Karyotype Analysis in Six Species of Lolium and Festuca (Poaceae)
Parisa Shafiee1, Fatemeh Amini1*, Ghader Mirzaghaderi2, Seyed Mohammad Mahdi Mortazavian1 and Seyed Ahmad Sadat Noori1
1 Department of Agronomy and Plant Breeding Sciences, University of Tehran, College of Aburaihan, P.O. Box: 3391653755 Tehran, Iran
2 Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Kurdistan, P.O. Box: 416, Sanandaj, Iran
Received June 15, 2020; accepted July 8, 2020
Summary Analysis of the ploidy levels between different plant species can help the breeder to select strategies for cross-based breeding programs. In this study, various ecotypes of Festuca pratensis, F. arundinacea, F. rubra, Lolium perenne, L. multiflorum, and L. hybridum were cytologically characterized and grouped based on their chromosomal and karyotypic parameters. Moreover, 5S and 45S rDNA loci were determined by fluorescence in situ hybridization (FISH) in four species. Based on the mitotic chromosome complements, all the F. arundinacea and F. rubra ecotypes were hexaploid, L. multiflorum ecotypes were tetraploid and both diploid and tetraploid ecotypes of L. perenne and L. hybridum were identified. All ecotypes were located in the 2A class of Stebbins’s asymmetry, except for Fa2 (F. arundinacea) from Iran, Fr1 (F. rubra) from Finland, and Lp3 (L. perenne) from Switzerland, which was classified as 2B. One or two B chromosomes were observed in several individuals of an Iranian F. pratensis. In a cluster analysis based on karyotype parameters, Lp2 (L. perenne) from Switzerland and Fp2 (F. pratensis) from Sweden showed the minimum distance, suggesting that their crossing might be success-ful. FISH identified three 5S and two 45S rDNA loci in F. arundinacea, one 5S, and one 45S rDNA loci in F. pratensis, three 5S and two 45S rDNA loci in F. rubra, and one 5S and three 45S rDNA loci in L. hybridum.
Festuca L. and it’s closely allied Lolium L. belongs to the grass family Poaceae, subfamily Pooideae and tribe Poeae (Soreng and Davis 1998). The Festuca is a primitive genus with more than 450 species and is con-sidered one of the main evolutionary lines in the tribe Poeae (Tzvelev 1983). Ten to 12 species of Festuca grow wildly throughout Iran (Bor 1970). Festuca and Lolium have diverse distribution and are considered as impor-tant components of grass ecosystems of the temperate zones (Ospina-Gonzalez 2016). Crossing of Festuca and Lolium and combining their desirable attributes has great potential for the production of appropriate forage crops (Jauhar 1993). Many species in Lolium and Fes-tuca can inter-cross and the chromosomal exchange and rearrangements in hybrids are the main factors of ge-netic variation and evolution. Hence cytogenetic studies can help to elucidate genetic relationships for appropri-ate selection in cross-breeding programs (Swanson et al. 1981, Płażek et al. 2017).
Festuca species vary substantially in C-value and ploidy levels, from diploid (2n=2x=14) to decaploid (2n=10x=70), most of them being allopolyploid (Lou-reiro et al. 2007, Smarda et al. 2008, Hand et al. 2010).
There are reports on cytogenetic studies of different accessions of Festuca. Bagheri Abyaneh et al. (2017) evaluated the karyotypic difference between species in Festuca and considered the chromosomal and karyo-typic variations as one of the main factors of speciation in this genus.
Due to the high resistance of Festuca to a wide range of environmental stresses and the palatability of Lolium species, hybrids from these two species have a high po-tential for forage production in crop and pasture sys-tems. Investigation of genetic diversity is important for effective management and conservation of germplasm resources (Singh 2003). Analysis of the genetic relation-ships among species may help to predict the crossable parental pairs for breeding programs by which various intergenomic combinations can be acquired (Buckner et al. 1979, Höglind et al. 2013).
Here, we studied the karyotypic variations and cyto-logical characteristics in Festuca and Lolium species to infer their evolutionary relationship and to get informa-tion useful in cross-breeding programs.
Materials and methods
Plant materialSeeds of five ecotypes of F. arundinacea (three eco-
types Fa1, Fa2, Fa5 from Iran and two ecotypes Fa3 and Fa4 from Switzerland), seven ecotypes of F. pratensis (four ecotypes Fp1, Fp4, Fp5, and Fp6 from Iran, two ecotypes Fp3 and Fp7 from Switzerland and one ecotype Fp2 from Sweden), two ecotypes of F. rubra (Fr2 from Iran and Fr1 from Finland) and three ecotypes of L. perenne (Lp1 from Iran and, Lp2 and Lp3 from Switzer-land), one ecotype of L. multiflorum (Lm) and four eco-types of L. hybrium (Lh1–Lh4) from Switzerland were received from ART (Agroscope Reckenholz-Tanikon) research center of Switzerland and the NORDGEN Re-search Center in Sweden. The Iranian ecotypes were collected from different geographical regions of Iran. Information about the ecotypes used in this study is pre-sented in Table 1.
Chromosome preparationFor root tip preparation, seeds were germinated at
room temperature (RT) in dark on moistened filter pa-per in Petri dishes for 3–4 days. Actively growing root tips of about 1.5–2 cm in length were cut and pretreated with nitrous oxide (N2O) gas at 10 bar pressure for 3 h. Roots were fixed in ice-cold 90% acetic acid for 10 min and then transferred to 75% ethanol, and stored at -20°C until use. Enzymatic digestion and chromosome prepa-ration from root tips were carried out as described in Abdolmalaki et al. (2019). The prepared slides contain-ing good metaphase chromosomes were stored in 70% ethanol at -20°C.
Estimation of chromosomal and karyotypic parametersSlides stored in 70% ethanol were taken out and dried
at RT. Chromosomes were stained by adding a drop of acetocarmine and a coverslip was applied. Slides were
inspected under a BX51 Olympus microscope and im-ages were captured from appropriate metaphase spreads with a DP72 digital camera. Idiograms of the ecotypes were generated using the IdeoKar software (Mirza-ghaderi and Marzangi 2015) by tracing the chromo-somes, defining centromere and 5S and 45S rDNA loci. Chromosomes of three high-quality metaphase spreads were obtained from three different seeds. Chromosomes from three choromosome spreads of each ecotype were traced simultaneously. The protocol proposed by Levan et al. (1964) was used for the description of chromosome morphology. Idiograms were further processed in Ado-be Photoshop software. Chromosomal parameters and karyotypic indices for the numerical characterization of the genomes were also calculated in IdeoKar for each ecotype including total chromosome length of the hap-loid complement (HCL), mean chromosome length (CL), and mean centromeric index (CI). Karyotype asymmetry was determined using the intra-chromosomal asymme-try index (A1) and inter-chromosomal asymmetry index (A2) (Romero-Zarco 1986), CVcl (=A2×100) and CVci [=(sci/xci)×100] of Paszko (2006), and categories of Steb-bins (1971).
DNA probes and FISH analysisThe DNA clones used as probes were pTa71 (Gerlach
and Bedbrook 1979) containing a 9 kb EcoRI fragment of the wheat 5.8S-18S-26S rDNA unit, and pTa794 (Ger-lach and Dyer 1980), which encloses a 410 bp BamHI fragment of the wheat 5S rDNA unit. pTa71 and pTa794 were extracted and labeled with Atto488 and Atto550 nick translation labeling kits, respectively, following the manufacturer’s instruction (Jena Bioscience, Germany). Probes were recovered by ethanol precipitation and used
Table 1. Sampling site information and chromosome number of the plant material used in the present study.
Species Ecotype codes Local collection Latitude Longitude Altitude (m) 2n
2020 Karyotype Analysis in Six Species of Lolium and Festuca 283
to detect 45S and 5S rDNA loci in FISH. FISH experi-ments were carried out according to Abdolmalaki et al. (2019).
Statistical analysisA data matrix of 22 ecotypes from six species×five
variables (S%, TF%, HCL, CVcl, and CL) was used as input data in SAS. Data were standardized and used to estimate the pair-wise euqlidian distance coefficients between genotypes. The resulting matrix was used to construct an unweighted pair group method with arith-metic averages (UPGMA) dendrogram in SPSS soft-ware. To evaluate the contribution of each karyotypic parameter to the ordination of species, the data matrix of individuals×karyotypic variables were also subjected to a principal component analysis in SAS software.
Results and discussion
Three different ploidy levels were observed among the studied ecotypes of the Festuca and Lolium (Figs. 1, 2).
All F. arundinacea and F. rubra ecotypes were hexa-ploid (2n=6x=42), F. pratensis ecotypes were diploid (2n=2x=14), L. multiflorum ecotypes were tetraploid (2n=4x=28) and in the L. perenne and L. hybridum, diploid and tetraploid ecotypes were detected. Different ploidy levels also were observed in the study of Malik and Thomas (1966) that F. pratensis, L. multiflorum, and L. perenne were diploid, F. arundinacea had three levels of tetraploid, hexaploid and octaploid and F. rubra had two levels of hexaploid and octaploid. In the study of Ceccarelli et al. (1992) all the populations of F. arundi-nacea were hexaploid. In the study of Özer et al. (2018) ploidy levels of different ecotypes of Lolium, were diploid. Seal (1983) reported tetraploid, hexaploid, octa-ploid, and decaploid levels for F. arundinacea ecotypes. In this research, we found a different ploidy level for Festuca and Lolium ecotypes. Two diploid and hexaploid levels for the Festuca and two diploid and tetraploid lev-els for the Lolium were observed.
In this study, most of the chromosomes of the evalu-ated species were ‘m’ and ‘sm.’ In the Festuca, CL varied
Fig. 1. Mitotic metaphase of the studied ecotypes of Festuca or Lolium spp. A) F. arundinacea (Fa1); B) F. arundinacea (Fa2); C) F. arundinacea (Fa3); D) F. arundinacea (Fa4); E) F. arundinacea (Fa5); F) F. rubra (Fr1); G) F. rubra (Fr2); H) L. hybridum (Lh1); I) L. hybridum (Lh2); J) L. hybridum (Lh3); K) L. hybridum (Lh4); L) L. multiflorum (Lm). Scale bar=10 µm.
284 P. Shafiee et al. Cytologia 85(4)
from 3.32 µm in Fp1 to 2.02 µm in Fa4 while in the Lo-lium, it ranged from 2.89 µm in Lp2 to 2.20 µm in Lh3 (Table 2). Karyotypic symmetry parameters showed that all the studied ecotypes were classified in the class 2A of the category of Stebbins (1971), except for Fa1, Fr1, and Lp3, which were located in class 2B (Table 2). Fr1, Fp3, Lm, and Lh4 ecotypes demonstrated low values of A1 while Fa4, Fp5, Lp1, and Lp3 ecotypes had more asym-metric chromosomes and hence obtained higher values of A1. A2 was 0.189 for Fp1 and Lp1 ecotypes, 0.130 for Fp7 and 0.159 for Lh2 (Table 2). A2 reflects inter-chro-mosomal variation in each ecotype and showed a more chromosome length uniformity for the Fp7 ecotype.
In general, Fp3 and Fr1 from the Festuca and Lm and Lh4 from the Lolium were the most symmetric, and Fa4 and Fp5 from the Festuca and Lp3 and Lp1 from the Lolium were the most asymmetric karyotypes among the studied ecotypes. Consequently, Fa4, Fp5, Lp1, and Lp3 ecotypes are more advanced than Fp3, Fr1, Lh4, and Lm. In herbaceous plants, increasing asymmetry is equivalent to decreasing the number of chromosomes
and increasing the level of specialization in some traits (Bennett 1987).
Fp7 ecotype demonstrated low values of CVcl while Fp1 and Lp1 ecotypes had higher values of CVcl. Chro-mosome length variation in Fp1 and Lp1 ecotypes is more than the Fp7 ecotype. Fr1 ecotype demonstrated low values of CVci while Fp1 and Lp1 ecotypes had ob-tained higher values of CVci (Table 2). In Fp4 ecotype that is native to Iran one or two B chromosomes were observed in some individuals (Fig. 2D). According to the CVcl and A2 the Fp1 and Lp1 have a more variation in chromosome length and Fp7 had a more chromosome length uniformity compare to other ecotypes.
A comparison of different ploidy levels ecotypes of Festuca and Lolium showed that the length of chromo-somes in ecotype with higher ploidy levels is smaller. Pandit et al. (2014) found that there was a relationship between the small genome size and the higher ploidy level, and also stated that this could affect the adaptation of plants in different ecological areas.
Analysis of variance showed that there was a sig-
Fig. 2. Mitotic metaphase of the studied ecotypes of Festuca or Lolium spp. A) F. pratensis (Fp1); B) F. pratensis (Fp2); C) F. pratensis (Fp3); D) F. pratensis (Fp4); E) F. pratensis (Fp5); F) F. pratensis (Fp6); G) F. pratensis (Fp7); H) L. perenne (Lp1); I) L. perenne (Lp2); J) L. perenne (Lp3). Arrows in D point to B chromosomes. Scale bar=10 µm.
2020 Karyotype Analysis in Six Species of Lolium and Festuca 285
nificant difference between ecotypes of the Festuca in all chromosomal characters at 1% level (data are not shown). In the ecotypes of the Lolium, all chromosomal traits had significant differences at 1% level except AR index which was significant at 5% level and r-value which was not significant. The results of the mean com-parison showed that in the Festuca, Fp1 and Fp3 had the highest mean and Fa4 had the lowest mean for short arm (S), long arm (L), and CL (Table 3). AR, A2, and CI traits somehow represent evolutionary relationships
through intra-chromosomal relationships. Mean com-parison results showed higher diversity in the Festuca compared to the Lolium, which could confirm the re-sults of the analysis of variance. The mean comparison showed that in the Festuca, Fa1, and Fp4 had the same amount for the evaluated characters. It seems that these ecotypes were cytogenetically similar compared to the other ecotypes. Fa5 in Festuca and Lp3 in Lolium were classified in a separate group for most of the traits, which indicates that they were significantly different
Table 2. Karyotypic parameters of Festuca and Lolium ecotypes used in this study.
Ecotype HCL (µm) TF AsK% S% Xci A XcA CVcl CVci AI A1 A2 ST KF
Values in columns followed by the same letters are not statistically significant according to Tuky’s approach.
286 P. Shafiee et al. Cytologia 85(4)
Fig. 3. Idiograms of the studied ecotypes of Festuca or Lolium spp. A) F. arundinacea (Fa1), (Fa2), (Fa3), (Fa4) and (Fa5); B) F. pratensis (Fp1), (Fp2), (Fp3), (Fp4), (Fp5), (Fp6) and (Fp7); C) L. hybridum (Lh1), (Lh2), (Lh3) and (Lh4); D) L. perenne (Lp1), (Lp2) and (Lp3); E) L. multiflorum (Lm); F) F. rubra (Fr1) and (Fr2). 5S rDNA and 45S rDNA loci were added from FISH experiments on the corresponding eco-types. Scale bar=10 µm.
Fig. 4. Genetic relationships are depicted among 22 Festuca and Lolium ecotypes by the first two components (PC1 and PC2) derived from principal component analysis of karyotype data.
Fig. 5. UPGMA-based dendrogram showing the genetic relation-ship among 22 ecotypes of Festuca and Lolium based on karyotypic asymmetry parameters.
Fig. 6. 5S (red) and 45S rDNA (green) loci were detected on the chromosomes in A) F. arundinacea (Fa2); B) F. pratensis (Fp4); C) L. hybridum (Lh4); D) F. rubra (Fr2). Arrowhead points to the B chromosome.
2020 Karyotype Analysis in Six Species of Lolium and Festuca 287
from other ecotypes (Table 3). Idiograms of the studied ecotypes of Festuca and Lolium is shown in Fig. 3. The principal component analysis was used to group eco-types based on karyotypic indices showed that 94% of the inter-species diversity was justified by the first four components. The ecotype distribution diagram based on principal component analysis is shown in (Fig. 4).
Based on chromosomal characteristics cluster analysis was performed to determine the genetic distance be-tween ecotypes (Fig. 5). Ecotypes with identical ploidy levels were classified into one group except for Fa3. Fac-tors such as the value of relative chromatin (VRC), arano index of karyotype asymmetry (ASK, Arano 1963), HCL with the highest value, and total form percentage (TF, Huziwara 1962) with the lowest value, could be caused this ecotype classified as a separate group. In a cluster analysis based on cytogenetic parameters, Lp2 from Switzerland and Fp2 from Sweden showed the minimum distance, therefor crossing of them and com-bining their desirable attributes has great potential for the production of the appropriate forage crop.
In this study FISH analysis using two rDNA showed the variation between species in the Festuca. FISH re-sults in the F. arundinacea ecotypes (Fa2) showed three 5S rDNA in chromosomes numbers 1, 4, and 10, and two 45S rDNA loci in chromosomes numbers 8 and 9 (Fig. 6A). All the 5S rDNA loci have been found in cen-tromeric regions. The location of the 45S rDNA was in centromeric and distal regions of the chromosome. In F. pratensis species (Fp4), the 5S rDNA loci were observed on chromosome number 3 and 45S rDNA loci on chro-mosome number 2 (Fig. 6B). The position of both rDNA loci was in the centromeric regions. FISH results in the L. hybridum (Lh4) showed three 45S rDNA loci in chro-mosomes numbers 2, 3, and 4. Chromosome number 2 had both 5S and 45S rDNA loci together (Fig. 6C). FISH in F. rubra (Fr2) showed three 5S rDNA loci on 2, 12, and 21 chromosome pairs, and two 45S rDNA loci on chromosomes 1 and 20. Chromosome 17 had both 5S and 45S rDNA loci together (Fig. 6D). The variation in the number and location of the rDNA is commonplace and is also found in different genera. Thomas et al. (1997) also reported for each of 5S and 45S rDNA two loci in F. arundinacea that were consistent with the results of our study. The results of Ezquerro-López et al. (2017) were consistent with the results of our study in terms of the number of rDNA loci. However, one of the 5S rDNA loci in our study was different from the study of Ezquerro-López et al. (2017), this location was in the telomeric area. The results of this research for F. pratensis species were similar to the results of Ksiaczyk et al. (2010).
Acknowledgements
Authors wish to express their thanks to Dr. Beat Boller from the Agroscope Reckenholz-Tanikon research
center of Switzerland and the NORDGEN Research Center in Sweden for providing foreign ecotype seeds.
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