Journal of Heredity Advance Access originally published online on July 9, 2007
Journal of Heredity 2007 98(4):311-316; doi:10.1093/jhered/esm047
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Introgression Mapping of Genes for Winter Hardiness and Frost Tolerance Transferred from Festuca arundinacea into Lolium multiflorum
uczak
sior
From the Institute of Plant Genetics, Polish Academy of Sciences, Strzeszy
ska 34, 60-479 Pozna, Poland (Kosmala, Zwierzykowski, Zwierzykowska, and uczak); the Department of Plant Physiology, Faculty of Agriculture and Economics, Agricultural University of Cracow, Pod
u
na 3, 30-239 Kraków, Poland (Rapacz and Gsior); and the Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion, SY23 3EB, UK (Humphreys)
Address correspondence to A. Kosmala at the address above, or e-mail: akos{at}igr.poznan.pl.
Genes for winter hardiness and frost tolerance were introgressed from Festuca arundinacea into winter-sensitive Lolium multiflorum. Two partly fertile, pentaploid (2n = 5x = 35) F1 hybrids F. arundinacea (2n = 6x = 42) x L. multiflorum (2n = 4x = 28) were generated and backcrossed twice onto L. multiflorum (2x). The backcross 1 (BC1) and backcross 2 (BC2) plants were preselected for high vigor and good fertility, and subsequently, a total of 83 BC2 plants were selected for winter hardiness after 2 Polish winters and by simulated freezing tests. Genomic in situ hybridization (GISH) was performed on 6 winter-hardy plants selected after the first winter and shown to be significantly (P < 0.05) more frost tolerant than the L. multiflorum control. Among the analyzed BC2 winter survivors, only diploid (2n = 2x = 14) plants were found. Five plants carried 13 intact L. multiflorum chromosomes and 1 L. multiflorum chromosome with a single introgressed F. arundinacea terminal chromosome segment. The sixth BC2 winter survivor appeared to be Lolium without any Festuca introgression capable of detection by GISH. A combined GISH and fluorescence in situ hybridization analysis with rDNA probes of the most winter-hardy (after 2 winters) and frost-tolerant BC2 plant revealed the location of an F. arundinacea introgression on the nonsatellite arm of L. multiflorum chromosome 2, the same chromosome location reported previously as a site for frost tolerance genes in the diploid and winter-hardy species Festuca pratensis.
The species within the Lolium–Festuca complex possess a range of complementary characteristics. Lolium multiflorum Lam. (2n = 2x = 14) and Lolium perenne L. (2n = 2x = 14) are considered to be Europe's most important forage grasses as they provide high productivity and quality, but their use is confined largely to regions where growth conditions in summer and/or winter are not severe as they generally have poor persistency under stress conditions. In comparison to Lolium, related Festuca species—Festuca arundinacea Schreb. (2n = 6x = 42) and its progenitors Festuca pratensis Huds. (2n = 2x = 14) and Festuca glaucescens Boiss. (2n = 4x = 28)—are more resistant to abiotic stresses and as such provide a potential source of genes for improved stress resistance for Lolium (Humphreys et al. 2005; Kosmala, Zwierzykowski, G
sior, et al. 2006). This is achievable as Lolium and Festuca species hybridize readily and their homologous chromosomes pair and recombine at high frequency during meiosis (Jauhar 1975; King et al. 1999; Naganowska et al. 2001; Kosmala, Zwierzykowski, and Zwierzykowski 2006). Two principal plant-breeding approaches have been used to harness together complementary genes from Lolium and Festuca, amphiploidy and introgression. However, a high level of genetic imbalance has been observed by different authors in advanced generations of amphidiploid (2n = 4x = 28) Lolium spp. x F. pratensis hybrids (Zwierzykowski et al. 1998, 2006; Canter et al. 1999; Pa
akinskien
and Jones 2003; Kopeck
et al. 2006). This has resulted in greater emphasis being placed on the alternative introgression breeding approach, made possible by incorporating marker-based technologies to monitor intergeneric gene transfer, where genome stability is better guaranteed.
The allohexaploid species F. arundinacea comprises 3 genomes, 1 from F. pratensis and 2 from F. glaucescens (Humphreys et al. 1995). There are 2 alternative approaches to transferring genes for stress resistance from F. arundinacea into L. multiflorum, either indirectly from the subgenomes of F. arundinacea (Humphreys 1989b; Humphreys and Thomas 1993; Humphreys and Paakinskien 1996; Humphreys et al. 1997) or directly from one or the other of its progenitors (Zwierzykowski et al. 1999; Morgan et al. 2001; Humphreys et al. 2005; Kosmala, Zwierzykowski, Gsior, et al. 2006).
Humphreys and Thomas (1993) described the indirect transfer of drought resistance from a genome of F. arundinacea into L. multiflorum using partially fertile, pentaploid (2n = 5x = 35) L. multiflorum (4x) x F. arundinacea (6x) hybrids. In contrast, Humphreys et al. (2005) described the direct transfer of Festuca genes to Lolium via a backcross-breeding program using a partially fertile tetraploid (2n = 4x = 28) L. multiflorum (4x) x F. glaucescens (4x) hybrid as the starting point. Genes governing resistance to severe drought stress were introgressed directly from F. glaucescens into diploid L. multiflorum. The authors showed that genomic in situ hybridization (GISH) combined with fluorescence in situ hybridization (FISH) with rDNA probes, and previously mapped chromosome markers allowed a single Festuca chromosome segment to be mapped successfully onto the NOR arm of Lolium chromosome 3 in the selected introgression forms.
Similarly, Kosmala, Zwierzykowski, Gsior, et al. (2006) described a backcross-breeding program where partially fertile triploid (2n = 3x = 21) F. pratensis (2x) x L. multiflorum (4x) hybrids were backcrossed onto diploid L. multiflorum cultivars in order to transfer frost resistance directly from F. pratensis to L. multiflorum. Kosmala, Zwierzykowski, Gsior, et al. (2006) using combined technologies of GISH and FISH with rDNA probes showed that a single Festuca chromosome segment localized either to Lolium chromosomes 2 or 4 gave improved frost tolerance to Lolium.
Presented here is the first example of a backcross-breeding program aimed at the transfer of winter hardiness and frost tolerance genes from F. arundinacea into winter-sensitive L. multiflorum using pentaploid hybrids between F. arundinacea (6x) and L. multiflorum (4x). The main objectives of the research were 1) to select BC2 introgression forms of L. multiflorum with F. arundinacea genes for winter hardiness and frost tolerance, 2) to identify the chromosome location of putative F. arundinacea genes for winter hardiness and frost tolerance introgressed into L. multiflorum using combined GISH and FISH with rDNA probes.
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Materials and Methods |
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Plant Materials
The intergeneric hybrids for the backcross program were produced by crossing allohexaploid (2n = 6x = 42) F. arundinacea Schreb. (cvs Terros and Stef) (designated Fa), used as female parents, with autotetraploid (2n = 4x = 28) L. multiflorum Lam. (cvs Polus and Gran) (designated Lm), used as male parents. Two partially fertile, pentaploid (2n = 5x = 35) F1-48/1/55 and F1-13/6/88 genotypes were backcrossed as male parents onto 2 diploid L. multiflorum cvs Abercomo-8 and Tur-5, respectively. As a result, 2 BC1 populations (nos. 92/1, 92/2, respectively) and a total of 64 BC1 plants were generated. Two BC1 plants—92/1/6 and 92/2/33—with high vigor and good fertility were backcrossed again as male parents onto 2 genotypes of L. multiflorum (cv. Tur-12 and Tur-10, respectively) to produce 2 BC2 populations—BC2-98/9 (43 plants) and BC2-98/12 (58 plants), respectively. Because of the "biennial" nature of L. multiflorum species, it was almost impossible to keep exactly the same L. multiflorum genotypes for backcross procedure more than several years. In our work, we used the best Lolium cultivars with excellent productivity and forage quality (data not shown). A total of 38 plants from the BC2-98/9 and 45 plants from the BC2-98/12 populations that were sufficiently large to be divided into the required 3 replicates were then tested for winter hardiness in field conditions.
Tests of the BC2 Plants for Winter Hardiness and Frost Tolerance
Tests for winter hardiness of 3 clones of each BC2 plant and the controls—Lm cv. Tur and Fa cv. Terros (these 2 cultivars were shown in our earlier work to be good indicators of both species behavior during low-temperature stresses) in the field were performed at opuszna Experimental Station (southern Poland) during 2 winters (2000/2001 and 2001/2002) and winter survival scored as in Rapacz et al. (2004). Meteorological data at the experimental site were published before (Rapacz et al. 2004). Mean scores for 3 clones of each genotype were calculated, and statistical significance for differences between plants in winter hardiness was determined using Duncan's multiple range test at P = 0.05. The BC2 genotypes that were significantly (P < 0.05) more winter hardy (after 2000/2001 winter) than the L. multiflorum control were then selected in the field during 2001/2002 winter, and 1 clone was taken to laboratory, divided into 30 independent parts with minimum size of 3 tillers, and used in simulated freezing tests. Four replicate clones of each genotype were planted randomly in each of the 3 boxes. As control, plants of 4 different genotypes of Lm cv. Tur, and Fa cv. Terros were used. Plants were established in the sand/peat mixture over 7 days (25 °C, 10:14 h day:night photoperiod, and 200 µmol m–2 s–1 photosynthetic photon flux density [PPFD], Philips AGRO sodium light source, Philips Lightning N.V., Turnhout, Belgium). Well-rooted plants were then transferred to a controlled environment (CE) chamber for prehardening (7 days at 12 °C, 8:16 h photoperiod, and 200 µmol m–2 s–1 PPFD) and then for cold acclimation (3 weeks at 2 °C, 10:14 h photoperiod, and 200 µmol m–2 s–1 PPFD). Plant frost tolerance was determined by a modified method of that described by Rapacz et al. (2004). Cold-acclimated plants were transferred for 24 h to a CE chamber at –2 °C, 10:14 h photoperiod, and 200 µmol m–2 s–1 PPFD and then for 20 h in the dark at –4 °C. Afterward, plants were frozen at a cooling rate of approximately 1 °C h–1 to the desired freezing temperature: box 1 to –7 °C, box 2 to –9 °C, and box 3 to –11 °C. After an 8-h exposure to the target temperature, each box was transferred to 2 °C, 10:14 h photoperiod, and 200 µmol m–2 s–1 PPFD to defrost, and 24 h later, plants were transferred to the CE chamber at 12 °C, 12:12 h photoperiod, and 300 µmol m–2 s–1 PPFD to recover over 23 days, which is normally sufficient for the regrowth of tillers (Larsen 1978). All plants were cut down to a height of 2–3 cm after hardening, and the regrowth of 4 clones of each genotype per particular freezing temperature was estimated using Larsen's (1978) visual score. This was 0 = dead, no sign of leaf elongation; 1 = dead, but leaves having previously elongated to approximately 5 mm; 2 = dead, but leaves having previously elongated to 1–2 cm; 3 = plant dying, but with leaves having elongated to >2 cm; 4 = plant likely to die, and with inner leaves brown; 5 = plant likely to survive, but badly damaged; 6 = plant surviving, but with severe damage to approximately 50% of the leaves; 7 = plant alive, but with visual signs of freezing injury; 8 = minimal freezing damage (leaf tips discolored or deformed); 9 = no visible sign of injury. Mean scores for 4 clones of each plant after recovery were determined, and the mean scores for each genotype at all the 3 freezing temperatures were calculated. Statistical significance for differences between plants in frost tolerance was determined using Duncan's multiple range test at P = 0.05. Freezing tests were performed in 2 independent experiments showing similar results. Thus, only data from one experiment were included, mainly due to smaller variation in freezing tolerance observed between plants.
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Cytogenetic Analyses |
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Genomic In Situ Hybridization
In order to determine the genomic structure of the winter-hardy and frost-tolerant BC2 forms, root-tip chromosome spreads were prepared by fixing in absolute ethanol:glacial acetic acid (3:1) mixture at 37 °C for 7 days and subsequently squashed in 45% acetic acid. To discriminate between Lm and Fa chromosomes, total genomic DNA of Lm cv. Tur was used as a probe and labeled with digoxigenin-11-dUTP by nick translation (according to Roche protocol). Total genomic DNA of Fa cv. Terros to be used as a blocker was prepared by boiling 400 µg DNA (1 µg µl–1) in 0.4 M NaOH for 45 min and then combined in the hybridization mixture at a ratio of 1:60 (probe:blocker). The GISH protocol was as in Masoudi-Nejad et al. (2002). The probe signal was detected by incubation with antidigoxigenin–fluorescein (Roche Applied Science, Mannheim, Germany) at a final concentration of 2 µg ml–1 at 37 °C for 40 min. Chromosome preparations were analyzed using a Nikon Optiphot-2 epifluorescence microscope and photographed on Fuji 800 films. For each genotype, the total number of chromosomes and the number of parental and recombined chromosomes were counted.
Fluorescence In Situ Hybridization
The FISH procedure was as described by Kosmala, Zwierzykowski, Gsior, et al. (2006). To detect 45S rRNA genes, an 18S-25S rDNA fragment from Arabidopsis thaliana in the plasmid SK+ was labeled with biotin-16-dUTP by nick translation (according to the manufacturer Roche). To detect 5S rRNA genes, plasmid clone pU5SLL that contained one unit of 5S rDNA from Lupinus luteus L. was also used (Nuc et al. 1993). DNA was labeled with digoxigenin-11-dUTP in a standard polymerase chain reaction using 20mer primers corresponding to the 5' and 3' ends of the 119-bp-long coding region, without the nontranscribed spacer. The preparations were examined using an Olympus BX-60 microscope (Olympus Optical Co., GmBH, Hamburg, Germany). An uncooled CCD Ikegami 47E camera was used, and images were collected and merged using the ANALYSIS 3.0 program (SIS Soft Imaging System GMBH, Münster, Germany). For karyotyping, the images of the same mitotic chromosome spreads after GISH and FISH were compared, and thereby, the identity of the Lm chromosome carrying Festuca introgressions was determined.
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Results and Discussion |
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Introgression of important agronomic traits from F. arundinacea into L. multiflorum has been demonstrated both from tetraploid (2n = 4x = 28) L. multiflorum (2x) x F. arundinacea (6x) and from pentaploid (2n = 5x = 35) L. multiflorum (4x) x F. arundinacea (6x) hybrids. Though tetraploid F1 hybrids between diploid L. multiflorum and allohexaploid F. arundinacea are male sterile, they usually have a low female fertility and can be used either for introgression of Festuca genes into L. multiflorum cultivars to provide improved persistency and abiotic stress resistance or alternatively for introgression of Lolium genes into F. arundinacea to improve palatability and digestibility (for review: Thomas and Humphreys 1991; Humphreys et al. 2003; Kopeck
et al. 2006). The pentaploid hybrid L. multiflorum (4x) 
x F. arundinacea (6x) has proved a useful source of Festuca genes for the introgression of drought tolerance into Lolium (Humphreys and Thomas 1993; Humphreys and Paakinskien 1996).
In the current study, F. arundinacea (6x) x L. multiflorum (4x) hybrids were used in the backcross program to generate 83 BC2 genotypes that were subsequently tested under field conditions during the harsh winter of 2000/2001 (see Rapacz et al. 2004 for meteorology details). Fifty-one (61.4%) plants survived the winter, but these varied greatly in winter hardiness ranging from 0.33 (11 plants) to 1.6 (1 plant). Only 6 (7.2%) of the tested genotypes showed significantly (P < 0.05) more winter hardiness than the L. multiflorum control (Table 1). The L. multiflorum cv. Tur used here as control was the same cultivar used previously to assess the benefits to accrue from transfer to L. multiflorum of F. pratensis genes for frost tolerance (Kosmala, Zwierzykowski, Gsior, et al. 2006). The plants with introgressed Festuca alleles described by Kosmala, Zwierzykowski, Gsior, et al. (2006) and here were exposed to the same winter conditions and grown at the same location and could be compared for winter hardiness. Despite finding in both populations, individuals with a significant improvement in frost tolerance compared with Lolium, the plants with introgressed F. arundinacea genes were not as resistant as other Lolium genotypes containing F. pratensis alleles for winter hardiness. It was reported previously (Humphreys et al. 1997) among natural populations of the 3 Festuca species, F. arundinacea and its 2 progenitors F. pratensis and F. glaucescens, that it is F. pratensis that is the most northerly distributed and is as a consequence considered among those 3 Festuca species to be the most effective donor of alleles for winter hardiness.
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Winter hardiness is a complex trait that includes the ability to survive the interacting factors of a winter environment, the intensity of each dependent on the year and the location (Humphreys 1989a). However, it is thought generally that frost tolerance is the main target trait for determining levels of winter hardiness (Pulli et al. 1996). This conclusion was supported by the present study as plants selected for good winter hardiness were all significantly (P < 0.05) more frost tolerant than Lolium, although the level of resistance varied greatly (from 0.5 to 1.5) between the BC2 plants (Table 1). Moreover, some suggestions of transgressive segregation were also observed in 3 BC2 genotypes (nos. 98/9/19, 98/12/15, 98/12/20) that were significantly more frost tolerant even than the Festuca control (Table 1).
GISH analysis was performed on 6 winter-hardy (selected after 1 winter) and frost-tolerant genotypes. All the selected plants were diploids (2n = 2x = 14) and primarily Lolium. Five plants carried 13 Lm intact chromosomes and 1 Lm chromosome with a single introgressed terminal Fa chromosome segment. One plant (BC2-98/12/38) had 14 Lm chromosomes without any Fa chromatin detectable by GISH. However, among the 6 frost-tolerant genotypes, this plant showed also the lowest value of tolerance (Table 1). Nevertheless, it may be the case that useful allelic variation for frost tolerance resides within some genotypes of the Lm cv. Tur. Another explanation would be that Fa alleles were introgressed into BC2-98/12/38, but any alien introgression was too small to be visualized by GISH. The lack of a visible Festuca-derived alien introgression in the genomes of certain Lm plants selected for improved drought resistance was reported previously by Humphreys and Paakinskien (1996), who analyzed using GISH derivatives of an equivalent L. multiflorum (4x) x F. arundinacea (6x) hybrid.
After field selection and exposure to 2 winters (2000/2001 and 2001/2002), only one BC2 genotype (no. 98/12/20) survived and was significantly (P < 0.05) more winter hardy compared with the Lm control (Table 1). The BC2-98/12/20 genotype was also shown to be the most frost-tolerant genotype among those 6 winter-hardy genotypes selected after 1 winter (Table 1). During the second winter, all the introgression plants (with the exception of 98/12/20), similarly to Lm control, were badly infected by snow mold, whereas Fa control was not infected (data not shown). What is characteristic, 98/12/20 genotype was also the most frost-tolerant plant, as it was mentioned above. Analyzing meteorological data, we can suppose that infection was the secondary effect of very low temperatures in January (with very a low snow cover) followed by the fast temperature increase, initially with huge snowfall (more than 60 cm in the field) and warm February (maximum temperatures about 10 °C).
Lm genome consists of 7 chromosome pairs—4 chromosomes (nos. 1, 4, 5, and 6) without secondary constrictions and the other 3 chromosomes (nos. 2, 3, and 7) with secondary constrictions and 45S rDNA loci. The chromosome no. 2 carries also a 5S rDNA locus on its short nonsatellite arm (Thomas et al. 1996). Therefore, the use of FISH analysis with rDNA probes could assist the Lm chromosome identification and karyotyping. In our work, for each plant cell analyzed, 6 signals for the 45S rDNA probe on secondary constructions of homologous pairs of chromosomes 2, 3, and 7 and 2 signals for 5S rDNA probe on a pair of homologues of chromosome 2 were observed (Figure 1b). After comparing GISH (Figure 1a) and FISH (Figure 1b) images of the same mitotic chromosome spreads, it was revealed that a Fa segment was located terminally on the short nonsatellite arm of chromosome 2 (chromosome with both 45S and 5S rDNA loci).
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Exactly at the same position, the single F. pratensis chromosome segment with genes for frost tolerance was found in the introgression forms derived from backcrosses of triploid (2n = 3x = 21) F. pratensis (2x) x L. multiflorum (4x) hybrids with diploid L. multiflorum cultivars (Kosmala, Zwierzykowski, G
sior, et al. 2006). Recently, the LpGRP1 gene, coding glycine-rich RNA-binding protein involved in the development of frost tolerance in perennial ryegrass, has been mapped on L. perenne chromosome 2 (Shinozuka et al. 2006). A number of genes of importance that are implicated in frost tolerance have been also identified at the orthologous position in the Triticeae species (for review: Cattivelli et al. 2002). Third backcross of the selected winter-hardy and frost-tolerant BC2-98/12/20 genotype into diploid cultivars of Lm has been performed and will include genotypes, where the Fa introgression has been "dissected" through chromosome recombination. |
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In the current study, we showed that it was quite efficient to transfer the genes for frost tolerance from F. arundinacea to L. multiflorum using the pentaploid F. arundinacea (2n = 6x = 42) x L. multiflorum (2n = 4x = 28) hybrids as initial plant materials in the backcrosses with diploid L. multiflorum. Furthermore, we confirmed once again that an appropriate single Festuca introgression in an otherwise undisturbed Lolium genome could provide increased winter hardiness and frost tolerance without compromise to the good growth and vigor found in Lolium. The combined use of GISH and FISH with 2 rDNA probes enabled the mapping of chromosome location of putative F. arundinacea genes for frost tolerance on L. multiflorum chromosomes 2. The chromosome location of frost tolerance genes identified herein was the same as the location of F. pratensis genes for frost tolerance found in our earlier work. This chromosome is known in the Triticeae species to contain genes involved in both winter hardiness and frost tolerance what should make further study of this chromosome a priority.
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European Union (QLK5-CT-2000-00764).
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Acknowledgments |
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We thank Dr Hanna Weiss for use of plasmid SK+ with 18S–25S rDNA and Drs Przemys
aw and Katarzyna Nuc for use of clone pU5SLL. We also thank Wodzimierz Zwierzykowski for technical support. |
Footnotes |
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Corresponding Editor: Reid Palmer
Received October 18, 2006
Accepted March 15, 2007
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  A. Kosmala, A. Bocian, M. Rapacz, B. Jurczyk, and Z. Zwierzykowski Identification of leaf proteins differentially accumulated during cold acclimation between Festuca pratensis plants with distinct levels of frost tolerance J. Exp. Bot., August 1, 2009; 60(12): 3595 - 3609. [Abstract] [Full Text] [PDF]
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