DNA profiling of Capsicum annum L. cultivars based on AFLP and ISSR markers

Transcription

1 DNA profiling of Capsicum annum L. cultivars based on AFLP and ISSR markers Ambika Baldev Gaikwad 1 *, Sunil Archak 2 and Dikshant Gautam 3 Division of Genomic Resources, National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi , India. (*author for correspondence) 1 Senior scientist (ambikabg@yahoo.co.in) 2 Senior scientist (sarchak@nbpgr.ernet.in) 3 Technical assistant(dikshant7874@rediffmail.com) Geneconserve: 12(49) 4:12 Abstract DNA fingerprints have been generated for thirty cultivars of Capsicum annum L. using six AFLP and four ISSR primers. Six AFLP primer pairs and four ISSR primers generated 353 and 53 markers respectively. The AFLP technique showed significantly higher power of genotype discrimination (Marker Index=14.62) compared to that of ISSR (MI=3.72). Whereas each of the AFLP primer pairs could individually distinguish all the 30 accessions, a combination of three ISSR primers, (GATA) 4, (CCTA) 4, (ACTG) 4 was required to discriminate all the 30 Capsicum accessions. The high level of heterozygosity detected by the ISSR markers indicates that this technique may be used effectively for diversity analysis and DNA fingerprinting of Capsicum. Key words Capsicum annum, Chilli cultivars, fingerprinting, molecular markers.

2 Abbreviations AFLP (Amplified Fragment Length Polymorphism), ISSR (Inter Simple Sequence Repeats) Introduction India is one of the leading producers of chilli (Capsicum annum L.) in the world. Though the plant is of Mexican origin (Pickersgill, 1969), it exhibits extensive diversity in India, especially in the hot pepper types (Singh and Singh, 1976). A large number of landraces are popular in different regions of the country. Besides, several advanced cultivars have been developed through selection and hybridisation. The recognition of intellectual property rights on plant varieties in the country s Plant Varieties Protection and Farmers Rights Act 2001 necessitates their efficient characterisation. Molecular techniques have been recognised as the method of choice for characterising plant genotypes due to their several advantages over the traditional morphology based characterisation (Karp et al., 1998). The present article reports the molecular characterisation of 30 cultivars of C. annum comprising of hot pepper, bell pepper and paprika types using the Inter-Simple Sequence Repeats (ISSR) and Amplified Fragment Length Polymorphism (AFLP) techniques and identification of a set of primers which would be useful not only for DNA fingerprinting of chilli, but also for diversity analysis studies. Materials and Methods Material Thirty cultivars of chilli representing the hot pepper, bell pepper and paprika (Table1) and including released varieties, local landraces and exotic collections were analysed using molecular markers.

3 Table 1: List of Capsicum annum cultivars analysed using AFLP and ISSR markers Hot peppers Agni Rekha Arka Abhir Arka Lohit Co 1 Co 2 G 3 G 4 Hybrid 1 K1 K2 Pant C1 Phule 5 Phule Jyoti Phule Suryamukhi PMK 1 Punjab Gucchedar Pusa Deepti hybrid Male parent of Pusa Deepti hybrid Pusa Sadabahar Ujjwala Sattur Samba Guntur local Mannaparai local Jalapeno type Bolivian Rainbow Bell peppers Yolo Wonder California Wonder Arka Gaurav Arka Mohini Paprika Kt Pl 19

4 Plant DNA extraction Seeds were sown in pots under greenhouse conditions. DNA was extracted as per the protocol of Saghai Maroof et al., 1984 with minor modifications. Two grams of young leaves were homogenized in 10 ml of homogenization buffer (100mM Tris, ph 8, 20mM EDTA, 1.4M NaCl, 2% CTAB, 0.2% 2-Mercaptoethanol) and incubated at 65 C for 1h. The homogenate was extracted twice with chloroform:isoamyl alcohol (24:1) and total cellular DNA precipitated with 0.6 volumes of isopropanol. The pellet was washed with 70% ethanol, air dried, dissolved in TE buffer (Tris 10mM, EDTA 1mM, ph 8.0), treated with bovine pancreatic RNase and extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1). Inter simple sequence repeat analysis Genomic DNA (10ng) was amplified in a final volume of 25 l containing 2.5mM MgCl 2, 10mM Tris-HCl ph 8.4, 50mM KCl, 0.2mM of each dntp, 1.0 M primer and 0.5unit of Taq DNA polymerase. Amplifications were performed using 4 microsatellite repeats as primers in a Perkin Elmer thermocycler for 35 cycles. Each cycle consisted of the following steps: 1 min at 95 C, 1 min at the annealing temperature of the primer and 5 min at 70 C. The annealing temperature was 50 C for (GACA) 4, (CCTA) 4 and (ACTG) 4, and 45 C for (GAAGTGGG) 2. The amplified products were separated on a 5% polyacrylamide gel in 1X Tris-Borate EDTA buffer and stained with 1X Gel Star Nucleic Acid Gel Stain (FMC Bioproducts). AFLP analysis The AFLP reactions were performed with the AFLP Plant Mapping Kit (regular genomes) of Perkin Elmer according to the manufacturers protocol. Six primer combinations were employed for generation of markers. These were MseI+CTC/EcoRI+ACG, MseI+CTC/EcoRI+ACT, MseI+CTC/EcoRI+ACC, MseI+CTG/EcoRI+ACA, MseI+CTG/EcoRI+AGC, MseI+CTG/EcoRI+AAG. The EcoRI primers were labeled with fluorescent dyes which allowed detection of amplification products that were resolved by an ABI Prism 310 Genetic Analyzer. Fragment sizes were determined in relation to the GenScan ROX size standard. The binary matrix was obtained

5 for the presence and absence of markers by sequential analyses using softwares GeneScan Version 3.1 and Genotyper version 2.5. Statistical analysis Amplicons were scored as discrete variables, using 1 to indicate presence and 0 for absence. The efficiency of the marker techniques in genotype discrimination was determined by evaluating marker index (MI) (Powell et al. 1996) as: MI = (N/P)H av ; H av = H N /N; H N = 1- pi 2 where N is the total number of loci, P is the number of primers/primer pairs, H av is the arithmetic mean heterozygosity, H N is the expected heterozygosity and pi is the frequency of i th allele. The efficiency of individual primers/primer pairs of a marker technique was compared on the basis of resolving power (Rp) (Provost et al. 1999) as: Rp = Ib; Ib = 1-(2x 0.5-p ) where Ib is the band informativeness and p is the frequency of presence of a band. Results and Discussion DNA based marker systems such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) have emerged as major tools for characterisation and assessing genetic diversity in plants. The arbitrary PCR based methods (RAPD and ISSR) of detecting DNA polymorphism have gained popularity over RFLPs due to the non-requirement of prior sequence based information and simplicity of experimental protocol. Whereas RAPD and inter simple sequence repeats (ISSR) detect variation in sites complementary to the particular primers, AFLPs also reflect variations in restriction sites. In the present study, all the 406 markers generated by four ISSR primers and six AFLP primer pairs (53 and 353 markers respectively) were polymorphic. Analysis of the same accessions using 30 RAPD primers generated only 91% polymorphism (unpublished data). Similar observations have been made in potato (Prevost et al. 1999), Eleusine

6 (Shalimath et al. 1995) and cotton (Noormohammadi et al. 2012)where ISSR analysis was found to generate more polymorphism than either RFLP or RAPD. Yolo Wonder I I I I I I I I I I I I I I I California Wonder I I I I I I I I I I I I I I Kt Pl 19 I I I I I I I I I I I I I I I I I PDH-male parent I I I Hybrid 1 I I I I I I I I I I I I I I Punjab Gucchedar I I I I I I I I I I I Phule Suryamukhi I I I I I Agni Rekha I I I I I I I I I I I I I I I I I I Phule Jyoti I I I I I I I I I I I I I I I Arka Lohit I I I I I I I I I I I I I Arka Mohini I I I I I I I I I I I I I I I I I Arka Gaurav I I I I I I I I I I I I I Phule 5 I I I I I I I I I I I I I I I Arka Abir I I I I I I I I I I I I I Pusa Sadabahar I I I I I I I I I I I I I I Pant C1 I I I I I Ujjwala I I I I I I I I I Pusa Deepti hybrid I I I I I I I I I I I I I I I G 3 I I I I I I I I I G 4 I I I I I I I I I I I I I I I Co 1 I I I I I I I I Co 2 I I I I I I I I I I I I I I I I I K1 I I I I I I I I I I I I I K2 I I I I I I I I I PMK 1 I I I I Satur Samba I I I I I I I I I I I I Mannaparai local I I I I I I I I I I I I I Guntur local I I I I I I I I I I I I I I Jalapeno I I I I I I I I I Bolivian rainbow I I I I I I Figure 1 - Barcode representation of DNA fingerprints of chilli cultivars based on AFLP primer-pair MseI + CTG/ EcoRI + ACA. Bars represent presence of fragments On the basis of the distribution of complete sets of markers, each technique could uniquely distinguish all the accessions. However, our purpose was to identify a minimum set of primers/primer pairs that would serve the same purpose. All the AFLP primer pairs could independently distinguish the present 30 accessions suggesting that any one of them could be used for fingerprinting Capsicum. Representative profiles of the accessions with the primer pair MseI+CTG/ EcoRI+ACA are given in Figure 1. The ISSR primers individually could not distinguish all

7 the accessions. The sets of primers with high discriminating efficiency were identified on the basis of the resolving power of the primers as detailed in Materials and Methods. Of the four ISSR primers used, a combination of three i.e. (GATA) 4, (CCTA) 4 and (ACTG) 4 with a resolving power of 10.11, 5.00 and 3.68, respectively could discriminate all the 30 accessions (Figure 2). Comparison of cultivar discrimination efficiency among the techniques was made on the basis of marker index. As detailed above, both AFLP and ISSR markers were 100% polymorphic. However, ISSR detected a higher level of heterozygosity (H av = 0.28) than AFLP (H av = 0.25), although the marker index in AFLP (MI = 14.62) was higher as compared to ISSR (MI = 3.72). This was due to the high multiplex ratio of AFLP, the mean number of markers generated per primer being 58.8 in AFLP and 13.3 in ISSR. Yolo Wonder I I I I I I I I I I I I I California Wonder I I I I I I I I I I I Kt Pl 19 I I I I I I I I I I I PDH-male parent I I I I I I I I I I I I I I I I I Hybrid 1 I I I I I I I I I I I I I I I I I Punjab Gucchedar I I I I I I I I I I I I I I I I I I I I I Phule Suryamukhi I I I I I I I I I I I I I I I I I I I Agni Rekha I I I I I I I I I I I I I I I I I I I I I I I Phule Jyoti I I I I I I I I I I I I I Arka Lohit I I I I I I I I I I I I I I Arka Mohini I I I I I I I I I I I I I I I I I I I I I I I I I Arka Gaurav I I I I I I I I I I I I I I I I I I Phule 5 I I I I I I I I I I I I I I I I I I I I I I I I I I Arka Abir I I I I I I I I I I I I I I I I I I Pusa Sadabahar I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Pant C1 I I I I I I I I I I I I I I I I I I I I Ujjwala I I I I I I I I I I I I I I I Pusa Deepti hybrid I I I I I I I I I I I I I I I I I I I I I I I I I I I I G 3 I I I I I I I I I I I I I I I I I I I G 4 I I I I I I I I I I I I I I I I I I I I I I I I I I Co 1 I I I I I I I I I I I I I I I I I I I I I I Co 2 I I I I I I I I I I I I I I I I I I I I I I I I I I K1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I K2 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I PMK 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Satur Samba I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Mannaparai local I I I I I I I I I I I Guntur local I I I I I I I I I I I I I I I I I I I I I I I I I I I I Jalapeno I I I I I I I I I I I I I I I I I I I I I I I I Bolivian rainbow I I I I I I I I I I I I I I I I I I I I I I Figure 2: Barcode representation of DNA fingerprints of chilli cultivars based on three ISSR primers. Bars represent presence of fragments.

8 The choice of a particular technique for characterisation of genotypes depends upon several factors besides the power of discrimination. These include reliability, speed, simplicity and economic feasibility. For fingerprinting Capsicum accessions, AFLP appears to be the technique of choice. Besides greater discrimination power, AFLP markers have high reproducibility, the lack of which is a serious drawback of RAPD (Karp and Edwards, 1997; McGregor et al., 2000). However AFLP is technically more demanding and cost per assay, particularly with the fluorescence based detection system is high. ISSR-PCR analysis has been shown to be a quick, reproducible and simple system that generates sufficient polymorphism with potential for large-scale DNA fingerprinting purposes (Prevost et al., 1999). Thus, although high marker informativeness and reproducibility make AFLP the technique of choice, ISSRs could provide important data to effectively fingerprint germplasm in laboratories with limited resources. Acknowledgements The funds and facilities provided at the National Bureau of Plant Genetic Resources, New Delhi, India under the project Technology development for DNA fingerprinting of horticultural crops are duly acknowledged. References Karp A and Edwards KJ (1997). Molecular techniques in the analysis of the extent and distribution of genetic diversity. In Ayad, W.G., T. Hodgin, A. Jaradat and V.R. Rao (eds.) Molecular genetic techniques for plant genetic resources. Report of an IPGRI workshop, 9-11 october 1995, Rome, pp International Plant Genetic Resources Institute, Rome. Karp A, Issac PG and Ingraam DS (1998). Molecular tools for screening biodiversity. Chapman and Hall, London.

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