Genetic Diversity and Taxonomic Status of Lotus glaber Mill populations in Fayoum Depression

MATERIALS AND METHODS

Plant material: A total of 90 accessions were collected to represent 7 L. glaber populations that are distributed in the 5 territories of Fayoum depression. The details of the locations and their description are shown in Fig. 1 and Table 1. For the morphological description of this species, 39 morphological characters were applied that include stem (4 characters), leaflets (12), inflorescence (2), flower (6), pod (3) and seed (2).

Seed germination: Seeds were surface sterilized by soaking in 70% (v/v) ethanol for 1 min, then rinsed several times with sterile distilled water. The seeds (25 seeds) were germinated for 7 days at 25°C in sterilized petri dishes with three moist filter papers.

Chromosome count: Root tips were soaked in 0.003% colchecine for 4 h then fixed in ethanol:glacial acetic acid (3:1 v/v) for 2 h and stained in HCl carmine for the appropriate time required for the root tips to become dark red. The deeply stained root tips were macerated with 45% glacial acetic acid on clean slides. Cells with well-spread metaphase chromosomes were count and photographed.

Isoenzyme analysis:
Extraction: Green leaves of 7-day-old seedlings were macerated in 5 mL saline solution (0.8% NaCl, 0.2% NaNO₃, 10% glycerol and 1% bromophenol blue) then centrifuged at 10000 g for 15 min. Supernatants were collected in pre-chilled tubes and stored at -20°C until use for electrophoretic separation of isoenzymes.

Electrophoresis: The isoenzyme separation was carried out using mini vertical slabs of 7.5% acrylamide concentration according to Laemmli (1970). Aliquots (20 μL) of extracts loaded onto the gels. Electrophoresis was carried out at 20 mA for 60 min. The gels were stained for 4 isoenzyme systems (acid phosphatase, amylase, esterase and malate dehydrogenase) according to Eduardo Vallejos (1983).

Data analysis: For each enzyme, gene loci and alleles were inferred and labelled following numerical and alphabetical sequence, respectively. Elementary descriptors of isozyme variation (allele frequency, number of alleles per locus (A), percentage of polymorphic loci (P) and expected heterozygosity (H)) and genetic distances (D) (Nei, 1978) were calculated using PopGen32 software (Yeh et al., 1999).

Table 1:	The studied locations and sites from which Lotus glaber were collected
Data on elevations, soil texture, salinity and drainage were adopted from Abd-El-Motaleb (1997 and 2002)

Values of Nei’s genetic distance were used for clustering of the collected populations using unweighted pair-group method, arithmetic average (UPGMA) (Sneath and Sokal, 1973) using MINITAB-13 for windows (Minitab Inc., 2000).

RESULTS

Morphological description:
Synonyms:
Lotus corniculatus subsp. tenuifolius (L.) P. Fourn., Quatre Fl. France: 564.1953.
Lotus corniculatus subsp.tenuis (Willd.) Berher in Louis, Dep.Vosges 2:72, 1887.
Lotus noeanus Boiss.,Diagn.Pl.Orient.ser.2,2:21.1856
Lotus tenuifolius Reichenb., Fl. Germ. Excurs.: 506.1832 [non Burm. fil. cap. prodr.: 22.1768 [nom. Illeg.]. Lotus tenuis Willd., Enum.Pl.Hort.Berol.:797.1809.

Perennial herbs, decumbent or ascending, stem glabrous with extremely variable stem branching (5 to more than 20 branches) and length (7-60) cm). Leaf compound of 5 glabrous or hairy leaflets, upper 3 leaflets oblanceolate to elliptic or obovate with acute to acuminate apex; the central leaflet length 7-11 mm long and width 2-4 mm; the peripheral leaflets measured 7-10 mm long and 2.0-2.7 mm width; lower pair of leaflets lanceolate to ovate, with acute to acuminate apex, slightly shorter than upper leaflets. Flowers axillary 2 to 3 forming an umbel, bright yellow, sometimes flushed with red, pink, orange or green veins; 7.5-8.5 mm long, about twice as long as the calyx. Axillary peduncle much longer than the subtending leaf, 4 to >6 mm long. Bract sessile of 3-leaflets, the 2 peripheral equal, 3-6 mm long, the central longer, 4-7 mm long. Pedicel very short and straight about 1.25-1.75 mm long. Calyx glabrous with sub-equal teeth nearly equal to the calyx tube. Corolla 7.5-8.5 mm long, about twice as long as the calyx. Standard suborbicular, longer than the keel and wings. Keel tip incurved, yellow with long beak. Wings broadly obvate, with obtuse apex. Style length 4.4-56.0 mm.

Fig. 2:	Metaphase plate of L. glaber accession collected from Qarun with chromosome number 2n = 12+2B

Pod straight, linear, broad, 2.5-4.0 cm long, usually tipped with the persistent style base; 7-20 seeded; valves brown. Seed rounded with smooth brown to dark brown test.

Chromosome count: The chromosome set of all studied accessions comprised of 12 chromosomes. Populations of Qarun and Ebshawai were characterized by two supernumerary (B) chromosomes (Fig. 2).

Isoenzyme analysis: A total of 14 loci were recorded collectively all over the studied populations (Table 2). The population of Etsa was characterized by the absence of the locus Aph1 while that of Tamia was distinguished by the absence of loci Amy4, Aph2, Est1, Est3 and Mdh2. Both populations shared the absence of loci Amy1 and Est3. At the loci Amy3, Aph3, Est1 and Mdh3 some populations exhibited the mono-allelic expression while remaining populations exhibited the bi-allelic expression of these loci (for example, the populations of Ebshaway, Tamia and Etsa at the locus ApH3, Table 3). At the remaining loci, the studied populations varied in the allele that having the higher frequency (the predominant allele), others showed a considerable balance in the frequency of the two alleles of the same locus.

Table 2:	Allele frequency at all observed isozyme loci of the collected poulations of L. glaber

Table 3:	Genetic diversity measures of the collected populations of L. glaber

Fig. 3:	UPGMA clustering of the studied population of L. glaber based on Nei’s genetic distance

The highest values of mean alleles per locus (A), percentage of polymorphic loci (P) and theoretical heterozygosity (H) were observed for the population of Fayoum (A = 1.929, P = 80%, H = 0.344; Table 3) followed by those collected from Qarun (A = 1.786, P = 66.67%, H = 0.313, Table 3), then Sennoris and Kom Oshim (similar values of A (1.714) and P (60%), H = 0.272 and 0.246, respectively), Ebshaway (A = 1.5, P = 40%, H = 0.181). The lowest values of the three genetic diversity measures were observed for the populations of Etsa followed by Tamia. The UPGMA clustering based on Nei’s genetic distance grouped the populations of Fayoum, Qarun, Sennoris and Ebshaway at distance 0.35 (G1, Fig. 3) while each of the remaining populations comprised a group of its own. At distance 0.25, the population of Fayoum was separated in a single subgroup (SG1, Fig. 3) while those of Qarun, Sennoris and Ebshaway comprised another subgroup (SG2, Fig. 3).

DISCUSSION

Knowledge of available plant genetic resources is fundamental to support programs for the development of new cultivars, as well as to protect existing natural resources. The assessment of genetic diversity should be based on sound taxonomic identification. The investigated accessions of all populations were observed to have short style (4.0-4.56 mm), short corolla (7.5-8.5 mm) and central leaflets 2.0-3.5 times longer than wide. Comparing these values with those of L. corniculatus (style 5-7 mm, corolla 10-18, central leaflet 1.5-3 times longer than wide; Kirkbride, 1999; El Hadidy, 2003) elucidated that the collected accessions from Fayoum depression should be treated as L. glaber.

The chromosome counts showed that all the studied accessions are diploid with 2n = 2x = 12. Several authors have affirmed the diploid conditions of L. glaber that is essential for its discrimination from the tetraploid L. corniculation (Raelson and Grant, 1988; Grant and Small, 1996; Grant, 1999; Steiner, 1999; Fjellstrom et al., 2001; Beuselinck et al., 2003). This added cumulative evidence in favour of L. glaber.

The remarkable feature of the metaphase plate in the present investigation was the occurrence of B chromosomes in accessions collected from Qarun and Ebshawai. The occurrence of B chromosomes (2n = 12 + 1B) was previously observed in some Egyptian accessions of L. corniculatus (now L. glaber) that doubled the quantities of seed proteins compared to those lacking B. (Sammour et al., 1991). It was also found that drought increased the proportion of survivors of Allium schoenoprasum with B chromosomes (Bougourd and Plowman, 1996). The relation between B chromosomes and environmental impact is not clear (Puertas, 2002). However, it could be possible that the occurrence of B chromosomes might help populations of Qarun and Ebshawai to tolerate highly saline soils.

The populations of Etsa, Tamia and Kom Oshim were characterized by relatively lower values of genetic diversity measures (Table 3). They were also separated in distinctive groups when UPGMA clustering based on Nei’s genetic distance was applied (Fig. 2). These populations were distinguished by their non- to moderately-saline soils with well to excessive drainage (Table 1). On the other hand, the populations collected from Fayoum, Qarun, Sennoris and Ebshaway showed the highest values of genetic diversity measures and were grouped in a single subgroup according to Nei’s genetic distance (SG1, Fig. 3). These populations were collected from saline to very highly saline soils with imperfect to poor drainage (Table 1). These findings are consistent with the previous observations that L. glaber is adopted to tolerate salinity and flooding (Blumenthal and McGraw, 1999; Maceira et al., 1999; Banuelos and Beuselinck, 2003; Striker et al., 2005; Striker et al., 2006). Since the response of a species to challenge an environmental stress correlates with its genetic variation, the populations of Qarun, Fayoum, Sennoris and Ebshaway provides a valuable genetic resource that can be used for the improvement of L. corniculatus.

In conclusion, the taxon collected from Fayoum depression was identified as the diploid L. glaber that was observed to tolerate salinity and flooding. The salt tolerant L. glaber populations were those characterized by higher genetic diversity and can be introduced for gene banks as valuable genetic resource. More research was initiated in our lab using DNA fingerprinting to find out DNA marker(s) that can be linked with salt and flooding tolerance of L. glaber.