Australian Journal of Crop Science Southern Cross Journals2009

3(3):122-128 (2009) www.cropj.com

DNA fingerprinting of rice (Oryza sativa L.) cultivars using microsatellite markers

1*
Muhammad Shefatur Rahman, 1Md. Rezwan Molla, 2Md. Samsul Alam and 3Lutfur Rahman
1
Plant Genetic Resources Centre, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur-1701,
Bangladesh
2
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh-2202,
Bangladesh
3
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202,
Bangladesh

*
Corresponding author: shefat@gmail.com

Abstract

Microsatellite combines several features of an ultimate molecular marker and they are used increasingly in
various plant genetic studies and applications. In this work, we report on the utilization of a small set of three
previously developed rice microsatellite markers for the identification and discrimination of 17 HYVs and 17
local rice cultivars including two wild rice cultivars. All analyzed microsatellite markers were found to be
polymorphic with an average number of 6.33 alleles per locus. These three markers were able to identify 15 local
rice cultivars and 11 HYVs. A total of three variety specific alleles, RM-11/147, RM-151/289 and RM-153/178
were identified for BR-11, Badshabhog and BR-19 cultivars respectively. DNA fingerprints of rice cultivars by
means of microsatellites provided meaningful data, which can be extended by additional microsatellite markers.
The data obtained can be used for the protection of plant genetic resources.

Key words: Rice; microsatellite; markers; variety identification

Introduction

Rice (Oryza sativa L.) (2n=24) belonging to the Plant scientists have long been used
family Graminae and subfamily Orazoidea is the morphological, pigmentation, quality or other
staple food for one third worlds population and characteristics to classify and distinguish plant
occupies almost one-fifth to the total land area genotypes within a species. With the advent of the
covered under cereals. It is grown under diverse plant variety protection act, the developer of a
cultural conditions and over wide geographical cultivar could protect it from commercial
range. Most of the worlds rice is cultivated and exploitation by others. While not having the same
consumed in Asia, which constitutes more than half exclusivity as a patent, plant variety protection,
of the global population. Approximately 11% of the nonetheless, offers the owner of a cultivar some
worlds arable land is planted annually to rice, and legal protection for the exclusive sale of a protected
it ranks next to wheat. Further scope of crop cultivar. In the case of Rice (Oryza sativa L.), and
improvement depends on the conserved use of most other cases leaf sheath color, inflorescence
genetic variability and diversity in plant breeding color, awn presence and other conventional
programme and use of new biotechnological tools. morphological traits, together with stress resistance,
There is wide genetic variability available in rice have been, and continue to being used to
among and between landraces leaving a wide scope distinguish the uniqueness of a new cultivar. As
for future crop improvement. more cultivar receives protection, and thereby
increases the size of the PVPO database, it becomes

122
more difficult to distinguish new cultivars from (1997) with modification. Fresh leaf samples of 22-
those in the database. Moreover, since new days-old seedling were used as the source of
cultivars normally arise from hybridizations genomic DNA. Leaf tissues were cut into small
between members of an elite group of genetically pieces, homogenized and digested with extraction
similar parents, the amount of genetic variability buffer (50 mM Tris-HCl, 25 mM EDTA, 300 mM
among newly developed cultivars is likely to NaCl and 1% SDS, pH 8.0). After incubation for 20
become even smaller. This will further complicate minutes at 650C with intermittent swirling, the
the task of unambiguously identifying new cultivars mixture was emulsified with an equal volume of
by the use of conventional characteristics alone. It phenol: chloroform: isoamyl alcohol (25 : 24 : 1,
is thus apparent that the use of molecular genetic v/v/v). DNA was precipitated using two volume of
markers would provide one solution to the problem absolute alcohol in presence of 0.3M sodium
of providing unique DNA profiles for the protection acetate and pelleted by centrifugation. The pellets
of new rice cultivars. Microsatellites are tandemly were then washed with 70% ethanol, air dried and
repeated nucleotide units of 1 to 6 base pairs and resuspended in an appropriate volume of TE buffer
alleles usually differ in the number of repeated (10 mM Tris-HCl, 1 mM EDTA, pH=8.0). DNA
units. These are generally co-dominant and highly quality was checked by electrophoresis in a minigel
polymorphic DNA based markers. Genetically and quantification was accomplished using a
mapped microsatellite markers cover the whole rice spectrophotometer (Spectronic Genesis, Spect-
genome with at least one microsatellite every 16 to ronic Instruments Inc., USA).
20 CM (Chen et al., 1997). This genome coverage
enables microsatellites to be used to anchor Microsatellite markers and PCR amplification
randomly generated PCR markers such as AFLPs to
known regions of the rice genome providing an Three previously developed rice microsatellite
economical means of producing genetic maps primer pairs (RM 11, RM 151 and RM 153) were
(McCouch et al., 1997). Rice microsatellites also used in the analysis (Table 2). Polymerase Chain
have a demonstrated utility for gene-tagging and Reactions were done in a volume of 10 l
marker-assisted selection (Chen et al., 1997 and containing 50 ng template DNA, 1 l 10X PCR
McCouch et al., 1997) and are polymorphic buffer containing 15mM MgCl2 , 0.25 mM each of
between (Akagi et al., 1996 and Panaud et al., the dNTPs, 0.25 M of each primer, 1 U ampli Taq
1996) and within rice varieties (Olufowote et al., DNA polymerase (GENEI Pvt. Ltd. Bangalore,
1997). Because of the high level of polymorphism, India) and a suitable amount of sterilize deionized
and therefore the greater informativeness of water. Amplification were carried out in a oil free
microsatellite markers, it seemed likely that these thermal cycler (Thermal cycler gradient,
markers would be particularly useful for developing Eppendorf, Germany) with the following program:
unique DNA profiles of rice genotypes. Such Initial denaturation at 94oC for 3 min followed by
profiles would be especially valuable to unambi- 35 cycles at 94 oC for 30 sec, 55 oC for 30 sec, and
guously distinguish rice cultivars in order to obtain 72 oC for 1 min and a final cycle at 72 oC for 7 min.
plant variety protection. The objective of the work PCR products were checked in 2% agarose gel.
reported here was to distinguish 34 diverse rice
genotypes of Bangladesh at a small number of loci Determination of microsatellite allele lengths
using microsatellite DNA markers.
PCR products were separated on 6% denatured
Materials and methods polyacrylamide gel containing 19:1 Acrylamide :
Bis acrylamide and 7M urea. Electrophoresis was
Plant materials carried out on Sequi Gen GT eletrophoresis cell
(Bio-Rad laboratories, USA). Gels were stained
Thirty four rice samples from Bangladesh, with silver nitrate using the Promega Silver
including 17 commercial varieties, 15 landraces and StquenceTM protocol (Gustavo et al., 1994).
2 wild rice accessions (Table 1) were used for
microsatellite analysis. Seeds were collected from Data analysis
Genetic Resources Centre (GRC), Bangladesh Rice
Research Institute (BRRI). Seeds were germinated The size (in nucleotides) of the most intensely
at aseptic condition and grown in glass house. amplified band for each microsatellite marker was
determined based on its migration relative to the
Genomic DNA isolation molecular weight (mw) size markers, 100bp DNA
ladder (GENEI Pvt. Ltd. Bangalore, India) using
Genomic DNA was isolated from the rice seedlings the software DNA frag Ver. 3.03 (Nash, 1991).
following protocol described by Aljanabi et al.

123
Table 1. Rice Cultivars used in the present study
A. High Yielding Varieties
Sl No Name Ecotype Pedigree
1 Binadhan-4 Aman BR 4 X Iratom 38 (Mutation)
2 Binadhan-5 Boro Dular x Iratom 24 (Mutation)
3 Binadhan-6 Boro Dular x Iratom 24 (Mutation)
4 Binasail Aman Nizersail (Mutation)
5 Iratom-24 Aus IR 8 (Mutant)
6 BR-11 Aman IR 20 X IR 5-47-2
7 BR-14 Aus IR 5 (D) X BR-3
8 BR-19 Boro IR 2180-2 X IR 2178-1
9 BR-21 Aus C 22 X IIT 1444
10 BR-26 Aus IR 18348-36-3-3 X IR 25863-61-3-2X IR 58
11 BRRI dhan-27 Aus KN 1 B-361-1-8-6-9X C 168
12 BRRI dhan-28 Boro IR-28 X Purbachi
13 BRRI dhan-29 Boro BG 92 X BR 51-46-5
14 BRRI dhan-30 Aman IR 2058-78-1-3-2-3 X BR 4
15 BRRI dhan-36 Boro IR 64 X IR 35293-125-3-2-3
16 BRRI dhan-38 Aman Bashmoti D x BR-5
17 BRRI dhan-40 Aman IR 4595-4-1-15 X BR 10
B. Local Cultivars
Sl No Name Ecotype Accession No.
18 Badshabhog Aman 4355
19 Dular Aus 0022
20 Hashikalmi Aus 3575
21 Hizaldigha Deep water Aman 1263
22 Kataktara Aus 2059
23 Kataribhog Aman 4362
24 Khaiyaboro Boro 0936
25 Maliabhanger Deep water Aman 0046
26 Nizersail Aman 0741
27 Nonasail Aman 0599
28 Pajam Aman 0646
29 Pakhbiroi Boro 5702
30 Pashusail Boro 0054
31 Razasail Aman 0784
32 Tepiboro Boro 0930
33 Wild rice-2 Deep water Aman 4338
34 Wild rice-7 Deep water Aman 4316

The polymorphism information content (PIC) was referred to as loci and DNA bands as alleles. All
calculated according to Neis statistics (Nei, 1973): three microsatellite markers were found to be
PIC=1-(pi2), where pi is the frequency of the ith polymorphic, revealing a total of 18 alleles with an
allele detected in the germplasm. Allele frequency average number of 6.33 alleles per locus in the
was determined using software POPGENE (version thirty four rice cultivars examined (Table 3). At the
1.31) (Yeh et al., 1999) RM-151 locus, a total of 9 different alleles were
identified among the 34 rice genotypes ranging in
Results size from 178 bp to 347 bp. Likewise, 5 alleles
(size ranging from 143 bp-180 bp) and 4 alleles
All thirty four rice cultivars were successfully (size ranging from 181 bp-223 bp) were detected at
amplified with the three microsatellite primer pairs the locus RM-11 and RM-153 respectively (Table
(RM 11, RM 151 and RM 153). Based on previous 3). Banding patterns generated by primer pairs RM
results (Sefc et al., 2000) primer pairs will be 151 and RM 153 in 15 rice cultivars are shown in
Fig 1.

124
Fig 1. Microsatellite profile of fifteen rice (Oryza sativa L.) cultivars at the locus RM 151 (A) and RM 153 (B). M: Molecular wt.
marker (100 bp ladder)
Lane Variety Lane Variety Lane Variety Lane Variety Lane Variety
1,2,3: BRRI dhan38 10,11,12: Dular 19,20,21: Kataktara 28,29,30: Maliabhanger 37,38,39: Pajam
4,5,6: BRRI dhan40 13,14,15: Hashikalmi 22,23,24: Kataribhog 31,32,33: Nizersail 40,41,42: Pakhbiroi
7,8,9: Badshabhog 16,17,18: Hizaldhigha 25,26,27: Khaiyaboro 34,35,36: Nonasail 43,44,45: Pashusail

Table 2. Details of the microsatellite markers used for rice genotype identification
Core motif and
Sl. Locus Chr. Forward Primer Reverse Primer Ann.T. Ref
Number of repeat
TC T CC T CTT CCC A TA G CG GG C G AG Panaud
1. RM 11 (GA)17 7 550C
CCG ATC GCT TAG et al, 1996
G GC TGC TCA TCA TCG G CA GTG GTA Temnykh
2. RM 151 (TA)23 1 550C
G CT G CA TGC G G AG TTT G AT C TG C et al, 2000
G CC TCG AG C ATC A TC AAC C TG CAC Temnykh
3. RM 153 (GAA)9 5 550C
A TC A TC AG TTG CCT GG et al, 2000

Among 18 alleles detected, three were specific to The PIC value which is the reflection of allele
three rice cultivars. One specific allele was detected diversity was also estimated. The average PIC
in the variety BR-11 (RM-11/147), BR-19 (RM- value was 0.69 and it ranged from 0.67 (RM11) to
153/178) and Badshabhog (RM-151/ 289) (Table 0.71(RM 151). The average PIC value for 17 HYVs
3). The three microsatellite primer pairs were able was 0.59 which was lower than that of 17 local
to identify and discriminate fourteen local cultivars cultivars (0.70) suggested that local cultivars are
and a wild cultivar, wild rice-7. The local cultivar more diverse than the HYVs.
Dular and wild rice-2 was not identified. Among
the HYVs, six varieties (Binadhan-4, Binadhan-6, Discussion
BRRI dhan30, Binasail, Iratom 24 and BRRI
dhan28) were not identified but the rest of the 11 Utilization of three microsatellite markers in the
HYVs were identified by the three microsatellite analysis of rice varieties revealed a high level of
markers (Table 3). genetic polymorphism which allowed unique

125
Table 3. Analysis of 3 microsatellite loci for 34 rice cultivars
No Cultivars Band positions due to primers (bp)
M RM11 RM151 RM153
Satellite A B C D E A B C D E F G H I A B C D
1. Binadhan-4 143 220 223
2. Binadhan-5 172 220 223
3. Binadhan-6 143 220 223
4. Binasail 180 220 223
5. Iratom-24 180 220 223
6. BR-11 147 220 212
7. BR-14 172 220 212
8. BR-19 172 178 212
9. BR-21 172 204 212
10. BR-26 180 220 212
11. BRRI dhan-27 165 220 212
12. BRRI dhan-28 180 220 223
13. BRRI dhan-29 143 234 223
14. BRRI dhan-30 143 220 223
15. BRRI dhan-36 172 234 223
16. BRRI dhan-38 143 244 212
17. BRRI dhan-40 143 220 212
18. Badshabhog 143 289 212
19. Dular 172 214 223
20. Hashikalmi 143 265 201
21. Hizaldigha 143 204 181
22. Kataktara 143 220 181
23. Kataribhog 143 347 212
24. Khaiyaboro 165 143 220 , 212 201
25. Maliabhanger 172 204 181
26. Nizersail 180 220 181
27. Nonasail 172 214 181
28. Pajam 172 143 244 223
29. Pakhbiroi 172 143 244 181
30. Pashusail 143 220 201
31. Razasail 165 347 212
32. Tepiboro 143 234 201
33. Wild rice-2 172 214 223
34. Wild rice-7 180 165 265 214 223
PIC Value 0.67 0.71 0.70
Number of Alleles 5 9 4

126
genotyping of 82.4% of the studied varieties and
only these three markers were sufficient for References
unambiguous identification of twenty eight rice
varieties which includes thirteen high yielding, Akagi H, Yokozeki Y, Inagaki A and Fujimura T
fourteen local and a wild rice cultivars. In the set of (1996) Microsatellite DNA markers for rice
twenty eight cultivars, eighteen alleles were chromosomes. Theoretical and Applied Genetics
detected which multiplied into a number of 93: 10711077
observed genotypes at each locus, giving high Aljanabi SM and Martinez I (1997) Universal and
discrimination value for varietal identification. rapid salt extraction of high quality gnomic DNA
Most of the unique genotypes used for varietal for PRC-based techniques. Nucleic Acids
identification were found at locus RM 151. Our Research 25: 4692-4993
results represent one of the first attempts to find out Chen X, Temnykh S, Xu Y, Cho YG and McCouch
a small set of microsatellite makers to discriminate SR (1997) Development of a microsatellite
rice cultivars of Bangladesh providing meaningful framework map providing genome-wide coverage
data that can be enlarged by additional rice in rice (Oryza sativa L.). Theoretical and Applied
cultivars and new microsatellite markers. Genetics 95: 553567
Microsatellites are not very demanding Dunja B, Jakse J and Javornik B (2002) DNA
technically, and a particularly important advantage fingerprinting of olive varieties by microsatellite
is that microsatellite data can be easily compared markers. Food Technology and Biotechnology 40:
among laboratories and are suitable for computer 185-190
databases, which is not always the case with other Gustavo CA and Grsshoff PM (1994) Staining
markers, such as RAPD. These results can be of nuclic acids with silver: an alternative to
practical use in rice variety identification. radioisotopes and fluorescent labeling. Promega
Microsatellites are considered appropriate for Notes 45: p13
variety identification because of their ability to McCouch SR, Chen X, Panaud O, Temnykh S, Xu
detect large numbers of discrete alleles repeatedly, Y, Cho YG, Huang N, Ishii T and Blair M (1997)
accurately and efficiently (Smith et al., 1996). In a Microsatellite marker development, mapping and
study, a minimum number of three microsatellite applications in rice genetics and breeding. Plant
markers was sufficient for rapid and unambiguous Molecular Biology 35: 8999
discrimination of olive varieties (Dunja et al., Nash JHE (1991) DNA frag, Version 3.03. Institute
2002). In another study by Olufowote et al. (1997), for Biological Sciences, 1991, National Research
as few as six, well chosen SSLPs were sufficient to Council of Canada, Ottawa, Ontario, Canada
discriminate between 71 related lines of rice. Nei M (1973) Analysis of gene diversity in
In the present experiment, larger number of local subdivided populations. Proceedings of the
cultivars was discriminated at the same loci National Academy of Sciences of the United States
compared with the modern high yielding rice of America 70: 3321-3323
varieties. It is due to the fact that local varieties Olufowote JO, Xu Y, Chen X, Park WO, Beachell
posses more genetic variability than that of the HM, Dilday RH, Goto M and McCouch SR
HYVs. (1997) Comparative evaluation of within-cultivar
In conclusion, the set of microsatellite markers variation of rice (Oryza sativa L.) using
used here provides a positive assessment to the microsatellite and RFLP markers. Genome 40:
ability of SSR marker to produce unique DNA 370378
profiles of rice genotypes. The data obtained can be Panaud O, Chen X and McCouch SR (1996)
used for varietal survey and the construction of a Development of microsatellite markers and
database of all rice varieties grown in Bangladesh, characterization of simple sequence length
providing also additional genetic information of the polymorphism (SSLP) in rice (Oryza sativa L.).
agronomic and quality characteristics of rice Molecular and General Genetics 252: 597607
varieties. Sefc KM, Lopes MS, Mendona D, Rodrigues M,
Santos D, Laimer M, Machado DC and Machado
Acknowledgements ADC (2000) Identification of microsatellite loci in
olive (Olea europaea) and their characterization
This research was undertaken as part of the in Italian and Iberian olive trees . Molecular
DANIDA funded project Genetic Fingerprinting Ecology 9: 11711173
of crop varieties of Bangladesh of the Seed Smith S, Helentjaris T (1996) DNA fingerprinting
Industry Development, Ministry of Agriculture, and plant variety protection. In : Paterson AH (ed)
Peoples Republic of Government of Bangladesh. Genome mapping in plants, Landes Cpmpany,
Texas, pp 95-110

127
Temnykh S, Park WD, Ayres N, Cartinhour S. Yeh FC, Yang RC and Boyle T (1999) POPGENE
Hauck N, Lipovich L, Cho YG, Ishii T and VERSION 1.31: Microsoft window-based free
McCouch SR (2000) Mapping and genome software for population genetic analysis.
organization of microsatellite sequences in rice ftp://ftp.microsoft.com/Softlib/HPGL. EXE
(Oryza sativa L.) Theoretical and Applied
Genetics 100: 697-7125

128