Genetic diversity of alcohol dehydrogenase 3 in wild barley population at the "Evolution Canyon" microsite, Nahal Oren, Mt Carmel, Israel

Li, YC, T. Krugman, T. Fahima, A. Beiles & E. Nevo

Institute of Evolution, University of Haifa, Mt. Carmel, Haifa 31905, Israel

Alcohol dehydrogenase (ADH, E. C. 1.1.1.1.) is a member of a small group of proteins expressed under hypoxic conditions. Previous studies proved that both Hordeum vulgare and H. spontaneum have three loci for alcohol dehydrogenases (Harbred & Edwards, 1983; Hanson & Brown, 1984), and that the ADH3 locus was highly polymorphic (Hanson & Brown, 1984). However, ADH3 is expressed only under extreme anoxic conditions, such as pure nitrogen atmosphere or prolonged root flooding (Harbred & Edwards, 1983; Hanson & Brown, 1984; Hanson et. al., 1984).

The "Evolution Canyon" microsite is located in lower Nahal Oren, Mt. Carmel, Israel, and provides a model for the effect of microclimatic contrast on biodiversity (Nevo, 1995; 1997). Higher solar radiation on the south-facing slope (SFS) than on the north-facing slope (NFS) makes it warmer, drier, more heterogeneous, and climatically more fluctuating and stressful than the NFS. The interslope microclimatic contrast causes a dramatic biotic interslope difference: a tropical Afro-Asian savanna on the SFS versus dense temperate Euro-Asian liveoak macquis forest on the NFS, even though the slopes are separated by only 100 m at the bottom and 400 m at the top. Spatiotemporally, the SFS represents a "broader-niche" (Van Valen, 1965), consisting spatially of more micro-habitat patches and subdivisions than the NFS, and involving a mosaic of habitats of open park forest of Ceratonia siliqua-Pistacia lentiscus plant association, savanna plant formations, and bushy islands. Notably, small variations in aridity in the savanna-like habitats amplify the biotic divergence of the SFS, both in space and over time. In contrast, the milder and more homogeneous NFS consists of lush and dense live-oak macquis forest with a few island openings (Nevo, 1995; 1997). On both slopes, soil water content in the upper and middle stations is lower than in the bottom because of water runoff and the higher solar radiation stress at the higher stations. Seven studied stations were established for long-term projects: 3 stations were assigned to the SFS, No.1 (upper), 2 (middle) and 3 (lower) at the elevations of 120, 90 and 60 m, respectively; 3 opposite stations were assigned on the NFS, No. 7 (upper), 6 (middle) and 5 (lower) at 120, 90 and 60 m, respectively. A valley bottom station (no. 4) is at 45 m elevation. Significant inter- and intraslope allozyme (Nevo et. al., 1997) and DNA (RAPD) (Owuor et. al., 1997) polymorphisms in H. spontaneum were found on the more variable and stressful SFS and upper stations, but no genetic polymorphisms were found in the allozyme analysis at both ADH1 and ADH2 loci (Nevo et. al., 1997).

In the present study, 43 genotypes of H. spontaneum, sampled from upper stations (No. 1 and 7) and middle stations (No. 2 and 6) on the SFS and NFS, respectively at 'Evolution Canyon' were analyzed to redetect spectrums of ADH isozymic bands in 72h-flooded roots of seedlings by horizontal starch gel electrophoresis.

Table 1 The alelle numbers and genetic diversity (He, Nei,1978) at upper and middle stations on SFS and NFS
The results confirmed that there were no genetic polymorphisms at both ADH1 and ADH2 loci, however, the ADH3 locus was very polymorphic with 3 variant alleles: the most common (M), null (N) and "I" whose product forms enzymatically inactive homodimers (see Hanson & Brown, 1984). Significant differences in allele frequencies at ADH3 locus were found between the two opposite geographically very close SFS and NFS. Allozymic polymorphism and allele diversity increased in parallel upward on both slopes. On the more stressful SFS, 2 functional alleles (M and I) were found in 88% of the 8 tested genotypes at the upper station (No.1) and 100% genotypes at the middle station (No.2); on the less stressful NFS, however, only 50% of the 10 tested genotypes at the upper station (No.7) showed M and I alleles, but no genotypes showed any functional allele at the ADH3 locus in the middle station (No.6, Table 1).

At the ADH3 locus, the genetic diversity was higher on the more stressful SFS (He =0.124, Nei, 1978) than on the NFS (He =0.109), and also higher at the more stressful upper stations (0.189 and 0.218 for station 1 and 7 on the SFS and NFS, respectively) than at the middle stations (0.058 and 0.000 for station 2 and 6 on the SFS and NFS, respectively) on both slopes. These results implied that the more stressful the slope and stations were, the more functional alleles appeared, and the higher were the genetic diversities. Natural microclimatic selection appears to be the major evolutionary force causing interslope and intraslope adaptive genetic divergence at the ADH3 locus in H. spontaneum as in other traits of this species and genetic differentiation in other organisms (Nevo, 1995; 1997). Fitness test by transplant experiments (Levie et. al., 1993) in H. spontaneum from both slopes suggested that microclimatic variations select genetic adaptations in the subpopulations on both slopes. And germination comparison study (Gutterman & Nevo, 1994) showed that ecological-genetic differentiation regulate the germination of caryopses of H. spontaneum; similarities in germination patterns were found between the high-radiation, hence warmer and more xeric SFS and the Sede Boqer site in the Negev desert, separated 220 km to the SFS, rather than with the H. spontaneum pattern from the 200 m opposite NFS. These results suggest that H. spontaneum display adaptive interslope divergence at the "Evolution Canyon" microsite mirroring regional divergence across Israel in fitness components.

References

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