression in Festuca-Lolium hybrids (Cao and Sleper, 2001). Mapping of tall fescue EST-SSRs in ryegrass LGs (Warnke et al., 2004) indicated the applicability of these markers for genome introgression studies in the Festuca × Lolium complex. Of 27 polymorphic EST-SSR loci, 15 were mapped to ryegrass LGs (Warnke et al., 2004). Seven of these marker loci were uniquely mapped on both male and female maps (NFFA031 and NFFA75 on LG 1; NFFA015, 036, and 048 on LG 6; NFFA019 and NFFA069 on LG 7).
Repetitive DNA sequences contribute significantly to the understanding of genomes of higher plants. Repetitive DNA sequences tend to be genome specific and represent 20 to 90% of a whole genome and, in most cases, have no known function. Genome-specific repetitive sequences were developed and characterized in Festuca species (Cao and Sleper, 2001). Two repetitive sequences (TF436 and TF521) were identified from a hexaploid tall fescue PstI genomic library (Xu and Sleper, 1994). The sequence of TF521 was P genome specific and hybridized only with meadow fescue and tall fescue, but not in tetraploid fescue (F. arundinacea var. glaucescens) or Lolium. The sequence of TF436 was tetraploid fescue specific and did not hybridize to diploid meadow fescue or Lolium. Further study suggested meadow fescue and tetraploid fescue as the probable progenitors of tall fescue. Hybridization results with repetitive DNA and non repetitive RFLP probes implied no significant DNA structural differentiation in the genomes of meadow and tetraploid fescue. Both genomic in situ hybridization (Humphreys et al., 1995) and RFLP hybridization (Cao and Sleper, 2001) experiments successfully differentiated meadow fescue from F. arundinacea var. glaucescens. Xu and Sleper (1994) also successfully used TF436 clone to detect alien chromatin introgression from F. mairei in the progenies of the hybrid F. mairei × L. perenne.
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