Prebreeding of Festulolium (Lolium x Festuca hybrids)
L. Østrem1, A. Larsen2, I. Pašakinskienė3 1 Norwegian Institute for Agricultural and Environmental Research, Fureneset, 6967 Hellevik i Fjaler, Norway; 2 Graminor Ltd., Vågønes, 8076 Bodø, Norway; 3Lithuanian Institute of Agriculture, Dotnuva-Akademija, LT58344 Kėdainiai Distr., Lithuania. Email:
[email protected] Keywords: Lolium perenne, Festuca pratensis, x Festulolium, introgression, prebreeding, winter hardiness, freezing tolerance, molecular analyses, GISH Introduction Existing cultivars of x Festulolium originate from tetraploid hybrids Lolium multiflorum L. (4x) x Festuca pratensis Huds (4x) or Lolium perenne L. (4x) x F. pratensis (4x), or they are developed from hybrids between L. multiflorum (2x) and Festuca arundinacea Shreb. (6x) with backcrosses to either F. arundinacea or L. multiflorum (4x) (Kopecký et al 2006, Zwierzykowski et al 2006, V. Černoch, pers. comm.). Alternatively, cultivar development can be undertaken at the diploid level using introgressed material described generally by Humphreys (1989) and for stress tolerance characteristics by e.g. Humphreys et al (1997) and Kosmala et al (2006). An introgression breeding programme was performed to transfer winter hardiness from the F. pratensis into L. perenne genome using partially fertile, triploid hybrids resulting from crossing L. perenne (4x) × F. pratensis (2x) as an initial material in backcrosses with L. perenne (2x). For Norwegian conditions cultivars with winter stress tolerances from the fescues combined with the growth characters and forage qualities of the ryegrasses would be advantageous. The study was initiated to investigate whether an introgression programme for development of x Festulolium diploid cultivars is a valid way for transferring winter hardiness from one species to another in new cultivars. The material must be genetic stable on the diploid level with sufficient amount of Festuca segments to ensure an increase in winter hardiness. Also, the optimal backcross generation should be identified as well as to look for possible relationships between freezing tolerance conducted in controlled freezing tests, observations under field conditions, and analyses on the genomic / genetic level. Materials and methods Plant material was comprised of 20 BC1 families and 40 BC2 families derived from two plant groups (assigned BC2-1 and BC2-2). For the BC1 and BC2-1 families 20 progenies from each backcross were cross pollinated within families in an isolation greenhouse (F1) followed by a second seed propagation (F2). The BC2-2 families originated from BC2 progenies that were cross pollinated within families, and seed harvested on individual plants followed by seed propagation of the half sib families in isolation (F2). Parental populations / varieties and standard cultivars of L. perenne and F. pratensis were also included in the investigations. Single plant trial was established in 2004 at Fureneset, Fjaler, West-Norway (61ºN) including 30 genotypes per family. Winter damage was observed on single plants in spring of 2005 and 2006 according to Nordic Gene Bank’s Descriptor list for Lolium perenne using a 1-9 scale where 1 defines plants with very low winter damage and 9 very high winter damage or dead plants, all plants compared to last year’s plant status. Plot trials were established at Fureneset and at Vågønes, Bodø, North-Norway (67ºN). Winter damage was observed as percentage ground cover in spring of 2005 and 2006 on plot basis. The freezing test was undertaken through the winter 2004-2005 at Vågønes including seed plants of all the 60 investigated families (BC1, BC2-1 and BC2-2) in addition to parents and standard cultivars. Two contrasting families (high and low freezing tolerance) in each of three backcross groups were selected for a second freezing test undertaken through the winter 2005-2006. The tested plants were ramets of the genotypes in the single plant trial at Fureneset. Plants were grown in a glasshouse and acclimated in growth chambers (one week at temperature 12/6°C and 12h photoperiod, followed by two weeks at +1°C and 16h photoperiod). Freezing was carried out in modified chest freezers (Gusta et al 1978) at gradually lowered temperature. The final temperature, ranging from –10 to –14°C for the various replicates, was kept constant for 24h and then gradually raised. Freezing tolerance was assessed twice, after two and three weeks at growth conditions, on the ability to regrowth according to a scale from 0 (dead) to 9 (without damage) (Larsen 1978). Analysis of variance for the field and freezing tests was carried out with AGROBASE98 (Agronomix Software INC. 1998). Attack from Microdoccium nivale was observed according per cent damage on leaves just after snow melt in spring 2005 at Vågønes. Chromosome preparation. Mitotic chromosomes from root-tips were prepared on objective slides after pretreatment in ice-cold water for 24h, followed by fixation in 1:3 acetic acid-ethanol. The roots were softened in a mixture of 0.1% pectolyase Y-23 and 0.1% cellulose R-10, and squashed in 45% acetic acid. Probes. F. pratensis genomic DNA was sonicated for 5 min in ELMA Transsonic T 460/H ultrasonic bath and labeled with rhodamine-11-dUTP (Roche) and fluorescein-12-dUTP (Fermentas). The pTA71 plasmid containing wheat 18S-5.8S-26S ribosomal DNA repeats was cleaved with restriction enzyme EcoRI to release the ribosomal DNA sequence and labeled with fluorescein-12-dUTP.
In situ hybridization. Slides were soaked in 45% acetic acid for 5 min at RT and for 3 min at 48-50°C. Denaturation of chromosome DNA was performed at 70°C in 70% deionized formamide 2xSSC for 2 min, followed by dehydration with cold ethanol series (70%, 90% and 100%), 2 min each and air-drying. Slides were incubated at 37°C with 25 µl of denatured (10 min at 70°C) hybridization mix (2 µg DNA probe, 60% formamide, 25% dextran sulphate, 10% 20 × SSC, 5% 5%SDS solution) for 16h in a moist chamber. After hybridization, slides were washed in 20% formamide in 0.1 × SSC twice for 5 min at 42°C, and 3 times for 3 min in 2 × SSC at 42°C. Slides were mounted with Vectashield antifade and DAPI (4,6-diamidino-2phenylindole) for counterstaining of DNA. Results Freezing tests. Among all backcross families significant differences for freezing tolerance were observed. Mean value for freezing tolerance decreased with backcross generation, while phenotypic variation increased from first to second backcross, and was highest in backcross group BC2-2 (Østrem et al 2005). Mean freezing tolerance of clones within contrasting (low and high frost tolerance in family means) families in the three backcross groups did not show the same differences as for testing of seed plants. Highest phenotypic and genetic variation among clones and broad sense heritability was observed within families selected for high freezing tolerances. Winter damage in single plants. Mean winter damage for the parent species and groups of backcross families in first and second year is shown in Figure 1. Mean winter damage for all plants after one winter was 1.98 and the corresponding figure for two winters was 4.25. Increase in winter damage was almost the same for the parent cvs. as for the backcross groups. In both years the three backcross groups were significantly different from each other. BC1 families were less and BC2-2 families most damaged both years and mean winter damage in the backcross groups were all higher than the parent species. In the 2nd year F. pratensis cvs. still had minor winter damage (1.8) and had significantly less winter damage than all other cvs. The two BC2-groups showed highest variation both years. No significant differences for winter damage were found between families with high and low freezing tolerance in any of the three backcross groups. There was an increase in winter damage from BC1, BC2-1 to BC22. The two families with low and high freezing tolerance within each BC-group showed small differences for winter damage with the exception of BC2-1 in 2005 where the winter damage was much lower in the high freezing tolerant compared to the low tolerant family. Winter damage
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Fig. 1. Winter damage in nursery field at Fureneset observed in spring in 1st (2005) and 2nd (2006) year after planting for parent species and three groups of introgression families using 1-9 scale (1 is very low winter damage, 9 is very high winter damage or dead plants). Vertical bars shows standard errors Winter damage in plot trial. The first winter resulted in only small damages on the sown plants. At Vågønes heavy attack of Microdoccium nivale on leaves were observed after snow melt in early spring, however, the attack had only minor effect on regrowth. F. pratensis was almost untouched by the fungus while especially diploid L. perenne was heavily attacked. Families in backcross group BC1 with highest amount of the Festuca genome had lower attack than families in the two BC2-groups (Table 1). Second winter led to heavy damage at Fureneset. Spring cover of F. pratensis and mean winter damage for the three backcross groups was 56% and 11%, respectively. Also the plot trial at Vågønes was damaged. Attack of Microdoccium nivale may have occurred in early winter, but wind, frost and ice had masked any sign of attack in spring, and the main cause for plant death seemed to be frost. Significant differences in spring cover occurred between backcross families at both locations. Table 2 shows mean values for spring cover after second winter and variation within each backcross group and of standard varieties at Vågønes. Among the backcross groups BC1 showed the highest and BC2-2 the lowest values for spring cover. F. pratensis had almost no winter damage, while L. perenne showed great variation between varieties. Both backcross groups BC1 and BC2-1 had some families nearly similar in winter hardiness to F.
pratensis. For the total material spring cover (winter survival) after the second winter was positively correlated with freezing tolerance measured under controlled conditions (r = 0.53) and negatively correlated with attack of snow mould on leaves the year before (r = -0.39). For the backcross families separately the correlation between spring cover and freezing tolerance was even higher (r = 0.89). Combining freezing tolerance and snow mould attack in a multiple regression explained nearly completely variation in spring cover of backcross families after the second winter (r² nearly 1). Freezing tolerance and attack of snow mould were not significantly correlated (r = -0.16). This is in agreement with earlier observations (Larsen 1994). Table 1. Mean and variation for per cent attack of snow mould in spring 2005 at Vågønes for groups of backcross families of x Festulolium and parent and control varieties of F. pratensis and L. perenne. Backcross No. Mean Lowest Highest family group BC1 20 65.8 50.8 79.2 BC2-1 20 74.5 29.2 100.0 BC2-2 20 70.5 50.8 96.7 F. pratensis 3 1.5 0.9 2.0 L. perenne 2n 5 66.3 54.2 73.3 L. perenne 4n 3 53.9 39.1 61.7 Table 2. Mean and variation for per cent spring cover 2006 at Vågønes for groups of backcross families of x Festulolium and parent and control varieties of F. pratensis and L. perenne Backcross No. Mean Lowest Highest family group BC1 20 72.0 17.9 100.0 BC2-1 20 57.3 7.9 100.0 BC2-2 20 28.2 1.0 86.3 F. pratensis 3 100.0 99.0 100.0 L. perenne 2n 5 60.5 18.1 92.7 L. perenne 4n 3 61.7 25.0 100.0 Molecular analyses. Using F. pratensis rhodamine labeled DNA plants of BC1, BC2-1 and BC2-2 introgression families were screened for the presence of Festuca chromosomal fragments. BC2-2-19 represented the most stress tolerant accession, and BC2-2-9 had the lowest tolerance to freezing. Festuca chromosomal segments were found in 34 plants out of 35 analyzed. No large Festuca fragments were detected. Most of the plants had 2 small interstitial Festuca fragments closely located to the rDNA sites. BC1 and BC2 plants had from 2 to 6 Festuca fragments located on different chromosomes. Both interstitial and terminal fragments were detected and some of these introgressions were quite significant in size. In total, number of Festuca introgressions in BC2-2 plants was lower and segments were smaller in comparison to earlier backcross generations BC1 and BC2-1. BC2-2-19 family (freezing-tolerant) had a slightly larger number of Festuca genome introgressions in comparison to BC2-2-9 (non-tolerant), 2.11 and 1.87, respectively. In BC2-219 line all plants had at least one Festuca introgression and in some genotypes this number reached up to 4. In BC2-2 -9 one plant occurred that had no Festuca segments at all. Some of the introgressions revealed could be F. pratensis chromosome segments which carry environmental stresses resistance genes. The number of rDNA sites varied within backcross families. It was found to be higher in family BC2-2-19, most of the plants had 7 – 8 sites, and one plant had 9. Therefore, mean rDNA site number was found slightly lower in BC2-2-9 than that in BC2-2-19 family, 6.80 and 7.71, respectively. Climatic data for the exp. period. At Fureneset total amount of precipitation in 2004 and 2005 was 2264mm and 2674 mm, respectively. The 30 year average (1960-1990) is 2010mm. Both years had wet autumns with frequent rain giving plants poor hardening conditions. In 2006 mid-winter was mild prior to a cold period in late winter and spring. Mean air temperature in March 2005 was 2.7ºC and for March 2006 –0.1ºC. In comparison total amount of precipitation at Vågønes was 1122mm and 1510mm for 2004 and 2005, respectively, which for 2005 was nearly 50 per cent over the normal. The first winter at Vågønes were mild but with snow cover. The second winter was very unstable with periods with hard wind and frost down to -10°C, combined with snow cover and some ice on the ground. Discussion Winter hardiness genes have been transferred from Festuca into the Lolium genome in the investigated prebreeding plant material. The increasing levels of winter damage in the three backcross groups BC1, BC2-1 and
BC2-2 observed in single plant and plot trials were in agreement with results from controlled freezing test on seed plants of the same lines in which the BC1 families showed on average a freezing tolerance between those of the initial parents (F. pratensis 2x and L. perenne 4x) (Østrem et al 2005). A second backcross to diploid L. perenne increased the winter damage as well as the variation among the BC2-1 families. Winter hardiness genes were lost during the second backcross, and for the winter hardiness characters one backcross may thus be recommended more than two, however, families with sufficient winter hardiness were present among the BC2 families. The investigation showed that slightly inbred families multiplied as progenies after single plants (here: BC2-2) is a good approach when looking for lines with characters of specific interest e.g. freezing tolerance and such a seed multiplication approach could also be used for progenies after first backcross. Additional cycles of seed multiplication will take place when synthetic varieties are made of selected families, and the implications of this process for the Festuca segments in a candidate cultivar are uncertain. GISH studies of amphiploid cultivars L. perenne or L. multiflorum x F. pratensis have revealed a change in genome balance in successive generations in favor of the dominant Lolium genome (Kopecký et al. 2006, Zwierzykowski et al. 2006). Results from the two freezing tests showed some disagreements, mainly because of great genetic variation in the investigated prebreeding material in which a random sampling of genotypes is difficult. Two of the families selected for low freezing tolerance showed significant lower broad sense heritability than the high tolerance families. A number of plants/ramets may have got low freezing tolerance of non genetic reasons, as diseases or physical causes, which have earlier been seen in low selected populations (Larsen 1979). The unusual high amount of winter damage at Fureneset (61ºN) compared to Vågønes (67ºN) after the second winter may be due to a very wet fall in the first harvest year followed by harsh winter and spring conditions. It may also be caused by the fact that the tested families originated from fairly northern Festuca and diploid Lolium material better adapted to Northern Norway. Genomes that are experimentally combined into hybrids and introgression families undergo changes in their organisation. These changes appear at the chromosome level and at the level of DNA sequences. Chromosome painting by GISH allows physical mapping of introgressed F. pratensis segments in the background of diploid genome of L. perenne. We detected interstitial and terminal introgressions in BC1 and BC2. Most of the introgression segments appear to be very small (Lideikytė et al 2006). Further, we are using ISSR fingerprinting method to estimate introgressions and changes at the level of DNA sequences. Acknowledgement The project is partially funded by The Nordic Gene Bank for 2004-2006. References Agronomix Software, Inc. 1998. AGROBASE 1997/1998. 171 Waterloo Street, Winnipeg, Manitoba, R3N 0S4 CANADA. Gusta L.V., M. Boyachek & D.B. Fowler (1978). A system for freezing biological materials. Hort. Sci. 13: 171172 Humphreys MW (1989). The controlled introgression of Festuca arundinacea genes into Lolium multiflorum. Euphytica 42:105-116. Humphreys M.W., H.M. Thomas, J. Harper, W.G. Morgan, A. James, A.G. Zare & H. Thomas (1997). Dissecting drought- and cold-tolerance traits in the Lolium-Festuca complex by introgression mapping. New. Phytol. 137:55-60. Kopecký D., J. Loureiro, Z. Zwierzykowski, M. Ghesquière & J. Doležel (2006). Genome constitution and evolution in Lolium x Festuca hybrid cultivars (Festulolium). Theor Appl Genet 113(4): 731-742. Kosmala A., Z. Zwierzykowski, D. Gasior, M. Rapacz, E. Zwierzykowska & MW Humphreys 2006. GISH/FISH mapping of genes for freezing tolerance transferred from Festuca pratensis to Lolium multiflorum. Heredity 96: 243-251. Larsen A. (1978). Freezing tolerance in grasses. Methods for testing in controlled environments. Meld. Norg. LandbrHøgsk. 57 (23), 56 pp. Larsen A. (1979). Freezing tolerance in grasses. Variation within populations and response to selection. Meld. Norg. LandbrHøgsk. 58 (42), 28 pp. Larsen A. (1994). Breeding winter hardy grasses. Euphytica 77: 231-237. Lideikytė L., I. Pašakinskienė & L. Østrem (2006). ’Chromosome painting’ by fluorescent in situ hybridization (FISH) in the hybrids and introgressions of Lolium and Festuca species. Biologija (in press). Østrem L., A. Larsen & I. Pašakinskienė (2005). Prebreeding of Lolium x Festuca hybrid derivatives. -In: Z. Zwierzykowski and A. Kosmala (eds.), Recent Advances in Genetics and Breeding of the Grasses. Institute of Plant Genetics PAS, Poznan, Poland 163-166. Zwierzykowski Z., A. Kosmala, E. Zwierzykowska, N. Jones, W. Joks & J. Bocianowski (2006). Genome balance in six successive generations of the allotetraploid Festuca pratensis x Lolium perenne. Theor Appl Genet 113(3): 539-547.
