Biotechnological

Communication

Biosci. Biotech. Res. Comm. 11(3): 496-504 (2018)

Optimization of sampling size for DNA-based PCR assay

for hybrid purity test in the brinjal,

Solanum melongena

Arpita Pattanaik

1,2

, Lakshmana Reddy D.C.

, T.H. Singh

, P. Pandiyaraj

and

Aswath Chennareddy

Division of Biotechnology, Centre for Post-Graduate Studies, Jain University, Bangalore, India

Division of Biotechnology, Indian Institute of Horticultural Research, Hesaraghatta Lake Post, Hesaraghatta,

Bangalore-560089, India

Division of Floriculture and medicinal Crops, Indian Institute of Horticultural Research, Bangalore-560089,

India

ABSTRACT

Farmers can harness the full potential of any hybrid only when they get genetically pure seeds of the hybrid. Hence,

ensuring the genetic purity of certi ed seeds of brinjal hybrids is mandatory in India, which is done through  eld

grow out test (GOT) based on the morphological characters of plants grown to maturity. GOT being land and labour

intensive, time consuming and in uenced by the environment, there is a need to identify rapid and reliable alterna-

tives like DNA based assays. Therefore, the present study was undertaken to identify the SSR markers that could be

used to test the hybrid purity of two commercial brinjal hybrids (viz., Arka Anand and Brinjal Asha) and to optimize

the minimum sample size that can be used for purity assessment of the brinjal hybrids. Among 120 SSR markers

studied, two markers were found to be suitable for testing the purity of these hybrids. The analysis of plant-to-plant

variation within the parental lines of all the hybrids, using the identi ed hybrid speci c markers, showed highly

homogenous SSR pro le, which further indicated the scope of application of these markers in maintenance and purity

testing of hybrids and parental lines. These two co-dominant markers can be used as referral markers for unambigu-

ous identi cation, seed purity testing and protection of the hybrids.

KEY WORDS: BRINJAL, CO-DOMINANT, GOT (GROW OUT TEST), HYBRID PURITY, SSR

496

ARTICLE INFORMATION:

*Corresponding Author: aswathiihr@gmail.com

Received 10

July, 2018

Accepted after revision 19

Sep, 2018

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA and

Crossref Indexed Journal

NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196

Online Contents Available at: http//www.bbrc.in/

DOI: 10.21786/bbrc/11.3/20

Arpita Pattanaik et al.

INTRODUCTION

Brinjal (Solanum melongena L.) identi ed as eudicot,

warm weather crop majorly grows in tropical and sub-

tropical regions of the world and is also known by name

eggplant (Schippers 2000; Daunay and Hazra, 2012).

Brinjal remains  fth most valued crop of Solanaceous

with commercial value of US $10 billion a year after

potato, tomato, pepper and tobacco (FAO, 2014). India

is the major Brinjal producer with annual production

reaching 13.55 million tonnes with a 19.1 mt/ha pro-

ductivity (NHB database, 2014). Brinjal also remains rich

in nutritional contents such as vitamins, minerals and

bioactive materials which supply number of bene ts to

human beings, (Raigon et al., 2008; Plazas et al., 2014b;

Docimo et al., 2016).

Although eggplant remains the promising crop for

productivity it remains susceptible to number of plant

pathogens such as bacterial wilt, fusarium wilt and many

others (Rotino et al., 1997). It is also being used as nutri-

ent source by number of pests such as shoot borer, white-

 ies, mites, aphids and others (Medakker and Vijayara-

ghavan, 2007; Rotino et al., 1997). In an attempt related

to crop improvement, number of breeding programmes

are involving new characters to create promising varie-

ties as F1 generation imparting better shelf life, disease

resistance and nutritional qualities (Daunay and Hazra,

2012). In many seed industries, Hybrid seed production

of the Brinjal is carried out by hand emasculation and

pollination; still brinjal undergoes cross-pollination to

change the genetic purity by involving foreign pollens.

The traditional Grow Out Test (GOT) is based on the

genetically induced morphological and biochemical fea-

tures expressed at suitable stages of development. Grow

out test (GOT) is an important method for genotypic

identi cation through adult plant phenotype (Arus,

1983). The physical features are studied in detail and all

the other plants i.e. off-types, pollen shredder, objection-

able and diseased plants, etc are traced out. GOT has got

certain limitations, as it is an expensive procedure. GOTs

take up a full growing season, which often results in

late entry of seed in market. In addition to GOT, number

of biochemical markers are used such as isozymes and

seed storage proteins, but they also remained restricted

in their usage (Dadlani et al., 1997; Mehetre and Dahat,

2001; Borle et al., 2007 and Rakshit et al., 2008). Keeping

in view, scienti c community is now moving towards

DNA markers which provides diversity in approach

with the type of methodology adopted such as simple

sequence repeats (Rana, 2003; Dongre and Parkhi, 2005;

Saravanan et al., 2007), Ampli ed fragment length pol-

ymorphism (Rana and Bhat, 2004), Random ampli ed

polymorphic DNA (Geng et al., 1995; Venu, 2001, Rao

et al., 2002). Restriction fragment length polymorphism

(Pendse et al., 2001; Dongre and Parkhi, 2005) and ISSR

marker (Dongre and Parkhi, 2005; Rana, 2006). These

are now widely used in hybrid purity check program.

Among all DNA markers, now SSR markers are found

to be promising with its co-dominant, polymorphic, dis-

criminative, reliable and repeatable features and can be

standardized for Distinctness, Uniformity and Stability

(DUS) testing (UPOV, 1997). This marker (SSR) undoubt-

edly overcome the problem of errors in morphological

based selections of hybrids which is occurring due to

plant growth stage and environmental  uctuation.Even

though molecular marker plays such an important role

in identifying the hybrid purity, there are no guidelines

available on the use of speci c number of markers &

sample size for 90, 95, & 99% purity in comparison with

the GOT (Grow-Out Test), where 400 seeds were used

for 95% purity. There is an urgent need for this method

to be standardized for large scale “Hybrid Purity Test”.

Hence, the present study was undertaken with the objec-

tive of optimizing the minimum sample size that can be

used for purity assessment of the brinjal hybrids. The

results obtained using SSR markers were then compared

with those from a GOT performed on the various sample

size and the percentage of genetic purity was calculated

for both GOT and SSR analysis.

MATERIALS AND METHODS

PLANT MATERIALS

The present study was carried out at Indian Institute of

Horticultural Research, Bangalore, India during 2014-

2016. Two commercial F

hybrid brinjal cultivars were

tested. Cultivar ‘Arka Anand’ and its parental (female

and male) lines were developed in Indian Institute of

Horticultural Research, Bangalore, India while ‘Brinjal

Asha’ and its related parental (female and male) lines

were provided by Noble Seeds Pvt. Ltd., Bangalore, India.

GROW OUT TEST (GOT)

To validate the conformity of the molecular marker-

based estimates of selfed or off-type or outcrossed plants

with the actual  eld morphological data, the experi-

ment was conducted by pooling 95% F

hybrids mixed

with 5% off-types / admixture individually in the fol-

lowing sets of 400, 300, 200 and 100. The parental and

four set of seed lots from each hybrid were sown in the

greenhouse with a day temperature of 24 + 3 ºC and a

night temperature of 18 + 3 ºC in the Indian Institute

of Horticultural Research, Bangalore. One-month-old

seedlings were transplanted to the polyhouse in 5m row

with 10cm plant to plant and 45cm row to row spacing.

Regular irrigation, fertilization, staking and crop protec-

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS OPTIMIZATION OF SAMPLING SIZE FOR DNA BASED PCR ASSAY 497

Arpita Pattanaik et al.

tion measures were adopted, and purity visual evalua-

tion was conducted based on the important morphologi-

cal characters throughout the growth period. The details

on morphological traits that have been recorded to dis-

tinguish the true hybrids from off types for all the two

hybrids along with their parents are furnished in table 1.

The genetic purity of hybrids was calculated as:

((Total number of plants-number of off types))

Hybrid purity (%) = ×100

(Total number of plants)

DNA EXTRACTION

Total genomic DNA was extracted from young and fresh

leaves using modi ed CTAB method (Doyle & Doyle,

1990). The quality and quantity of isolated genomic

DNA was checked using 0.8% Agarose gel electropho-

resis and Gene Quant UV Spectrophotometer (GE Health

Care Bio-Sciences Ltd., Bengaluru, India) respectively.

After quanti cation, the  nal concentration of DNA was

adjusted to 20ng/l and used as template DNA for PCR

ampli cation.

PCR AMPLIFICATION PROFILE

A total of 120 SSR markers were screened for the detection

of polymorphism within the parents of Arka Anand and

Brinjal Asha. Later the polymorphic SSR markers were

tested on F

population to determine the discriminatory

and stability of F

plants. The PCR assay was performed

in a 25l volume containing 2.5l of 10X PCR buffer, 2.5

µL of 20mM MgCl

, 2.5µL of 1mM dNTP mix, 2.5µL of

5pmol of each (F&R) primer, 2.5µl of 20ng/µl of template

DNA, 0.3µl of 3 units of Taq DNA polymerase and 9.67µL

of double distilled water to a total volume of 25µL. The

ampli cation was carried out in a thermocycler (model

TC-5000; Bibby Scienti c (Asia Limited, Hong Kong). A

touch down PCR protocol was optimised in brinjal with

a temperature program consisting of the initial dena-

turation at 95 °C for 30 minutes followed by 10 cycles

with a decrease of 1 ºC per cycle of denaturation at 94

°C for 30 seconds, annealing at 60-55 °C for 30 seconds

and polymerization at 72 °C for 1 minute followed by 30

cycles of denaturation at 94 °C for 30 seconds, annealing

Table 1. Morphological characters used to identify the selfed/offtypes during the Grow- out Test.

Brinjal (Arka Anand)

Sl. No. Morphological characters Female parent Male parent Hybrid (Brinjal Asha)

1. Plant: Growth habit Semi erect Semi erect Erect

2. Stem hairiness Present Slightly present Present

3. Leaf hairiness Present Absent Slightly Present

4. Leaf colour Dark green Light green Light green

5. Flower:colour Dark purple Dark purple Light purple

6. Flower:petal no. 5 6 6

7. Calyx:spininess Present Absent Absent

8. Fruit:colour of calyx Purplish Greenish Light Greenish

9. Fruit: General shape Obovate Ovoid Ovoid

10. Fruit: Colour Dark purple Dark purple Light purple

11. Fruit: Stripes Absent Present present

Brinjal (Asha)

Sl. No. Morphological characters Female parent Male parent Hybrid

1. Plant: Growth habit Semi erect Semi erect Erect

2. Stem hairiness Present Slightly present Present

3. Leaf hairiness Present Absent Slightly Present

4. Leaf colour Dark green Light green Light green

5. Flower:colour Dark purple Dark purple Light purple

6. Flower:petal no. 5 6 6

7. Calyx:spininess Present Absent Absent

8. Fruit:colour of calyx Purplish Greenish Light Greenish

9. Fruit: General shape Obovate Ovoid Ovoid

10. Fruit: Colour Dark purple Dark purple Light purple

11. Fruit: Stripes Absent Present present

498 OPTIMIZATION OF SAMPLING SIZE FOR DNA BASED PCR ASSAY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Arpita Pattanaik et al.

at 55 °C for 30 seconds and polymerization at 72 °C for 1

minute. Final elongation was at 72 °C for 5 min.

The ampli cation products were analyzed by electro-

phoresis in a 3% (w/v) agarose gel containing 0.5 µg/ml

ethidium bromide in 45 mM Tris-borate- (1mM) EDTA

buffer, pH 8.0. 25 µl of PCR products was loaded into

the well after adding 3µl of loading dye (50% (w/v) glyc-

erol and 50% (w/v) BPB). The 100bp DNA ladder (3B

bioscience, Spain) was used to calculate PCR product

size. Electrophoresis was carried out at 70V for 1hr. The

ampli ed fragments were visualized with UV transillu-

minator (Syngene, USA) and documented using UV-Pro

gel documentation system.

RESULTS AND DISCUSSION

In the Grow-out Test, purity evaluation was conducted

based on morphological traits. In the present study,

plants from four different sample sizes (400, 300, 200

and 100) of ‘Arka Anand and Brinjal Asha’ were studied

individually to determine if they were true-to-type for

ten morphological characters in the GOT (Table 1). In case

of Arka Anand, out of the ten morphological characters

analysed; Flower petal no., leaf colour, leaf length, fruit

shape and fruit colour exhibited the maximum variation

(Figure 1). The percentage of hybrid purity in GOT assay

for 400, 300, 200 and 100 sample sizes of Arka Anand

was calculated to be 95% (Table 2). In case of Brinjal

Asha, out of the ten morphological characters analysed;

 ower colour,  ower petal no., calyx spininess, fruit

shape, colour of calyx, fruit colour and presence of fruit

stripes exhibited the maximum variation (Figure 2). The

characters of few individuals shown deviation from the

standard characters were identi ed as off-type and they

were similar to those of the female parental type. The

percentage of hybrid purity in GOT assay for 400, 300,

200 and 100 sample sizes of Brinjal Asha was calculated

to be 93%, 95%, 95%, 94% respectively (Table 2).

In the PCR-based assay, a total of 120 pairs of

SSR markers were tested, with twenty-one markers

FIGURE 1. Variability in morphological parameters of Brinjal hybrid ArkaAnand

and its parents.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS OPTIMIZATION OF SAMPLING SIZE FOR DNA BASED PCR ASSAY 499

Arpita Pattanaik et al.

FIGURE 2. Variability in morphological parameters of Brinjal hybrid Asha and its parents.

(Table 3) showing polymorphism between the parental

lines. Out of 21 polymorphic markers, two SSR markers

(eme08D09 & emb01F16) was found to be Co-dominant.

Using eme08D09, we were able to determine hybrid

purity of two Brinjal hybrids (Arka Anand & Brinjal

Asha) resulted in ampli cation of 210bp female-speci c

amplicon (FSA) as well as 230bp male-speci c ampli-

con (MSA). On the other hand, emb01F16 was only use-

ful for determining hybrid purity of one hybrid (Brinjal

Asha) resulted in ampli cation of 150bp female- speci c

amplicon (FSA) as well as 170bp male-speci c amplicon

(MSA). Further, these co-dominant markers (eme08D09

& emb01F16)

was tested on all the four-sample size of

each hybrids to detect the heterozygosity of the hybrids.

In 400 hybrid sample size of Arka Anand, the percent-

age of hybrid purity in SSR analysis was calculated to

be 95% (Table 2). Similarly, in the case of 300, 200 and

100 hybrid sample size of Arka Anand, the percentage

500 OPTIMIZATION OF SAMPLING SIZE FOR DNA BASED PCR ASSAY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Arpita Pattanaik et al.

Table 2. Comparison of hybrid purity assessment based on GOT and SSR analysis with different

sample size in Brinjal hybrids.

Brinjal (Arka Anand)

Sample size GOT (%) Putative false hybrid

individuals

SSR Analysis (%) Putative false hybrid

individuals

400 95 20 95 20

300 95 15 94.7 16

200 95 10 95 10

100 95 5 95 5

Brinjal (Asha)

Primer 1

Sample size GOT(%) Putative false hybrid

individuals

SSR Analysis (%) Putative false hybrid

individuals

400 95 20 95 20

300 95 15 94.3 17

200 95 10 95 10

100 95 5 95 5

Primer 2

Sample size GOT(%) Putative false hybrid

individuals

SSR Analysis (%) Putative false hybrid

individuals

400 95 20 95 20

300 95 15 94.3 17

200 95 10 95 10

100 95 5 95 5

FIGURE 3. Ampli cation pro le of brinjal hybrids ‘Arka Anand’and ‘Asha’ and its parents with Co-dominant

SSR maker eme08D09 resolved on 3% agarose gel; M=100bp DNA ladder, P1= Male parent, P2= Female par-

ent, Lane 1-5 = individual F1 plants.

of hybrid purity was calculated to be 94.7%, 95% and

95%, respectively (Table 2). In 400 hybrid sample size

of Brinjal Asha, the percentage of hybrid purity in SSR

analysis was calculated to be 95% (Table 2). Similarly, in

the case of 300, 200 and 100 hybrid sample size of Brin-

jal Asha, the percentage of hybrid purity was calculated

to be 94.3%, 95% and 95%, respectively (Table 2).

Based on the results obtained from present study, it is

clear that there is a need to critically assess the hybrid

purity of popular cultivars of premium quality at each

and every stage of seed multiplication and processing

with the help of molecular markers so that, seeds culti-

vated by farmers are true-to-type and fetches premium

price. In the present study where, simultaneous labora-

tory and  eld study has been done for hybrid purity

check of brinjal hybrids, that to in the past number of

similar approaches gain promising results and elimi-

nated any bias factor in laboratory analysis (Smith and

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS OPTIMIZATION OF SAMPLING SIZE FOR DNA BASED PCR ASSAY 501

Arpita Pattanaik et al.

Wych, 1986; Orman et al., 1991). As Singh and Singhal

(1999); Tunwar and Singh (1988) recommended to use

minimum 400 individual seeds for purity testing as per

Indian minimum seed certi cation standards, we have

observed that by using SSR primers (eme08D09 and/

or emb01F16) with only 100 seeds sample size; hybrid

purity could be calculated upto 95% percentage which

was in agreement with the results obtained via, 400

sample sizes tested in GOT. Hence results indicated that

instead of handling 400 samples in GOT, use of only 100

seeds in SSR analysis proving useful with less time and

cost concerned approach.

The present study also recorded the same results with

SSR markers when tested with sample size of 400, 300

and 200. Hence instead of using 70 days long protocol

involved with GOT, by applying few days protocol of SSR

with the given primers; it will assure the early releases

of hybrid seeds. As per study results of  eld grow out

tests and SSR markers of individual seeds; highly sig-

ni cant correlation does exist between two studies and

more reliability to the latter technique. As the results

were fairly consistent; hence SSR markers study can be

recommended as a supplementary technique for making

speedy decision to accept or reject hybrid seed lots based

on contamination.

GOT certainly delays the whole process of decision

making, packaging and marketing of the commercial

seed. Thus, farmers did not get the hybrid seeds at right

time for sowing, resultant precluding the immediate cul-

tivation of the hybrid seed produced. In addition to cap-

ital invested on hybrid seed production and additional

expenditure incurred on storage of hybrid seed, GOT

ultimately increases the hybrid seed cost (Nandakumar et

al, 2004). Similar to present study, use of SSR markers

for genetic purity testing has been demonstrated in rice

(Nandakumar et al., 2004); in maize (Wang et al., 2002)

and in sun ower (Pallavi et al., 2011) and overall data

remained comparable with  eld grow out test. Similar

to SSR marker applied in present study with minimum

sample size; number of workers with other plants have

applied different sample size of seeds for RAPD primers

for example: 400 seeds in chicory (Bellamy et al., 1998),

120 in canola (Marshall et al., 1994), 40 in tomato (Rom et

al., 1995), 30 in Chinese cabbage (Meng et al., 1998) and

10-20 in pepper (Ballester and Vicente, 1998) to assess the

genetic purity of the hybrid seeds.

Overall data of the present study have showcased that

SSR markers are quick, reliable and results are mostly

consistent with morphological analysis in the  eld study.

Markers reported in the study could be involved for rou-

tine genetic purity testing of Arka Anand and Brinjal

Asha hybrids. The SSR markers developed through this

study will be useful for seed industry to select appropriate

marker combinations and assess genetic purity of the crop.

Table 3. List of polymorphic SSR markers used in the

study.

Sl.

no.

Primer Name Primer Sequence 5’ - 3’

1 emi04H10 F ATCGGAGCAAGAGACATTAGATGC

R GTTTCCAACACAGTCCCCAATACAACAA

2 emb01D10 F AAGAATCGGTCCTCTTTGCATTGT

R TGCTTTTCACCTCTCCGCTATCTC

3 eme08D09 F ATGGATTAGCATGTGGAGGACTGAA

R GTTTCATGGTAGGTGGAGACAGAACCA

4 emf21K08 F ATCAATGACACCCAAAACCCATTT

R GTTTGAAAACCCAATACAAATCCGA

5 emg11M21 F ATAGCCTACTGCCTTCAAGACCAT

R GTTTCCTACGTCCAGTCCCCTTAGGT

6 emb01J19 F GACAGGGATAGGGGTACGGATAGG

R ATCCATGTGATGCCTCGATTTTCT

7 emb01F16 F AAAACAGAAGCAAAGTCGGCAGTC

R GTCCACCAACACCTTACCATCCTC

8 emi06A04 F ATTTGGGACAAATGTGGGTGAGAC

R GTTTCCACGCTACTTAGGGGACTCAA

9 eme03F04 F ATATGACGACAGACGTAAAGCGACC

R GTTTCAGAGTTTTGCCATCTGTGTCGAG

10 emf11B07a F ACGAGAGTTGCTACAGTTAAGGGG

R GTTTGGGGACCAAAGTGTATTTTCAAGG

11 emk04H07 F ATTTGGCTGGGTTGTTGGTCTAGT

R GTTTGGCCCAATTACTCAAATACCCTG

12 emf21C11 F AGGTTGGAGCCATGATTACTTGAA

R GTTTGCTACCTATCAAACAGGCGGAA

13 emf01O04 F ATCCGTTGATACTAGCCGTTGCCT

R GTTTCACCCGGTATGAGTGTATCCC

14 emk04N11 F ATCTCCCCCTCAACTTTGAACAAT

R GTTTGTGTGATATAGCCCAACAATTCAC

15 emh11N11 F ATTCAGTTCTTCGCTTTGGAGCTT

R GTTTCCAAACCCGACCCATCCTAAATAA

eme05G05 F ACAAGAAAGAGGAGCTGGGGAAATTG

R GTTTCCTTCTTGGGAAGACAACTTATCA

17 eme01D03 F ACAAGAATCGGTCCTCTTTGCATTGT

R GTTTGCTTTTCACCTCTCCGCTATCTC

18 emg11P03 F ACTCGCCTCTCTCAATCTTTCTTG

R GTTTCAATATAACCTCGGCTATGAGACCC

19 emd01C04 F ACCTGCATGAAATGTGTTTGAGTG

R GTTTGGGTCTTTTCATCTCAAAATGGG

20 emh05H12 F AGTCACTGCTCTTAGTTTCTGCAA

R GTTTCAGAGCAGCGATCCTTTCTTCATT

21 emd01A01 F ACAGCAAAATGCCTAATGACAGCACA

R GTTTATGCCTGACTCTGCTTGTGCCTA

502 OPTIMIZATION OF SAMPLING SIZE FOR DNA BASED PCR ASSAY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Arpita Pattanaik et al.

ACKNOWLEDGEMENTS

This research was supported by, Indian Institute of Hor-

ticulture Research, Bangalore. Thanks to Dr. C. Aswath

and Dr. D. C. L Reddy for their continuous guidance and

support in the completion of this research.

REFERENCES

Arus P. (1983). Genetic purity of commercial seed lots. In

Isozymes in Plant Genetics and Breeding (Part A), (eds. S.D.

Tanksley and T.J. Orton), Elsevier, Amsterdam, Pp 415–423.

Ballester J, and Vicente M.C. (1998). Determination of F1 hybrid

seed purity in pepper using PCR-based markers. Euphytica,

Vol. 103(2), Pp 223-226.

Bellamy A, Vedel F and Bannerot H. (1996). Varietal identi -

cation in Chicorium intybus L. and determination of genetic

purity of F1 hybrid seed samples, based on RAPD markers. The

Journal of Plant Breeding, Vol. 115, Pp 128-132.

Borle U.M.,. Mehetre S.S. and Harer P.N. (2007). Seed protein

banding pattern studies on different cytosteriles, their main-

tainers, fertility restores and hybrids of upland cotton. Jour-

nal of Maharashtra Agricultural University, Vol. 32, Pp 208–

210.

Dadlani M, Vashist V, Singh D.P. and Varrier A. (1997). A com-

parison of  eld Grow out test and electrophoresis methods for

testing genetic purity of cotton hybrid seed. Seed Research,

Vol. 24, Pp 160–167.

Daunay M.C. and Hazra P. (2012). “Eggplant,” in Handbook of

Vegetables, eds Peter KV and Hazra P (Houston, TX: Studium

Press), 257–322.

Docimo T, Francese G, Ruggiero A, Batelli G, De Palma M, Bas-

solino L, Toppino L, Rotino G.L., Mennella G, Tucci M. (2016).

Phenylpropanoids accumulation in eggplant fruit: characteri-

zation of biosynthetic genes and regulation by a MYB tran-

scription factor. Frontiers in Plant Science, Vol. 6, Pp 1233.

Dongre A and Parkhi V. (2005). Identi cation of cotton hybrid

through the combination of PCR based RAPD, ISSR and micro-

satellite markers. Journal of Plant Biochemistry and Biotech-

nology, Vol. 14, Pp 53–55.

Doyle JJ and J. L. Doyle. (1990). Isolation of Plant DNA from

Fresh Tissue. Focus, Vol. 12(1), Pp 13-15.

FAO. (2014). FAOSTAT Production Databases. Available online

at: http://www.faostat.fao.org (Accessed January 30, 2017).

Geng C.D., Gong Z.Z., Huang J.Q. and Dang Z.L. (1995). Iden-

ti cation of differences between cotton cultivars (G. hirsutum)

using RAPD method. Jiangsu Journal of Agricultural Science,

Vol. 11 Pp 21–24.

Marshall P, Marchand M.C., Lisieczko Z and Landry B.S. (1994).

A simple method to estimate the percentage of hybridity in

canola (Brassica napus) F 1 hybrids. Theoretical and Applied

Genetics, Vol. 89(7-8), Pp 853-858.

Medakker A and Vijayaraghavan V. (2007). Successful com-

mercialization of insect-resistant eggplant by a public–private

partnership: reaching and bene ting resource-poor farmers.

In: Intellectual Property Management in Health and Agricul-

tural Innovation: A Handbook of Best Practices, Krattiger A,

Mahoney RT, Nelsen L, Thomson JA, Bennett AB, Satyanaray-

ana K, Graff GD, Fernandez C, and Kowalski SP (Oxford: MIHR

and Davis; PIPRA), Available online at: www.ipHandbook.org.

Mehetre S and Dahat D. (2001). Page of seed proteins as a tech-

nique for varietal identi cation of Gossypium spp.: A review,

Journal of Maharashtra Agricultural University, Vol. 26, Pp

233–238.

Meng, J, S. Shi, L. Gan, Z. Li and X. Qu. (1998). The produc-

tion of yellow seeded Brassica napus (AACC) through crossing

inter-speci c hybrids of B. campestris (AA) and B. carinata

(BBCC), with B. napus. Euphytica, Vol. 103, Pp 329-333.

Nandakumar N, Singh A.K., Sharma R.K., Mohapatra T,

Prabhu K.V. and Zaman F.U. (2004). Molecular  ngerprinting

of hybrids and assessment of genetic purity of hybrid seeds

in rice using microsatellite markers. Euphytica, Vol. 136, Pp

257–264

NHB database. (2014). http://nhb.gov.in/statistics/Publica-

tion/Horticulture%20At%20a%20Glance%202017%20for%20

net%20uplod%20(2).pdf

FIGURE 4. Ampli cation pro le of brinjal hybrid ‘Asha’and its parents

with Co-dominant SSR maker emb01F16 resolved on 3% agarose gel;

M=100bp DNA ladder, P1=Male parent, P2= Female parent, Lane 1, 2,

4-17=individual F1 plants, Lane 3= Selfed/Offtype

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS OPTIMIZATION OF SAMPLING SIZE FOR DNA BASED PCR ASSAY 503

Arpita Pattanaik et al.

Pallavi H.M., Rame Gowda, Vishwanath K, Shadakshari Y.G.

and Bhanuprakash K. (2011). Identi cation of SSR markers for

hybridity and seed genetic purity testing in sun ower (Heli-

anthus annuus L.). Seed Science & Technology, Vol. 39, Pp

259-264.

Pendse R, Malhotra S, Pawar S and Krishna T. (2001). Use of

DNA markers for identifying inbreds and hybrid seeds in cot-

ton (Gossypium hirsutum L.). Seed Science & Technology, Vol.

29, Pp 503–508.

Plazas M, Prohens J, Cunat A.N., Vilanova S, Gramazio P,

Herraiz F.J. and

Andujar I. (2014). Reducing capacity, chlo-

rogenic acid content and biological activity in a collection of

scarlet (Solanum aethiopicum) and gboma (S. macrocarpon)

eggplants. International Journal of Molecular Sciences, Vol.

15(10), Pp 17221–17241.

Raigon M.D., Prohens J, Munoz-Falcon J.E. and Nuez F. (2008).

Comparison of eggplant landraces and commercial varieties

for fruit content of phenolics, minerals, dry matter and pro-

tein. Journal of Food Composition and Analysis, Vol. 21, Pp

370–376.

Rakshit A, Vashisht V, Rakshit S, Dadlani M. (2008). Electro-

phoresis technique for varietal identi cation and genetic purity

in hybrid cotton (Gossypium hirsutum L.). Seed Research, Vol.

36, Pp 28–32.

Rana M.K. (2003). C.F. Use of DNA markers for assessing purity

of parental lines and hybrids in various crops, IARI, New Delhi.

Rana M.K., Singh S and Bhat K.V. (2006). RAPD, STMS and

ISSR markers for genetic diversity and hybrid seed purity test-

ing in cotton. Seed Science & Technology, Vol. 35, Pp 709–

721.

Rao R.G.S, Dadlani M and Mahapatra T. (2002). F1 hybrid seed

analysis by RAPD markers in cotton hybrid (Gossypium spp.),

Seed Technology News, 32, 162.

Rom M, Bar M, Rom A and Polowsky. (1995). Purity control of

F1 hybrid tomato cultivars by RAPD markers. Plant Breeding.

Vol. 114, Pp 188-190.

Rotino G.L., Perri E, Acciarri N, Sunseri F, Arpaia S. (1997).

Development of eggplant varietal resistance to insects and dis-

eases via plant breeding. Advances in Horticultural Science,

Vol. 11, Pp 193–201.

Saravanan N, Ram S, Thiruvengadam V, Ravikesavan R and

Raveendran T. (2007). Production and fertility restoration of

an interspeci c Hybrid between Gossypium hirsutum L. and G.

raimondii U. CytologIia, Vol. 72, Pp 195–203.

Schippers R.R. (2000). African indigenous vegetables, an over-

view of the cultivated species, Natural resources Institute,

Chatham, UK.

Singh D.P. and Singhal N.C. (1999). Optimum size for grow out

test of cotton hybrids. Seed Research, Vol. 27 (2), Pp 135-139.

Smith J.S.C. and Wych R.D. (1986). The identi cation of female

selfs in hybrids of maize using electrophoresis and morphol-

ogy. Seed Science & Technology, Vol. 14, Pp 1-8.

Tunwar N.S.and Singh S.V. (1988). Indian Minimum Seed Cer-

ti cation Standards published by, The Central Seed Certi ca-

tion Board, Department of Agriculture and Corporation, Min-

istry of Agriculture, Government of India.

UPOV. (1997). http://www.upov.int/edocs/tgdocs/en/tg080.pdf

Venu A. (2001). Development of molecular marker-based seed

identi cation procedure for cotton parents and hybrids (Gos-

sypium spp.). M. Sc. Thesis, Tamil Nadu Agricultural Univer-

sity, Coimbatore, India.

Wang J, Zhong G.Y., Chin E.C.L., Register J.C., Riley R.D., Nie-

ber W.S. and Smith J.S.C. (2002). Identi cation of parents of F1

hybrids through SSR pro ling of maternal and hybrid tissue.

Euphytica, Vol. 124, Pp 29-34.

504 OPTIMIZATION OF SAMPLING SIZE FOR DNA BASED PCR ASSAY BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS