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Published in Crop Sci. 43:2154-2161 (2003).
© 2003 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA

TURFGRASS SCIENCE

Genome Introgression of Festuca mairei into Lolium perenne Detected by SSR and RAPD Markers

J. P. Wanga, S. S. Bughrara*,a and D. A. Sleperb

a Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824
b Department of Agronomy, University of Missouri-Columbia, Columbia, MO 65211

* Corresponding author (bughrara{at}msu.edu).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The genera of Lolium and Festuca belong to the same tribe Poeae, of subfamily Pooideae, but offer a range of complementary characteristics of agronomic importance. Intergeneric hybridization between the two genera is expected to combine the desirable characteristics of Lolium (good turf quality and good establishment) with Festuca species (drought and heat tolerance and disease resistance) in turfgrass breeding. The use of molecular markers to trace genome introgression in intergeneric hybrids has been reported for many crops, but not for Lolium and Festuca. The objective of this study was to use simple sequence repeats (SSR) or microsatellite markers developed from Lolium perenne L. and random amplified polymorphic DNA (RAPD) markers to assess genomic introgression of Festuca mairei St. Yves (Fm) into L. perenne (Lp). Out of the 40 SSR markers tested, nine markers covering seven linkage groups, fully discriminated the Fm and Lp parents and revealed that the Fm genome was transferred into the backcross progenies of Fm and Lp. In 13 backcross derivatives detected by SSR markers, 11 individuals showed that the Fm genome was introduced. Forty-one RAPD primers were chosen to detect genome introgression of the backcross progeny. A total of 188 parent-specific markers were obtained. Ninety-two (49%) are Fm-specific markers. The 13 backcross progenies showed a range of introgression of Fm-specific markers (5.4–60.9%). The introgression levels of the backcross progeny revealed by SSR and RAPD markers were significantly correlated (P <= 0.0116). The SSRs and RAPD markers were informative and effective in detecting Fm genome introgression into the Lp genome.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Festuca and lolium are two important genera that contribute to turf and forage in the temperate regions of the world. Both belong to tribe Poeae, subfamily Pooideae, in which the basic number of chromosome (x) is 7. These two genera express complementary traits (Jauhar, 1993). For both forage and turfgrass breeders, it has long been a goal to combine the complementary traits of Festuca and Lolium species. Lp is a diploid (2n = 2x = 14, LL) commonly known as perennial ryegrass. It is one of the most important grasses used extensively for both turf and forage. Lp has desirable agronomic features for turfgrass such as rapid establishment and good turf quality. Fm, found in northwestern Africa, is a xeromorphic tetraploid (2n = 4x = 28, M1M1M2M2) species, and is commonly known as atlas fescue. Fm is tolerant to high temperature and drought, which are valuable assets for turfgrass improvement; however, it lacks good turf quality. Combining the desirable attributes of Lp and Fm has great potential in turfgrass breeding.

Wide hybridization can be used to introduce agriculturally important genes from one species to another and create new species or novel allopolyploids for breeding purposes. Many intergeneric hybrids have been generated between L. perenne and some Festuca species (Morgan and Thomas, 1991; Crowder, 1953). Some cultivars derived from hybridization between Lolium and Festuca have been released (Buckner et al., 1977; 1983).

With the goal of introducing stress tolerance from Fm into Lp, Chen (1996) obtained triploid (3x) and tetraploid (4x) hybrids between Fm and Lp. The close genomic relationships between the two species were revealed by genomic in situ hybridization (GISH), where the L chromosomes of Lp paired with M (M1 and M2) chromosomes of Fm, as well as M1 paired with M2 in hybrids (LM1M2) (Cao et al., 2000).

Understanding genome segment introgression in intergeneric hybridization would help to identify the lines introgressed with desirable traits and develop new cultivars in breeding programs. The development of molecular markers has recently raised expectations for their application in tracing genome introgression in intergeneric hybridization. However, molecular markers for tracing introgression between Festuca and Lolium have lagged behind most other crops. Cao and Sleper (2001) identified one genome-specific repetitive DNA sequence, which was used as a probe to monitor the chromatin introgression by Southern blotting. However, one repetitive DNA sequence covers a very limited region of the whole genome, and it is not informative for detecting DNA fragment introgression in all progeny.

Microsatellites, or SSR, are short tandemly repeated DNA sequences that are abundant in eukaryotic genomes. Microsatellites are hypervariable and therefore able to distinguish among closely related plant cultivars (Olufowote et al., 1997; Davila et al., 1998; Udupa et al., 1999). The SSR markers are codominant and have become powerful, ideal tools for genotype assignment, marker assisted breeding, genetic mapping, and diversity assessment (Paetkau, 1997; Gupta and Varshney, 2000; Hearne et al., 1992; Powell et al., 1996). Recent studies have shown that SSRs are conserved in related species, and may allow for the analysis of different species by the same microsatellite loci. The SSR loci are randomly dispersed in the genome, and suitable for wide comparisons (Morgante and Olivieri, 1993; Röder et al., 1995; Macaulay et al., 2001). Furthermore, SSR polymorphism can be detected easily and robustly by polymerase chain reaction (PCR) and DNA sequencing. The primers could be labeled fluorescently with three different fluorochromes to allow simultaneous examination and detection of up to nine different SSR loci on an ABI automated DNA sequencer (Applied Biosystems, Foster City, CA) (Waugh et al., 1999). Recently, a large number of SSR markers for perennial ryegrass have been isolated and constitute a valuable resource of markers for the molecular breeding of ryegrass (Jones et al., 2001). Up to this time, SSR markers have not been utilized to monitor genome introgression of Lolium and Festuca species.

RAPD markers (Williams et al., 1990; Welsh and McClelland, 1990) can generate a large number of polymorphisms. They have been successfully used in examining genetic variability in various plant species (Schierenbeck et al., 1997), linkage map studies in tomato (Klein-Lankhorst et al., 1991), cultivar identification (Caetano-Anolles et al., 1991), evolution (Arnold et al., 1991), and population genetics (Chalmers et al., 1992). RAPD profiles were shown to provide enough information to identify Lolium–Festuca hybrid genomes (Wiesner et al., 1995; Siffelova et al., 1997; Charmet et al., 1997).

The objective of this study was to use SSR and RAPD markers to assess the introgression of Fm genome into Lp in the progeny of a backcross population.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Materials
One clone (#2) of F. mairei (Fm) collected from Morocco was used as a donor parent in the hybridization with L. perenne (Lp). Two genotypes of Lp from turfgrass ‘Citation II’ and ‘Calypso’ were used as recurrent parents (Fig. 1a). Thirteen backcross individuals (Fig. 1b) were derived from interpollination of BC1F1 progeny (Chen and Sleper, 1999). All plants were greenhouse grown.



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  		Fig. 1. The phenotypes of the plant materials. a. the parents (F. mairie and L. perenne) and F1 hybrid; b. the backcross progeny.

 
DNA Isolation
Total genomic DNA was extracted from young growing leaves of the plants from each genotype. Collected leaf tissues were immediately ground in liquid nitrogen. Lysis of cells was initiated by adding the extraction buffer (0.1 M Tris-HCl, 0.05 M EDTA-Na and 0.25 NaCl, PH = 8.0). Sodium dodecyl sulfate (SDS) was added to lyse the cells. Potassium acetate (5 M) was used for deproteinization and recovery of DNA. Nucleic acid was precipitated by isoproponol followed by RNase treatment to degrade the RNA. The DNA in solution was reprecipitated by sodium acetate and redissolved in TE buffer. The DNA concentration was detected by spectrophotometer readings at a wavelength of 260 nm. The DNA quality was checked by loading 100 ng DNA in 1% (w/v) agarose gel by electrophoresis at 72 V for 2 h.

SSR Characterization, PCR, and Polymorphism Analysis
From perennial ryegrass, 101 SSR markers were developed and characterized by Jones et al. (2001). In this study, 40 primer pairs were selected to detect polymorphism between Fm and Lp. These markers cover seven linkage groups of perennial ryegrass with two to four SSR markers per linkage group, and have previously been shown to amplify DNA fragments of Festuca species (Jones et al., 2001). The SSR work conducted at the Plant Biotechnology Center, La Trobe University, Bundoora, Victoria, Australia, and the sequences are not available. The primers showing polymorphism between the parents were utilized to perform SSR analysis of all plant materials.

PCR amplification for SSRs was conducted following the procedure of Jones et al. (2001) with small adjustment according to the Tm value of each primer pair. Polymorphism was detected by means of automated capillary electrophoresis of fluorochrome-labeled PCR products. The forward primers were labeled with one of three fluorochrome moieties [FAM = 6-carboxyfluorescein, HEX = hexachloro-6-carboxyfluorescein, or NED = 7', 8'-benzo-5'-fluoro-2, 4, 7-trichloro-5-carboxyfluorescein (Applied Biosystems)]. Triplex PCR products were separated with an ABI 3700 96-channel DNA sequencer (Applied Biosystems) and the fragments were sized by means of a ladder labeled with a fourth fluorochrome (ROX = 6-carboxy-X-rhodamine).

RAPD Reaction
Fifty-six decamer oligonucleotides (Operon Technologies, Alameda, CA) were tested to detect the high complexity and maximum polymorphism between the parents, Fm and Lp. The suitable primers (see Table 1 for sequences) were then applied for RAPD analysis of all progenies against the parents.


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  		Table 1. The sequence of 41 RAPD primers and the number of fragments amplified.

 
The RAPD reaction mixture (25 µL) contained 10 mM Tris-HCl (pH = 8.3), 4 mM MgCl2, 0.24 µM of each dNTP, 1.2 µM of primer, 30 ng of template DNA, and 1U Taq polymerase (Gibco Invitrogene, Grand Island, NY). Amplification was conducted in a PTC-100 programmable thermal controller (MJ Research, Waltham, MA). Amplification conditions were as follows: 3 preamplification cycles, each consisting of a denaturation step of 1 min at 94°C, an annealing step of 1 min at 35°C, and an extension step of 2 min at 72°C. After initiation of the reaction, 35 amplification cycles were conducted (94°C for 20 s, 40°C for 20 s, and 72°C for 2 min). The last cycle was followed by 5 min at 72°C to ensure that primer extension reactions proceeded to completion. The RAPD profiles were generated in 2% (w/v) agarose gel with 0.003% (w/v) ethidium bromide. A 1-kb ladder was used to mark the size of fragments. The images of RAPD were obtained through an Eagle Eye II still video system V3.2 (Stratagene, La Jolla, CA).

Data Analysis
Peaks of SSR markers and bands of RAPD profiles were scored as 1 (present) and 0 (absent). The Fm genome introgression levels in the progeny from SSR data equaled the number of loci showing Fm alleles in the backcross individual divided by total number of polymorphic SSR loci. Similarly, the Fm genome introgression levels in the progeny from RAPD data were calculated as follows: the number of Fm-specific markers present in the backcross individual/total number of the Fm-specific markers. The correlation analysis of genome introgression levels between SSR and RAPD data was conducted with SAS system V8 (SAS Institute, Cary, NC). The Proc Corr procedure was run to obtain the correlation coefficient and the P value.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Genome Introgression Detected by SSR Markers
Forty primer pairs were used to detect polymorphism between the Lp and Fm parents. Of these, 19 primer pairs failed to produce any amplification product. Three primer pairs could not distinguish between species due to similarly-sized amplified products (Fig. 2a). Four SSR primers shared alleles between the parents, and could not fully discriminate Fm from Lp (Fig. 2b). The shared alleles may be associated with closely positioned heterozygous alleles between Fm and Lp parental genotypes. Amplification of the same size bands in both Fm and Lp parents indicated a close relationship or certain homeology between these two genomes. One marker was unscorable because of multiple products being amplified or too many alleles segregating. The remaining 13 markers covering seven linkage groups (Table 2) could fully discriminate Fm and Lp by amplifying completely different sizes of SSR products from the parents (Fig. 2c). This result indicated that there was a significant DNA structural differentiation in the genomes of the two species on the basis of these SSR markers. These 13 SSR markers were used to detect Fm genome introgression in progeny derived from hybridization between Fm and Lp.




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  		Fig. 2. The SSR profiles detected by automated capillary electrophoresis of fluorochrome-labeled PCR products. a. A single monomorphic allele of 191 bp was amplified from all the parental genotypes by primer LPSSRK14B01, one of the three primer pairs that could not distinguish between species. b. The shared allele (246 bp) between the parents was amplified from the parents by LPSSRH11G05, which is one of the 4 primer pairs that could not fully discriminate Fm and Lp. c. The SSR pattern amplified from the primer pair LPSSRK11E11, that could fully discriminate Fm and Lp. The fragment sized 125 bp is the Fm specific allele and 108 bp is the Lp specific allele. d. The SSR profile amplified from the primer pair LPSSRK10F08, that could confirm the hybrid nature of F1 plants by presence of parental bands (Fm specific-alleles: 76 and 110 bp and Lp specific-alleles: 115 and 136 bp) and could also detect the segregation of alleles in backcross population.

 

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  		Table 2. The SSR markers that revealed F. mairei genome introgression into the backcross progeny.

 
Out of the 13 markers, two did not amplify expected fragments in the F1 and backcross progeny. Another two markers amplified expected alleles in the F1 but not in the backcross population (Table 2). The remaining nine markers could confirm the hybrid nature of F1 plants by presence of SSR parental bands and could also detect the alleles' segregation in the backcross population (Fig. 2d).

All nine markers detected that the Fm genome had introgressed in at least one place of the genomes of the backcross individuals, because each of the nine markers amplified the Fm-specific alleles in one or more backcross individuals (Table 2). Out of the 13 backcross individuals analyzed, 11 showed that the Fm genome fragments were introduced and that the introgression levels ranged from 0 to 66.7% (Table 3). The introgression of Fm alleles in G11a could not be detected by current SSR markers, because they did not show any Fm-specific alleles at the nine loci. Another backcross individual, G6 had amplification from only one primer pair and did not detect any Fm genome introgression. More SSR markers would be needed for a more completed genome analysis of those two individuals, and also, for a more accurate estimation of genome introgression of the progenies because of the low number of SSR markers employed.


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  		Table 3. Number of markers that revealed the F. mairei genome introgression into backcross individuals and the introgression level.

 
In this study, not all markers at any one linkage group detected genome introgression in the individuals simultaneously. For example, at linkage group 7, three SSR primer pairs (LPSSRK15F05, LPSSRK11E11, and LPSSK14F07) (Table 2) were used and only one primer (LPSSRK11E11) revealed the Fm fragment in G8. Two primers (LPSSRK11E11 and LPSSRK14F07) showed the Fm allele in G15, but not all of the three markers detected Fm alleles in G8 and G15. Such introgression suggested that the mechanism of alien chromosome segments transmission is probably due to genetic recombination through crossover rather than substitution of whole or large segments of chromatin from Fm. Otherwise, all the SSR markers at the same linkage group should have detected the Fm genome introgression in the same individual at the same time.

Genome Introgression Detected by RAPD Markers
Fifty-six RAPD primers were selected on the basis of their ability to amplify the polymorphic bands in Festuca and Lolium (Xu et al., 1993; David, 1997; Charmet et al., 1997; Siffelova et al., 1997). Forty-one primers could successfully discriminate the parents by amplifying the polymorphic bands. These 41 RAPD primers were used to detect the genome introgression in the backcross progeny. A total of 222 polymorphic bands were amplified. Among these 222 RAPD markers, 34 (15.3%) were not amplified in Fm and Lp parents or the F1 hybrid, but were present in some progeny (Fig. 3). These new bands could come from the parental chromosome crossovers and recombination in F1. The other 188 markers were parent-specific markers. Ninety-two (48.9%) were Fm-specific markers and 96 (51.1%) were Lp-specific markers. These 92 Fm-specific markers (Fig. 3) were scored in the backcross progeny to detect Fm alleles. The presence of the Fm-specific markers in the progeny was treated as introgression of Fm DNA or genome fragments in the progeny.



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  		Fig. 3. The RAPD banding pattern (negative image) of F. mairei (lane 1), L. perenne (lanes 2 and 3), F1 hybrid between Fm and Lp1 (lane 4), and backcross progeny (lane 5-17). The whole arrows point to the Fm-specific bands (lane 1). The half arrow point to the new bands (lanes 8 and 15). M = 1-kb size ladder.

 
Thirteen backcross progeny were detected and all of them showed Fm genome introgression. Genome introgression levels ranged from 5.4 to 60.9% (Table 3). The correlation coefficient of the genome introgression levels assessed by SSR and RAPD markers was 0.698 (introgression level of G6 detected by SSR marker was ignored). The correlation between these two sets of markers in detecting genome introgression was significant (P = 0.0116).

The molecular marker detection results were also consistent with morphological characteristics (Fig. 1b). Individuals resembling Fm were detected by more markers to show that more Fm genome segments were transferred. For example, in G27a, which resembled Fm with rigid, long leaves as compared to Lp with softer and shorter leaves, at five SSR loci, Fm DNA segments were introgressed, out of the nine loci detected. The genome introgression level was as high as 66.7%. More than half of 92 RAPD markers (introgression level was 60.9%) revealed Fm chromatin introduced in G27a. While in G11a, which resembled Lp with soft and short leaves, none of the Fm genome was introgressed at the nine SSR loci, and only five out of 92 RAPD markers (5.4% introgression level) showed introgression of Fm chromatin.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Intergeneric hybridization is broadly used to produce new cultivars by the introgression of alien genomes into elite cultivated crops. It has long been a goal for turfgrass breeders to introduce the stress tolerance of Festuca species into the high value cultivated Lolium species (Jauhar, 1993). However, their distant genetic relationship complicates genome introgression by excluding the Festuca genome rapidly in the hybrids or in backcross progenies (Jauhar, 1993; Thomas et al., 1994). Recent advances in cytogenetic techniques and molecular markers provide opportunities for understanding and manipulating the Lolium–Festuca complex through breeding. The fluorescent in situ hybridization (FISH) and GISH, were used to monitor chromosome pairing and introgression for distant hybrids (Chen and Sleper, 1999; Cao et al., 2000). However, cytogenetic analysis could not pinpoint which genome segment was transferred at the DNA level. Restriction fragment length polymorphism (RFLP) based markers were also difficult to detect alien DNA in the hybrid if only a small portion of a chromosome or few chromosomes were transferred (Chen and Sleper, 1999).

Simple sequence repeat polymorphism is produced by variations in the number of tandem repeats within DNA microsatellites at certain loci. It is highly sensitive in detecting DNA structural differentiation in plant genomes. Cregan et al. (1994) was able to identify near isogenic lines of soybean [Glycine max (L.) Merr.)] that were not distinguishable by other markers. In this study, nine SSR markers were distributed in 7 linkage groups. The three markers in linkage group 7 in our study did not show the same introgression results (Table 2), which would suggest that the whole chromosome from Fm was not substituted, but probably some genome fragments were transferred though chromosome crossovers in the individuals tested. This study demonstrated that SSR markers developed from Lp could be effective and informative in detecting Fm genome introgression. Furthermore, SSRs can be readily applied to commercial genetic and breeding programs as they do not require the use of radioactive isotopes and also exclude the use of ethidium bromide, if checked in the sequencer. With further development, SSR markers could be used to assess introduction of whole alien genomes or chromatin into Lolium.

The RAPD method allows for the identification of a large number of polymorphic DNA markers distributed throughout the genome. The genetic validity of RAPD markers has been questioned, because the homology of RAPD bands of the same molecular weight is uncertain. However, several authors (Thormann et al., 1994; Lanner et al., 1996; Benabdelmouna et al., 1999) have checked the homology of RAPD bands by hybridizing with RAPD fragments used as probes and found low error rates, which do not have significant effects on estimates of genetic relatedness. Southern and FISH confirmed the usefulness of RAPD as introgression markers in Petunia (Benabdelmouna et al., 1999). A significant proportion of RAPD markers were species specific and had the ability to detect introgression of alien chromatin–DNA fragments in intergeneric hybrids and their advanced generations (Bommineni et al., 1997; Benabdelmouna et al., 1999). Random amplified polymorphic DNA is the current method of choice for routine fingerprinting of breeding germplasm or cultivar lots. In our study, the introgression level revealed by RAPD concurred with that assessed by SSR markers and morphological characters. This result demonstrated that RAPD could be equally effective and informative in monitoring the introgression of alien DNA fragments, as compared to SSR markers. Random amplified polymorphic DNA markers are also efficient in detecting genome introgression because of its small tissue sample requirements, rapid analysis, low cost, no previous knowledge of DNA sequence requirement, and easy establishment. The potential for genome labeling and chromosome tagging could be increased because of the possibility of converting RAPD markers into sequence characterized amplified regions (SCARs) (Paran and Michelmore, 1992).

In this study, it was discovered that the introgression levels detected by SSR and RAPD markers were significantly correlated. Genome introgression levels were favorably consistent with morphological characteristics. These results demonstrated that the two molecular techniques were efficient in assessing alien genome introgression. Backcross progenies with the expectation of obtaining some stress tolerance from Fm were detected by SSR and RAPD markers. The data showed that these backcross individuals achieved different introgression levels of Fm DNA fragments. These results could be highly useful for further improvement of the breeding materials.


   ACKNOWLEDGMENTS
 
We especially wish to thank Ms. Leonie Hughes, Drs. Elizabeth S. Jones and John W. Forster, of the Plant Biotechnology Center, La Trobe University, Bundoora, Victoria, Australia for their assistance in conducting the SSR procedures in this study. We would also like to thank the Michigan Turfgrass Foundation and the Michigan Agricultural Experiment Station for their support of this research.

Received for publication August 31, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 





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