GENETIC DIVERSITY OF LEMNA GIBBA L. AND L. MINOR L. POPULATIONS IN NILE DELTA BASED ON BIOCHEMICAL AND ISSR MARKERS

Egypt. J. Exp. Biol. (Bot.), 11(1): 11 – 19 (2015) © The Egypt ian Society of Experimental Biology

ISSN: 1687-7497 On Line ISSN: 2090 - 0503 http://my.ejmanger.com/ejeb/

R E S E A R C H A R T I C L E

Aziza S. El -Kholy Moham ed S. Youssef Ebrahem M. Eid

GENETIC DIVERSITY OF LEMNA GIBBA L. AND L. MINOR L. POPULAT IONS IN NILE DELTA BASED ON BIOCHEMIC AL AND ISSR MARKERS

ABSTRACT: The genetic diversity of nine populations of Lemna , seven for Lemna gibba and two for L. minor was evaluated. The populations were col lected from different water courses in the Ni le Delta, Egypt; agricultural drainage, mixed (agricultural and industr ial) drainage and irrigation canals. The genetic relationship among the examined populations was estimated based on recorded di fferences in SDS-PAGE profiles, five isozyme systems and ISSR f ingerprinting. Construction of distance trees illustrat ing the genetic distance among the studied populations was performed using the UPGMA method. The results revealed a high level of inter and intra-species level of geneti c diversi ty. The two species were delimited as two clusters at a distance of 1.52 in a UPGMA tree and Limna g ibba populations were separated into two subclusters at a distance of 1.20 reflecting the samples col lected from Kafr El-Sheikh were separated from those col lected from Gharbia. Geographical distribut ions of the samples appear to have obvious influence on the geneti c diversity of the studied populations.

KEY WORDS: Genetic diversi ty; ISSR; isozyme; SDS-PAGE; Lemnaceae

CORRESPONDENCE: Aziza S. El -Kholy Botany Department, Faculty of Science, Kafr El-Sheikh University, Kafr El-Sheikh, Egypt E-mail: [email protected] [email protected]

Moham ed S. Youssef Ebrahem M. Eid Botany Department, Faculty of Science, Kafr El-Sheikh University, Kafr El-Sheikh, Egypt ARTICLE CODE: 02.02.15

INTRODUCTION: Lemnaceae, commonly called duckweed,

comprise 37 worldwide species and five genera of aquatic monocotyledonous plants which include the world's smallest angiosperms that float on the surface of fresh water (Landolt, 1986; Appenroth et al., 2013). Duckweeds are small, widely distributed around the globe ranging from temperate to tropical regions (Wang et al., 2010). Duckweed plants lack true stems and leaves. The plant body generally consists of expanded, flat, leaf-like called fronds that float on the water surface or are slightly submerged and involved in photosynthesis and reproduction. The roots attach themselves on the lower surface of the fronds and its maximum number is species specific (Stomp, 2005). Generally, duckweeds reproduce vegetatively by generating daughter fronds from the meristematic tissue on the pocket cleft towards the base of the mother frond and form a colony, then dissociate upon maturation. Lemna species are aroused attention as animal feed due to its high productivity and its high protein content (Appenroth et al., 2013). These aquatic plants have been used to remediate wastewater from both municipal and industrial sources (Appenroth et al., 2013; Gadallah and Sayed, 2014). These plants could be employed in reducing salt concentrations in irrigation water (Abou El-Kheir et al., 2007; Balla et al., 2014). Salinity is an important factor in limitation of growth of Limna gibba (Yilmaz, 2007). The starch content of duckweed is highly variable and has already been demonstrated for the production of ethanol following saccharification and fermentation of the resulting glucose (Qian et al., 2012).

Much of the classification of duckweeds is based on integrative methods including morphological variation, flavonoid types, isozyme polymorphism and DNA markers however, the resolution between closely related species and the intraspecific diversity are not often addressed (Appenroth et al., 2013). The genus Lemna is represented in Egypt by four species i.e. L. aequinoctialis Welw., L. gibba L., L. minor L., and Lemna trisulca L. (Tackholm, 1974; Boulos, 2009; Azer, 2013).

Egypt. J. Exp. Biol. (Bot.), 11(1): 11 – 19 (2015)


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Electrophoretic data analysis of native or denatured proteins of different species have been widely used to provide information concerning the genetic variability and systematic relationships at the subspecies and varietal levels as in plants/examples include Trifolium L. (Badr, 1995) and soybean (El-Kholy, 2013). The electrophoretic detection of protein polymorphism as markers for genetic and ecological variability has been also studied through determination of isozyme forms as in Ipomea carnea (Hamoud et al., 2005). Allozymes reflect discrete genetic differences and thus represent a potentially useful method for assessing whether one or two gene pools exist within what has been treated as two taxa. Allozymes provided useful data for distinguishing species in Lemnaceae; particularly in the genera Spirodela (Crawford and Landolt, 1993), Wolffia (Crawford and Landolt, 1995), Wolffiella (Crawford et al., 1997) and Lemna (Crawford et al., 1996; 2001; 2005). It has been also used to distinguish populations of L. minor (Vasseur et al., 1993; Cole and Voskuil, 1996).

The molecular markers generated by the new possibilities of genome genotyping provide a useful molecular evidence for evaluating genetic diversity of plants (Badr et al., 2012). Inter-simple sequence repeat (ISSR) is a molecular marker technique that was developed by Zietkiewicz et al. (1994). ISSR markers are highly polymorphic and are useful in studies on genetic diversity, phylogeny, gene tagging, genome mapping and evolutionary biology (Reedy et al., 2002). A high level of genetic differentiation among populations of Lemna and Spirodela were produced by ISSR markers (Xue et al., 2012).

It is quite apparent that the special properties of Lemnaceae family of aquatic plants are now capturing more interests from the scientific community worldwide than ever before (Appenroth et al., 2013). So far, there are no reports on the genetic diversity of the duckweed species in Egypt. The aim of this study was to assess the genetic diversity and the genetic

relationships between two different populations of two species of Lemna (L. gibba and L. minor) distributed along different water courses in Nile Delta based on biochemical markers and ISSR-PCR.

MATERIAL AND METHODS: The Sampling sites:

The sampling area lies in the Nile Delta bounded by the two branches of the Nile: Rosetta to the west and Damietta to the east. Most of these water courses were dug in the last 200 years (Hurst, 1952). According to UNESCO (1977), the annual mean air temperature decreases from 20.7C at the north of the Nile Delta (Baltim, Kafr El-Shiekh Governorate) to 19.9C at its middle (Tanta- El-Gharbiya Governorate). The relative humidity decreases in the same direction from 69% to 65%. Sample collection:

For measuring the phytomass, sampling of L. gibba and L. minor was carried out using 24 cylindrical plot sampler of diameter (16 cm) distributed along 7 drainage and irrigation canals within the study area. The plants within a sampled plot were collected and transferred to the laboratory in polyethylene bags. In the laboratory, the phytomass was measured after oven drying at 60°C to constant weight. All biomass values were determined as gram dry matter per square meter (g DM m−2). For biochemical and molecular studies, 9 populations were sampled; 7 for L. gibba and 2 for L. minor. Six Populations (numbered 1-4, 8, and 9) were selected from El-Gharbiya governorate and three populations (numbered 5, 6, and 7) were selected from Kafr El-Sheikh governorate. The GPS positions for collection sites, the distribution of the studied populations and type of water courses are listed in table 1. Water samples were brought to the laboratory, filtrated through a Whatman 3-mm filter paper, and stored at 4°C before assaying. Each water sample was assayed for pH and electric conductivity.

Table 1.The distribution of studied populations of Lemna, type of water course, dry matter per square meter, pH, and EC

No. Species Site of collection Type of water course Location GPS Biomass (g DM m−2 ) pH EC

(µScm−1)

1 L. gibba El-Gharbiya Governorate

Agricultural and industrial drainage canal

30° 48' 41.25'' N, 30° 53' 52.79''E 17.30 7.43 1083.3


Agricultural and industrial drainage canal

30° 48' 54.53'' N, 30° 54' 12.81'' E 79.30 7.88 1850.0


Agricultural drainage canal

30° 53' 20.55'' N, 30° 52' 15.67'' E 107.35 7.45 916.7



30° 54' 26.90'' N, 30° 51' 32.84'' E ----- 8.42 1033.3

5 L. gibba Kafr El-Sheikh Governorate Irrigation canal 31° 01' 23.49'' N,

30° 57' 43.72'' E 180.75 8.46 766. 7

6 L. gibba Kafr El-Sheikh Governorate Irrigation canal 31° 01' 23.49'' N,

30° 57' 43.72'' E 81.69 8.46 766. 7

7 L. gibba Kafr El-Sheikh Governorate


31° 01' 34.43'' N, 30° 57' 44.75'' E 275.61 7.7 1116. 7

8 L. minor El-Gharbiya Governorate


30° 53' 42.02'' N, 30° 52' 16.80'' E 40.82 7.51 1266. 7

9 L. minor El-Gharbiya Governorate


30° 54' 26.90'' N, 30° 51' 32.84'' E 77.32 8.42 1033.3

El-Kholy et al., Genetic Diversity of Lemna Gibba L. & L. Minor L. Populations in Nile Delta Based on Biochemical and Issr Markers


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Biochemical analysis and ISSR-PCR:

The col lected samples of L. gibba and L. minor for biochemical analysis and ISSR-PCR fingerprint ing were washed several times by tape water, then by dist i lled water, f rozen by l i quid ni trogen and kept at - 20°C. Samples were prepared for protein extract ion by homogenizing 0.5 g in 1 ml of 0.125 M tris borate buf fer pH 8.9 with 2% SDS, the extracts were centr ifuged at 10000 rpm for 20 min and supernatants were stored at – 20ºC unt i l used. Extracts were denatured before being loaded on gel in sample buffer containing 5% 2-mercaptoethanol and heat ing at 100°C for 4 min. One dimensional SDS-PAGE was performed using 12% (w/v) polyacrylamide gel (Laemmli, 1970). The gel was stained wi th Coomassie brili ant blue R250 and visual i zed in whi te f luorescent li ght.

For isozymes detect ion, 0.5 g of young fronds from each populat ion was homogenized in 1 m l tris borate buffer pH 8.9. The homogenate was centr ifuged at 15000 rpm for 20 min at 4°C. The supernatant was immediately used for nat ive electrophoret ic separat ions or stored at – 20°C unt i l use. Separat ing gels of 10% polyacrylamide were prepared (Pasteur et a l. , 1988). Then the gels were stained in speci f ic substrates for each enzyme according to (Sol tis et al. , 1983). Five enzymatic systems were assayed: Peroxidase (POX), α- and β-Esterase (EST), Catalase (CAT), and Glutamate oxaloacetate transaminase (GOT).

Genomic DNA was ext racted from frozen fronds of the examined populat ions of L. gibba and L. minor using Gene JET Plant Genomic DNA Puri ficat ion Mini kit #k0791 (Thermo Fisher Scient i fic, Inc) according to manufacturer 's instruct ions. The isolated DNA was quanti f ied by 1% agarose gel electrophoresis in 1X Tri s-borate-ethylenediaminetet raacet ic acid (TBE) buffer using Thermo Scient ific Gene-Ruler DNA ladder 1 kb of known concentrat ion (100 ng µl–1) . Ethidium bromide-stained gel was visualized and documented by using the Gel Documentat ion System (CFW-1312M; BioRad).

Five UBC set no. 9 ISSR primers were synthesized by the Biosearch Technologies, Inc. USA were independently used in the PCR react ions. The ISSR fingerprinting was performed by using each of the used primers (1.0 µM) in 25 µl react ion volume containing genomic DNA (10 ng), ddH2O (8 µl) and 12.5 µl Dream Taq Green PCR Master Mix #k1081 (Thermo Fisher Scient if i c, Inc.). PCR ampli f icat ion was performed using a Primus 25 advanced® cycler machine and performed as fol lows: initi al denaturat ion at 95°C for 5 min, 40 cycles at 95°C for 1 min; 50°C for 30 Sec and 72°C for 1 min followed by an

extension for 8 m in at 72°C. On complet ion of PCR, 10 µl of the PCR products were el ectrophoresed in 1.6% agarose gels containing ethidium bromide in 1X TBE buf fer in a submarine gel apparatus at 100 V. The gels were visual i zed under ul traviolet l ight, and photographed using a gel documentat ion system (CFW-1312M; Bio-Rad). All react ions were performed at least twice, and only stable products were scored. Data analysis:

For i sozyme data analysis, relat ive mobili t y (Rf) of bands was calculated. Bands having very close Rf values were grouped as members of a dist inct zone ( locus). W hen these zones are expressed independently, they were considered as an expression of a single locus and the bands within this zone are dealt as an expression of al leles within that locus (Pasteur et a l. , 1988). Calculation of allele frequency was carr ied out to f ind a correlat ion of individual trai ts.

The protein and DNA bands wer e scored in binary matrices, where 0 stands for the absence and 1 stands for the presence of bands in the prof ile of each individual populat ion. Similari ti es between populat ions and species were est imated using Dice coef f icient of similar ity (Dice, 1945) and the data were analyzed by the NTSYS-pc software (Rohlf, 2002). Construction of the trees i llustrating the geneti c distance among the studied populat ions was performed using the unweighted pai r group method using the ari thmetic average (UPGMA) (Sokal and Mickener, 1958) as implemented in the NT-SYS-pc. The genetic relat ionship among examined populat ions was est imated based SDS-PAGE data and isozymes data, and ISSR data separately and in combinat ion.

RESULTS:

The dry matter of the studied populations of L. gibba ranged from 17.30 to 275.61 g DM m -2 recorded for populations 1 and 7, respectively. Populations 8 and 9 of L. minor recorded 40.82 and 77.32 g DM m -2

(Table 1). Recorded pH values ranged from 7.43 in the si te of populat ions 1 and 8.46 in site of populat ions 5 and 6. On the other hand, EC ranged from 766.7 µS cm −1 in si te of populations 5 and 6 to 1850.0 µS cm − 1 in si te of population 2. Significant negative correlat ion was evident between EC values and estimated dry matter of both L. gibba and L. minor populations (Table 2). pH values showed non-signif icant correlation with dry matter (r = 0.17) of L. gibba populat ions. In contrast, i t showed highly significant correlat ion with L. minor populations (r = 0.97).



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Table 2. Correlation coefficients between dry matter of L.gibba and L. minor and pH and EC of water courses

Species pH EC

L. gibba, Dry Matter

0.17ns

− 0.604*

L. minor, Dry Matter

0.97***

− 0.96***

*: P < 0.05, ***: P < 0.001, ns: non-significant (P > 0.05). The analysis of protein banding patterns

of the studied populations showed geneti c polymorphism (Fig. 1). The overall polymorphism was 92%; while the intra-

species polymorphism was 88% and 24% for L. gibba and L. minor , respecti vely. The total number of recorded bands was 26 and for each species 17 bands were detected. From al l investigated populations, 2 common bands (120 and 47 KDa) and 6 species-specific bands to L. minor were detected. The polymorphic bands include 4 posit ive bands unique to 4 populations; 2 bands (54, 38 KDa) are unique to populat ion 9, band (23 KDa) is unique to population 8 and band (29 KDa) is unique to population 4.

KDa M 1 2 3 4 5 6 7 8 9

200 150 120 100 85 70 60

50

40

30

25 20

Fig. 1. Protein banding patterns produced by SDS-PAGE analysis in 9 populations of Lemna spp. For

localit ies and sites of the populations see table 1. From the examined populat ions, 11

isozyme loci were resolved from the enzymes which assayed and showed 19 al leles. The POX zymograms reveal 4 zones of act ivi ty and the second zone is species-speci f ic to L. minor and shows heterozygosity for i ts populat ions. The act ivi ty of both α-esterase and β-esterase were demonstrated. The two loci (α-Est-1 and β-Est-1) at the anodal area are speci f ic for L. minor and can be considered as biochemical markers for the di fferent iat ion between the two species. They exhibi t heterozygosity for populat ion 8, homozygosity for populat ion 9 and no act ivi ty in other L. g ibba populat ions. The α-Est-2 locus showed 3 polymorphic alleles which were detected only in L. g ibba populat ions col lected from Kafr El -Sheikh (5, 6, and 7) ref lect ing heterozygous genotypes of these populat ions. On the other hand, β-Est-2 locus showed a homozygous genotype for the above three populat ions and no act ivi ty was detected for other invest igated populat ions. Figure 2 i l lustrates the patterns of activity of the previous isozyme systems.

A- Peroxidase

B- α-esterase



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C- β-esterase Fig. 2. Patterns of three different isozyme systemes (POX,

α-EST and β-EST) in 9 populations of Lemna spp. For localities and sites of the populations see table 1.

The zymograms of catalase are characterized by one zone of activity and polymorphism was observed between populations with 2 alleles producing 2 different allozymes. The gel treated for manifestation of GOT activity displays 2 loci at the cathodal area. The loci displayed species-specificity, where the locus GOT-1 is expressed only in L. gibba populations and the activity of the locus GOT-2 is detected only for L. minor.

The ISSR primers used in the present study are l isted in table 3. Al l primers generated 100% polymorphism for all populat ions. The number of bands generated per pr imer varied from 4 (UBC 861) to 13 bands (UBC 857). Each of the studied populat ions showed a unique ISSR genotype. Among L. g ibba populat ions, 12 posi tive unique bands were detected. Populat ion 6 expressed 3 posi tive unique bands, 3 negat ive unique bands, and 9 unique posi tive bands were distributed in other populat ions. Results of ISSR fingerprinting of all primers with regard to the number of total , polymorphic, monomorphic and unique bands are summarized in table 4. Table 3. List of primers used for ISSR amplification.

No Primer Sequence (5'–3') 1 UBC 856 ACA CAC ACA CAC ACA CCA 2 UBC 855 ACA CAC ACA CAC ACA CCT 3 UBC 861 ACC ACC ACC ACC ACC ACC 4 UBC 857 ACA CAC ACA CAC ACA CTG 5 UBC 845 CTC TCT CTC TCT CTC TGG

Table 4. Number and type of protein bands generated by SDS-PAGE and amplified bands generated by five primers used in ISSR fingerprinting of the examined 9 populations of Lemna

Protein data ISSR data Type of bands All L. gibba L. minor All L. gibba L. minor

Monomorphic 2 2 13 - 1 7 Polymorphic 24 15 4 47 33 17 % Polymorphism 92% 88% 24% 100% 97% 63% Unique 4 4 4 16 15 17 Total 26 17 17 47 34 24

Figure 3 illustrates an example of ISSR fingerprinting by primer UBC 857. The number of recorded bands was 13 with 100% polymorphism. The unique bands (950 bp, 450 bp and 550 bp) were observed in populations 1, 3, and 6, respectively. There were 2 species-specific bands to L. minor with molecular size of 750 bp and 300 bp. This primer differentiated between the two populations of L. minor by a band of molecular size 500 bp. Primer UBC 856 produced 10 bands, from which 2 unique bands in

populations 1 and 4 of L. gibba and 2 unique bands in populations 8 and 9 of L. minor. The profiles produced by primer UBC 855 showed 11 polymorphic bands from which 2 bands were unique to population 1 and 2 bands unique to population 8. The bands generated by primer UBC 861 were 4; one band is species- specific to L. gibba and another one to L. minor. The fifth primer UBC 845 produced 9 bands; 5 bands were unique to population 9 and one unique band to population 3.

Fig. 3. Example of ISSR fingerprinting revealed by primer UBC 857 in 9 Populations of Lemna spp. For localities and

sites of the populations see table 1.

M 1 2 3 4 5 6 7 8 9 size bp

250

500

750

1000

1500 2000



16

Three UPGMA trees based on biochemical data, ISSR data and combination of both of them were constructed (Figs 4, 5, and 6). All trees were found to have simi lar topology and divided al l populat ions into two main clusters at about 1.52 dissimilari ty distances. One cluster included the populations of L. minor (8 and 9). The second cluster involved the populations of L. gibba (1-7) and was separated as two sub-clusters

at distances of about 1.20; the fi rst sub-cluster compressed the populat ions 1, 2, 3, and 4 which were collected from El -Gharbia Governorate. Populations 5, 6 and 7 that were collected from Kafr El-Sheikh Governorate, were del imited together at the second sub-cluster. However population 6 was distinguished from all other populations of the same species.

0.46 0.72 0.98 1.24 1.50

1

2

3

4

5

6

7

8

9

Genetic dissimilarity distance

Fig. 4. Dendrogram of 7 populations of Lemna gibba (1-7) and 2 populations of L. Minor (8 and 9) based on SDS-PAGE protein data and allele frequencies at different loci of three isozyme systems (POX, α-EST and β-EST) using the UPGMA method. For localities and sites of the populations see table 1.


Fig. 5. Dendrogram of 7 populations of Lemna gibba (1-7) and 2 populations of L. minor (8 and 9) based on ISSR fingerprinting using the UPGMA method. For localities and sites of the populations see table 1.



17


Fig. 6. Dendrogram of 7 populations of Lemna gibba (1-7) and 2 populations of L. minor (8 and 9) based on biochemical analysis ISSR fingerprinting using the UPGMA method. For localities and sites of the populations see table 1.

DISCUSSION: The genetic diversi ty encountered in the

Lemna populations might be due to numerous factors. The adaptation of the examined populations to different types of water courses could be the main reason. The variation of the aquatic habitats was ref lected in the recorded ranges of pH (7.43-8.46), EC (766.7 – 1850 µS cm− 1) and plant yield (17.30 - 275.61 g DM m -2). According to previous studies pH value plays a significant role in toxicity assessment (Martin, 1987). Waste treatment using L. g ibba becomes impossible at pH levels above approximately 9.8 depending on the temperature (Korner et al., 2001). The pH values in these studies are considered within the permissible limit that lies between 6 and 8.4 according to (MWE, 2006).

Populations 1 and 2 that were col lected from mixed agriculture and industrial drainage with the highest EC values (1083.3) and (1850 µS cm−1), respecti vely, produced the lowest dry matter (17.3 and 79.3 g DM m -2). The estimated variation of EC and pH values might be due to dif ferent levels of water pollution in the sites. Industrial effluents are a main source of direct and often continuous input of pollutants/toxicants into aquati c ecosystems with long-term implications on ecosystem functioning (Smolders et al. , 2004).

Considerable level of genet ic variation was revealed by electrophoretic analysis of protein and five enzyme systems. Other studies showed signif icant amounts of geneti c variability in local populations of L. minor in Canada (Vasseur et al. , 1993) and in 11 Minnesota populations of L. minor (Cole and Voskuil, 1996). Also, Al lozymes were used to

study taxonomical relationships and genetic variations between and within different species in Lemnaceae all over the world (Crawford et al. 1997, 2005). These results are in contrast to the reports of Wain et al. (1985) which concluded that aquatic plants contained relati vely litt le variabili ty at the species level and their constituent populations were poorly di fferent iated.

Theoret ically, the diversity of al lozyme appears to be correlated with speci fic polluted environments (Mukherjee et al. , 2004). The previous author found that esterase enzyme di fferentiated between metal exposed and non-metal exposed L. minor populations by three loci and was considered a potential biomarker of heavy metal pollution. Also, biochemical changes and the enzyme activity in L. minor plants under lead and cadmium stresses is considered as an indicator of heavy metal toxicity (Mohan, 1997).

Al l ISSR primers generated 100% polymorphism for nine L. populations collecting from Ni le Delta. The same primers accounted 100% polymorphism for 16 Lemna populations col lecting from di fferent regions of Vietnam and West China (Xue et al., 2012). There are 7 species-specific bands from 47 bands in ISSR prof iles indicating the ef ficiency of ISSR marker to di fferentiate between L. g ibba and L. minor. Using UPGMA trees based on biochemical data, ISSR data and combination between them, the populations of L. minor were del imited as a separate cluster from the populations of L. gibba. Azer (2013) divided Lemna species in Egypt into 3 clusters based on morphological traits; L. gibba and L. minor ; were clearly distinguished from L. aequinoct ial is and L. tr isulca , respectively. Populations 5, 6 and 7




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of L. gibba (Kafr El-Sheikh Governorate) were separated in a sub cluster from the other populations (El -Gharbiya Governorate) except that tree based on ISSR fingerprinting separated population 6 alone. Xue et al. (2012) reported geographic distribution had significant influence on the population's geneti c diversity.

Ecotypic dif ferent iation within aquatic plant species was observed over small distances suggesting that it is caused by selection pressures rather than by reduced dispersal (Santamaria, 2002). Isolation by distance at various scales has been demonstrated previously in some aquati c macrophytes; Eichhornia panicu lata (Husband and Barrett, 1995) and Zostera marina (Reusch, 2000). Other aquatic species, however, have not shown this correlation as in Minnesota populations of L. minor (Cole and Voskuil, 1996) and L. gibba populations col lected from dif ferent regions all over the world (Crawford et al. , 2005). Also, Cluster analysis by RAPD markers failed to reveal

intraspecific differentiation l inked to the geographic distribution of the Lemnaceae accessions and this may be conditioned by the absence of territorial i solation due to possible dispersal of Lemnaceae along river beds and with migrating water birds (Marti rosyan et al. , 2008).

In Conclusion, the collection sites have obvious influence on the geneti c diversity of Lemna species. Lemna minor populations were separated from L. gibba populations. The populations of the latter species that were collected from El-Gharbiya were separated from those collected from Kafr El-Sheikh.

ACKNOWLEDGEMENT We pay genuine gratefulness towards

Prof. Dr. Abdelfattah Badr for his valuable suggestions in the manuscript and his productive revision.

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بین عشائر عدس الماء المحبب وعدس الماء الصغیر من دلتا النیل باستخدام طرق التنوع الوراثى كیمیائیة ومؤشرات التكرارات البینیة البسیطة

عزيزة سلیمان الخولى ، محمد سمیر يوسف ، ابراھیم محمد عید ، مصرجامعة كفر الشیخ، كلیة العلوم، قسم النبات

وقد تم فصل عشائر . ةعالیة من التباين بین العشائر المختلفعدس الماء المحبب عن عشائر عدس الماء الصغیر فى مجموعتین وكذلك فصل عشائر عدس الماء المحبب المجمعة من محافظة كفر الشیخ عن تلك المجمعة من محافظة الغربیة فى شجرة تمثل العالقات الوراثیة بین العشائر المختلفة والتى

.أجريت علیھا الدراسة

:ونالمحكم حلوانمعبد الفتاح بدر قسم النبات، علو. د.أ عادل الشنشوري قسم النبات، علوم طنطا. د.أ

أجريت ھذه الدراسة لتقدير التباين الوراثى بین تسع عشائر لنباتى عدس الماء المحبب وعدس الماء الصغیر من

صادر مائیة وقد تم تجمیع النباتات من م. فصیلة عدس الماءمتنوعة من دلتا النیل فى مصر منھا قنوات رى وقنوات صرف . زراعى وقنوات مختلطة من صرف زراعى وصناعى

واستخدمت طريقة التفريد الكھربى للبروتینات والنظائراستريز، - استريز ، بیتا- بیرأوكسیديز ، ألفا(االنزيمیة لالنزيمات

وتباين ) كاتالیز، جلوتامیت أوكسالوأسیتات ترانسفیريزمؤشرات التكرارات البسیطة للحمض النووى ذلك لتقدير

وقد أظھرت النتائج الخاصة . التباين بین ھذه العشائر النووى درجة بالبروتینات والنظائر االنزيمیة ومؤشرات الحمض

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