64 Advances64-69, in Life2016 Sciences 5(1), 2016 Advances in Life Sciences 5(1), Print : ISSN 2278-3849,
Assessment of Genetic Diversity in Garden cress [Lepidium sativum L.] Using SSR Markers K. C. BHALALA*1, V. N. KAPADIA2, V. B. KUNDARIYA3 AND M. A. PATEL4 1, 2 & 3
Department of Genetics and Plant Breeding, B. A. College of Agriculture, Anand Agricultural University, Anand – 388 110. 4 Medicinal and Aromatic Plants Research Station, Anand Agricultural University, Anand – 388 110. *email:
[email protected]
ABSTRACT Garden cress [Lepidium sativum L.] is an important indigenous medicinal plant which is used in various systems of medicines. It is therefore, essential to understand the pattern of genetic diversity in this important medicinal crop species. Out of 40 SSR primers, only 8 primers were showed amplification of genomic DNA which indicated 20% crosstransferability in garden cress. The study was undertaken to identify the efficient SSR primers that could differentiate a set of 32 garden cress genotypes. The PIC value ranged from 0.176 to 0.375. Clustering pattern of dendrogram generated by SSR data revealed two clusters. The similarity coefficient value ranged from 0.000 and 0.375. The average coefficient similarity for all the genotypes was found 0.102, which indicates the low distribution of genetic variability. Key words
Genetic diversity, SSR, Lepidium sativum L.
Garden cress has been widely used to treat a number of ailments in traditional system of medicine throughout India and it is a native of Ethiopia and introduced in Europe, Asia and USA. It belongs to the family Brassicaceae and commonly known as garden cress in English and chandrasur in Hindi. This is a small, herbaceous, glabrous annual plant having 15-45 cm height and cultivated for seed purpose and salad supplement throughout India. Seeds are pungent, odourless and mucilaginous (Khory, 1999). Garden cress is an allogamous plant with self-compatible and self-incompatible forms and cut the various degrees of tolerance to prolonged autogamy. There are diploid form 2n=16 and tetraploid form 2n=32. (Wadhwa et al., 2012). Garden cress seeds are good tonic when given daily for feeding to animals after calving to meet the strain and drain of calving for the first two weeks (Anonymous, 1952-53). Paranjape and
Mehta (2004) elucidated the usefulness of garden cress in the form of traditional tonic to increase height of children, to increase the milk content in female and a tonic for eyes. The plant is being used for the treatment of Amavata, Sandhivata, and Katishula successfully in ayurveda (Raval and Pandya, 2011). The alkaloids of Lepidium sativum L. are member of the rare imidazole alkaloids that is known as lepidine (Maier et al., 1998). Garden cress seeds have good amount of fat, therefore, it can be a promising new oil seed crop in addition to medicinal importance (Angelini et at., 1997). Garden cress cultivars were distinguished by morphological characters such as plant height, leaf length, seed yield per plant, test weight and oil content. This method is slow and unreliable and phenotypic identification based on morphological traits is subjected to environmental variation (Nielsen, 1985) and are unsuitable for correct assessment of the genetic diversity. These limitations can be largely overcome by the use of molecular markers, which are unlimited in number and are not influenced by the environment. The Simple Sequence Repeats (SSRs) have been proven as an excellent tool for cultivar identification, pedigree analysis and the evaluation of genetic distances among many plant species (Priolli et al., 2002). In the present study 8 primers were utilized for genetic diversity assessment of 32 garden cress genotypes.
MATERIALS AND METHODS SSR analysis of 32 garden cress accessions was conducted in Centre of Excellence, Department of Agricultural Biotechnology, Anand Agricultural University, Anand, Gujarat. The genomic DNA was extracted using the CTAB (Cetyltrimethylethyl Ammonium Bromide) method with some modifications (Murray and Thompson, 1980). The reagents and buffers for DNA isolation were
BHALALA et al., Assessment of Genetic Diversity in Garden cress [Lepidium sativum L.] Using SSR Markers
65
Table 1. List of SSR primers Sr. No
Primer name
1
BRMS 01
2
BRMS 07
3
Ol10-D03
4
Ol10-D10
5
Ni4-G08
6
BRMS 16
7
BRMS 33
8
BRMS 37
Sequence (5’-3’) Forward (F), Reverse (R)
Source species of primers
Repeat motif
Annealing Temp. (Ta) °C
F: GGTGGCTCTAATTCCTCTGA
Brassica rapa
(GA)25
55.0
Brassica rapa
(CT)24
50.0
Brassica oleracia
(CT)20
62.0
Brassica oleracia
(GT)13
62.0
Brassica napus
(CT)87
62.0
Brassica rapa
(TC)20
63.9
Brassica rapa
(CA)11
62.7
Brassica rapa
(CA)10
55.9
R: ATCTTTCTCTCACCAACCCC F: AAATTGTTTCTCTTCCCCAT R: GTGTTAGGGAGCTGGAGAAT F: GCCAAAGACCTCAAAGATGG R: AAGCCACGTGAAGAAAGTCC F: TGAGACCCTAATCAGCTGGC R: AGTTGGGTCGAGAGACCAGG F: ATTTGACGGACTCCTCTTGC R: CACTTGGTAACTCTATGGATGCC F: TCCCGTATCAATGGCGTAACAG R: CGATGGTGACATTATTGTGGCG F: GCGGAAACGAACACTCCTCCCATGT R: CCTCCTTGTGCTTTCCCTGGAGACG F: CTGCTCGCATTTTTTATCATAC R: TACGCTTGGGAGAGAAAACTAT
prepared as per Sambrook et al. (1989). Quality of extracted DNA was determined using a Spectrophotometer (Eppendorf, Germany). The PCR amplification was carried out in a thermal cycler (Biorad, USA). Each SSR reaction mixture (20 ìl) composed of 13.5 ìl nuclease free water, 2.0 ìl 10 X PCR buffer, 1.1 ìl MgCl2, 0.4ìl dNTPs, 0.8 ìl primer: 0.4ìl (Forward) + 0.4ìl (Reverse), 0.2 ìl Taq DNA Polymerase (2.5U), 2.0 ìl DNA Sample (50ng). Cycling condition used for amplification were as follows; 94°C : 5 min (Initial
denaturation), 94°C : 1min (Denaturation), Annealing temperature specific for a particular primer pair : 1min (Annealing), 72°C : 1min (Elongation), 72°C : 8min (Final elongation), 4°C : Hold (infinite) followed by 30 cycles. The PCR products were separated by electrophoresis in a 6% denaturing polyacrylamide gel in 1x TBE buffer (pH 8.0) at 110 V for 3-4 hrs, stained with silver nitrate (0.2%, W/V) and photographed using a gel documentation system (Fig.-1). The Product sizes were determined in comparison to a 100 bp DNA
Table 2. Results of SSR analysis Sr. No.
Primer Name
Molecular Band Size (bp)
Total No. of Bands
Total No. of Alleles
Polymorphism (%)
PIC value
1
BRMS 01
180
32
1.00
0.00
0.000
2
BRMS 07
215
32
1.00
0.00
0.000
3
Ol10-D03
167, 170
32
2.00
18.75
0.258
4
Ol10-D10
175, 181
32
2.00
18.75
0.258
5
Ni4-G08
180, 186
64
2.00
9.37
0.375
6
BRMS 16
207, 210
37
2.00
0.00
0.176
7
BRMS 33
220, 226
64
2.00
0.00
0.375
8
BRMS 37
175, 190
64
2.00
0.00
0.375
Total
-
357
14.00
46.87
-
Average
-
44.62
1.75
5.86
0.227
66
Advances in Life Sciences 5(1), 2016
Fig. 1. SSR profile of primer Ol10-D10
ladder (Invitrogen). The PIC value for each SSR primer was calculated according to the formula PICi = 2fi (1- fi) where, fi is the frequency of the marker
fragments that were present and 1- f i is the frequency of the marker fragments that were absent. Allele molecular weight (base pairs) were used to
Fig. 2. UPGMA cluster analysis of SSR data generated for thirty two genotypes of garden cress depicting patterns of genetic diversity
MLS 1001
1
0.00
0.00
0.00
0.00
0.00
0.00
0.25
0.125
0.00
0.312
0.00
0.062
0.187
0.00
0.062
0.00
0.125
0.062
0.25
0.00
0.125
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.00
0.00
SLS 1
1
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.25
0.125
0.00
0.312
0.00
0.062
0.187
0.00
0.062
0.00
0.125
0.062
0.25
0.00
0.125
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.00
0.00
SLS 1
MLS 1001
MLS 1007
MLS 1016
HLS 4
HLS 5
ALS 3
ALS 4
ALS 6
ALS 7
ALS 8
ALS 9
ALS 10
ALS 11
ALS 12
ALS 13
ALS 14
ALS 15
ALS 16
ALS 17
ALS 18
ALS 19
ALS 20
ALS 21
ALS 22
ALS 23
ALS 24
ALS 25
ALS 26
ALS 27
ALS 1
GA 1
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.125
0.00
0.25
0.062
0.125
0.00
0.062
0.00
0.187
0.062
0.00
0.312
0.00
0.125
0.25
0.00
0.00
0.00
0.00
0.00
1
MLS 1007
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.125
0.00
0.25
0.062
0.125
0.00
0.062
0.00
0.187
0.062
0.00
0.312
0.00
0.125
0.25
0.00
0.00
0.00
0.00
1
MLS 1016
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.125
0.00
0.25
0.062
0.125
0.00
0.062
0.00
0.187
0.062
0.00
0.312
0.00
0.125
0.25
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.125
0.00
0.25
0.062
0.125
0.00
0.062
0.00
0.187
0.062
0.00
0.312
0.00
0.125
0.25
0.00
0.00
1
5
4
1
HLS
HLS
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.125
0.00
0.25
0.062
0.125
0.00
0.062
0.00
0.187
0.062
0.00
0.312
0.00
0.125
0.25
0.00
1
3
ALS
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.125
0.00
0.25
0.062
0.125
0.00
0.062
0.00
0.187
0.062
0.00
0.312
0.00
0.125
0.25
1
4
ALS
0.25
0.25
0.25
0.25
0.125
0.375
0.125
0.25
0.25
0.125
0.25
0.00
0.312
0.125
0.25
0.312
0.25
0.187
0.312
0.25
0.062
0.25
0.125
1
6
ALS
0.125
0.125
0.125
0.125
0.25
0.25
0.25
0.125
0.125
0.00
0.125
0.125
0.187
0.25
0.125
0.187
0.125
0.062
0.187
0.125
0.187
0.125
1
7
ALS
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.125
0.00
0.25
0.062
0.125
0.00
0.062
0.00
0.187
0.062
0.00
0.312
1
8
ALS
0.312
0.312
0.312
0.312
0.187
0.312
0.187
0.312
0.312
0.187
0.312
0.062
0.25
0.187
0.312
0.25
0.312
0.125
0.25
0.312
1
9
ALS
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.125
0.00
0.25
0.062
0.125
0.00
0.062
0.00
0.187
0.062
1
ALS 10
0.062
0.062
0.062
0.062
0.187
0.062
0.187
0.062
0.062
0.187
0.062
0.312
0.00
0.187
0.062
0.00
0.062
0.125
1
ALS 11
0.187
0.187
0.187
0.187
0.312
0.187
0.312
0.187
0.187
0.062
0.187
0.187
0.125
0.312
0.187
0.125
0.187
1
ALS 12
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.125
0.00
0.25
0.062
0.125
0.00
0.062
1
ALS 13
0.062
0.062
0.062
0.062
0.187
0.062
0.187
0.062
0.062
0.187
0.062
0.312
0.00
0.187
0.062
1
ALS 14
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.125
0.00
0.25
0.062
0.125
1
ALS 15
0.125
0.125
0.125
0.125
0.00
0.25
0.00
0.125
0.125
0.25
0.125
0.125
0.187
1
ALS 16
0.062
0.062
0.062
0.062
0.187
0.062
0.187
0.062
0.062
0.187
0.062
0.312
1
ALS 17
0.25
0.25
0.25
0.25
0.125
0.375
0.125
0.25
0.25
0.125
0.25
1
ALS 18
Table 3. Jaccard’s similarity coefficient based on SSR of thirty two genotypes of garden cress
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
0.00
0.125
1
ALS 19
0.125
0.125
0.125
0.125
0.25
0.25
0.25
0.125
0.125
1
ALS 20
0.00
0.00
0.00
0.00
0.125
0.125
0.125
0.00
1
ALS 21
0.00
0.00
0.00
0.00
0.125
0.125
0.125
1
ALS 22
0.125
0.125
0.125
0.125
0.00
0.25
1
ALS 23
0.125
0.125
0.125
0.125
0.25
1
ALS 24
0.125
0.125
0.125
0.125
1
ALS 25
0.00
0.00
0.00
1
ALS 26
0.00
0.00
1
ALS 27
0.00
1
ALS 1
1
GA 1
BHALALA et al., Assessment of Genetic Diversity in Garden cress [Lepidium sativum L.] Using SSR Markers 67
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Advances in Life Sciences 5(1), 2016
determine the genetic distance for phylogeny reconstruction using Neighbor Joining (NJ) method (Nei, 1973) in POWERMARKER v.3.25 (Liu and Muse, 2005) with the tree viewed using TREEVIEW (Page, 1996) and by Un-weighted Pair Group Method with Arithmetic Mean (UPGMA).
RESULTS AND DISCUSSION Available literature on use of molecular markers to detect genetic diversity in garden cress is very few. Only ISSR and RAPD primers were used by some research workers in garden cress. Total 40 SSR primers were used for screening 32 genotypes of garden cress, out of which 8 primer were amplified which gave total number of 14 loci. 6 primers gave polymorphism among 8 amplified primers (Table 1). Out of 40 SSR primers, only 8 primers were showed amplification of genomic DNA which indicated 20% cross-transferability in garden cress. Similarly, Singh et al. (2012) also reported 18.6% cross-transferability in Lepidium sativum L. PCR amplification of genomic DNA of 32 garden cress genotypes generated 357 scorable bands with average of 44.62 bands per primer. The size of bands ranged from 167 to 226 bp (Table 2). In present investigation, total 14 alleles were generated with average of 1.75 alleles per primer. The SSR primers tested in present investigation produced fragments of different size. The PIC value ranged from 0.176 to 0.375 with average of 0.227. The primers Ol10-D03 and Ol10-D10 were showed 18.75% polymorphism, while Ni4-G08 was showed 9.37% polymorphism. Average polymorphism was observed using SSR markers was 5.86%. However, in contrast to this, Bansal et al. (2012) and Kumar et al. (2012) were reported higher percentage of polymorphism. The Dendrogram based on Jaccard’s similarity coefficients was created using UPGMA. Two clusters were found where, cluster-I have 13 genotypes and cluster-II consist of the remaining genotypes (Fig.-2). The cluster analysis showed very low genetic variation among the garden cress accessions studied, with a similarity coefficient varying between 0.000 and 0.375 with the average of 0.102. Maximum similarity coefficient was observed between ALS 6 or ALS 18 and ALS 24 (Table 3). The results obtained in the present investigation are in the agreement with the results of Bansal et al. (2012) that cluster analysis of the genotypes based on UPGMA divided the 18 genotypes into two main clusters.
In the present study, SSR primers that roughly differentiated 32 garden cress genotypes into two major clusters in which first cluster shows polymorphism amongst varieties while another cluster shows non-significant difference between varieties. This low amount of genetic diversity is due to the use of SSR markers from different species of Brassicaceae family which shows species transferability and conservation of microsatellite markers. Results clearly illustrated the need of development of microsatellite markers in garden cress as well as acquisition of variability at genetic level by artificial methods like mutagen treatments for various garden cress breeding programs.
LITERATURE CITED Angelini, L. G.; Moscheni, E.; Colonna, G.; Belloni, P. and Bonari, E. 1997. Variation in agronomic characteristics and seed oil composition of new oil seed crops in central Italy. Industrial Crops and Products, 6(34): 313-323. Anonymous 1953. Final report submitted to ICAR, Western Regional Animal Nutritional Research Station, G. A. U., Anand, Gujarat. Bansal, D.; Bhasin, P.; Yadav, O. P. and Punia, A. 2012. Assessment of genetic diversity in Lepidium sativum L. a medicinal herb used in folklore remedies in India using RAPD. Journal of Genetic Engineering and Biotechnology, 10: 39-45. Khory, R. 1999. Materia Medica of India and Their Therapeutics, Komal Prakashan, New Delhi, pp. 63. Kumar, S.; Goyal, R.; Sheorayan, A.; Kajla, S.; Yadav, O. P. and Mangal, M. 2012. Assessment of Genetic Diversity in Lepidium sativum L. Using RAPD and ISSR Markers. Annals of Biology, 28(2): 93-97. Liu, K. and Muse, S. V. 2005. PowerMarker: An integrated analysis environment for genetic marker analysis. Bioinformatics, 21: 2128-2129. Maier, U. H.; Gundlach, H. and M. H. Zenk, 1998. Seven imidazole alkaloids from Lepidium sativum L. Phytochemistry, 49: 1791. Murray, M. G. and Thompson, W. F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res., 8: 4321-4326. Nei, M. 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci., U. S. A., 70: 33213323. Nielsen G., 1985. The use of isozymes as probes to identify and label plant varieties and cultivars.In: M. C. Rattazzi, Scandalios, J. G. & Whitt, G. S. (Eds), Isozymes: Current Topics In Biological and Medical Research. 12: 1-32. Page, R. D. 1996. Treeview: An application to display phylogenetic trees on personal computers. Comput. Mol. Biol., 12: 357-358.
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Singh, B. K.; Thakur, A. K. and Rai, P. K. 2012. Genetic diversity and relationships in wild species of Brassica and allied genera as revealed by cross-transferable genomic STMS marker assays. Australian Journal of Crop Sciences, 6(5): 815-821.
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Accepted on 11-01-2016
