Published in Crop Sci. 43:1659-1662 (2003).
© 2003 Crop Science Society of America
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CROP BREEDING, GENETICS & CYTOLOGY
Cytogenetic Analysis of Festuca Species and Amphiploids between Festuca mairei and Lolium perenne
Mingshu Caoa,
Suleiman S. Bughrarab and
David A. Sleper*,a
a Dep. of Agronomy, Univ. of Missouri, Columbia, MO 65211
b Dep. of Crop and Soil Sci., Michigan State Univ., East Lansing, MI 48824
* Corresponding author (sleperd{at}missouri.edu).
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ABSTRACT
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Intergeneric hybridization between Lolium and Festuca has been an interest of forage and turfgrass breeders because most agronomic and economically important traits found in Lolium and Festuca are complementary. However, low fertility of intergeneric hybrids has often been present and difficult to overcome. Synthesis of amphiploids from F1 hybrids was proven to be an effective way in restoring fertility of these intergenic hybrids. Amphiploids were used as bridging germplasm to improve forage quality and persistence in these two genera. In this report, amphiploids (LLM1M1M2M2) between F. mairei St. Yves. (M1M1M2M2, 2n = 4x = 28) and L. perenne L. (LL, 2n = 2x = 14) were produced from the F1 hybrid (F. mairei x L. perenne) through the treatment of colchicine and dimethyl sulfoxide (DMSO). Cytological analyses of pollen mother cells (PMCs) indicated that the amphiploids were unstable in meiosis with chromosome configurations of 9.75 I + 15.32 II + 0.43 III + 0.10 IV at metaphase I (MI). The pollen potassium iodide (KI) stainability of the amphiploids was 21%, while the F1 had only 0.1% pollen stainability. Interspecific hybrids were made through crosses between F. arundinacea Schreb. (6x) and F. mairei (4x). The pentaploid hybrids (PG1M1G2M2) had mean chromosome configurations of 6.47 I + 13.40 II + 0.30 III + 0.19 IV + 0.01 V at MI, indicating a close genomic relationship between F. arundinacea Schreb. (PPG1G1G2G2) and F. mairei (M1M1M2M2). The amphiploids and pentaploid hybrids were proposed to be used for improving drought persistence of tall fescue with incorporation of genes from drought-tolerant F. mairei.
Abbreviations: DMSO, dimethyl sulfoxide • GISH, genomic in situ hybridization • KI, potassium iodide • MI, metaphase I • PMCs, pollen mother cells
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INTRODUCTION
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INTERGENERIC HYBRIDIZATION between species of Lolium and Festuca has been a continual interest to forage and turfgrass breeders because the two genera offer a range of complementary characteristics of agronomic and economic importance (Jauhar, 1993). Close genomic relationships between Lolium and Festuca have been revealed by extensive conventional cytogenetic analysis (Jauhar, 1993) and most recently through molecular cytogenetic techniques (Thomas et al., 1994; Zwierzykowski et al., 1998; Cao et al., 2000). Although intergeneric crosses between Festuca and Lolium are possible, embryo rescue is often necessary for enhancing opportunities of obtaining viable hybrids. Intergeneric hybrids are usually of low fertility or male-sterile, an obstacle that needs to be overcome to make intergeneric hybridization effective for plant improvement. Pollen fertility of F1s can be partially restored by the production of amphiploids through chromosome doubling. Synthesized amphiploids have been used either as bridging germplasm or as varieties in forage production after several rounds of selection for improved agronomic performance.
Research on synthesis and cytogenetic analysis of amphiploids between Lolium and Festuca has been extensively documented. Amphidiploids between L. multiflorum Lam. (or L. perenne) and F. pratensis Huds. have been popular in the grasslands of several countries, including Poland, Canada, and Australia, such as ‘Elmet’ and ‘Prior’ as documented by Jauhar (1993). Amphiploids (LLG1G1G2G2, 2n = 6x = 42) between L. multiflorum (LL, 2n = 2x = 14) and F. arundinacea var. glaucescens auct. [= F. arundinacea subsp. fenas (Lag.) Arcang.] (referred to as F. glaucescens thereinafter) (G1G1G2G2, 2n = 4x = 28) were synthesized to improve palatability of tall fescue by introducing the L genome from L. multiflorum (Cao et al., 1994). Amphiploids (2n = 8x = 56) between ryegrass (L. multiflorum) and tall fescue (F. arundinacea, 2n = 6x = 42) were produced with the objective of transferring the nutritive quality of the Lolium species into tall fescue while retaining the adaptive qualities of tall fescue (Buckner et al., 1961, 1985; Buckner, 1965). The amphiploid (2n = 56) of ryegrass and tall fescue has been subjected to extensive cytological analyses and determined to be a good bridging material for improving forage quality of tall fescue. The amphiploids had unstable chromosome pairing behavior with a varied number of univalents, such as 2.16 (Buckner et al., 1961), 1.73 to 5.46 (Lewis, 1966), 2.27 to 2.90 (Zwierzykowski, 1980), and 2.20 to 9.79 (Kleijer and Morel, 1984). The highest frequency of univalents (9.79) was observed in the amphiploid of L. multiflorum cv. ‘Lior’ and F. arundinacea cv. ‘Mayers’. Jenkins and Jimenez (1995) suggested B chromosomes appear to promote bivalent formation in the Lolium amphiploids. However, no B chromosomes were observed in the reports mentioned above.
Festuca mairei St. Yves. (M1M1M2M2, 2n = 4x = 28) is a xeromorphic species that is found in northwest Africa and has tolerance to higher temperature and drought (Borrill et al., 1971). It has been reported that the photosynthetic rate per unit leaf area of F. mairei is 
30% higher than that of F. arundinacea (Xu, 1998). The long-term goal of our research is to incorporate the genomes of F. mairei into tall fescue to improve its drought persistence. The production and characterization of the intergeneric hybrids (F1) between F. mairei and L. perenne were reported previously (Chen et al., 1995). The objectives of this study were: (i) to synthesize amphiploids of F. mairei and L. perenne from their hybrids and present cytogenetic characterizations of the synthesized amphiploids; (ii) to produce and characterize hybrids between F. arundinacea (6x) and F. mairei, providing genetic materials for improvement of drought tolerance of tall fescue.
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MATERIALS AND METHODS
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Plant Materials
Two intergeneric triploid hybrids of F. mairei and L. perenne (Chen et al., 1995) were used for producing amphiploids. Festuca mairei (2n = 4x = 28) (PI 283313) and F. arundinacea (2n = 6x = 42) (‘Kentucky 31’) were used for interspecific hybridizations. All plant materials were initially maintained in the greenhouse at the University of Missouri-Columbia and are currently maintained at Michigan State University.
Chromosome Doubling of F1 Hybrids
Individual vegetative tillers were detached from vernalized F. mairei x L. perenne F1 hybrids and incubated in a solution of 0.2% (w/v) colchicine + 2% (v/v) DMSO for 24 h at 18°C in a growth chamber. After treatment, tillers were rinsed thoroughly in running tap water for 8 h before planting in pots. The tillers were then placed in the greenhouse and grown in the absence of direct sunlight during the first week. Seeds were harvested from the interpollination of these tillers. Resulting plants were subjected to cytological analysis in the following year.
Preparation of Meiotic Slides
Anthers at MI were collected and fixed in 3:1 (v/v) of ethanol:acetic acid for 1 wk. After treatment in 1N HCl at 60°C for 10 to 15 min, anthers were stained using the Feulgen staining technique. Chromosome preparations were made from PMCs by squashing a piece of anther on a slide in a drop of 45% (v/v) acetic acid.
Other methods, such as conventional crossing and pollen staining, followed the description by Chen et al. (1995) with minor modifications. Immature embryo culture was not used in this study. Potassium iodide was used for pollen staining instead of using a mixture of acetocarmine and glycerine.
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RESULTS
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Synthesis and Cytogenetic Analysis of Amphiploids
The 3x intergeneric F1 plants of F. mairei (2n = 4x = 28) x L. perenne (2n = 2x = 14) were male-sterile and had nondehiscent anthers (Chen et al., 1995). After vernalization, 132 tillers were detached from the F1 plants and treated with 0.2% (w/v) colchicine + 2% (v/v) DMSO. Ninety-five tillers (72%) survived this treatment, which was designated as the C0 population. A significant increase of pollen KI stainability was observed among the C0 population with an average of 30%, and most anthers dehisced normally, while the F1 plants had only an average of 0.1% pollen KI staining. Plants of the C0 population were isolated to allow interpollination. Two seeds (C1) were produced, and both germinated and resulted in flowering plants.
The two C1 plants had 2n = 6x = 42 chromosomes indicating that they had been successfully doubled. The amphiploids appeared intermediate in morphology between their parents. Leaves in the seedling stage were tender like ryegrass but as erect and rigid as F. mairei. Of 82 cells examined, the number of univalents ranged from 4 to 11. The high frequency of chromosome associations was expected, which averaged 15.32 II with the presence of trivalents and quadrivalents (Fig. 1). Seventy-five percent of bivalents were rod bivalents. Mean chromosome configurations were 9.75 I + 15.32 II + 0.43 III + 0.10 IV. Irregular meiosis was observed with large numbers of lagging chromosomes (Fig. 2) and micronuclei in the tetrads. A chromosome bridge shown in Fig. 2 indicated that translocations or paracentric inversions occurred in the amphiploid. Overall high frequency of univalents (9.75 I) indicated cytogenetical instability of these newly synthesized amphiploids. Pollen KI staining of C1 was 21%, which was 9% less than that of the C0 population. The reason for this was unclear. However, a similar situation was reported by Buckner (1965) in that male-fertility of G2 (equivalent to C2 in this report) progenies varied from 47 to 84% (based on the percentage of anther dehiscence), while G3 progenies had lower male-fertility, varying from 39 to 62%.
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Fig. 1. Festuca mairei x Lolium perenne amphiploid pollen mother cell at metaphase I showing 9 I + 11 rod II + 2 ring II + 1 III + 1 IV.
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Fig. 2. Festuca mairei x Lolium perenne amphiploid pollen mother cell at anaphase I showing laggards and a chromosome bridge.
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It is worthy to note that colchicine and DMSO treatments can cause variations in plant vigor, leaf color, and leaf thickness. These types of variations were observed in the C0 population, even for those individuals whose chromosomes were not doubled. Some colchicine-induced variations were heritable (Francis and Jones, 1989; Francis, 1990).
Hybridizations between F. arundinacea (6x) and F. mairei (4x)
Kentucky 31 (2n = 6x = 42) is a tall fescue (F. arundinacea, PPG1G1G2G2) cultivar widely used for forage and turf. PI 283313 is a wild species of F. mairei (M1M1M2M2 2n = 4x = 28), collected from a drought-prone area of Morocco, North Africa. Two viable seeds were successfully harvested from the cross combination of F. arundinacea x F. mairei (from 120 emasculated florets), while no seeds were obtained from the reciprocal cross combination of F. mairei x F. arundinacea (110 emasculated florets). The hybrid plants had 35 chromosomes (2n = 5x = 35) (Fig. 3). The mean chromosome configurations were 6.47 I + 13.40 II + 0.30 III + 0.19 IV + 0.01 V, based on 65 observed cells. The F1 meiotic behavior was mostly irregular with large numbers of laggards and with a number of chromosome bridges and fragments present in anaphase I. Anthers failed to dehisce and only 12% of pollen stained using KI. Genotypes used in the interspecific crosses of Festuca species apparently affected chromosome pairing. Chandrasekharan and Thomas (1971) obtained hybrids from the same cross combination (F. arundinacea x F. mairei) using different genotypes, and they reported a mean of 9.23 bivalents and a high frequency of trivalents (1.17) and quadrivalents (0.60). A mean of 13.40 bivalents were observed in the present study. The frequency of univalents was below seven in the hybrid (PG1G2M1M2) and a low frequency of quadrivalents, as shown in Fig. 4, also occurred in the pentaploid.
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Fig. 4. Festuca arundinacea x F. mairei hybrid PMC at metaphase I with 7 I + 4 rod II + 8 ring II + 1 IV.
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DISCUSSION
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Breeding Implication of the Amphiploids
Fungal endophytes [Neotyphodium coenophialum (Morgan-Jones and Gams) Glenn, Bacon, and Hanlin comb. nov.] produce ergot-like alkaloids in tall fescue that cause animal toxicosis, but the presence of endophytes provides tall fescue competitive advantages in terms of drought persistence and insect resistance. The development of endophyte-free tall fescue that maintains the persistence of tall fescue is a goal of many researchers (West, 1994). The amphiploid (LLM1M1M2M2) synthesized in this study might avoid this problem by improving persistence of tall fescue through chromatin introgression of the drought tolerant species F. mairei, which would lead to persistence without the presence of the endophyte.
Two turf cultivars of L. perenne, Citation II (Rose-Fricker et al., 2002) and Calypso (Burr et al., 2001), were used in the initial crosses with F. mairei (Chen et al., 1995). The morphology of the synthesized amphiploids (LLM1M1M2M2) was more like a turfgrass having narrow leaves with many vegetative tillers. Their seedling vigor was not as great as that of forage grasses. Fertility of the amphiploids has been improved considerably compared with the F1 hybrids. The amphiploids could be backcrossed with F. arundinacea to potentially improve drought persistence of turf-type tall fescue.
The fertility and drought tolerance of amphiploids are expected to improve with selection. High frequency of recombinations between F. mairei and L. perenne genomes (Cao et al., 2000) make the introgression of drought tolerance quite feasible. In another amphiploid, ‘Prior’ of L. perenne and F. pratensis, genomic in situ hybridization (GISH) revealed that L. perenne accounted for 62.1% and F. pratensis for 37.9% of the total chromatin (Canter et al., 1999), indicating that extensive recombinations must have occurred between the two intergeneric genomes. Traditional taxonomic and cytogenetic evidence suggested that there is a close genomic relationship between Festuca and Lolium species. Darbyshire (1993) has proposed the realignment of the Schedonorous section of Festuca into Lolium. However, the activation of pairing control genes in intergeneric hybrids may complicate meiotic analyses and confound attempts to determine species relationships as pointed out by Thomas et al. (1997). Current GISH results make it more perplexing to draw a conclusion about the genomic relationships between Festuca and Lolium. For instance, Festuca and Lolium chromosomes are easily differentiated from each other by GISH (Thomas et al., 1994; Zwierzykowski et al., 1998; King et al., 1999; Cao et al., 2000), which suggests a clear distinction between their genomes. However, the chromosomes of L. perenne were readily paired with the chromosomes of F. mairei with 50% of heterogeneous bivalents observed in their hybrids (Cao et al., 2000), which indicates the two genomes readily recombine with each other. With the ability of GISH to differentiate Festuca and Lolium chromosomes, it would be interesting to record Festuca and Lolium chromosome pairing behavior in the amphiploids or hybrids at all stages of meiosis, and to investigate how different genotypes affect the chromosome pairing affinity in the intergeneric amphiploids. The GISH techniques would provide an opportunity to understand the control mechanisms of genetic recombination between Lolium and Festuca genomes.
Genetic Relationships between F. arundinacea and F. mairei
The hybrid of F. arundinacea x F. mairei (PG1G2M1M2) could serve the same breeding purpose as the amphiploids (LLM1M1M2M2) to improve drought persistence of tall fescue. The high percentage of bivalents in this hybrid indicated the close relationship between F. arundinacea (PPG1G1G2G2) and F. mairei (M1M1M2M2). In another pentaploid hybrid between F. arundinacea x F. glaucescens, Malik and Thomas (1967) observed a mean of 11 bivalents and other chromosome associations including 0.78 III + 0.43 IV + 0.05 V. The frequency of univalents averaged 7.0 with a range from 2 to 14. Only 0.08% of the mature pollen stained normally. The chromosome pairing data from the pentaploid hybrids of F. arundinacea x F. mairei and F. arundinacea x F. glaucescens do not indicate whether F. mairei or F. glaucescens has the closer genomic relationship with F. arundinacea, because determining which chromosomes contributed to bivalents and quadrivalents is difficult. Usually, preferential pairing of genomes from the same species is anticipated; that is, between G1 and G2, and between M1 and M2. The presence of quadrivalents could be a result of either heterogeneous pairing of chromosomes from G and M genomes or a chromosome structural variation such as an inversion or translocation. The ability of using GISH to differentiate between the G and M genomes is critical to solve the puzzle. However, an initial effort suggested that F. mairei and F. glaucescens could not be differentiated from each other by GISH (Cao and Sleper, 2001), which might be an indication of their close genomic relationships in terms of similar content of repetitive DNA sequences.
Received for publication July 15, 2002.
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ABSTRACT
INTRODUCTION
