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Genomic in situ hybridization (GISH) reveals high chromosome pairing affinity between Lolium perenne and Festuca mairei. Mingshu Cao, David A. Sleper, ...
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Genomic in situ hybridization (GISH) reveals high chromosome pairing affinity between Lolium perenne and Festuca mairei Mingshu Cao, David A. Sleper, Fenggao Dong, and Jiming Jiang

Abstract: Intergeneric hybridizations have been made between species of Lolium and Festuca. It has been demonstrated, largely through conventional cytogenetic analysis, that the genomes of the two genera are related, however, much information is lacking on exactly how closely related the genomes are between the two species. We applied genomic in situ hybridization (GISH) techniques to the F1 hybrids of tetraploid Festuca mairei with a genomic constitution of M1M1M2M2 and diploid Lolium perenne with a genomic constitution of LL. It was shown in the triploid hybrids (LM1M2) that the chromosomes of M1 and M2 from F. mairei could pair with each other, and it was further discovered that L chromosomes of L. perenne paired with M1 and M2 chromosomes. Our results showed that meiocytes of Lolium–Festuca are amenable to GISH analysis, and provided direct evidence for the hypothesis that the chromosomes of Lolium and Festuca may be genetically equivalent and that reciprocal mixing of the genomes may be possible. Key words: Lolium, Festuca, in situ hybridization, meiosis. Résumé : Des hybrides intergénériques entre des espèces appartenant aux genres Lolium et Festuca ont été produits. Il a été démontré, principalement au moyen d’analyses cytogénétiques conventionnelles, que les génomes des deux genres sont apparentés. Cependant, il manque encore beaucoup d’information sur le degré véritable de parenté des génomes de ces deux espèces. Les auteurs ont employé l’hybridation génomique in situ (GISH) sur des hybrides F1 provenant du croisement entre un Festuca mairei tétraploïde (formule génomique M1M1M2M2) et un Lolium perenne diploïde (formule génomique LL). Il a été montré, dans les hybrides F1 triploïdes (LM1M2), que les chromosomes M1 et M2 du F. mairei s’appariaient les uns avec les autres et il a été observé que les chromosomes L du L. perenne s’appariaient également avec les chromosomes M1 et M2. Ces résultats ont montré que les méiocytes de l’hybride Lolium–Festuca se prêtent à l’analyse GISH et ont procuré des évidences à l’effet que les chromosomes des genres Lolium et Festuca pourraient être génétiquement équivalents et que le brassage réciproque des génomes pourrait être possible. Mots clés : Lolium, Festuca, hybridation in situ, méiose. [Traduit par la Rédaction]

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Introduction The grass genera Lolium and Festuca belong to the same tribe Poeae (formerly Festuceae) of subfamily Pooideae (formerly Festucoideae) (Jauhar 1993). Lolium is a small genus of eight species which are all diploid with 2n = 2x = 14 chromosomes. Festuca is a large, diverse genus. It comprises approximately 450 species (Clayton and Renvoize 1986) that range from diploid (2n = 2x = 14) to decaploid (2n = 10x = 70). The two genera offer a range of complementary characteristics of agronomic importance. Intergeneric hybridizaCorresponding Editor: J.P. Gustafson. Received April 15, 1999. Accepted November 3, 1999. M. Cao and D.A. Sleper.1 Department of Agronomy, University of Missouri, Columbia, MO 65211, U.S.A. F. Dong and J. Jiang. Department of Horticulture, University of Wisconsin, Madison, WI 53706, U.S.A. 1

Author to whom all correspondence should be addressed (e-mail: [email protected]).

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tions between Lolium and Festuca have been studied by many researchers to combine rapid establishment, good forage production, and palatability of Lolium species with winter hardiness, drought tolerance, and persistence of Festuca (Crowder 1953; Morgan 1990; Jauhar 1993). Some cultivars derived from the intergeneric hybridizations between Lolium and Festuca have been released, such as ‘Kenhy’ (Buckner et al. 1977) and ‘Johnstone’ (Buckner et al. 1983). Based on geographical distribution, crossability, fertility, and mostly chromosome pairing affinity in hybrids, the genomic relationships between Lolium and Festuca have been partly established. The L genome from L. perenne L. (LL) is very close to the P genome from diploid F. pratensis Huds. (PP) and more closely related to one of the genomes (PP) than other genomes in hexaploid Festuca arundinacea Schreb. (PPG1G1G2G2) (Kleijer 1984; Sleper 1985; Jauhar 1993). Lolium and Festuca are very close and they may be congeneric on the evolutionary scale. It may be as easy to obtain intergeneric hybrids between the two genera as it is to obtain interspecific hybrids within Lolium or Festuca (Jauhar 1993). Although in most cases, intergeneric hybrids were male-sterile and immature embryo culture was required to © 2000 NRC Canada

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rescue F1 hybrids (Cao et al. 1994; Chen et al. 1995; Jauhar 1993). Festuca mairei St. Yves is an allotetraploid (2n = 4x = 28) with genomes of M1M1M2M2. F. mairei is found in northwest Africa and persists under conditions of high temperature and drought (Borrill et al. 1971). It also has a photosynthetic rate significantly higher than that of F. pratensis (2x) and F. arundinacea (6x) (Randall et al. 1985). The goal of our project was to improve the drought tolerance and persistence of L. perenne by introgressing appropriate genes of these desirable traits from F. mairei into L. perenne. The crosses of L. perenne with F. mairei had been proven to be difficult, as rescue of the embryo hybrids was necessary (Chen et al. 1995). All triploid intergeneric hybrids (LM1M2) were male-sterile and had non-dehiscent anthers. The hybrids had an average of 6.60 bivalents and 0.38 trivalents, which made up 68.3% of the total number of chromosomes, and fitted the 2:1 model (Chapman and Kimber 1992). These data indicated that there were two genomes that were closely related and another was distant in the hybrids (LM1M2). Meiotic analysis of both 3x and 4x hybrids suggested that a close relationship existed within F. mairei (M1 and M2) and a more distant relationship was observed between the L. perenne and F. mairei genomes (Chen et al. 1995). Genomic in situ hybridization (GISH) has been successfully used to differentiate genetically close genomes such as L. multiflorum Lam. and F. pratensis (Thomas et al. 1994). This technique was used to discriminate among the ancestral genomes in the polyploids of Festuca (Humphreys et al. 1995) and to identify alien chromosomes and chromosome segments (Schwarzacher et al. 1992; Jiang and Gill 1994; Humphreys and Pasakinskiene 1996; Zwierzykowski et al. 1998). In the hybrids of Vulpia and Festuca, heterogeneous bivalents were observed by using GISH techniques (Bailey et al. 1993). The objectives of our research were to determine if GISH could be used to distinguish the genomes of L. perenne (LL) and F. mairei (M1M1M2M2) in their F1 hybrids, and to observe their chromosome pairing behavior.

Materials and Methods Plant materials Two accessions of F. mairei (Fm1: PI 283313 and Fm2: F. mairei #2) and two accessions of L. perenne (Lp1 and Lp2) were used in this investigation. Lp1 and Lp2 were vegetative clones from two turf-types of perennial ryegrass. Lp1 was a vegetative clone from the cultivar ‘Citation II’ and Lp2 was from the cultivar ‘Calypso’. The intergeneric triploid hybrids (LM1M2) from the cross combination of Fm2 × Lp1, obtained by Chen et al. (1995), were used in this study. All plant materials were maintained in a greenhouse. For meiotic analysis, the plant materials were moved into the field during the fall and returned to the greenhouse after vernalization.

Preparation of genomic DNA Genomic DNA extractions were based on previous procedures (Xu et al. 1991) by using CTAB (mixed alkytrimethylammonium bromide) buffer and purification with 1:1 volume of phenol and chloroform–octanol. Blocking DNA was sheared by adding with the final concentration of 0.4 M NaOH and boiling for 40–45 min.

399 Sheared blocking DNA was then precipitated twice with ethanol and dissolved in a final volume of TE (pH 8.0).

Preparation of meiotic slides Anthers at metaphase I (MI) were collected and fixed in 3:1 ethanol and acetic acid. Chromosome preparations were made from pollen mother cells (PMCs) by squashing pieces of anther in 45% acetic acid. Slide preparations were examined under a phasecontrast microscope. Slides with MI cells were placed on a CO2 block to flip the cover glass. After air-drying, slides can be stored at –20°C for up to several months. Alternatively, slides with cover glasses can be stored at –80°C for at least one year.

Fluorescence in situ hybridization (FISH) FISH and probe detection followed the method of Jiang et al. (1995) with modifications. Approximately 1 µg genomic DNA from F. mairei was labeled with biotin-16-dUTP in a 50-µL reaction mixture according to routine protocol of nick translation (Boehringer Mannheim). Slides were dehydrated in a series of 70%, 90%, and 100% ethanol and slide-bound chromosomal DNA was denatured in 70% formamide (in 2× SSC) at 80°C for 1.5 min. The hybridization mixture contained 20 ng of labeled genomic DNA, 30–50% formamide, 10% dextran sulfate, 2× SSC, and 10 µg of salmon sperm DNA. Genomic DNA of L. perenne was sheared to about 500 bp and added to the hybridization mixture to block cross-hybridization of F. mairei DNA to L. perenne chromosomes. To each slide, 10 µL of denatured hybridization mixture was applied and sealed under a coverslip (22 × 22 mm) with rubber cement. Hybridization was conducted at 37°C overnight. After hybridization, slides were washed at room temperature in 2× SSC for 5 min, at 45°C in 2× SSC for 10 min, at room temperature in 2× SSC for 5 min, and at room temperature in 1× PBS (phosphatebuffered saline) for 5 min. The biotin-labeled probes were detected with a fluorescein isothiocyanate (FITC) conjugated anti-biotin antibody (Vector). Propidium iodide (PI) in an antifade solution (Vector) was used to counterstain chromosomes. Slides after FISH were examined by using an epifluorescent microscope. The images were captured using a digital CCD (charged-coupled device) camera, merged with Adobe Photoshop (version 5.0) and printed by using a Fuji Pictography 3000. All of the original pictures can be stored electronically for future reference.

Results The genomic DNA of F. mairei was labeled as a probe with biotin-16-dUTP. After detection with a FITCconjugated antibody, the F. mairei chromosomes showed green hybridization signals, while the L. perenne chromosomes were red because propidium iodide (PI) was used for counter-staining. In the intergeneric hybrid LM1M2, (2n = 3x = 21), 21 chromosomes could be counted, and pairing patterns could be recorded after PI staining (Fig. 1A). The missing parts of chromosome pairing complexes were readily identified under a FITC filter. In Fig. 1B, arrows are pointed to that missing part of a bivalent and a trivalent, indicating that some chromosomes could not be hybridized with F. mairei genomic DNA. A composite picture (Fig. 1C) allowed us to clearly differentiate the chromosomes of F. mairei (green-yellow) with chromosomes of L. perenne (red) in meiosis. The L. perenne chromosomes are numbered 1 to 7 in Figs. 1C through 1F. GISH signals were readily generated when the concentration of blocking DNA was 50 times higher than the concentration of the probe DNA, however, signals were very weak © 2000 NRC Canada

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Fig. 1. (A) Chromosome configurations at metaphase I of meiosis with 5 I + 2 rod II + 3 ring II + 2 III (2n = 3x = 21). Propidium iodide (PI) used as background stain. (B) GISH signals with F. mairei genomic DNA used as a probe. Arrows point to the part of the pairing complex without hybridization signals. (C) A composite picture of Figs. 1A and 1B. The numbers indicate a chromosome configuration with a red chromosome from the L genome. Configuration 1 is a rod II of L + M; 2, 5, 6 are ring II of L + M; 3 and 4 are two trivalents, each is involved in two chromosomes from M and one chromosome from L. Seven is a univalent from L. (D) Chromosome configurations of 5 I + 1 rod II + 4 ring II + 2 III. Configuration 1 is a rod II of L + M; 2 and 5 are two trivalents; 3, 4, 6 are ring II of L + M, and 7 is a L univalent. (E) Chromosome configurations of 5 I + 2 rod II + 6 ring II. Chromosomes 1 and 7 are L univalents. Chromosome configurations 2 and 3 are rod II of L + M; 4, 5, 6 are ring II of L + M. (F) Chromosome configurations of 5 I + 6 ring II + 1 IV. All five univalents (1, 2, 3, 6, 7) are L chromosomes; 6 ring II of M + M; and one IV (arrow) with configurations of L + M + M + L.

when the blocking DNA concentration was increased 100 times higher. Different genotypes of L. perenne (Citation II and Calypso) and F. mairei (Fm1: PI 283313 and Fm2: F. mairei #2) have been used as blocking DNA and probes, respectively. Fm1 and Fm2 produced FISH signals equally, and Lp1 and Lp2 were effectively used as blocking DNA at similar concentrations. Unexpectedly, the L. perenne and F. mairei chromosomes were observed to readily pair with each other at metaphase I

of meiosis (Figs. 1C–F). The intergeneric pairing was presented as heterogeneous bivalents and trivalents. The chromosomes of L. perenne paired with one of the F. mairei genomes (M1 or M2) as well as M1 and M2 chromosomes paired with each other. The pairing between L and M chromosomes is defined as L–M (red and green-yellow bivalents), and the pairing between M1 and M2 is defined as M–M (green-yellow bivalents). Twenty-six cells have been examined with a total of 187 bivalents, of which 53% were © 2000 NRC Canada

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involved in intergeneric chromosome associations (L–M) and 47% were from the pairing between M1 and M2 (M–M) (Table 1). Approximately 4.65 univalents were observed with equal number of unpaired chromosomes from the L and M genomes, respectively (Table 1). Four of the five univalents presented in Fig. 1D were from F. mairei rather than from L. perenne, as expected. All trivalents observed had the same chromosome configurations with two chromosomes from F. mairei and one chromosome from L. perenne (Figs. 1C and 1D). GISH revealed the identity of chromosomes participating in the association of a quadrivalent (Fig. 1F) in which two chromosomes from L. perenne and two chromosomes from F. mairei formed a pairing complex at MI of meiosis. The mean chromosome configuration of the hybrid was 4.65I + 7.19II + 0.57III (Table 1), which was slightly different for the average configuration of 6.66I + 6.60II + 0.38III reported by Chen et al. (1995). Twenty-six PMCs were analyzed in this study. More extensive investigations will give rise to more accurate estimations when this procedure becomes more routine.

Discussion Our results strongly indicated that meiocytes of Lolium– Festuca are amenable to GISH analysis. A close relationship was revealed between the genomes of L. perenne and F. mairei. The results were unexpected, and challenged us to contemplate further the genomic relationships between these two grass genera. Genomic relationship of Lolium and Festuca Festuca mairei, a member of the scariosae section, was regarded as an allotetraploid based on the observation of 14 bivalents at MI (Malik and Thomas 1967). Its genomes were designed as M1M1M2M2 (Chandrasekharan and Thomas 1971). Another tetraploid, F. arundinacea var. glaucescens (G1G1G2G2), a member of the bovinae section, is considered as one of the donors of hexaploid tall fescue (F. arundinacea) (PPG1G1G2G2), one of the most economically important Festuca species (Chandrasekharan and Thomas 1971; Sleper 1985). Diploid donors for both tetraploid Festuca species are not clear; neither are the genomic relationships between F. mairei and F. arundinacea var. glaucescens. The interspecific hybrids between the two tetraploid Festuca species showed that most chromosomes formed bivalents (Chandrasekharan and Thomas 1971). The numerical analysis of the hybrids confirmed the 2:2 interpretation of meiosis, however, they could not conclude whether the pairing was autosyndetic or allosyndetic (Kopyto et al. 1989). The close relationships between species of Lolium and Festuca have been well documented, based on conventional cytogenetic (Jauhar 1993) and RFLP analysis (Xu et al. 1992). Lolium genomes are closely related to that of F. pratensis (PP). All reciprocal hybrids (LP) of L. perenne and F. pratensis were male-sterile in spite of regular pairing. The crosses between L. multiflorum or L. perenne and F. arundinacea (6x) (PPG1G1G2G2) have received the most attention because of the potential in practical plant breeding programs. Their F1 (LPG1G2) hybrids were also male-sterile, but there was indeed some female fertility to enable their backcrossing to the parental species. Because of the high

401 Table 1. Chromosome configurations of F1 hybrids (LM1M2) of F. mairei × L. perenne at metaphase I of meiosis. Twenty-six PMCs were analyzed.

Total No. Mean L (%)* L–M (%)** M–M (%)***

I

Rod II

Ring II

Ring III

Ring IV

121 4.65 51.2

64 2.46

123 4.73

15 0.57

1

43.7 56.2

57.2 42.3

*Percentage of univalents (L. perenne chromosomes). **Percentage of heterogeneous bivalents (one L chromosome pairs with one M chromosome). ***Percentage of homoeologous bivalents.

frequency of bivalents in the hybrids (LPG1G2), the intergenomic pairing (L and P), and the pairing between the other two genomes (G1 and G2) of F. arundinacaea were inferred (Jauhar 1975, 1993; Springer and Buckner 1982; Kleijer 1984). More than 7 bivalents formed in the hybrids (LG1G2) of L. multiflorum and F arundinacea var. glaucescens indicating partial homoeologous pairing between L and G1 (G2) in addition to the pairing between G1 and G2 (Cao et al. 1994). In the analysis of triploid hybrids (LM1M2), homoeologous pairing between M1 and M2 have been taken for granted because they come from the same species. Without direct evidence, the pairing of L and M could be deduced only when the bivalents are more than 7 or a certain frequency of trivalents is present. Conventional cytogenetic analysis was not able to pinpoint pairing events involving different genomes until the fluoresence in situ hybridization technique was applied. GISH analysis from our results showed that the percentage of heterogeneous bivalents (L–M) was approximately 53% of all 187 bivalents. Ring bivalents have been naturally thought of as resulting from homoeologous pairing, but in the hybrids of the F. mairei and L. perenne, the percentage of heterogeneous pairing (L–M) was even slightly greater than homoeologous pairing (M–M) in the form of ring bivalents (Table 1). These results suggest that conclusions drawn from traditional pairing data might need to be reevaluated. One question remains, considering the fact that species between the two genera are readily crossed and some hybrid cultivars have been released: How close is the relationship between Lolium and Festuca? More recently, GISH analysis of mitotic chromosomes in the intergeneric hybrids between tetraploid F. pratensis and L. multiflorum revealed that the Lolium genome can be readily incorporated into the Festuca genome (Zwierzykowski 1998). A majority of chromosomes in each cell analyzed were “hybrid chromosomes” composed of segments of chromatin from both parents. The number of detected translocation breakpoints ranged from 22 to 38 per cell and from 0 to 7 per chromosome. The authors were surprised by the extent of genome recombinations (Zwierzykowski 1998). From our observations, the F1 hybrids (LM1M2) had enough female fertility to be backcrossed with L. perenne despite male-sterility. Combined use of a genome-specific probe along with chromosome counting allowed us to identify potential addition lines and translocation lines (unpublished © 2000 NRC Canada

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data) even in the BC1F1 generation. Our results also suggested that despite considerable taxonomic distance in morphology, the chromosomes of L. perenne and F. mairei are genetically equivalent and reciprocal mixing of the genomes is possible, as concluded by Zwierzykowski (1998). We suggest that the intergeneric hybrids of (LG1G2) and (LPG1G2) might need to be re-examined using GISH to identify the chromosome pairing partners. It might be shown that the L genome could pair and readily recombine with any genome in Festuca, including genomes of P, G, and M. The value of using GISH to assess genome relationships The analysis of chromosome pairing has been critical to genome analysis of allopolyploids and determination of the genomic relationships between species. However, the collection and interpretation of data varies considerably from one study to another. Some of the early work concluded genomic relationships based on the mode of the number of bivalents. Numerical approaches to the analysis of meiosis in polyploid hybrids have been developed (Alonso and Kimber 1981; Chapman and Kimber 1992) and applied to hybrids of Lolium and Festuca species (Kleijer 1984; Crane and Sleper 1989; Kopyto et al. 1989). However, numerical analysis can not differentiate between heterologous pairing and homoeologous pairing in most circumstances. Our results show that GISH offers a reliable means to discriminate the identity of chromosomes involved in pairing. This is particularly valuable when the chromosomes of parents are of similar sizes and shapes. Either using genomic DNA or genome-specific repetitive DNA as a probe, FISH provides a great opportunity to unravel the secret of chromosome pairing between different species (Cuadrado et al. 1997; Benavente et al. 1996). In this study, F. mairei can be paired as 14 bivalents at meiosis MI. The incorporation of the L genome from L. perenne apparently disrupted the diploid-like meiosis in F. mairei. Evans and Aung (1986) reported that genotypes of L. perenne were capable of suppressing the activity of the pairing control genes of F. arundinacea, and the presence of B chromosomes may limit homoeologous chromosome associations between chromosomes of L. perenne and those of F. arundinacea. No B chromosomes were observed in L. perenne in this study. GISH is promising with respect to the investigation of how different genotypes of L. perenne will affect the chromosome affinity between the two genomes of F. mairei, and between the genomes of F. mairei and those of L. perenne, because autosyndetic and allosyndetic pairing can be clearly distinguished by using the GISH technique. GISH analyses of wheat and its relatives showed that the frequency of recombination is fairly low. For instance, only terminal translocations were observed between wheat and rye (Benavente et al. 1996). Usually, radiation or alternation of the pairing control systems (mutants of Ph genes) has to be used to facilitate the recombination between wheat and its relatives (Jiang and Gill 1994). Wheat is a highly selfpollinated species and has strict genetic control of chromosome pairing. However, the situation is distinct in the openpollinated Lolium and Festuca species. Application of GISH to the genome analysis of the Lolium–Festuca complex suggests that the relationships between Lolium and Festuca genomes are close. The genomes of the two genera may

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have introgressed with each other during evolution. The exchange of genetic information between the two genera could be unimpeded. With the availability of efficient selection tools, we are apt to introduce genes from F. mairei to L. perenne more efficiently.

Acknowledgements We acknowledge the counsel and assistance of Dr. Perry Gustafson for completion of this research. Thanks also go to Ms. Kathleen Ross for her assistance.

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