STUDIES ON EFFICIENCY OF RAPD PRIMERS IN DEVELOPINGMOLECULAR PROFILES FOR GENETIC PURITY STUDIES IN...

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Corresponding author: Sanjay Kumar, Genetics Laboratory, Department of Zoology, Banaras

Hindu University, Varanasi 225001, India (Phone: +91 7860448788; E-mail:

[email protected])

UDC 575:633

DOI: 10.2298/GENSR1403681K

Original scientific paper

STUDIES ON EFFICIENCY OF RAPD PRIMERS IN DEVELOPING MOLECULAR

PROFILES FOR GENETIC PURITY STUDIES IN SOYBEAN (Glycine max L.)

CULTIVARS

Sanjay KUMAR

Genetics Laboratory, Department of Zoology, Banaras Hindu University,

Varanasi, India

Kumar S. (2014): Studies on efficiency of RAPD primers in developing

molecular profiles for genetic purity studies in soybean (Glycine max L.) cultivars. -

Genetika, Vol 46, No. 3, 681- 692.

Major advances have recently been made in our understanding of soybean

genetics and of the application of new technologies to soybean improvement. Estimates of

genetic relationships on the basis of the enzymes and molecular markers have been shown

to be consistent with expectations based on origin and pedigree information. To identify

efficient markers, that are to be used for genetic purity studies, polymorphism is the basic

criterion. RAPD has been found to be an effective and efficient tool to evaluate and reveal

genetic polymorphism in several crop species. In present study a total of 80 RAPD

primers were screened, out of which 37 gave amplification and only 30 primers showed

unambiguous DNA profile. Out of these 30 primers, 22 gave polymorphic banding

patterns. It is evident from the result that, 30/80 primers tried (38%) provided

unambiguous amplification and out of 30 primers, 22 primers (73%) were found to be

polymorphic. Scorable 30 RAPD primers led to amplification of 120 fragments out of

which 81 (67.5%) bands were found to be polymorphic. On an average, we got 4 bands

per primer and 15 primers (50%) have been found to produce more number of bands than

the average value which is encouraging. 8 primers were found to give 100%

polymorphisms. Our results are indicative of the efficiency of RAPD primers towards

development of molecular profiles.

Key words: Glycine max, Polymorphism, PCR, RAPD, UPGMA

INTRODUCTION

Soybean (Glycine max (L.) Merr.) belongs to the family Leguminosae. It is an annual

herbaceous leguminous crop, with a high rain protein content and easy adaptation to diverse soil-

682 GENETIKA, Vol. 46, No.3, 681-692, 2014

climatic conditions. Being one of the main oil crops of the world, in India it is grown in all its

major states viz. Madhya Pradesh, Uttar Pradesh, Maharashtra, Rajasthan and Gujarat. It is also

grown on a small scale in Himachal Pradesh, Punjab and Delhi. Major advances have recently

been made in our understanding of soybean genetics and of the application of new technologies to

soybean improvement. Thus it is now possible, using molecular methods, to alter the protein and

oil composition of soybean, as well as to produce other foreign proteins in the plant. Traditionally,

the study of genetic diversity has fallen within population genetics which has focussed on

measuring its extent in natural populations, in comparing levels of genetic diversity within and

among populations and in making references on the nature and intensity of evolutionary processes

from the observed patterns of genetic diversity. Hence, there is a long tradition as well as a wealth

of conceptual tools in population genetics for analyzing, measuring and partitioning genetic

diversity (KUMAR and SINGH 2014a; b).

Several thousand soybean accessions from the USDA Soybean Germplasm Collection

(Urbana, IL) have been evaluated for agronomic and seed composition traits as well as disease

resistance (NELSON et al., 1987; NELSON et al., 1989; JUVIK et al., 1989; BERNARD et al., 1989)

although a large number of G. max lines are available to soybean breeding programs, discovering

and transferring novel, favourable genes from G. soja into G. max could be a successful

approach

for enhancing the genetic variability and improving cultivated soybean (CARPENTER and FEHR,

1986). Identifying useful

diversity is a challenge. Molecular characterization of soybean

germplasm has been performed with isozymes, proteins, and DNA markers. Estimates of genetic

relationships on the basis of the enzymes and molecular markers have been shown to be consistent

with expectations based on origin and pedigree information (GRIFFIN and PALMER, 1995;

MAUGHAN et al., 1995, 1996; DOLDI et al., 1997; THOMPSON et al., 1998). KEIM et al. (1989)

surveyed 58 G. max and G. soja accessions with 17 restriction fragment length polymorphism

(RFLP) markers and found that the molecular diversity was the least among cultivated

soybeans

and greatest between species. Using simple sequence repeat (SSR) and amplified sequence length

polymorphism (ASLP) markers MAUGHAN et al. (1995) reported that five microsatellite

markers

detected a total of 79 alleles in a sample of 94 accessions of wild and cultivated soybean. Allelic

diversity for the SSR loci was greater in wild soybean than in cultivated soybean.

Overall, 43 more

SSR alleles were detected in wild than in cultivated soybean. MAUGHAN et al. (1996) evaluated 23

accessions of wild and cultivated soybean using amplified fragment length polymorphism

(AFLP).

Among the 759 AFLP fragments scored, 17% were polymorphic in G. max and 31% were

polymorphic in G. soja. Their results also indicated that AFLP phenotypic variation was greater in

wild soybean than in cultivated soybean. GRIFFIN and PALMER (1995) screened more than 1200 G.

max and G. soja plant introductions with eight enzymes. The numbers of alleles per locus and

average gene diversity were greater in the G. soja samples than in the

G. max samples.

MATERIALS AND METHODS

Plant material

A total of 24 elite released cultivars of soybean grown across India and representing

different morphological variations, agro climatic zones and parentage were selected for molecular

polymorphism studies (Table 1). All the cultivars were collected from concerned breeder working

on soybean in SAUs or ICAR institute in India.

S. KUMAR: RAPD MARKER PROFILS OF SOYBEAN CULTIVARS 683

Table 1. Name, parentage and morphological descriptions of the soybean varieties used in the present study

Accessions

No.

Genotype Maturity

Duration

(Days)

Yield Reaction to insect-pest &

diseases

Salient features

S1 MAUS 61-2 100-105 20-31 Resistant to rust leaf

spot,bud bligh, soybean

mosaic virus and laef

eating caterpillar

Semi determinate, purple flowers,

glabrous leaves, yellow seeds

with light brown hilum, resistant

to pod shattering.

S2 PUSA 24 110-115 25-30 Resistant to bacterial

pustules, tolerant to

Rhizoctonia, YMV and

major insect-pests.

White flowers, tawny

pubescence, yellow seed coat,

black hilum, compact plant.

S3 NRC 2 103-106 25-30 Resistant to Rhizoctonia,

pod blight, green mosaic

virus, bacterial blight and

tolerant to Cercospora

leaf spot and

Anthracnose



grey to black hilum, good

germinability, determinate.

S4 PUSA16 105-115 25-30 Resistant to Rhizoctonia

& bacterial pustules,

tolerant to YMV, tolerant

to major insect-pests.

Purple flowers, tawny


grey hilum, compact plant, good

germinability.

S5 JS-335 95-100 25-30 Resistant to bacterial

pustule, bacterial blight

and tolerant to green

mosaic. Susceptible to

YMV.

Purple flowers, semi-determinate,

resistant to shattering, black

hilum. Performs well in Eastern

and Southern states.

S6 MACS 58 90-100 25-30 Resistant to bacterial

pustules & leaf spot,

tolerant to YMV.



light brown hilum, tall semi-

determinate, suitable for

mechanical harvesting.

S7 JS-71-05 90-95 20-24 Resistant to tobacco

caterpillar and jassid.

Tolerant to green

semilooper, girdle beetle

and stem fly

Purple flowers, yellow seed coat,

black hilum, semi-dwarf, plant

height 30 to 40 cms, determinate,

poor seed longevity.

S8 MAUS-61 95-100 26-28 Resistant to major

diseases and pests

Semi determinate, Violet flowers,

glabrous leaves, pods covered

with leaves, non shattering

S9 JS-72-280 102-105 20-22 Tolerant to bacterial

pustules.



black hilum, tall and

indeterminate, fast growing.

684 GENETIKA, Vol. 46, No.3, 681-692, 2014

S10 KALITURE 120-130 18-20 Susceptible to soybean

mosaic, tolerant to

bacterial pustules

Purple flowers, tawny pubescen,

black seed coat, black hilum,

small seeded semi indeterminate

S11 PUNJAB 1 95-100 25-30 Susceptible to bacterial

pustules, less susceptible

to blue beetle.

Purple flowers, yellow pods with

brown pubescence, yellow seed

coat, small seeds, light brown

hilum, high pod-shattering, early,

good germinability, suitable for

food uses.

S12 INDIRA

SOYA 9

106 22-23 Resistant to rust.

Moderately resistant to

stem tunneling and girdle

beetle and leaf folder.

Performs well under low

to moderate plant

densities

Light grey pubescence

throughout the plant parts, broad

light medium size green leaves,

yellow seeds of medium size with

black hilum and intermediate

lustre.

S13 JS-80-21 105-110 25-30 Tolerant to bacterial

pustules, viral diseases

and foliar insect-pests.



brown/black hilum, determinate,

high seed germinability. Perform

well in eastern states.

S14 JS-2 90-95 18-20 Resistant to bacterial

pustule, tolerant to

Macrophomina


pubescence, pods with dense

brown pubescene, yellow seed

coat, light brown hilum,

determinate, highly shattering

S15 MAUS 71 93-100 18-30 Not known Semi determinate, purple flowers,

glabrous leaves, yellow seed with

black hilum, resistant to pod

shattering.

S16 SHYAMA 105-110 20-25 Resistant to bacterial

pustules and seed and

seed ling rot, tolerant to

Anthracnose

Purple flowers, brown pubescen,

black seed coat, buff coloured

hilum, medium tall plants, suited

to low input conditions.

S17 MACS 124 95-105 25-30 Resistant to bud blight,

soybean mosaic and

bacterial pustules.

Purple flowers,

tawny pubescence, yellow seed

coat, dark brown hilum, semi-

determinate resistant to lodging.

S18 MACS 450 90-95 25-40 Resistant to leaf spot,

bud blight, yellow

mosaic, soybean mosaic,

bacterial pustule. Highly

resistant to stemfly and

defoliators.

Purple flowers, medium tall,

semi-determinate, tawny

pubescence, yellow seed, black

hilum.


S19 BRAGG 112-115 15-20 Resistant to bacterial

pustules, susceptible to

YMV.

White flowers, grey pubescence,

yellow seed coat, black hilum,

brown pods, determinate.

S20 PK 416 115-120 30-35 Resistant to YMV &

bacterial pustules,

tolerant to Rhizoctonia.



brown hilum, semi-

determinate. Performs well in

Punjab, Haryana and also in

central zone.

S21 SL 295 120-130 20-25 Resistant to yellow

mosaic virus

White flower, tawny pubescence,

determinate, yellow seed coat,

black hilum.

S22 NRC 37 96-102 35-40 Moderately resistant to

collar rot, bacterial

pustule, pod blight and

bud blight like

syndrome. Moderately

resistant to stem fly and

leaf miner. Non lodging

under optimum plant

population, non

shattering behaviour upto

10 days after harvest

maturity

Erect, determinate plants with out

anthocyanin colouration in the

hypocotyl, white flowers, tawny

pubescence, small to medium,

spherical yellow seeds with light

to dark brown hilum.

S23 PUSA 20 105-120 25-30 Resistant to Rhizoctonia

& bacterial pustules,

tolerant to YMV and

major insect-pests.



black hilum, compact

plant. Performs well in central

zone.

S24 NRC 7 90-99 25-35 Resistant to bacterial

blight, green mosaic

virus, bacterial pustules,

phyllody, soybean

mosaic, Myrothe-

cium and Cercospora

leaf spots, tolerant to

stem fly, girdle beetle,

green and grey

semilooper, leaf miner

and defoliators.

Determinate, grey pubescence,

purple flowers, yellow seed coat,

brown hilum, high oil content,

resistant to pod-shattering.

Isolation of genomic DNA

DNA was isolated followed by CTAB method (MURRAY and THOMSON, 1980). Three

grams of fresh leaf sample was ground using the extraction buffer (1.4 M NaCl; 20 mM EDTA,

pH 8.0; 100 mM Tris, pH 8.0; 2 % CTAB), incubated at 65 °C for 30 min and centrifuged at

686 GENETIKA, Vol. 46, No.3, 681-692, 2014

10,000 rpm for 10 min. The upper aqueous phase was extracted 2-3 times with fresh chloroform:

iso-amyl alcohol (24:1). The final aqueous phase was transferred to other centrifuge tubes. To

these, 0.6 volume of ice cold iso-propanol was added and mixed gently by inverting the tube. DNA

complex was spooled out with a bent pasture pipette and 20 ml of washing solution was added to

it. The pellet was gently agitated for few minutes and collected by centrifugation at 40C, dried and

an appropriate volume of TE buffer was used to dissolve the pellet. Further the RNaseA @

10µg/ml was added into the DNA which was then incubated at 370C for 30 minutes. Equal volume

of phenol: chloroform: iso-amyl alcohol (25:24:1) was added into it and centrifuged at 10000 rpm

for10 minutes. Aqueous phase was taken and equal volumes of chloroform: iso-amyl alcohol

(24:1) was added and centrifuged at 10000 rpm for 10 min. To the aqueous phase, 1/20th

volume

of sodium acetate (3M, pH-5.2) and 2.5 volume of ethanol was added. Next it was incubated at -

200C for 1h (or-70

0C for 30 min). Then the solution was centrifuged at 10000 rpm for 10 min.

Thereafter, pellets were washed with 70% ethanol (10000 rpm, 5 min), air dried and dissolved in

distilled water. Yield of DNA was estimated using DNA markers with known λDNA – uncut,

through electrophoresis.

PCR amplification and gel electrophoresis

The random amplification was performed following a modified method of WILLIAMS et

al. (1990). The reaction was carried out in a thermal cycle (Mastercycler, Eppendorf). A total of 80

RAPD primers were screened, out of which 37 produced amplifications and only 30 primers (viz.

OPA 01, 03, 04, 08, 13 ; OPJ 01, 05, 09, 11, 12, 13, 16, 17, 18, 19; OPP 03, 04, 05, 06, 07, 08, 09

and OPT 01, 05, 07, 12, 13, 15, 16, 20, Operon Technologies, Canada, USA) showed

unambiguous DNA profile (Table 2). Polymerase chain reaction mixture of 25 µl contained 25 ng

of genomic DNA template, 0.6 U of Taq DNA polymerase (Bangalore Genei, Bangalore, India),

0.3 M decamer primer (Operon Technologies, Alameda, CA, USA), 2.5 µl 10 X PCR assay buffer

(50 mM KCl, 10 mM Tris–HCl, 1.5 mM MgCl2) and 0.25 µ1 pooled dNTPs (100 mM each of

dATP, dCTP, dGTP and dTTP from Promega, USA). PCR cycle conditions were as follows:

initial denaturing step at 94°C for 3 min followed by 44 cycles of 94°C for 1 min, 37°C for 1 min

and 72°C for 2 min. In the last cycle, primer extension at 72°C for 7 min was provided. PCR

products separation was done through 1.5% agarose gel electrophoresis alongside O’Gene

RulerTM 100 bp DNA Ladder Plus (Fermentas Life Sciences) as molecular mass marker. The

amplified products were documented under UV light source.

Data analysis

Clearly observed bands were scored manually for their presence (1) and absence (0)

across the genotypes and assembled in a binary data matrix table. Data on clearly resolved bands

generated by 30 primers were used to estimate genetic similarity(s) among the genotypes based on

Jaccard’s coefficient. This matrix was analysed using UPGMA (Unweighted Pair Group Method

using Arithmetic averages) following the SHAN (Sequential Agglomerative Hierarchical Nested)

cluster analysis module to derive a dendrogram. All these computations were carried out using

software NTSYS-pc (ROHLF, 2000).


Table 2. Name, sequence and details of the polymorphic RAPD primers used in the present study

RESULTS

Out of total 80 primers (Kits OPA, OPP, OPT and OPJ) tried in this study 37 primers

showed clear and unambiguous amplification (Table 3). Scorable 30 RAPD primers led to

amplification of 120 fragments ranging from about 3,300 bp (OPT 1) to 300 bp (OPT 12), out of

which 81 (67.5%) bands were found to be polymorphic. The level of polymorphism ranged from

33.4% (OPT 16, where 1/3 bands was found to be polymorphic) to 100% (OPA1, 4, OPP 5, 7, 8,

9, 15 and OPT 13). Maximum number of 7 amplified products were obtained by primer OPT 01

and 13. Eight primers were found to be monomorphic and the primer OPA 03 amplified a

Primer Sequence (5'-3') Molecular

weight

No. of bands

amplified

No. of

polymorphic

band

%

polymorphism

OPA-01 CAGGCCCTTC 2964 6 6 100

OPA-03 AGTCAGCCAC 2997 1 0 0

OPA-04 AATCGGGCTG 3068 5 5 100

OPA-08 GTGACGTAGG 3108 2 1 50

OPA-13 CAGCACCCAC 2942 5 2 40

OPJ-01 CCCGGCATAA 2997 2 1 50

OPJ-05 CTCCATGGGG 3044 2 0 0

OPJ-09 TGAGCCTCAC 2988 1 0 0

OPJ-11 ACTCCTGCGA 2988 6 3 50

OPJ-12 GTCCCGTGGT 3035 5 4 80

OPJ-13 CCACACTACC 2917 3 1 33.3

OPJ-16 CTGCTTAGGG 3059 1 0 0

OPJ-17 ACGCCAGTTC 2988 3 0 0

OPJ-18 TGGTCGCAGA 3068 5 5 100

OPJ-19 GGACACCACT 2997 3 2 66.67

OPP-03 CTGATACGCC 2988 5 2 40

OPP-04 GTGTCTCAGG 3059 1 0 0

OPP-05 CCCCGGTAAC 2973 5 5 100

OPP-06 GTGGGCTGAC 3084 5 4 80

OPP-07 GTCCATGCCA 2988 4 4 100

OPP-08 ACATCGCCCA 2957 7 7 100

OPP-09 GTGGTCCGCA 3044 4 4 100

OPT-01 GGGCCACTCA 3013 7 6 85.7

OPT-05 GGGTTTGGCA 3099 3 0 0

OPT-07 GGCAGGCTGT 3084 5 2 40

OPT-12 GGGTGTGTAG 3139 5 4 80

OPT-13 AGGACTGCCA 3037 7 7 100

OPT-15 GGATGCCACT 3028 6 5 83.3

OPT-16 GGTGAACGCT 3068 3 1 33.3

OPT-20 GACCAATGCC 2997 3 0 0

688 GENETIKA, Vol. 46, No.3, 681-692, 2014

minimum of 1 band. On an average we obtained 4 bands per primer and 22 primers used in the

study produced polymorphic banding pattern. RAPD product (750 bp) produced by OPT 07 was

found to be specific for MAUS-61 , while OPJ 18 produced a 2350 bp band only in Indira Soya 9.

DNA amplification pattern as detected by some of the RAPD primers in the soybean cultivars has

been provided in Fig. 1.

Figure 1. RAPD profile of soybean varieties obtained with primers OPA-04, OPJ-12 and OPT-13.

Serial number of the varieties corresponds to Table 1. M = Standard DNA marker, 100 bp

DNA Ladder Plus

OPA-04

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

OPJ-12

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

OPT-13

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24


Table 3. Similarity matrix of 24 soybean cultivars

Figure 2. Genetic relatedness based on RAPD profiles in selected cultivars of soybean

690 GENETIKA, Vol. 46, No.3, 681-692, 2014

Thus, it is evident that, 30/80 primers tried (38%) provided unambiguous amplification

and out of 30 primers, 22 primers (73%) were found to be polymorphic.

DISSCUSSION

RAPD has been found to be an effective and efficient tool to evaluate and reveal genetic

polymorphism in several crop species like rice (VIRK et al., 1995; RAY-CHOUDHURY et al., 2001),

wheat (CAO et al., 2000), maize (PEJIC et al., 1998), barley (YU et al., 2002) and soybean

(BARANEK et al., 2002; BARAKAT, 2004). In our present study 67.5% fragments were found to be

polymorphic as compared to 46% obtained by BARANEK et al. (2002) in 19 Czech National

Collection of soybean genotypes. Further, THOMPSON et al. (1998) were able to identify only 34%

polymorphism using RAPD. Very high degree of polymorphism detected in our present study

indicated an accurate selection of the polymorphic RAPD primers. The 30 RAPD primers used in

the present study has been selected after screening of 80 primers, 22 out of 30 were found to be

polymorphic. Thus, quite a high percentage of primers (73%, as mentioned earlier). Two

genotypes, were able to generate specific unique band. However, validation of the identified

primers and reproducibility of those bands are required for proper identification of the genotypes.

Moreover, the unique bands could be converted into SCAR (Sequence Characterized Amplified

Region) markers for specificity. In this regard, few more RAPD primers could be tried for

identification of unique bands in newer varieties. On an average, we got 4 bands per primer.

Further, 8 primers were found to give 100% polymorphism among the genotypes studied. 15

primers (50%) have been found to develop more number of bands than the average value which is

encouraging (Table 2) and indicates the efficiency of RAPD primers towards development of

molecular profiles.

ACKNOWLEDGEMENTS

Author is very grateful to Directorate of Seed Research, Kushmaur, Mau. India, to provide

facilities for this work. Special thanks are due to Dr. P. RAY-CHOUDHURY and Mr. S.P. TRIPATHI,

for his guidance during this work.

Received December 16th, 2013

Accepted September 05th, 2014

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ISPITIVANJE EFIKASNOSTI RAPD MARKERA U RAZVOJU MOLEKULARNOG

PROFILA ZA ISPITIVANJE GENETIČKE ČISTOĆE (Glycine max L.)

Sanjay KUMAR

Genetička laboratorija, Odelenje za zoologiju Banaras Hindu Univerzitet, Varanasi, Indija

Izvod Glavni napredak u istraživanjima soje je u razumevanju genetičkih osobina i primena

novih tehnologija u njenom poboljšanju. Utvrđivanje genetičkog odnosa aktivnosti enzima i

molekularnih markera je pokazalo da je konzistentno sa očekivanjima zasnovanim na poreklu i

informacijama pedigrea. Da bi se identifikovali efikasni markeri koji će biti korišćeni u

ispitivanjima genetičke čistoće polimorfizam je osnovni kriterijum. U ovom radu vršena je

evaluacije 80 RAPD markera od kojih je moglo da se amplifikuje 37 a samo 30 prajmera je dalo

nedvosmislene profile. Od tih 30, 22 je imalo polimorfnu sliku fragmenata DNK (banding).

Analiziranih 30 RAPD markera je dovelo do umnožavanja 120 fragmenata od kojih je 81 (67.5 %)

fragmenata bilo polimorfno. U proseku dobijena su 4 fragmenta po prajmeru i 115 prajmera (50

%) je dalo veći broj fragmenata od prosečnog broja što je ohrabrujuće. 8 prajmera je imalo 100%

polimorfizam. Rezultati su indikativni za efikasnost RAPD prajmera u razvoju molekularnih

profila.

. Primljeno 16. XII. 2013.

Odobreno 05. IX. 2014.

STUDIES ON EFFICIENCY OF RAPD PRIMERS IN DEVELOPINGMOLECULAR PROFILES FOR GENETIC PURITY STUDIES IN...

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