IJERD – International Journal of Environmental and Rural Development (2022) 13-1

Research articleerd

DNA Fingerprinting of Selected Maize (Zea mays L.)
Genotypes using SSR Markers
SAN KYI*Yezin Agricultural University, Nay Pyi Taw, Myanmar
Email: sankyi747@gmail.com

KYAW KYAW WIN
Pro-Rector’s Office of Administration, Yezin Agricultural University, Nay Pyi Taw, Myanmar

HLA THAN
Pro-Rector’s Office of Academic, Yezin Agricultural University, Nay Pyi Taw, Myanmar

SOE WIN
Department of Plant Breeding, Physiology and Ecology, Yezin Agricultural University,Nay Pyi Taw, Myanmar

NYO MAR HTWE
Advanced Center for Agricultural Research and Education, Yezin Agricultural University,
Nay Pyi Taw, Myanmar

AYE LAE LAE HLAING
Plant Molecular Biology Laboratory, Biotechnology Research Section,
Department of Agricultural Research, Nay Pyi Taw, Myanmar
Received 28 January 2022

Accepted 2 May 2022(*Corresponding Author)

Abstract Molecular marker has been used for variety identification, genetic diversity ofgenotypes and additional using intellectual property protection in DUS testing. DNA
fingerprinting of fourteen maize genotypes had been studied at Plant Biotechnology
Laboratory, DAR, Myanmar during 2020-2021. Nine hybrids and five inbreds wereanalyzed using 24 polymorphic SSR markers, resulted a total number of 101 alleles with arange from 2 to 9 alleles per locus. Polymorphism has been sufficiently detected with theaverage of 0.66 per SSR locus. Cluster analysis separated all the maize genotypes as five
major groups and indicated the existence of genetic variation among the observed inbreds
and hybrids. Six SSR primer pairs (dupssr12, bnlg1940, umc1248, umc1586, bnlg1518 andbnlg1028) were selected as final marker set for variety identification with the selection
criteria such as detection rate of the SSR fragment, the presence of rare allele, PIC value,and reproducibility and PCR band pattern of SSR fragments. In this study, all tested
genotypes have been fingerprinted with unique profile identity (ID) to support the DNA
fingerprint catalogues of Myanmar Maize Molecular DUS test guidelines.
Keywords DNA fingerprinting, maize SSR markers, varietal identification

INTRODUCTION
Maize (Zea mays L.) is the most diverse crop species characterized at both morphological andmolecular levels (Anderson and Cutler, 1942; Buckler et al., 2006; Zheng et al., 2008). Genetic
identification of maize is essential in the process of crop improvement, crop quality estimation andthe finding of parent components and their crosses.
SSR markers were able to discriminate maize genotypes depending upon their hybridizationlevel and their genetic relationships among the different inbred lines (Kamal et al., 2010). It is themost powerful biotechnological tool, suitable for detecting of genetic purity status of maize hybrids.
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SSR also used in DNA fingerprinting analysis for identification of individuals, populations, inbredlines, and hybrids, making the description of the species at the molecular level, to protect thebreeders’ Rights and its protection.
Plant varietal protection and patents are the most important end products of plant breeding
institutes to get back their research investment by mean of intellectual property protection. DNA
fingerprinting technology is useful for preventing counterfeit and fake varieties in the market andthis fingerprinting information is additional information for DUS characterization (Wang et al.,2013). UPOV (2010) recommends SSRs for current construction of DNA fingerprint databases thathave been well-defined and tested. In the present study, the SSR primers set were used todifferentiate hybrids and inbreds, and to create the DNA fingerprint catalogues for supporting ofMyanmar Maize DUS test guidelines for identifying maize genotypes in infringement case, and for
utilizing as a source of parental line for future breeding programs.
METHODOLOGY
Plant Materials and Genomic DNA Extraction
Fourteen maize genotypes (Table 1) were used to study molecular characterization at Plant
Biotechnology Laboratory, Department of Agricultural Research (DAR), Myanmar during 20202021. Nine hybrids (imported by private companies) and five inbred lines (developed by DAR)
were used. Genomic DNA extraction, DNA quantification and qualification were carried out
according to the procedure of Hlaing et al. (2017).
Table 1 List of selected maize genotypes for molecular characterization
No.

Genotypes

Type

SourceNo.

Genotypes

Type

SourceV1

Asia Seed (A.55)

HybridChina

V8NK-621

HybridSyngenta

V2

Asia Seed (A.99)

HybridChina

V9TSF-1633

HybridThailand

V3AA-737

HybridThailand

V10

YZI-10-054

InbredDAR

V4GT-722

HybridThailand

V11

YZI-10-095

InbredDAR

V5NK-625

HybridSyngenta

V12

PAC-999

InbredThailand

V6

KMHE-3550

HybridIndia

V13

C7

InbredDAR

V7

CP-111HybridThailand

V14

YZCI-16-019

InbredDARPCR Amplification and Gel Electrophoresis
Twenty-four SSR primer pairs were used to identify the studied maize genotypes. These highly
polymorphic primer sets, PCR amplification and agarose gel electrophoresis were conductedfollowing the methods reported by previous research findings (Hlaing et al., 2017).
Statistical Analysis on Marker Data
The SSR allele segregation data were used to construct Nei distance dendrogram (Nei, 1972) using
NTSYSpc 2.1 software (Rohlf 2000). Polymorphism Information Content (PIC) for each SSR wascalculated by the formula PIC=1-ΣX2k/n developed by Ni et al. (2002) where, X2k represents thefrequency of the kth allele, and n represents the number of genotypes.
Development of Core SSR Marker Set for DNA Fingerprinting (DUS testing)Ⓒ ISERD

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Based on their specific discrimination capacity of SSR markers, the core marker set was developedwith the following criteria: (1) PCR banding pattern of SSR fragments (2) the presence of rare
allele (3) PIC values and (4) marker’s reproducibility. Furthermore, the genetic fingerprint map oftested maize genotypes was constructed using the coding-based system with the original allele size
coded by the assigned numeric values which set in two numeric codes from “01”, “02”, “03”, etc.
The transformation was generated to all cores SSR ID set based on allele size (bp) range. The final
SSR fingerprinting map of maize genotypes was produced to support variety identification andDUS testing on PVP system.
RESULTS AND DISCUSSION
Amplification Profile, SSR Polymorphism and Cluster Analysis on Diversity Assessment
SSR markers have been proven to be powerful tools in the assessment of genetic variation within
and among the species. A total of 24 SSR primers were used for identification of maize genotypes
through evaluation of DNA fragment polymorphisms. A total number of 101 alleles were detectedwith a range from 2 to 9 alleles per locus. These varied number of alleles indicated that there is ahigh level of genetic diversity among tested genotypes. The primer pairs bnlg1028 showed thehighest number of 9 alleles per locus, umc1248 showed 8 alleles in all tested genotypes andumc1586, bnlg1940 and dupssr12 showed 6 alleles in all samples (Table 2).
Table 2 Data on number of alleles, total number of alleles, and PIC value
obtained among 14 maize genotypes for 24 SSR markers

1.

SSRmarker
umc1397

No. ofalleles
4

Total no.
of alleles
20PIC
value
0.63

13.

SSR
markerbnlg1617

No. ofalleles4

Total no.
of alleles
14PIC
value
0.72

2.

umc22343

200.6214.

umc15205220.713.

dupssr12

630

0.77

15.

umc1248

838

0.85

4.

umc1542

3

5.

umc2372

4

20

0.62

16.

umc13933

150.66190.7017.

umc1671

524

0.59

6.

bnlg1940

628

0.74

18.

umc23953

160.537.
8.

bnlg1160

3230.6319.

bnlg1065

3200.65

umc15014

160.7320.

umc15866480.80

9.

umc2061

4160.72

21.

umc23592140.41

10.

umc11013190.6122.

umc19625340.77

11.

umc17522140.50

23.

bnlg1518

4180.67

12.

umc1792

2150.4824.

bnlg1028

9360.78No.

No.

The Polymorphism Information Content (PIC) value reflects the evidence of allele diversity
and allele frequency among the tested genotypes (Pervaiz et al., 2009). In present study, PIC value
ranged from 0.41 to 0.85 with an average value of 0.66 (Table 2). The highest PIC value wasobtained for umc1248 followed by umc1586, bnlg1028, dupssr12, umc1962, bnlg1940,respectively. The PIC values of umc1248, umc1586, bnlg1028, dupssr12, and umc1962 werehigher than 0.75 that were considered as the best markers for studied genotypes.
The SSR markers with PIC value of 0.5 or higher indicate that these are highly informativeand extremely useful in distinguish the polymorphism rate of a marker at a specific locus(DeWoody et al., 1995). In this study, the genetic diversity of each SSR locus appeared to be
associated with number of alleles detected per locus, i.e. the higher the PIC value of a locus, thehigher the number of allele detected.
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Moreover, the amplification profile of the 14 maize genotypes using 24 SSR markers sets
showed alleles of different molecular weight, and displayed the higher polymorphism among thetested genotypes (Fig. 1). Our study highlighted the detection of genetic variability among thetested maize genotypes and also agreed the finding of polymorphic SSR markers by Liu and Muse(2005).
Cluster Analysis is a classification to determine whether the genotypes could be regarded asconsisting of a number of partially dissociated groups. Genetic dissimilarities obtained from SSR
marker data of 9 hybrids and 5 inbred maize genotypes were used to create a cluster diagram using
Nei’s genetic distance and the unweighted pair group method (UPGMA). Clustering analysis for 14maize genotypes could be fully distinguished from one another and grouped into five major clusters(Fig. 2).

Fig. 1 Amplification of DNA profiles of the 14 maize inbreds and hybrids
generated by SSR primers(a) dupssr 12 (b) bnlg1940 (c) umc 1248 (d) umc 1586 (e) bnlg 1518 and bnlg 1028.
Lane M = 100bp DNA Ladder.
V1V4V2V3V5V12V14V10V6V8V7V9V10V11V13
0.29

0.44

0.60

0.75

0.91

CoefficientFig. 2 UPGMA clustering tree of 14 maize genotypes based on Nei’s genetic distance
Development of Core SSR Marker Set for DNA Fingerprinting (DUS testing)
The goal of development of core SSR marker set for genotype identification is to maximize
distinguishing ability of each marker with a minimum number of markers set in DNA
fingerprinting. In this study, the core marker candidate was decided with the selection criteria suchas detection rate of the SSR fragment, the presence of rare allele, PIC value, and reproducibility
and PCR band pattern of SSR fragments. As a result, six SSR primer pairs were selected as a final
marker set for variety identification, i.e., dupssr12, bnlg1940, umc1248, umc1586, bnlg1518 andbnlg1028 due to their characterization and discrimination capacity. These polymorphic markerⒸ ISERD

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pairs revealed that there were four to nine differentiated loci among all genotypes with a fragment
length of 50-57 bp to 600 bp across loci (Table 3).
Table 3 Coding system of selected six SSR marker set based on allele size (bp) range
Codedupssr12

bnlg1940

umc1248

umc1586

bnlg1518

bnlg1028

01

110-115

60-64

50-57

166-182

162

75

02

120-128

106-118

100

237

180

100

03

135-140

121-125

112-120282-310190-21311004

150-160

212-220125341-358

225-23813805

170-175

225-234150

400-41516206

197-200

237-240

175-180

600

150

07

200-210

175-18708

230-23220009

246-250

Table 4 SSR fingerprinting map of 14 maize genotypes based on their allele size codes
Genotype namedupssr12

bnlg1940

umc1248

umc1586

bnlg1518

bnlg1028

Asia Seed (A.55)

0204060205

050608

03

03

0103

Asia Seed (A.99)

03

0205

050608030506040104

AA-7370206

0205

05060801020405

04020409

GT-7220206

0206

05060801030403020609

NK-6250405060206

0102070102030405

03060709

KMHE-3550

0203

0204

01020701030405

04060709

CP-1110302050103

03040504060709

NK-6210206

0204

01020401040503060809

TSF-1633

0206

0205

0102030102030405

040609YZI-10-054

0106

0204

0104

01020304

020609YZI-10-095

0103040204

0102040305060102

060709

PAC-9990205060103

0104

01030406

0304

0609

C7

0406

0102

0103070304

030509YZCI-16-019

0206

0103

0103

01030405

020304060709This allele size can be assigned as a specific DNA fingerprinting band of the genotypes andcan be used as the maize variety identification. The primer bnlg1028 was found the best marker for
the identification of studied genotypes as revealed by higher PIC values and showed the highest
polymorphism. In other study, DNA fingerprinting with SSR set of tested maize genotypes werefully distinguished from one another compared to the specific SSR markers which differentiatedmaize hybrids from their parental inbreds (Jhansi et al., 2015). In this study, the genetic fingerprint
map of 14 maize genotypes was constructed using the coding-based method with the original allele
size coded by the assigned numeric values and the specific marker’s allelic fragments weretransformed into combine codes as final identity of the genotype, shown in Table 3 and Table 4. By
using the core marker set, the results of assigning numeric code or ID described a specific identityof the variety (genotype) that can quantitatively differentiate it from the others. In this study, 14maize varieties had unique profile ID to support plant variety protection and breeder right,
complementing tools for DUS testing purpose. It is noted that these findings not only met therequirements for the minimum number of primers but were also sufficient to fully distinguish the14 varieties from one another.
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CONCLUSION
The goal of this research is to develop DNA fingerprinting catalogue of maize genotypes (bothinbreds and hybrids) using SSR polymorphic information and their genetic variance supporting
Myanmar maize DUS guidelines. In this study, the six pairs SSR sets generate the geneticfingerprint map (coding system) of typical maize genotypes which can be used in DUS testing for
identification of hybrids and inbreds at any stage of crop growing cycle. Furthermore, molecular
based SSR testing provide a higher degree of detection efficiency in DUS testing than traditional
morphological based DUS method with regard to the verification of new varieties or genotypes.
ACKNOWLEDGEMENTS
The authors are grateful to the responsible persons from DOA, DAR and YAU for their kindpermission and collaborating to conduct this study. We acknowledge all the staffs of Biotechnology
Research Section, DAR for helping us.
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