Published in Crop Sci 39:1676-1679 (1999)
© 1999 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
Crop Science 39:1676-1679 (1999)
© 1999 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
FISH and RFLP Marker-Assisted Introgression of Festuca mairei Chromosomes into Lolium perenne
C. Chena and
D.A. Sleperb
a Harvard Institute of Human Genetics, Harvard Medical School, 4 Blackfan Circle, Boston, MA 02146 USA
b Dep. of Agronomy, Univ. of Missouri-Columbia, Columbia, MO 65211-0001 USA
sleperd{at}missouri.edu
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ABSTRACT
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Plant breeders and geneticists have attempted for several decades to combine perennial ryegrass (Lolium perenne L.) with fescue (Festuca spp.) to create novel forage grasses containing both high forage quality and good drought tolerance. Difficulty in selecting true hybrids with alien chromatin or chromosome addition–substitution has been a major barrier in Festuca x Lolium breeding programs. In this investigation, fluorescence in situ hybridization (FISH) and restriction fragment length polymorphism (RFLP) markers were used to monitor transfer of Festuca mairei St. Yves chromosomes into L. perenne through intergeneric hybrids. Among 64 hybrid plants of the BC1G1 generation (intercrossed progeny of first backcross to L. perenne), chromosome addition and substitution of F. mairei were identified by FISH using total genomic DNA of F. mairei as a probe. Forty-two clones from a Pst I-genomic DNA library of F. arundinacea Schreb. were used to screen for the presence of F. mairei DNA in the hybrid plants. These RFLP probes rapidly identified presence of the F. mairei genome in F1, F2, BC1, but not in BC2 plants. In contrast, genomic FISH on meiotic cells effectively detected any F. mairei chromosomes as well as chromosomal pairing relationships in any hybrid. By monitoring and selectively introducing F. mairei chromosomes into ryegrass, these molecular markers may accelerate the Festuca x Lolium breeding for improvement of ryegrass.
Abbreviations: FISH, fluorescence in situ hybridization • RFLP, restriction fragment length polymorphism • PBS, phosphate-buffered saline • FITC, fluorescein isothiocyanate
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INTRODUCTION
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PERENNIAL RYEGRASS
is a diploid
species widely used for turf and forage in the temperate zone of the USA (Terrell, 1966; Jauhar, 1993). However, drought susceptibility reduces persistence following moisture and heat stress (Riewe and Mondart, 1985). Introduction of alien germplasm from Festuca may be valuable for turf and forage improvement in Lolium (Riewe and Mondart, 1985; Thomas et al., 1994).
Festuca mairei is a drought-tolerant tetraploid
(Borrill et al., 1971). Intergeneric F. mairei and L. perenne hybrids display morphological and drought-tolerant characteristics of both parents (Chen et al., 1995). Their distant genetic relationship complicates transfer of desirable genes via chromosomal crossing-over because Festuca chromosomes are rapidly excluded in hybrids within a few backcross generations, often resulting in a Lolium-like population (Jauhar, 1993; Thomas et al., 1994). Thus, marker-assisted selection should be applied to maintain desirable traits of the Festuca genome in the intergeneric hybrid.
There are two widely used approaches for marker-assisted introgression. FISH, using total genomic DNA from alien species, has been widely used to identify alien chromatin in genetic backgrounds of cultivated species (Lapitan et al., 1986, Jiang and Gill, 1994). However, only a few studies have reported the use of FISH with forage grasses (Thomas et al., 1994; Humphreys et al., 1995). DNA markers, such as restriction RFLPs, have also been used. A genomic DNA library from tall fescue (F. arundinacea) was constructed by Xu et al. (1991). Approximately 57 and 68% of the probes had dimorphic hybridization patterns in L. perenne and F. mairei, respectively (Chen and Sleper, 1997). Both dimorphic and Festuca-specific probes could be used to detect alien chromosomes in F. mairei x L. perenne hybrids.
Our objective was to determine whether FISH or Festuca-specific RFLP markers could effectively monitor introgression of 4x F. mairei chromosomes into 2x L. perenne in a backcross breeding program.
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Materials and methods
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Two accessions of L. perenne (Lp1 and Lp2 /Lp3) and one of F. mairei (Fm1) were used. Single plants each of Lp1 and Lp2 were from turf cultivars Citation II and Calypso, respectively. Lp3 is another single plant from Calypso. Fm1 was a single plant from F. mairei #2, a clonal selection from a population of F. mairei.
Procedures of intergeneric hybridization between tetraploid F. mairei and diploid L. perenne have been described previously (Chen et al., 1995). A partially fertile 4x F1 hybrid was backcrossed as a female to diploid L. perenne. Eleven 21-chromosome and three 28-chromosome BC1 plants were obtained by hand pollination. The BC1 plants were intercrossed in isolation to generate the BC1G1 generation (Fig. 1)
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Fig. 1 Crossing scheme for introgression of Festuca mairei genomes (MMM'M') into Lolium perenne (LL). The 4x F1 hybrid, F. mairei (4x) x L. perenne (2x), was used as the female in backcrossing to L. perenne. Fourteen BC1 plants were interpollinated to produce BC1G1 progeny containing from
chromosomes
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Young roots (0.5–1.5 cm long) were produced from detached vegetative tillers 3 to 5 d after replanting. Excised root tips were placed in 
0°C water for 26 h and then fixed for 4 to 24 h in 3:1 100% (v/v) ethanol: acetic acid. After fixation, root tips were washed in 70% ethanol and in deionized water for 20 min each. Cleaned root tips were treated with 1% (w/v) cellulase and 3% (v/v) pectinase for 25 to 35 min at room temperature. Treated root tips were again rinsed in deionized water for at least 30 min, transferred to 45% acetic acid for not more than 30 min, and squashed in 45% acetic acid. After the cover glass was removed by freezing, slide preparations were placed in 45% acetic acid for 5 to 20 min and air dried. Preparations were stored at -20°C for up to 3 mo.
Anthers at meiotic metaphase I were fixed in 3:1 (v/v) 100% ethanol: acetic acid for 2 to 4 h, transferred to 70% ethanol for 10 to 20 min, rinsed in deionized water at least 30 min, and squashed in 45% acetic acid. Slides were prepared as previously described.
In situ hybridization and probe detection followed the method of Jiang et al. (1995) with minor modifications. One hundred microliters of hybridization mix contained 50 µL of 100% deionized formamide, 10 µL of 20x SSC (3 M sodium chloride, 0.3 M sodium citrate, pH 7), 20 µL of 50% (w/v) dextran sulfate, 2 to 5 µL of sheared salmon sperm DNA (1 mg/mL), 5 µg of sheared L. perenne DNA as a block, and 0.2 to 0.4 µg of F. mairei DNA as probe. The probe mix was denatured at 80°C for 10 min. After chilling on ice for at least 5 min, approximately 20 µL of the mixture was applied to each slide and covered with a 22- by 22-m coverslip. Hybridization was conducted overnight at 37°C, followed by stringent washes in 2x SSC twice at room temperature for 5 min each, once at 37°C for 10 min, and then with 1x phosphate-buffered saline (PBS) for 5 min. Sites of probe hybridization were detected by applying 100 µL of rabbit anti-biotin antibody (1:100 dilution with 2 mg/mL bovine serum albumin in 1x PBS) at 37°C for 45 min, followed by using 100 µL fluorescein isothiocyanate (FITC) labeled goat anti-rabbit antibody (Enzo Diagnostics, New York; 1:100 dilution). Chromosomes were counterstained with propidium iodide (1 µg/mL). Slides were mounted in antifade mountant and analyzed on a Zeiss epi-fluorescent microscope.
Total genomic DNA was extracted from young leaves of F. mairei and L. perenne as described by Xu et al. (1991). Genomic probes were prepared by mechanically shearing DNA of F. mairei to approximately 5 to 10 kb by repeated passes through a hypodermic needle and then labeling it by nick translation with Bio-11-dUTP (Enzo Diagnostics).
Forty-two clones were chosen from a tall fescue genomic DNA library (Table 1)
. RFLP procedures were described by Chen et al. (1995).
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Table 1 Presence (+) or absence (-) of alien Festuca DNA in F. mairei x Lolium perenne generations (BC1G1,BC1F1) determined with 42 Pstl–genomic DNA clones of tall fescue as probes
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Results and discussion
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Genomic Fluorescence In Situ Hybridization
Using total genomic DNA of F. mairei as a probe, meiotic FISH could identify chromosomal origin and relationship in intergeneric F. mairei x L. perenne hybrids (Fig. 2)
. When labeled genomic DNA of F. mairei was hybridized in situ to denatured metaphase chromosomes, hybridized Festuca chromosomes fluoresced green, while Lolium chromosomes fluoresced red as a result of counterstaining with propidium iodide. Yellow-green fluorescence was due to a combination of the red fluorescence of propidium iodide mixed with the green fluorescence of FITC. Fourteen chromosomes from F. mairei were found in F. mairei x L. perenne 4x F1. Most F. mairei chromosomes paired among themselves. Of 214 cells analyzed, only 32 showed homoeologous chromosome pairing (an average of 0.6 homoeologous bivalents and 0.8 multivalents per cell), suggesting a distant genomic relationship between F. mairei and perennial ryegrass. This was consistent with previous results of meiotic chromosome pairing (Chen et al., 1995).
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Fig. 2 Genomic FISH using labeled F. mairei DNA as a probe effectively detected substitution of a pair of Festuca mairei chromosomes in a Lolium-like hybrid plant
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A total of 64 BC1G1 plants with 14 to 32 chromosomes were obtained by intercrossing BC1 plants
. Most BC1G1 plants contained 14, 21, and 28 chromosomes representing 28, 22, and 31% of the population, respectively (Fig. 3)
. Thirty-five Lolium-like and relatively drought-tolerant (in visual comparisons to perennial ryegrass plants growing in the same area) BC1G1 plants were selected to determine presence of F. mairei chromosomes by genomic FISH. A total of 0 to 14 F. mairei chromosomes were retained among these BC1G1 plants. We were particularly interested in those 14-chromosome plants because of the possibility of addition or substitution of F. mairei chromosomes. However, most 14-chromosome hybrids lacked fluorescent signals on either mitotic or meiotic chromosomes, resulting from the loss of F. mairei chromosomes within three generations. One of these plants showed very strong hybridization signals on a pair of chromosomes, suggesting that genome constitution of the hybrid included one pair of F. mairei chromosomes and six pairs of L. perenne ones (Fig. 2). This Lolium-like hybrid survived well during the hot summer (in visual comparisons to perennial ryegrass growing in the same vicinity) and recovered rapidly later at Columbia, MO. We hypothesized that this pair of chromosomes might contain genes related to drought tolerance in the hybrid. Our next step is to develop a stable chromosome substitution line from this hybrid for breeding drought-tolerant ryegrass.
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Fig. 3 Distribution of the chromosome number among a total of 64 BC1G1 plants derived from Festuca mairei x Lolium perenne hybrids
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RFLP Detection
Polymorphic probes were chosen from a PstI DNA library of tall fescue (Xu et al., 1991), to evaluate the presence of F. mairei DNA in the hybrids (Chen and Sleper, 1997). Of 42 probes, only one (tf515) showed F. mairei-specific hybridization in a 14-chromosome BC1G1 plant. Genomic FISH demonstrated that this Lolium-like plant contained substitution of a pair of F. mairei chromosomes (Fig. 2; Table 1). This result suggested that RFLP probes had difficulty detecting alien DNA in the hybrid if only a small portion of chromatin or few chromosomes were transferred. However, RFLP probes could easily detect a set of F. mairei genomes in F1, F2, and BC1 generations (Fig. 4)
. Tall fescue probe tf109 strongly hybridized to HindIII-digested genomic DNA and showed distinct RFLP banding patterns between L. perenne and F. mairei. This probe could easily detect presence of the F. mairei genome in F1, F2, and even BC1 generations with 
21 chromosomes (Fig. 4A). Clone tf515 only hybridized to F. mairei DNA. It was considered as a Festuca genome-specific probe. Interestingly, this RFLP probe detected the presence of F. mairei DNA in the 14-chromosome hybrid plant. The result confirmed the presence of F. mairei chromosomes initially identified by genomic FISH (Fig. 4B).
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Fig. 4 Southern hybridization of the HindIII- or EcoRI-digested genomic DNA of Lolium perenne (Lp), Festuca mairei (Fm), and their hybrid derivatives with (A) TF109, identifying the presence of F. mairei genome in F1, F2, and BC1 plants; (B) TF515, a Festuca genome-specific probe, showing strong hybridization only in F1, BC1, and some BC1G1 plants (M = labeled  DNA/HindIII fragments)
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Breeding Applications
Limited success has been reported in the literature regarding introgression from wild to cultivated Lolium or Festuca species, mainly because alien chromosomes in the Festuca-Lolium hybrid derivatives are rapidly lost during early backcross generations (Jauhar, 1993). Being smaller in size and similar in morphology, alien chromosomes are not identified in the introgressed lines by conventional chromosome staining (Thomas et al., 1994). This study demonstrated that FISH was a suitable technique in identifying alien chromatin of F. mairei in the Lolium background. The ability to differentiate genomes through the use of in situ hybridization could prove valuable in both turf and forage grass breeding programs.
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NOTES
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Journal series number 12,798.
Received for publication July 23, 1998.
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ABSTRACT
INTRODUCTION
