This paper reports the results of a genetic survey on population structure of
Eastern plane (
In Bulgaria
Populations of this species in Bulgaria are located in three regions over four river basins. Since river basins are separated by relatively high mountain ridges and some of the populations are also rather distant geographically, it is of particular interest to study the genetic variation and the differentiation between and within populations of
The objective of the present study was to assess the genetic variation and population structure of
The material for the study was collected from nine populations, representing the whole area of distribution of the species in Bulgaria (
Enzymes extraction was done after grinding the bud tissue in Tris-HCl extraction buffer pH 7.3. Ten milligrams of Polyvinil-polypyrrholidone (PVPP-40) were added as a stabilizing agent in the plastic vessels, where the extraction took place. Before extraction, 15 mg dithiothreitol (DTT), 500 mg saccharose, 150 mg Polyvinil-pyrrholidone (PVP-40) and 5 mg Na2EDTA were dissolved in 15 ml extraction buffer, and 100 μl β-mercaptoethanol were also added to the solution.
Standard 12 % starch gel electrophoresis was applied to separate the isoenzyme variants, in two buffer systems: Lithium-borate - Tris-citrate pH 8.1 discontinuous buffer system (
The loci were numbered according to their position from the anode,
Allele frequencies were calculated from diploid genotypes. Genetic diversity within populations was characterized by the following parameters: mean number of alleles (
Nine of the enzyme systems studied were polymorphic - LAP, GOT, GDH, PGI, PGM, MNR, ADH, MDH, and SKDH. The allele frequencies are available from authors upon request. The predominant allele was the same in all populations, which corresponds to the so-called “minor polymorphism” (
The percent of polymorphic loci ranged from 53.8 (Melnik) to 76.9 % (Topolovo - 0.05 criterion). These results are somewhat higher than the average values summarized in
Usually the genetic differentiation is measured by comparing the pairwise genetic distances (
The putative number of migrants calculated by using the
Locus MNR again showed different patterns of variation. It was the least differentiating locus, and with high inbreeding. As its visualization and interpretation was the easiest one, we can exclude possible misinterpretation of the electrophoregram as an explanation; we thus hypothesize that there were some mechanisms of selection most affecting this locus.
The genetic distances were of magnitude 0.005 to 0.085, and the most different population again appeared to be Ivaylovgrad, which was also the most distant and isolated one (
The Multidimensional Scaling (
The analysis of the genetic variation and population structure of
Multidimensional scaling of the populations on the basis of the genetic distances.
Bulgarian populations of
Population (abbreviation) | Geographic coordinates | Altitude (m a.s.l.) |
---|---|---|
Kresna (KR) | 41º 44’ N23º 08’ E | 300 |
Sandanski (SA) | 41º 36’ N23º 20’ E | 200 |
Slavyanka (SL) | 41º 26’ N23º 33’ E | 500 |
Petrich (PE) | 41º 24’ N23º 03’ E | 400 |
Melnik (ME) | 41º 30’ N23º 24’ E | 250 |
Goce Delchev (GD) | 41º 37’ N23º 52’ E | 300 |
Assenovgrad (AS) | 41º 58’ N24º 52’ E | 150 |
Topolovo (TO) | 41º 54’ N25º 00’ E | 150 |
Ivaylovgrad (IV) | 41º 35’ N26º 06’ E | 300 |
Investigated enzyme systems. (A): Lithium borate (pH 8.1) Tris Citrate (pH 8.1) discontinuous buffer system (
Enzyme system (abbreviation and EC code) | No of loci scored | Buffer system |
---|---|---|
Glutamate dehydrogenase ( |
1 | A |
Glutamate-oxaloacetate transaminase ( |
2 | A |
Leucine aminopeptidase ( |
2 | A |
Malate dehydrogenase ( |
4 | TC |
Menadione reductase ( |
1 | A |
Phosphoglucomutase ( |
1 | TC |
Phosphpoglucose isomerase ( |
2 | A |
Shikimate dehydrogenase ( |
2 | TC |
Alcohol dehydrogenase ( |
2 | TC |
Characteristics of genetic diversity and polymorphism. (
Population |
|
P | F | ||||
---|---|---|---|---|---|---|---|
Kresna | 52.7(1.9) | 2.3(0 .2) | 1.314 | 61.5 | 0.239(0.072) | 0.237(0.059) | -0.008 |
Sandanski | 49.2(1.9) | 2.2(0 .1) | 1.312 | 61.5 | 0.238(0.060) | 0.260(0 .053) | 0.085* |
Slavyanka | 53.8(2.7) | 2.5(0 .3) | 1.406 | 69.2 | 0.289(0.066) | 0.289(0.055) | 0.000 |
Petrich | 56.0(0.5) | 2.3(0.2) | 1.331 | 61.5 | 0.249(0.066) | 0.245(0.055) | -0.016 |
Melnik | 67.5(1.6) | 2.2(0.2) | 1.297 | 53.8 | 0.229(0.064) | 0.229(0.058) | 0.000 |
Goce Delchev | 63.5(0.3) | 2.2(0.3) | 1.349 | 76.9 | 0.259(0.079) | 0.241(0.057) | -0.075 |
Asenovgrad | 38.5(1.4) | 2.2(0.2) | 1.294 | 61.5 | 0.227(0.063) | 0.249(0.056) | 0.088* |
Topolovo | 51.3(2.4) | 2.3(0.2) | 1.295 | 76.9 | 0.228(0.070) | 0.283(0.053) | 0.194* |
Ivaylovgrad | 49.6(1.0) | 2.2(0.3) | 1.337 | 69.2 | 0.252(0.069) | 0.242(0.055) | -0.041 |
Shannon index of diversity (I’).
Locus | |
---|---|
Lap-1 | 0.2201 |
Lap-2 | 0.1930 |
Got-2 | 1.1594 |
Gdh | 0.6158 |
Pgi-1 | 0.2655 |
Pgi-2 | 0.8955 |
Mnr | 0.1372 |
Adh-1 | 0.6392 |
Adh-2 | 0.6121 |
Pgm | 0.2390 |
Mdh | 0.4290 |
Skdh | 0.3049 |
Mean | 0.4545 |
F-statistics and putative migration. (
Locus |
|
|
|
|
---|---|---|---|---|
Lap-1 | 0.306 | 0.324 | 0.026 | 9.1949 |
Lap-2 | 0.351 | 0.429 | 0.121 | 1.8230 |
Got-1 | 0.328 | 0.366 | 0.057 | 4.1396 |
Got-2 | -0.206 | -0.168 | 0.031 | 7.7110 |
Gdh | -0.161 | -0.059 | 0.088 | 2.5750 |
Pgi-1 | 0.450 | 0.473 | 0.042 | 5.6915 |
Pgi-2 | 0.385 | 0.533 | 0.240 | 0.7901 |
Mnr | 0.379 | 0.388 | 0.015 | 16.6564 |
Adh-1 | -0.031 | 0.010 | 0.040 | 6.0737 |
Adh-2 | -0.323 | -0.281 | 0.032 | 7.5851 |
Pgm | -0.056 | 0.022 | 0.073 | 3.1635 |
Mdh | 0.140 | 0.168 | 0.032 | 7.4486 |
Skdh | 0.154 | 0.189 | 0.040 | 5.9241 |
Mean | 0.019 | 0.094 | 0.077 | 2.9710 |
Genetic distances between the population pairs.
Population | SA | SL | PE | ME | GD | AS | TO | IV |
---|---|---|---|---|---|---|---|---|
Kresna (KR) | 0.012 | 0.050 | 0.035 | 0.017 | 0.052 | 0.019 | 0.052 | 0.028 |
Sandanski (SA) | - | 0.034 | 0.033 | 0.014 | 0.042 | 0.012 | 0.040 | 0.033 |
Slavyanka (SL) | - | - | 0.008 | 0.019 | 0.008 | 0.017 | 0.018 | 0.069 |
Petrich (PE) | - | - | - | 0.010 | 0.005 | 0.013 | 0.010 | 0.055 |
Melnik (ME) | - | - | - | - | 0.018 | 0.005 | 0.029 | 0.022 |
Goce Delchev (GD) | - | - | - | - | - | 0.014 | 0.013 | 0.078 |
Asenovgrad (AS) | - | - | - | - | - | - | 0.023 | 0.038 |
Topolovo (TO) | - | - | - | - | - | - | - | 0.085 |
Ivaylovgrad (IV) | - | - | - | - | - | - | - | - |