Assessment of Hybrid Purity in Maize (Zea mays L.) Using RAPD and SSR Markers

Abstract

Maize (Zea mays L.; 2n=20) is third most important cereal crop and referred as the "Queen of Cereals" and "Miracle Crop" due to its high productivity potential as compared to the other cereals. Maize is native to Mexico and Central America. Maize is the most important crops in the world that can be grown in diverse seasons, ecologies it is used mainly for human food, animal feed and industry. This work was carried out to assess the hybrid purity of maize hybrid Hyd.18227 × Hyd.10306 (Tall) and their parental line Hyd.18227 (Dwarf) and Hyd.10306 (Tall) using RAPD and SSR markers. DNA extracted from young leaves and PCR were conducted using 24 RAPD and 18 SSR markers. Out of fifteen RAPD markers and five SSR makers showed the polymorphism between the maize parental lines and remaining primer produced monomorphic banding pattern. The SSR primer Umc-1858 identified 64 % hybrid seeds of total cross.

Full text

~ 870 ~
The Pharma Innovation Journal 2021; 10(4): 870-874
ISSN (E): 2277- 7695
ISSN (P): 2349-8242
NAAS Rating: 5.23
TPI 2021; 10(4): 870-874
© 2021 TPI
www.thepharmajournal.com
Received: 05-02-2021
Accepted: 08-03-2021
NA Shinde
Vilasrao Deshmukh College of
Agricultural Biotechnology,
Latur, Maharashtra, India
AA Bharose
Vilasrao Deshmukh College of
Agricultural Biotechnology,
Latur, Maharashtra, India
DK Sarode
Vilasrao Deshmukh College of
Agricultural Biotechnology,
Latur, Maharashtra, India
RS Swathi
Vilasrao Deshmukh College of
Agricultural Biotechnology,
Latur, Maharashtra, India
PA Pimpale
Vilasrao Deshmukh College of
Agricultural Biotechnology,
Latur, Maharashtra, India
SS Shinde
Vilasrao Deshmukh College of
Agricultural Biotechnology,
Latur, Maharashtra, India
Corresponding Author:
AA Bharose
Vilasrao Deshmukh College of
Agricultural Biotechnology,
Latur, Maharashtra, India
Assessment of Hybrid Purity in Maize (Zea mays L.)
Using RAPD and SSR Markers
NA Shinde, AA Bharose, DK Sarode, RS Swathi, PA Pimpale and SS
Shinde
Abstract
Maize (Zea mays L.; 2n=20) is third most important cereal crop and referred as the “Queen of Cereals”
and “Miracle Crop” due to its high productivity potential as compared to the other cereals. Maize is
native to Mexico and Central America. Maize is the most important crops in the world that can be grown
in diverse seasons, ecologies it is used mainly for human food, animal feed and industry. This work was
carried out to assess the hybrid purity of maize hybrid Hyd.18227 × Hyd.10306 (Tall) and their parental
line Hyd.18227 (Dwarf) and Hyd.10306 (Tall) using RAPD and SSR markers. DNA extracted from
young leaves and PCR were conducted using 24 RAPD and 18 SSR markers. Out of fifteen RAPD
markers and five SSR makers showed the polymorphism between the maize parental lines and remaining
primer produced monomorphic banding pattern. The SSR primer Umc-1858 identified 64 % hybrid seeds
of total cross.
Keywords: Maize, F1 hybrids, hybrid purity, RAPD, SSR
1. Introduction
Maize is third most important cereal crop after rice and wheat in the world. Maize is referred
as the “Queen of Cereals” and “Miracle Crop” due to its high productivity potential as
compared to the other cereals. Maize is native to South America. Maize is diploid with
chromosome number 2n=20. Maize (Zea mays L.) is popularly known as corn. The important
maize growing countries are USA, China, Brazil, Mexico, India, Philippines, South Africa and
Indonesia. In India, maize has been grown in an area of 9.2 million ha with production of
24.17 MT and average productivity of 2.56 t/ha (USDA 2017-18). In Maharashtra the area
under maize is increasing every year. The area under maize in Maharashtra is about 2 million
ha with a production of 3.45 MT and productivity of 3.24 t/ha.
Conventionally, purity of F1 hybrids is assessed by grow-out test (GOT) at the field. This test
is time consuming and resource intensive (Elci and Hancer, 2014) [8]. To overcome this
disadvantage, biochemical markers are being used in many crops. However, repeatability and
accuracy of these results on biochemical markers are subject to question. This made a way for
the use of DNA markers particularly the co-dominant markers. Hence, it is essential to develop
a more rapid, accurate and cost effective method for the identification of maize hybrids.
Considering the disadvantages of grow out test, rapid and reliable methods using biochemical
and molecular markers are getting attention for genetic purity testing. Now several
biochemical and molecular markers i.e., RAPD, ISSR, AFLP, SSR etc. are being used.
Molecular marker technology provides effective, fast, accurate and appropriate tool for crop
improvement. DNA markers such as RFLP (Restriction Fragment Length Polymorphism),
SSR (Simple Sequence Repeats), CAPS (Cleaved Amplified Polymorphic Sequences), RAPD
(Randomly Amplified Polymorphic DNA), ISSR (Inter Simple Sequence Repeats), AFLP
(Amplified Fragment Length Polymorphism), SNPs (Single Nucleotide Polymorphisms),
Sequence characterised amplified regions (SCAR), Expressed sequence tag (EST) and
sequence tagged sites (STS) have been used for varietal identification, seed purity testing,
genetic similarity analysis and marker-assisted selection of crops in many species. SSRs, also
known as microsatellites, are repeated sequences of DNA and they can easily detect both
parental alleles, confirmation of F1 hybrid purity and diversity in maize hybrids by using
molecular markers are more accurate because of their co-dominancy. Also, SSR marker
efficiency was analyzed for further studies on maize.
The objective of this study was assessment of hybrid purity in F1 populations.

~ 871 ~
The Pharma Innovation Journal http://www.thepharmajournal.com
2. Materials and Methods
Seeds of maize hybrid namely Hyd.18227 × Hyd.10306 (Tall)
and parental line Hyd.18227 (Dwarf) and Hyd.10306 (Tall)
was collected from Department of Botany. The experiment
was conducted at Department of Plant Biotechnology,
Vilasrao Deshmukh College of Agricultural Biotechnology,
Latur (M.S.) during the year 2017-18.
2.1 DNA extraction and Quantification
DNA was extracted by Cetyl Trimethyl Ammonium Bromide
(CTAB) protocol given by Doyle and Doyle (1990) with
some modifications and quantification was done by
Spectrophotometer. DNA was diluted in 0.1 T.E buffer to a
concentration of 50 ng/micro liter for PCR analysis.
2.2 Primer Screening and polymerase chain reaction
Twenty four random RAPD primers (GeNei) were used for
the present investigation. The list of the primers with their
sequences is given in the below in Table No. 1. PCR reaction
were performed using a 25 μl reaction mixture containing
10X PCR buffer with MgCl2, 10 mM dNTPs, 10 pmol primer,
1 U of Taq DNA polymerase, 50 ng/µl template DNA and
sterile distilled water. For DNA amplification the DNA
thermal cycler (Sensoquest Labcycler, Germany) was
programmed as follows: incubation at 94⁰C for 5 min; 35
cycles at 94⁰C for 30 sec (denaturation), 37⁰C for 30 sec
(annealing) and 72⁰C for 30 sec (extension), followed by one
final extension cycle of 10 min at 72⁰C. After completion of
the cycles the samples were kept at 40C till electrophoresis.
Eighteen SSR primers (Eurofins Genomics, India) were used
for the present investigation. The list of the primers with their
sequences is given in the below in Table No. 2. PCR reaction
were performed using a 20 μl reaction mixture containing
10X PCR buffer with MgCl2, 10 mM dNTPs, 10 pmol of each
primer (both forward and reverse), 1U of Taq DNA
polymerase, 50 ng/µl template DNA and sterile distilled
water. For DNA amplification the DNA thermal cycler
(Sensoquest Labcycler, Germany) was programmed as
follows: incubation at 94⁰C for 10 min; 40 cycles at 94⁰C for
30 sec (denaturation), 58⁰C for 50 sec (annealing) and 72⁰C
for 1:30 min (extension), followed by one final extension
cycle of 10 min at 72⁰C. The annealing temperature was
adjusted based on the specific requirement of each primer
combination.
The amplified products were resolved on 2 % agarose gel for
SSR markers, 1.5% for RAPD marker at 5V/cm for 1 to 1.5
hr. After electrophoresis, the gel was taken out for
observation of banding pattern and photographed on a Gel
Documentation System (Alpha-Innotech, USA).
2.4 Genetic Purity Test of Hybrid Seeds
The percentage of hybrid genetic purity were calculated based
on banding pattern that appears on the individual plant
samples, with the following formula:
Where: TS (total sample) = number of samples/individual
plants were tested
NH (non-hybrid) = number of samples/individual plants
having the same banding pattern with female or male parents.
3. Results and Discussion
3.1 Development of F1
In present investigation, two parental maize genotypes were
used for crossing of which one genotype was dwarf and
remaining genotype was tall. The Hyd.18227 (Dwarf) used as
female and Hyd.10306 (Tall) used as male parent. These
genotypes were crossed successfully to obtained tall hybrid.
The F1 hybrid seeds were harvested from particular cross after
seed set and maturity.
3.2 DNA isolation and quality analysis
The genomic DNA was extracted from young leaves of maize
hybrid and their respective parents by Cetyl Trimethyl
Ammonium Bromide (CTAB) DNA extraction method given
by Doyle and Doyle (1990) [7] with some modifications. This
method yielded qualitatively as well as quantitatively pure
genomic DNA. The quantification of extracted DNA was
done by measuring absorbance at 260 nm wavelengths. Purity
of DNA was checked by reading absorbance ratio of
A260/280 for protein contamination. The quantitative and
qualitative analysis was done by resolving DNA on 0.8%
agarose gel. The concentrations of all samples were ranged
between 500-1400 ng/μl. Working samples were prepared by
diluting with sterile nuclease free water to obtain final
concentrations of 50 ng/μl for RAPD and SSR analysis.
3.3 Hybrid confirmation based on RAPD fingerprint
profile analysis
RAPD analysis would be very useful in breeding for rapid and
early verification of hybrid population and even purity testing
of different seed lots, allowing the detection of true hybrids
and verification of parentage of the hybrids and
lines/cultivars. RAPD analysis has been successfully used for
hybrid and parentage verification of other crop plants. RAPD
marker fingerprinting data was used for hybrid confirmation.
The present study utilized total 24 RAPD markers for
identification of maize hybrid (Hyd.18227×Hyd.10302) along
with their female parental line (Hyd.18227) and male parental
line (Hyd.10302). The hybrid confirmation discussed here
under was done by comparing banding patterns of hybrids
with their respective parents as described by Akhare et al.
(2008) [1]. Different types of markers have been designated for
hybrid confirmation as per convenience in Coffea arabica
(Mishra et al., 2011) and cotton (Mehetre et al., 2004) [11, 10].
For hybrid purity analysis, the total of six types of banding
patterns was observed in the parents and hybrids (Table No.
3).
Table No 1: List of RAPD primers used for hybrid purity assessment
Sr. No.
Primer ID
Sequence
Sr. No.
Primer ID
Sequence
1
RPI 1
5’AAAGCTGCGG3’
13
RPI 13
5’ACGGCAAGGA3’
2
RPI 2
5’AACGCGTCGG3’
14
RPI 14
5’ACTTCGCCAC3’
3
RPI 3
5’AAGCGACCTG3’
15
RPI 15
5’AGCCTGAGCC3’
4
RPI 4
5’AATCGCGCTG3’
16
RPI 16
5’AGGCGGCAAG3’
5
RPI 5
5’AATCGGGCTG3’
17
RPI 18
5’AGGCTGTGTC3’

~ 872 ~
The Pharma Innovation Journal http://www.thepharmajournal.com
6
RPI 6
5’ACACACGCTG3’
18
RPI 19
5’AGGTGACCGT3’
7
RPI 7
5’ACATCGCCCA3’
19
RPI 20
5’AGTCCGCCTC3’
8
RPI 8
5’ACCACCCACC3’
20
RPI 21
5’CACGAACCTC3’
9
RPI 9
5’ACCGCCTATG3’
21
RPI 22
5’CATAGAGCGG3’
10
RPI 10
5’ACGATGAGCG3’
22
RPI 23
5’CCAGCAGCTA3’
11
RPI 11
5’ACGGAAGTGG3’
23
RPI 24
5’CCAGCCGAAC3’
12
RPI 12
5’ACGGCAACCT3’
24
RPI 25
5’GAGCGCCTTC3’
Table No 2: List of SSR primers used for hybrid purity assessment
S. No.
Primer ID
Forward
Reverse
1
Phi053
CTGCCTCTCAGATTCAGAGATTGAC
AACCCAACGTACTCCGGCAG
2
Phi057
CTCATCAGTGCCGTCGTCCAT
CAGTCGCAAGAAACCGTTGCC
3
Phi080
CACCCGATGCAACTTGCGTAGA
TCGTCACGTTCCACGACATCAC
4
Phi96100
AGGAGGACCCCAACTCCTG
TTGCACGAGCCATCGTAT
5
Phi328175
GGGAAGTGCTCCTTGCAG
CGGTAGGTGAACGCGGTA
6
Phi034
TAGCGACAGGATGGCCTCTTCT
GGGGAGCACGCCTTCGTTCT
7
Phi299852
GATGTGGGTGCTACGAGCC
AGATCTCGGAGCTCGGCTA
8
pumc1064
GTGGGTTTTGTCTGTAGGGTGGTA
TCCATCCACTCGACTTAAGAGTCC
9
pumc1013
TAATGTGTCCATACGGTGGTGG
AGCTGGCTAGTCTCAGGCACTC
10
pumc1746
ACACGAGCATCCTACATCCTCCTA
ACCTTGCCTGTCCTTCTTTCTCTT
11
pumc1071
AGGAAGACACGAGAGACACCGTAG
GTGGTTGTCGAGTTCGTCGTATT
12
pumc1040
CATTCACTCTCTTGCCAACTTGA
AGTAAGAGTGGGATATTCTGGGAGTT
13
pumc1035
CTGGCATGATCACGCTATGTATG
TAACATCAGCAGGTTTGCTCATTC
14
pumc1066
ATGGAGCACGTCATCTCAATGG
AGCAGCAGCAACGTCTATGACACT
15
Umc-1858
GTTGTTCTCCTTGCTGACCAGTTT
ATCAGCAAATTAAAGCAAAGGCAG
16
Umc-1600
CATATTGATAGGCTAGGCAAATGGC
CAATACAAGTTTGGTCCCAAATAAGC
17
Umc1605
CCAGGAGAGAAATCAACAAAGCAT
GGAGAAGCACGCCTTCGTATAG
18
Umc1331
TTATGAACGTGGTCGTGACTATGG
ATATCTGTCCCTCTCCCACCATC
Table No 3: Types of RAPD markers observed in hybrid and their parents
+ indicates presence of band while – indicates absence of band.
MPS-Male parent specific band
FPS-Female parent specific band
Out of twenty four RAPD primers fifteen primers were found
to be polymorphic and nine primers were found to be
monomorphic. Confirmation of hybrid was achieved by six
primers amplified the specific allele size of RPI-1 (370 bp),
RPI-8 (900 bp), RPI-10 (1200 bp), RPI-18 (400 bp), RPI-15
(700-500 bp), and RPI-20 (390 bp) in hybrid and male parent
but not amplified in female parent. These six primers were
used to confirm the true hybrid. These primers produced MPS
band of Type 2 maker (Fig. No 1). The two primers RPI-2 and
RPI-25 amplified the specific allele of size 1200 bp and 280
bp respectively in hybrid and female parent but not amplified
in male parent. These primers produced FPS band of Type 3
maker and used for hybrid confirmation (Fig. No.2). Three
primer viz., RPI-5, RPI-6 and RPI-7 amplified the specific
allele size (1100 bp), (600 bp) and (1000 bp) respectively in
male parent but not amplified in hybrid and female parent.
These primers produced Type 5 maker and good markers to
identify off types (Fig. No.3). Two primers viz., RPI-4 and
RPI-19 produced female parent specific band ̴1200 bp and ̴
600 bp respectively. These primers produced Type 4 marker
and good markers to identify self and off types (Fig. No.3).
RPI-24 and RPI-21 produced the cross specific band size 250
bp and 300 bp respectively. These primers produced Type 6
marker and useful for identification of specific cross (Fig.
No.3).
Similar investigations based on RAPD analysis have been
successfully employed for parentage verification, hybrid
confirmation, cultivar identification and purity testing in
maize (Asif et al, 2006; Mrutu 2015) [2, 12] and other crops
such as sorghum (Akhare et al., 2008), rice (Deshmukh et al.,
2013) [1, 5] and cotton (Asif et al, 2009 and Dongre et al. 2005)
[3, 6].
3.5 Hybrid confirmation based on SSR fingerprint profile
analysis
From eighteen primers tested five primers were found
polymorphic and thirteen primers were found monomorphic.
Primer Umc-1858 produced polymorphic bands and was
capable to distinguish parental line of maize hybrid.
Microsatellite marker Umc-1858 was specific used for testing
genetic purity of Hybrid (Hyd.18227×Hyd.10306). The SSR
primer Umc-1858 had amplified allele of size 170 bp in
female parent (Hyd.18227).On the other hand the male parent
(Hyd.10306) had an amplicon at 180 bp. However, hybrid
(Hyd.18227×Hyd.10306) exhibited the alleles of both parents
confirming the heterozygosity of the hybrid by having two
bands at 170 and 180 bp. These primers produced both MPS
Type 2 maker and FPS Type 3 marker (Fig.No.4). Thus, it
Markers Type
Female (F)
Male (M)
Cross (H)
Nature
Remark
1
+
+
+
Monomorphic
Good marker to confirm hybrid of its respective
parents (male and/or female
2
-
+
+
MPS
3
+
-
+
FPS
4
+
-
-
-
Good markers to identify self and off types
5
-
+
-
-
6
-
-
+
Cross specific
Useful for identification of cross

~ 873 ~
The Pharma Innovation Journal http://www.thepharmajournal.com
confirmed that the presence of both female and male parent
alleles was observed as a resultant of crossing between two
parents (F1 hybrid). The Phi-328175 and Pumc-1746 primers
amplified a specific allele size of 270 bp and 130 bp
respectively in cross and female parent but not in male parent.
These primers produced female specific band of Type 3
marker (Fig. No.4). The primer Pumc-1013 produced the
female parent specific allele size 170 bp and male parent
specific allele size 130 bp but not amplified in cross. These
primer produced Type 4 and 5 markers (Fig. No.4). The
primer Pumc-1066 amplified allele size 190 bp and 170 bp in
cross but allele size 190 bp not amplified in female and male
parents. These primer produced Type 6 marker and these band
is useful for identification of specific cross (Fig. No.4).
The banding pattern of all these hybrids showed both the
amplicons present in female as well as male parent, thus
confirming the genuine crossing and heterozygotic condition
of the hybrid. The use of SSR markers for genetic purity
testing has already been demonstrated in maize (Daniel et al.,
2012; Mrutu, 2015; Wu et al., 2010; Sudharani et al., 2013 [4,
12, 14, 13]. In all the above studies SSR markers were used for
germplasm identification, cultivar fingerprinting, true hybrid
identification, genetic purity testing. Parentage confirmation
of hybrids and identification of heterotic pattern in hybrids
both female and male specific bands and are useful in genetic
purity testing.
Fig No 1: Banding Profile of hybrid and parents obtained by Primer RPI-1, RPI-8, RPI-10, RPI-18, RPI-15 and RPI-20
Fig No 2: Banding Profile of hybrid and parents obtained by Primer RPI-02, RPI-25, RPI-05, RPI-06 and RPI-07
Fig No 4: Banding Profile of hybrid and parents obtained by Primer RPI-14, RPI-19 RPI-24 and RPI-21
Fig No. 4: Banding Profile of hybrid and parents obtained by Umc-1858, Pumc1013, Pumc-1746, Phi-328175 and Pumc-1066

~ 874 ~
The Pharma Innovation Journal http://www.thepharmajournal.com
Fig 5: Banding profile of hybrid and parents obtained by Umc-1858
3.6 Genetic Purity Test of Hybrids
Thirty leaf samples of F1 plants were taken for genetic purity
testing with SSR markers. The percentage of hybrid genetic
purity was calculated based on banding pattern that appears
on the individual plant samples.
All 30 samples of maize cross were screened by using Umc-
1858 of which 11 samples resembles male type band (Type 2
maker) and remaining sample produced male and female
specific bands (Type 2 and Type 3 makers). Overall the total
sample contained 64 % of the maize seeds that were
genetically pure and 36 % of the maize seeds that were
genetically impure (Fig. No.5).
The genetic purity test of maize hybrid was demonstrated by
Hipi et al., (2013) [9] on selected 40 hybrid samples of cv.
Bima-4 identified using phi96100 marker, showed that seven
samples which similar to the male parent bands, and one
sample which similar female parent band. Overall the total
sample contained 20% of the cv. Bima-4 seeds that were not
genetically pure.
Thus, the present study concluded that the PCR based
molecular markers are likely to be promising for
identification, registration and protection of commercial
sample and will gain more and more influence on plant
breeding in future and will speedup breeding processes
considerably. The primer Umc-1858 screened with 30 maize
hybrid samples contained 64% of the maize seeds that were
genetically pure and 36% of the maize seeds that were
genetically impure.
4. Acknowledgment
The author would like to thank the Department of
Biotechnology, Government of India, New Delhi for
providing grants to conduct research work.
5. References
1. Akhare AA, Sakhare SB, Kulwal PL, Dhumale DB,
Kharkar, A. RAPD profile studies in sorghum for
identification of hybrid and their parents. Intl. J.
Integrative Biol 2008;3:1-18.
2. Asif M, Rahman MU and Zafar Y. Genotyping analysis
of six maize (Zea mays L.) hybrid using DNA
fingerprinting technology. Pak. J. Bot 2006;38:1425-
1430.
3. Asif M, Rahman MU, Mirza JI and Zafar Y. Parentage
Confirmation of Cotton Hybrids Using Molecular
Markers. Pak. J. Bot 2009;41(2):695-701.
4. Daniel IO, Adetumbi JA, Oyelakin OO, Olakojo SA,
Ajala MO and Onagbesan SO. Application of SSR
Markers for Genetic Purity Analysis of Parental Inbred
Lines and Some Commercial Hybrid Maize (Zea mays
L.). American Journal of Experimental Agriculture
2012;2(4):597-606.
5. Deshmukh CU, Saxena RR, Xalxo SM, Sharma D and
Verulkar SB. Hybrid purity testing in rice (Oryza sativa
L) using microsatellite markers. Elec. J. Plant Breed
2013;4(1):1021-1026.
6. Dongre A and Pakhi V. Identification of cotton hybrid
through the combination of PCR based RAPD, ISSR and
microsatellite markers. Jr. Plant Biochem. Biotechnol
2005;14:53-55.
7. Doyle JJ and Doyle JL. Isolation of plant DNA from
fresh tissue. Focus 1990;12:13–15.
8. Elci and Hancer. Genetic Analysis of Maize (Zea mays
L.) Hybrids Using Microsatellite Marker. Journal of
Agricultural Science 2014,192-198.
9. Hipi A, Surahman M, Ilyas S and Giyanto. Seed Genetic
Purity Assessment of Maize Hybrid Using Microsatellite
Markers (SSR). International Journal of Applied Science
and Technology 2013;3:5.
10. Mehetre SS, Gomes M and Eapen S. RAPD analysis of
hybrid nature of the offspring of Gossypium hirsutum ×
G. raimond. Current Sci 2004;87:25-28.
11. Mishra MK, Narayana S, Bhat AM, Nayani S, Kumar SS,
Kumar A and Jayarama. Genetic molecular analysis of
Coffea arabica (Rubiaceae) hybrids using SRAP markers.
Rev. Biol. Trop 2011;59(2):607-617.
12. Mrutu BA. Assessment of seed genetic purity of hybrid
maize variety UH6303 in southern highlands of Tanzania
by random amplified polymorphic DNA (RAPD)
markers. African J. Agric. Res 2015;10:2911-2918.
13. Sudharani M, Rajya Lakshmi K, Vishnuvardhan AR,
Bharthi K and Rama Raju N. Use of DNA polymorphism
for characterization and assessment of genetic purity
testing in rice hybrids (Oryza sativa L.). Electronic
Journal of Plant Breeding 2013;2:1113–1118.
14. Wu M, Jai X, Tain L and Baochun LV. Rapid and
reliable purity identification of F1 hybrid of maize (Zea
mays L.) using SSR markers. Maize Genomics Genet
2010;1:1-4.