Genetic analysis of greenbug populations from Argentina and Chile based on enzyme variability

SALDÚA, L. et al. Greenbug genetic variability

Genetic analysis of greenbug populations from Argentina and Chile

based on enzyme variability.

SALDÚA Luciana**, María S. TACALITI*, Erica TOCHO**, Anthony F. G.

DIXON *** and Ana M. CASTRO*

* Faculty of Agronomy Sciences, CC31, (1900) La Plata, Argentina;

e-mail: [email protected]

** INFIVE-CONICET, CC 327, (1900) La Plata, Argentina

*** School of Biological Sci. University of East Anglia, Norwich,

U.K., NR4, 7TY, U.K.

ABSTRACT

Twenty-nine Schizaphis graminum (Rondani) populations and sixty clones

collected from very contrasting regions of Argentina and Chile

were investigated electrophoretically. A high degree of enzymatic

polymorphism was found. The enzymatic structure was descript for

the estearase system, founding nine different loci. Latitudinal

stratification was determined and populations were associated into

three groups, according to the latitude there were collected from.

The 90% of loci resulted polymorphic in the first group and 100%

of loci were polymorphic in the rest. Observed heterozygosity was

lower than the expected. None group had fixed alleles according to

Fsr values; group one had a slight heterozygous excess, while the

other groups showed slight positive Fsr values, because there was

a homozygous excess. According to Frt third group showed the

highest value that was in concordance with the highest gene flow

value. There were three populations that could not been joint to

any of the groups. The -carboxyl-estearase was always present,

1


also in clones and populations which were located in non

agriculture zones, implicating that the insecticide resistance is

well spread throughout the Argentinean and Chile territory. Non

relationship was found between the enzymatic patterns and the

biotype nor the host species where the aphids were collected from.

KEY WORDS. Schizaphis graminum. Enzyme. Genetic flow. Genetic-

structure. Geographical association.

Análisis genético de poblaciones de pulgón verde colectadas en

Argentina y Chile basado en su variabilidad enzimática.

RESUMEN

Veintitrés poblaciones de Schizaphis graminum (Rondani) y sesenta

clones colectados en regiones muy contrastantes de la Argentina y

Chile fueron investigadas electroforéticamente. Se encontró un

alto grado de polimorfismo enzimático. La estructura enzimática

fue descripta para el sistema estearasa, encontrando nueve loci

diferentes. Se determinó que existe estratificación latitudinal y

las poblaciones fueron asociadas en tres grupos, de acuerdo a la

latitud donde fueron colectadas. El 90% de los loci resultaron

polimórficos en el primer grupo y el 100% de los loci fueron

polimórficos en el resto. La heterocigosidad observada fue menor

que la esperada. Ningún alelo fue fijado, de acuerdo con el valor

del Fsr.. El primer grupo tuvo un ligero exceso de heterocigosis,

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mientras que los demás grupos mostraron valores ligeramente

positivos de Fsr, debido al exceso de homocigosis. Con respecto a

Frt, el tercer grupo mostró un valor alto que estuvo en

concordancia con el alto valor de flujo génico. Tres poblaciones

no pudieron ser incluidas en ningún grupo. Las -carboxil-

estearasas siempre estuvieron presentes, incluso en clones y

poblaciones colectadas en zonas no agrícolas, lo que implica que

la resistencia a insecticidas está ampliamente extendida a lo

largo del territorio argentino y chileno. No se encontró relación

entre el patrón enzimático y el biotipo, así como tampoco con la

especie hospedera de donde fueron colectados los áfidos.

PALABRAS CLAVE. Schizaphis graminum. Enzima. Flujo génico. Estructura

genética. Asociación geográfica

INTRODUCTION

Schizaphis graminum (Rondani) is the most wide spread aphid pest in

Argentina, and has received much attention because of its history

of overcoming host plant resistance and for its wide range of host

species. Greenbug was first detected in 1914 in La Pampa province

in Argentina, currently the aphid is spread throughout the cereal

region. Important outbreaks were recorded in the center of Santa

Fe, Córdoba, La Pampa and south-west of Buenos Aires provinces

(Noriega et al., 2000).

Greenbug damages their hosts producing chlorosis, reduction of

root volume in wheat (Burton, 1986), barley (Arriaga, 1969;

Castro, 1988; Gerloff & Orttman, 1971) oat and sorghum (Castro,

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1987; 1990). It also reduces the aerial biomass of wheat (Burton,

1986), barley and rye (Arriaga, 1956). When early attacks occur in

oats and barley a decrease in the protein of reserve movement from

seed to aerial part of the plant has been recorded (Castro et al.,

1987 and Castro and Rumi, 1987).

Biotypes have been defined as populations within an arthropod

species that differ in their ability to utilize a particular plant

genotype (Smith, 2005). Since 1961, eleven different greenbug

biotypes had been identified in USA by means of injury levels

determined in differential cultivars and species (Puterka &

Peters, 1988; Wood el at., 1961; Teetes et al., 1975; Porter et

al., 1982; Harvey & Hackerrot, 1969). These biotypes had given the

letter designations A-K (Shufran et al. 2000), and the origin and

evolution are still controversial subjects (Porter et al., 1997).

Greenbug biotype C and E were detected in 1988 in Argentina (Ves

Losada, 1987). Noriega et al (2000) identified B and C biotypes in

Buenos Aires and Córdoba provinces, they also reported consistent

evidence that biotype E is present in Buenos Aires. Finally,

biotype F and A were found infesting wheats (Almaraz et al.,

2001).

Genetic variation of aphid aggressiveness has been studied among

clones within ecotypes by means of the coleoptile tests (Giménez

et al., 1991a and b, 1992; Castro et al., 1991, 1994). Greenbug

populations collected from very contrasting ecological regions of

Argentina showed significant differences in reproductive behavior,

lifespan, aggressiveness and host preference (Ramos et al., 1998,

Giménez et al., 1991a,b; Clúa et al 2004).

The relationship between aphid resistance to insecticides and

estearases has been thoroughly confirmed. A major cause of

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insecticide resistance in Myzus persicae is the amplification of

one or two closely related genes encoding the

carboxilesterases E4 and FE4, which hydrolyse and sequester

insecticides (Field & Devonshire, 1998). A wide range of copy

numbers can exist for both genes, which give a proportionate

increase in esterase level and consequent resistance (Field

et al, 1999; Devonshire et al., 1998).In order to obtain indirect information about the existence and

intensity of genetic exchange between different regions and about

the presence of insecticide resistance, the purpose of the current

research is to determine the variability in estearase activity in

greenbug populations collected from very contrasting regions of

Argentina and Chile in autumns and springs since 1987.

MATERIALS AND METHODS

Aphid material.

Aphids were collected from contrasting regions of Argentina and

Chile (Clúa et al., 2004) “Table 1”. They were reared on

susceptible barley (‘Malteria Eda’) and maintained at 16:8 LD

photophase, 20ºC of temperature and 50% HR in growth cabinets.

Clones were initiated by allowing a single, isolated female to

reproduce by parthenogenesis. Populations and the derived clones

were kept separately by covering pots with transparent plastic

glass with voile on the top and ladder. These aphids were

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collected on different hosts in the field and maintained during

several months isolated in the insectary.

Approximately 200 individuals from every clone was separated and

collected into an eppendorf tube where they were smashed with a

capillary tube sealed with heat on its tip. The samples were

diluted 1:1 with trisglicina buffer (pH 8,3) and centrifuged at

13.500 rpm for 3 minutes. The supernatant was collected and

diluted 1:1/4 with glycerol and frozen at -20ºC.

Procedures.

In order to determinate the genetic base of different isozyme and

to separate these from allozymes, several specific substrates were

used simultaneously. Activity of estearase was performed in

poliacrylamide gel using 10% acrylamide with both tris-chlorhydric

(pH 8,65) and tris-borate (pH 7,9) buffers in a vertical

electrophoresis system, running at 16 volts for 15 minutes and

subsequently at 8 volts for 2 hours.

The gels were developed for alfa and beta estearase activity,

several subtracts were used to differentiated isozymes from

allozymes (20 ug alfa-naftil acetato, 20 ug beta-naftil acetato,

20 ug alfa-naftil-mirstate, 20 ug beta-naftil-miristate, 20 ug

alfa-naftil-butirate, 20 ug beta-naftil-butirate , 20 ug alfa-

naftil-laurate and 20 ug beta-naftil-laurate dissolved in acetone)

added to 50 mg of fast blue RR salt and diluted in 100 ml 0.1M

sodium phosphate buffer (pH 7,2). The gels were scanned for

analytical studies.

Esterase patterns were determined and identified the allelic

forms. These models were determined by means of high significant

correlation coefficients between electrophorms (PROCOR, SAS, 1998)

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and the genotypes. Data was analyzed with POPGENE 3.2 (Yeh et al.,

1997) and obtained Shanon’s index, observed and calculated

heterozygosities, Fsr, Frt, Fst and gene flow were obtained. The

average Hardy-Weinberg expected and observed heterozygosities were

calculated among clones within populations, populations within

regions and populations within total area.

Preliminary analysis showed that there was a regional structure

(Castro, 1994), consequently the inbreeding effect of population

substructure was assessed with the fixation index (F). The F index

is a useful index of genetic differentiation because it allows an

objective comparison of the overall effects of population

substructures among different clones. This index explains the

reduction in the expected heterozygosity with random mating at any

level of a population hierarchy, related to another, more

inclusive level of the hierarchy. Fsr is the fixation index of the

populations relative to the regional aggregates. Frt is the

proportional reduction in heterozygosity of the regional

aggregates relative to the total combined population. Finally, Fst

compares the least inclusive to the most inclusive levels of the

population hierarchy measuring all effects of population

substructure combined. F values varies from 0 to 1, the last value

indicates fixation for alternative alleles in different

populations (Hartl-Clark, 1997).

Table 1

RESULTS

Bands in the gels were distributed in seven regions (A-G) every

representing different loci “Fig. 1” and a total of ten different

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electrophenotypes were revealed. All studied populations and

clones showed esterase activity.

The first region (A), comprised by A1, A2, A3, revealed a brownish

coloration that did not show enzymatic variability. Three bands

appeared in all the studied genotypes “Fig. 1”. This enzyme

represented a dimorphic protein, being every individual

heterozygous.

The second region (B) is represented by presence or absence of

bands. Two alleles were detected, being one of them null. The B1

band represented the null allele. The B2 bands stained brownish.

The model that matches this pattern was a monomorphic protein.

The third region (C) comprised C1 to C4 bands, stained reddish and

presented seven different patterns composed by none, one or two

electrophorms each. A monomorphic protein with four alleles (C1,

C2, C3 and C4) could explain the activity of this enzyme. The

homozygous individuals presented one or none band, while

heterozygous individuals revealed two bands. All possible

combinations between alleles were presented in the current survey.

(Fig. 1, genotype 5 has C2- C3, genotype 6 has C3- C4, genotype 7

showed C2- C4).

The fourth region, D, (D1 to D5) revealed the activity of alfa

esterase bands by a brownish coloration. Every genotype with none,

one or two bands showed five different allelic forms within all

the homozygous individuals. Ten electrophoretic patterns were

found, that indicated the activity of monomorphic proteins with

five alleles (Figure 1, genotypes: 1, 2, 3, 4 and 5). Although,

two band patterns were also registered, only five combinations of

alleles were present in our clones, representing heterozygous

individuals (Fig. 1, genotype 6- 10).

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Similarly, the fifth region (E) ranged from E1 to E4, revealed

none, one or two bands. Seven different patterns were detected.

The activity of a monomorphic protein with four alleles, one of

them considered null, explained this pattern. Those clones with

none activity represented the null homozygous individuals. Other

clones have only one band (Fig. 1, genotypes 2- 4), while

heterozygous individuals presented two bands combining alleles E2,

E3 and E4 (Fig. 1, genotypes 5, 6, 7).

The sixth region, F (F1 to F3), showed also none, one or two

bands, indicating the activity of monomorphic proteins. The zone

stained light reddish. Probably, homozygous individuals were

represented by none or one band and heterozygous by two bands, by

combining allele F2 and F3.

The last region (G), comprised G1 to G7, revealed by dark-brownish

bands characteristic of alfa carboxyl estearase activity. This

region was present in all the studied genotypes with only one

band. Seven different allelic forms were found representing the

activity of a monomorphic protein.

Ten clones not shared the pattern commonly found in regions E and

F. These two new patterns were considered as two new proteins

different from the previously reported above. In region E1 six

clones (Bordenave 8, Gonzales Chaves 2, Gonzales Chaves 1, Villa

María 2, Teka 4 and Bavio 2) showed a three band pattern that

represented the activity of a dimorphic protein. These individuals

were heterozygous (E122- E123- E133). In region F1, four clones

(Gonzales Chaves 2, 4, 1 and Bordenave 9) revealed a three band

pattern (F122- F123- F133), being all individuals heterozygous.

Fig. 1

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The allelic frequencies of all the studied regions were estimated

“Table 2”. Hardy-Weinberg equilibrium was only found for region F1.

All studied loci were found polymorphic, nonetheless not all the

possible allelic combinations were found in our study.

Table 2

First analysis of genetic distance showed that populations could

be joint into three groups by means of similitude index values

“Table 3”. First group was conformed by populations: Salta,

Tucumán, Santiago del Estero, Frías, La Cumbre, Rafaela, and

Córdoba Norte (that were collected from 26º 50´ SL to 31 40´ SL)

“Fig. 2A”. Second group joint the following populations: Paraná,

Córdoba Sur, Villa María, Baradero, Bavio, Malargüe and Temuco

(from 31º 52´SL to 35º 30´SL) “Fig. 2B”. The third group was

represented by Ayacucho, Balcarce, Bordenave, Gonzales Chaves,

Osorno, Miramar, Tres Arroyos, La Dulce, Lonquimay, Cabildo, Bahía

Blanca, Los Alerces, Teka, and Castro (from 37º 08´ SL to 43º 28

´SL) “Fig. 2C”. In the first group, La Cumbre showed significant

differences with the rest of the populations. Baradero belongs to

the first group according to its geographical location, however it

joint the third group. Similarly, Lonquimay joint the rest of the

third group at high genetic distance. Temuco fit into the second

group but is geographically located in the strip corresponding to

the third group (38º 20´LS).

Fig. 2A

Fig. 2B

Fig. 2C

The second and third groups revealed 100% of the loci polymorphic

while the first group has a 90% of polymorphic loci “Table 3”.

Consistently with the values of polymorphic loci the lowest

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Shannon’s index was found in the third group, followed by the

first group and the second raised 0.90. In all the studied groups

the observed heterozygosity was lower than the expected

heterozygosity, this could be explained by the effect of the

homozygous individuals that were the most frequent genotype and

consequently, the observed heterozygosity values dropped due to

the lack of heterozygous individuals.

None group had fixed alleles according to Fsr values “Table 3”;

group one had a slight heterozygous excess, while groups two and

three showed slight positive Fsr values, because there were a

homozygous excess.

Comparing Frt values “Table 3” the first and third groups showed

the highest values, instead the second group differed

significantly from the total, showing the lowest values of Frt.

Fst values “Table 3” showed significantly differences related to

the total expected heterozygosity (higher than 0.25) of the total

populations in all the studied populations. These results agreed

with the calculated gene flow values. The second group had the

highest value contrasting with first group which had the lowest

value, and simultaneously showed the highest Fst with very great

differences to the total populations based on the expected

heterozygosity under Hardy-Weinberg equilibrium.

Table 3

DISCUSSION

The present study revealed the existence of variability for

esterase enzyme in Schizaphis graminum contrasting with previous

results (Steiner et al., 1985; Beregovoy & Shark, 1986; Abid et

11


al., 1989) and in agreement with Giménez et al. (1991a) and Castro

et al. (1996). The enzymatic structure of greenbug populations of

Argentina and Chile was descript for the esterase system. Nine

different loci were found, only one showed none variability. These

loci were found to be reliably resolved and polymorphic in

preliminary surveys of isozyme variation (Castro et al., 1987).

Kephart (1990) provided helpful information for interpreting

isozyme patterns. Infrequent multimeric association or low enzyme

activity may generate faint bands. In plants with null alleles

that lack enzymatic activity, homozygotes and heterozygotes become

visually indistinguishable in monomorphic proteins, heterozygotes

show unexpected two or four banded phenotypes in dimorphic and

tetramorphic proteins. Furthermore, under certain experimental

conditions, extra ghost bands that show primary bands are present

(Kephart, 1990). Their proposal of the existence of a null allele

in the heterozygous individual is based on the intensity of the

bands in the gels. In the current study it is not possible to

confirm the enzymatic patterns from regions B, C, D, E, F and F1,

because it is not possible to differentiate whether the

individuals that showed only one band are homozygous for this gene

or heterozygous carrying the null allele. In region A the model is

not adjusted to the presence of three monomorphic proteins, due to

the lack of discontinuity between the bands.Instead this could be

a dimorphic protein.

Kephart (1990) proposed that the genetic basis of observed

patterns should be verified by isozyme analysis of progeny arrays

from controlled crosses. In fact, crosses in our aphid populations

are extremely difficult because the number of sexual individuals

is not enough to successfully carry out them.

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Populations and clones were associated into three groups

latitudinal structured, similar results has recently found

by:::::::::(….). It would be possible that day-light conditions

(photoperiod), presence of bottlenecks, regimes of temperature,

etc. could be conditioning the genetic similitude of the studied

populations. There were four exceptions, La Cumbre, Baradero,

Lonquimay and Temuco populations. La Cumbre and Lonquimay did not

have significant similitude to the rest of the studied

populations. La Cumbre could be considered as a different group

and a correlation between the absence of eggs (Clúa et al, 2004)

and the isolation from the rest of the studied populations could

be considered. Lonquimay was collected over 2800 msl with a high

rainfull and very cold winter with wide range between day-night

temperature, although non discontinuity of host species was found

between the valley and the agriculture parcels situated high in

the Andes. This population was characterised to reproduce sexually

by Clúa et al (2004). Nonetheless, there were parthenogenetic

individuals together with others that induced sexual individuals.

Temuco population genetically joint with the second group (from

31º 52´SL to 35º 30´SL), but geographically it is located into the

third group (from 37º 08´ SL to 43º 28´SL). Temuco is located into

the typical cereal winter production region of Chile (central

valley) with very dry summers and a rainfull bellow 700 mm. Insect

populations pass through drastic bottlenecks due to the strong

chemical control and the lack of summer cereal crops.

In the other hand populations from Baradero and Parana were highly

similar to each other due to its geographical location, although

they joint different groups. It was not possible to collect any

greenbug sample in those location situated in-between those

13


places, probably there would have been a common origin for these

aphids or a high genetic flux.

It is also important to mention that all the populations were

collected from a very great variety of host species including

cultivated and non-cultivated host species. Non relationship was

found between the host specie where the insects were collected

from and the current genetic analysis of sampled populations.

Clones and populations have been maintained in insectary

conditions reared on susceptible barley since their collection

date, this fact could have conditioned the enzymatic patterns and

probably as it was found by Anstead et al. (2002) a relationship

between recent captured population of Schizaphis graminum and its

hosts might be found.

Studying the biotypes of S. graminum by a molecular phylogenetic

analysis based on the cytochrome oxidase I mitochondrial gene,

Shufran et al (2000), combined with earlier studied of mDNA (Power

et al., 1989), rDNA (Black, 1993) and RAPD (Black et al., 1992),

concluded that the biotypes of S. graminum are probably host-adapted

races. The biotype C was the predominant agricultural biotype

infesting sorghum and wheat found in our populations (Noriega et

al., 2000; Clua et al., 2004). By analyzing the relationship

between the isozymes profiles and greenbug biotypes distinctive

patterns were found, agreeing with Abid (1989), Ramos et al.

(1998) and Noriega (2000). On the contrary, Beregovoy and Starks

(1986) established that few isozyme differences exist between

biotypes B, C, or E. Biotype C that gathered Balcarce population,

Bavio 6, La Dulce 9, Bahia Blanca 1 and 2, Bordenave 6 and 9

(Noriega et al., 2000; Clúa et al., 2004), showed different

14


electrophenotypes represented in Figure 1 (i.e. genotypes 2, 1, 1,

3, 2, 5, 5 for the C esterase, respectively).

It is also worth to note that -carboxyl-esterase (Field &

Devonshire, 1998; Field et al, 1999; Devonshire et al., 1998)

was present, also in clones and populations which were

located in non agriculture zones, implicating that the

insecticide resistance is well spread throughout the

Argentinean and Chile territories. Current results show there is genetic variability on the greenbug

populations collected in Argentina and Chile. Every population

revealed alfa carboxyl estearase activity pointing out the

existence of insecticide resistance in our local populations. It

was also possible to identify latitudinal stratification of

greenbug populations. Finally, there was association between the

biotype and the ellectrophoretic pattern of estearases.

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Brasil, p 300.

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Table 1: Region of collection, geographical coordinates (GPS Data)

and host plant of each greenbug (Schizaphis graminum) population

Population CoordinatesS. L. W.L.

Host Plants

1. Salta 24°40´ 65°03´ Sorghum halepense2. Tucumán 26°50´ 65°12´ Sorghum halepense3. Santiago delEstero

27°48´ 61º 35´ Sorghum halepense

4. Frías 28°39´ 65°09´ Sorghum halepense5. La Cumbre 30°59´ 64°29´ Hordeum sp

Bromus sp.6. Rafaela 31°10´ 61°28´ Sorghum sp.7. Paraná 31°52’ 60° 29’ Triticum sp.

Sorghum sp.Bromus sp.

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8. Córdoba Norte

31°40’ 62° 20’ Sorghum sp.Avena sp.

9. Córdoba Sur 32°40’ 60° 53’ Hordeum vulgareAvena sp.

10. Villa María 32°25’ 63° 15’ Avena sp.11. Baradero 33°49´ 59°30´ Sorghum sp.

Avena sp.12. Los Hornos 34°55’ 57° 57’ Avena sp.

Hordeum vulgarePoa sp

13. B. Bavio 35°05’ 57° 44’ Avena sp. Sorghum sp.Hordeum sp.

14. Malargüe 35°30’ 69° 35’ Secale cerealeBromas sp.

Hordeum sp.15. Ayacucho 37°08’ 58° 29’ Hordeum sp.

Poa sp.16. Balcarce 37°50’ 58° 17’ Triticum sp.

Hordeum vulgareTriticosecale

17.Bordenave 37°51’ 63° 01’ Hordeum vulgareAvena sp.

Secale cerealeTriticosecale

Triticum sp.Bromus sp.

18. G. Chaves 38°02’ 60° 05’ Avena sativaHordeum vulgareTriticum aestivumTriticum durum

19. Osorno (CH)1

38°13’ 72° 20’ Secale cerealeTriticum sp.

20. Miramar 38°15’ 57° 50’ Triticum sp.Hordeum vulgare

Bromus sp.21. Tres Arroyos

38°23’ 60° 17’ T.aestivumT. durum

Triticosecale Hordeum vulgare

22. La Dulce 38°25’ 58° 42’ Avena sp.Hordeum vulgare

Triticum sp.23. Lonquimay (CH) 1

38°26’ 71° 21’ Avena sp.

24.Temuco (CH)1 38°20´ 72°20´ Triticum sp.

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Avena sp.25. Cabildo 38°30’ 61° 54’ Triticum sp.

Tritordeum26. Teka 43°28’ 70° 50’ Triticum sp.

Bromus sp.27. Bahía Blanca

42°29’ 61° 55’ Triticum sp.

28. Castro (CH)1

42°29´ 73°45´ Dactylis sp.

29. Los Alerces 42°55’ 71° 20’ Triticum sp.1CH: Chile

Table 2: Allelic frequencies of estearase loci determined for

populations and clones of Schizaphis graminum Regio

n

Null

Allele

Allel

e 1

Allel

e 2

Allel

e 3

Allel

e 4

Allel

e 5

Allel

e 6

Allele

7

A 0 0.5 0.5 0 0 0 0 0

B 0.28 0.71 0 0 0 0 0 0

C 0.24 0.34 0.34 0.08 0 0 0 0

D 0.24 0.16 0.28 0.16 0.16 0 0 0

E 0.23 0.19 0.41 0.16 0 0 0 0

E1 0 0.5 0.5 0 0 0 0 0

F 0.25 0.26 0.40 0 0 0 0 0

F1 0.74 0.233

8

0.024 0 0 0 0 0

G 0 0.015 0.09 0.22 0.22 0.317 0.015 0.11

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Table 3: Percentage of gene polymorphism, observe and expected

heterozygocity, Fsr, Frt, Fst and gene flow for the three groups

of Schizaphis graminum, following Nei (1987).

Group %Gene

Polymorphi

sm

Shannon

Index

Obser.

Hetrozygosity

Expec.Heterozygo

sity

Fsr Frt Fst Gene

Flow

1 90 0.82 0.28 0.47 -

0.1

5

0.4

9

0.4

1

0.2

6

2 100 0.90 0.28 0.50 0.1

8

0.2

3

0.3

8

0.8

0

3 100 0.95 0.28 0.54 0.0

7

0.4

6

0.4

1

0.3

5

Figure 1: Enzymatic patterns for nine loci of Schizaphis graminum with

the description of the found genotypes in sampled populations and

clones.

Electrophenotypes Region 1 2 3 4 5 6 7 8 9 10

A ------A1------A2

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------A3

B B1 B2

C C1

C2

C3

C4

------C2------C3

------C3------C4

------C2

------C4

D D1 D2

D3

D4

D5

------D2------D3

-------D2

-------D4

------D2

------D5

-------D3-------D4

-------D3

-------D5

E E1 E2

E3

E4

-------E2-------E3

-------E2

-------E4

-------E3-------E4

E1 ------E122------E123------E133

F F1 F2

F3--------F2--------F3

F1 F11

------F122------F123------F133

G

G1

G2

G3

G4

G5

G6 G7

In this figure, and ---- indicate different band intensities

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Figure 2: Genotypes grouping of greenbug clones and populations

collected in Argentina and Chile, by means of similitude index

values A: Sampled collected between 26º 50´ SL to 31° 40´ SL. B:

Aphids collected between 31° 52´SL to 35º 30´SL. C: Aphids

collected between 37º 08´ SL to 43º 28´SL.

Figure 2A: Group 1

Pop3 Pop6 Pop8 Pop2 Pop1 Pop11 Pop4 Pop5

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Figure 2B: Group 2

Pop14 Pop9 Pop13 Pop24 Pop10 Pop7 Pop12

Figura 2C: Group 3

Pop22 Pop26 Pop17 Pop18 Pop29 Pop21 Pop27 Pop28 Pop15 Pop19 Pop16

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Pop20 Pop25

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Genetic analysis of greenbug populations from Argentina and Chile based on enzyme variability

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