Printer Friendly

ASSESSMENT OF GENETIC DIVERSITY WITHIN GERMPLASM ACCESSIONS IN.

Byline: S. Naz, A. Zafrullah, K. Shahzadhi and N. Munir

ABSTRACT

A total of 25 high yielding tomato accessions were selected for the characterization that helped in the reliable varietal selection programme for breeding. All tomato accessions were analyzed by two parameters e.g. morphological and molecular parameters. For morphological characterization the plant height, shape, size, leaf length and width, and fruit size, colour and shape were selected and for molecular characterization RAPD markers were used. A total 25 RAPD decamer primers were selected for the genetic analysis of all tomato accessions. Only 15 polymorphic RAPD primers were accessed for the genetic distance calculation to find out the phylogenetic relationship among 25 tomato accessions under study. A total of 130 loci were generated outof which 98 were polymorphic by 15 primers with 05-14 loci/primer having fragment's size range from 400to 2500bp maximum. The Nie and Lie's Coefficients was used to calculate the genetic similarity.

The extent of genetic diversity and construction of phylogenetic tree was done by DNAMANN software. The average genetic similarity observed across all the genotypes was 75.6% with 24.4% polymorphismin 25 tomato accessions. Although RAPD study supports the morphological characters but not upto 100%.

Key words: Randomly amplified polymorphic DNA, Polymerase chain reaction, Lycopersiconesculentum.

INTRODUCTION

Tomato is an herbaceous plant belongs to family Solanaceae or Nightshade family. It is one of the significant vegetable crops of special economic importance in the horticultural industry, originating in South America and its many varieties are now commonly grown in greenhouses in cooler climates(He et al.,2003).Itis the most popular garden vegetable belongs to the genus Lycopersicon, the resemblance between leaves and flowers of potato and tomato plants seems to certify this taxonomic grouping (Wang et al., 2005 and Shidfar et al., 2011).

The popularity of tomato and its products continue to rise as it is a good source of vitamin A and C in significant amount. It is extensively used in salad as well as for culinary purposes and also used for various processed formsinclude pastes, sauces, pulps, juices, ketchup and as flavoringingredients in soups, meat or fish dishes(Gosselin and Trudel, 1984).The fruit contains significant amounts of lycopene, beta-carotene, magnesium, iron, phosphorus, potassium, riboflavin, niacin, sodium and thiamine. It has antioxidant properties and potential beneficial health effects (Zhang et al., 2009). Tomato intake is reduced entire cholesterol, LDL cholesterol and triglyceride levels in white blood cells, reducing cardiovascular risk related with type 2 diabetes also decreased risk of breast cancer, neck cancers and strongly protective against neurodegenerative diseases (Freedmanet al., 2008).

Tomato is grown worldwide for its edible fruitshaving bisexual flowers, although often grown outdoors intemperateclimates as they do not tolerate frost.The tomato species cultivate as annuals in colder regions while they are perennial in warmer regions, it is a self-pollinated crop but in some cases as high as 30% cross-pollination. The plant has compound leaves, made up of leaflets which are arranged along with 2 to 6 opposite or sub-opposite pairs of petiolate and subsessileleaflets. (Peralta et al., 2005).Tomato cultivars produce red, yellow, pink, green, black or white fruit and they have been selected with varying fruit typesand for optimum growth in differing growing conditions. The plants normally grow to 1-3 meters (3-10 ft) in height and having slender and herbaceous but weak stem that sprawls over the ground and vines over other plants.(Rico-Garcia et al., 2009).

Tomato is the well-studied crop species forbreeding, genetics and genomics. It is one of the initial crop plants for which a genetic linkage map was constructed, presently there are several molecular maps based on crosses between the cultivars and many wild species of tomato. Genetic analysis of tomato is essential to enhance the genetic yield potential and maximum utilization of the desirable characters for synthesizing of any ideal genotypes (Kumar et al.,2003).The assessment of genetic diversity within and between populations of tomato varieties is measuredby using morphological, biochemical and molecular characterization (Garcia et al.,2004).Morphological markers have several defacts that reduce the ability to estimate genetic diversity in plants as it highly dependent on the environment for expression.

Molecular markers can give an effective tool for efficient selection of desired agronomic traits because theyare based on the plant genotypes and also are independent of environmental variation (Francoet al., 2001). Researchers have been calculated genetic variation in tomato landrace and cultivar collections using several molecular techniquesincluding amplified fragment length polymorphism (AFLP), restricted fragment length polymorphism (RFLP), simple sequence repeats (SSR) and random amplified polymorphic DNA (RAPD) (Bredemeijer et al., 1998, Villand et al., 1998, Park et al., 2004 and Garcia-Martinez et al., 2006).

The RAPD is the first PCR-based molecular markers technique to develop DNA marker for detecting and monitoring pedigree breeding record of inbred parents or varieties evaluation in test crosses and determining genetic relationships among genotypes (Dongre and Parkhi, 2005). It is an efficient method for varietal identification, study of polymorphism, gene mapping, biodiversity, genetic map construction, hybridization and phylogenetic relationship in tomato varieties (Sharma and Sharma, 1999).

The aim of present study is to observethe morphological characters, genetic diversity within different tomato accessions by using random amplified polymorphic DNA (RAPD) markers and development of phylogentic tree by using bio informatics tools. Moreover, enterprise the protocol for DNA extraction and PCR amplification and development of phylogentic tree of different tomato accessions by using bio informatic tools.

MATERIALS AND METHODS

Seeds of tomato accessions obtained from National institute of Genetic resources (Islamabad, Pakistan) were grown in green house for germination and growth. After two weeks of sowing sprouting started in almost all accessions and after 4-5 weeks of regular watering these plants were grown at a fair height of 60-70 cm.

Table 1. Name of Tomato accessions for DNA extraction

1.###Tm 006233###2.###Tm 017856###3.###Tm 017860###4.###Tm 017869###5.###Tm 017870

6.###Tm 017872###7.###Tm 017873###8.###Tm 017874###9.###Tm 017875###10.###Tm 017876

11.###Tm 017877###12.###Tm 017878###13.###Tm 019842###14.###Tm 019843###15.###Tm 019844

16.###Tm 019846###17.###Tm 019849###18.###Tm 019851###19.###Tm 019852###20.###Tm 019853

21.###Tm 019855###22.###Tm 019856###23.###Tm 019857###24.###Tm 019860###25.###Tm 101159

Morphological Characterization of Tomato accessions:Morphological characters were studied in selected tomato accessions by already set standards for morphological characters by IPGRI (International Plant Genetic Resources Institute) tomato descriptor (Darwin et al., 2003). These Characterizations include the plant growth type and size, leaf shape, size and arrangement, plant height and fruit morphology i.enumber of fruits per plant, immature and mature colure. For Molecular characterizationRAPD (Random Amplified Polymorphic DNA) analysis selected for the estimation of genetic diversity and phylogeny among these tomato accessions.

DNA extraction method: Total genomic DNA was isolated from fresh and healthy leaves using the CTAB (hexadecyltrimethylammonium-bromide) method (Murray and Thompson, 1980) with few modifications. Briefly, 1g of leaves was ground in liquid nitrogen to a fine powder. The powder was added to 3 mL of extraction buffer (100 mMTris-HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl, 2% (wv-1) CTAB, 2- mercaptoethanol 2% and incubated at 65 degC for 30 min. The DNA was extracted with chloroform - octanol (24: 1). The DNA was washed with 70% ethanol and dissolved in 100-400 ml of T.E (10 mMTris-HCl pH 8.0, 1 mM EDTA and 0.2 -1 mg/ml RNAase). DNA is quantified by spectrophotometer by using a comparison of the optical density values of the solution at A260/A280 wavelengths. Stock DNA samples were stored at -20 degC and diluted to 20 ng mL-1 when it use.

RAPD analysis: The RAPD primers were purchased from Invitrogen product (Invitrogen, USA). A total of 25 decamer oligonucleotides of arbitrary sequence were tested for PCR amplification. The basic protocol reported by William et al. (1990) for PCR was performed in a total volume of 15 ml, containing 20 ng mL of template DNA, 0.4 mM of single primer, 0.6 U Taq DNA polymerase (Invitrogen, USA) 0.20 mM of dNTPs, 1.5 mM MgCl2, 10 mMTris-HCl,and 50 mMKCl.

DNA amplification was carried out in advanced Primus-96 Thermal cycler and the thermal cycler conditions for PCR reactions were an initial denaturation cycle of 94degC for 4 minutes was followed by 35 cycles comprising 30 sec at 94degC, 1 min at 36degC and 2 min at 72degC. An additional cycle of 7 min at 68degC was used for final extension. Amplification products were separated by electrophoresis in 1.0 % agarose gels and stained in ethidium bromide. A photographic record was taken under UV illumination.

Statistical analysis: Only clear and repeatable amplification products were scored as 1 for present bands and 0 for absent ones. The specific bands useful for identifying different varieties were named with a primer number followed by the approximate size of the amplified fragment in base pairs. Polymorphism was calculated based on the presence or absence of bands. The 0 or 1 data matrix was created and used to calculate the genetic distance and similarity using DNAMANN software. The dendrogram was constructed by using a distance matrix using Nie and Lie's Coefficients to access the genetic similarity and dissimilarity among all accessions.

RESULTS

Morphological characterization:All25 selected tomato accessions were characterized morphologically in this study by comparing the height of plant, leaf length, shape and arrangement, fruit shape and size.

This study revealed that maximum height of plant was 80 cm in Tm 019856 while minimum height of plant was 20 cm in Tm 019843.The maximum leaf length (7.8 cm)was noted in Tm 019856and minimum length(5 cm) was found in Tm 019846.In fruit morphology fruit colour was light green (at immature stage) and red (at mature stage) was observed with rounded in shape. Similarly Maximum fruit size (4.5 cm) was obtained by Tm 019853 while minimum fruit size (2 cm) was found in Tm 017870.

RAPD-PCR characterization of varieties:

Genetic polymorphism amongtomato accessions: After screening 25 primers, 15 primers produced polymorphic and repeatable products. The banding profile and polymorphism generated using the primer (TP-05) (Figure 4.2.3) are shown. PCR amplification of the DNA isolated from 25 selected tomato accessions yielded a total of 130 amplified products out of which 98 were polymorphic and 32 were monomorphic (Table 2). The total no. of DNA bands amplified varied between 05 (Primer TP- 09 andTP-13) and 14 (Primer TP-01) with the average of 8.6 bands per primer. The maximum no. of polymorphic bands (14) was obtained with primers TP-01 and the minimum number (05) was obtained with primer TP-09 and primer TP-13.The polymorphism percentage ranged from 42.85% (Primer TP-07) to as high as 91.66% for primer (TP-05). Average polymorphism across 25 selected tomato accessions was found to be 72.6%. Overall size of the PCR amplified product bands ranged from 400bp to 2500 bp.

Polymorphism analysis was done for 25 tomato accessionsOut of these25, 15tomato accessionswere monomorphic and 10 were polymorphic. Primer TP 05 generated maximum polymorphic bands and Primer TP 08 produced the minimum number of bands. The highest level of polymorphism was detected with primer CP 05 (91%) whereas primer TP 08 detected the least polymorphism (42%). (Table 2).

Multiple sequence alignment by DNAMAN software: The multiple alignments of the sequences generated by using 0-1 matrix method for gel scoring. DNAMAN Software aligned the similar sequences and showed a sequence similarity index of 75.60% in the DNA of 25 selected tomato accessions with dissimilarity index of 24.4%. Phylogenetic tree wasgenerated for phylogenetic studies among 25 tomato accessions in the present study.

DISCUSSION

The main aim of present study was to characterize different tomato accessions in Pakistan through morphological and molecular markers and then the generation of phylogenetic tree for these varieties on the basis of RAPD fingerprints. Morphological characterization included the stem and plant growth type, plant height (before transplanting), leaves type, size and arrangement, inflorescence type and exterior colour of immature and mature fruit, shape and size were studied.

Molecular characterization was carried out through RAPD molecular technique by using 25 decamer primers, out of which 15 primers showed polymorphism. Genomic DNA was amplified by 15 polymorphic primers that generated a total 130 loci in all varieties. Maximum number of loci 14 was obtained in the genome of Tm 019851 and minimum number of loci 05 in the genome of Tm 017872.

Polymorphism was estimated between 25 tomato accessions by 25 decamer primers with different sequence out of which 15 primers showed about 72.6% polymorphism in all accessions of tomato. By using 08 decamer RAPD primers, 228 DNA bands were obtained among 36 tomato cultivars (Huhet al., 2011). They also found total of number of bands generated by eachprimer was range from 2 to 11 with an average of 6.3 bands per primer. Bernardette et al., (2006) also estimated the variability of 35 tomato accessions in which 257 reproducibly bands were obtained from 20 primers with 78.6% polymorphic. Seventy-four (74) amplified products were scored with 62.2% of polymorphism in 14 tomato varieties were reported byEzekielet al., (2011).

Phylogenetic dendrogramme was constructed among selected varieties by using RAPD fingerprints through DNAMAN computerizes software. Elham et al., (2010) also reported the phylogenetic diversity and relationships of some tomato varieties by using RAPD analysis and Ezekiel et al., (2011) studied the genetic diversity in 14 tomato varieties by RAPD-PCR technique.

Table 2. Identity, sequence, number of total bands, range of fragment size, polymorphic bands and polymorphic percentage for 10 RAPD decamer polymorphic primers used on 25 selected tomato accessions.

###Total number of###Range of band###Total number of###Rate of

Primer###Primer Sequence

###loci produced###size(bp)###polymorphic bands###Polymorphism %

01###AGTCAGCCAC###14###500 -1400###11###78

02###GTTGCCAGCC###12###400 - 1500###10###83

03###GGGGTGACGA###07###400 - 2500###04###57

04###GAGACGCACA###05###500 -1200###03###60

05###AACGGCGACA###12###400 - 2000###11###91

06###CAGAAGCGGA###06###400 -1000###04###66

07###GACACGGACC###09###500 - 1500###08###88

08###CTCACCGTCC###07###500 - 1300###03###42

09###CCACAGCAGT###07###500 - 1200###05###71

10###ACGACCGACA###11###500 - 1300###09###81

11###AGGGAACGAG###09###500-1400###07###77

12###AGGTGACCGT###06###400-1200###04###66

13###CTGCTGGGAC###06###500-1500###05###83

14###GTGAGGCGTC###10###400-2000###08###80

15###AATCGGGCTG###09###600-1500###06###66

Total###15###130###98###72.6%

REFERENCES

Bernardette, P.C., L.T. Sheng, F.G. Grazziotin and E. Sergio (2006). Genetic Diversity among Brazilian Cultivars and Landraces of Tomato Lycopersiconesculentum Mill. Revealed by RAPD Markers. Genetic Resources and Crop Evolution, 53(2), 395-400.

Bredemeijer, G. M. M., P. Arens, D. Wouters, D. Visserand. Vosman (1998). The use of semi- automated fluorescent microsatellite analysis for tomato cultivars identification. Theor. Appl. Genet., 97, 584-590.

Darwin, S.C., S. Knapp and I.E. Peralta (2003).

Taxonomy of tomatoes in the Galapagos Islands: Native and introduced species of Solanum section Lycopersicon (Solanaceae). Syst. Biodiversity, 12, 29-53.

Dongre, A. and V. Parkhi (2005). Identification of cotton hybrid through the combination of PCR based RAPD, ISSR and microsatellite markers. J. Plant Biochem. Biotechnol.,14, 53-55.

Doyle, J. J. and J. L. Doyle (1990). A rapid total DNA preparation procedure for fresh plant tissue. Focus, 12, 13-15.

Elham, A.A., A.E. Hady, A.A. Atef, S. Haiba, R. Nagwa, A.E. Hamid and A. Aida (2010). Phylogenetic Diversity and Relationships of Some Tomato Varieties by Electrophoretic Protein and RAPD analysis. J. American Sci., 6(11), 434-441.

Ezekiel, C.N.1., C. Nwangburuka, O.A. Ajibade and A.C. Odebode (2011). Genetic diversity in 14 tomato (Lycopersiconesculentum Mill.) varieties in Nigerian markets byRAPD-PCR technique. African J. Biotech., 10(25), 4961-4967.

Franco, J., J. Crossa, J.M. Ribaut, J. Betran, M.L. Warburton, M. Khairallah (2001). A method for combining molecular markersand phenotypic attributes for classifying plant genotypes. Theor Appl Genet., 103,944-952.

Freedman, N.D., Y. Park and A.F. Subar (2008). Fruit and vegetable intake and head and neck cancer risk in a large United States prospective cohort study. Intl J. Cancer, 122 (10), 2330-6.

Garcia, A.A.F., L.L. Benchimol, A.M.M. Barbosa, I.O.

Geraldi and C.L. Souza (2004). Comparison of RAPD, RFLP, AFLP, and SSR markers for diversity studies in tropical maize inbred lines. Genet Mol Biol., 27,579-588.

Garcia-Martinez, S., L. Andreani, M. Garcia-Gusano, F. Geuna and J.J Ruiz (2006). Evaluation of amplified fragment length polymorphism and simple sequence repeats for tomato germplasm fingerprinting: utility for grouping closely related traditional cultivars. Genome, 49,648-656.

Gosselin, A. and M.J. Trudel (1984). Interactions between root-zone temperature and light levels on growth, development and photosynthesis of Lycopersiconesculentum Mill. Cultivar 'vendor'. Sci. Hortic., 23, 313-321.

He, C., V. Poysa, and K. Yu (2003). Development and characterization of simple sequence repeat (SSR) markers and their use in determining relationship among Lycopersiconesculentum cultivars. Theor. Appl. Genet., 10, 363-373.

Huh. M.K., S.J. Youn and S.C. Kang (2011).

Identification and Genetic Diversity of Korean Tomato Cultivars by RAPD Markers. J. Life Sci.,21(1),15-21.

Kumar, S., R. Kumar, S. Kumar, M. Singh, M.K. Banerjee and M. Rai(2003). Hybrid seed production of Solanaceous vegetables. A Practical Manual, II VR Technical Bulletin, 9, 1- 34.

Murray, M. G. and W. F. Thompson (1980). Rapid isolation of high molecular weight plantDNA. Nuclic acid research., 8 (19), 4321-4326.

Park, Y. H., M.A.L. West, A. Stiford. and D. Clair (2004). Evaluation of AFLPs for germplasm fingerprinting and assessment of genetic diversity in cultivars of tomato (Lycopersiconesculentum L.). Genome, 47, 510- 518.

Peralta, I.E. and D.M. Spooner (2005). Morphological characterization and relationships of wild tomatoes (Solanum L. Section Lycopersicon) Monogr. Syst. Bot., Missouri Bot Gard.,104, 227- 257.

Rico-Garcia, E., F. Hernandez, G. Soto-Zarazuaand G. Herrera-Ruiz (2009). Two new Methods for the Estimation of Leaf Area using Digital Photography. Int. J. Agric. Biol., 11(4), 397-400.

Sharma, A.K. and A. Sharma (1999). Plant Chromosomes Analysis Mainpulation and Engineering. ISBN.,90(3), 5702-387.

Shidfar, F., N. Froghifar, M. Vafa, A .Rajab, S. Hosseini, S. Shidfar and M. Gohari (2011). The effects of tomato consumption on serum glucose, apolipoprotein B, apolipoprotein A-I, homocysteine and blood pressure in type 2 diabetic patients. Int J Food Sci Nutr., 62(3), 289-94.

Villand, J., P. Skroch, W. Lai, P. Hanson, C.G. Kuosand J. Nienhuis (1998). Genetic variation among tomato accessions from primary and secondary centers of diversity. Crop Sci., 38, 1339-1347.

Wang, X.F., R. Knoblauch and N. Leist (2005). Varietal discrimination of tomato (Lycopersiconesculentum L.) by ultrathin-layer isoelectric focusing of seed protein. Seed Sci. Technol., 28, 521-526.

Zhang, C.X., J.H. Fu, S.Z. Cheng and F.Y. Lin (2009).

Greater vegetable and fruit intake is associated with a lower risk of breast cancer among Chinese women. Intl. J. Cancer, 125 (1), 181-8.
COPYRIGHT 2013 Asianet-Pakistan
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2013 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Publication:Journal of Animal and Plant Sciences
Date:Aug 31, 2013
Words:3140
Previous Article:ANTIBODIES RESPONSE OF BROILERS TO LOCALLY PREPARED OIL BASED MYCOPLASMA GALLISEPTICUM VACCINE.
Next Article:PATTERNS OF MORPHOLOGICAL DIVERSITY AND CHARACTER ASSOCIATION IN CHICKPEA GENOTYPES THROUGH MULTIVARIATE APPROACH.
Topics:

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters