IJERD – International Journal of Environmental and Rural Development (2018) 9-1

Research article
erd

Genetic Identification of Myanmar Sein Ta Lone Mango(Mangifera indica L.) Landrace from the Different EcoGeographic Regions using Microsatellite Markers
MOE MOE OO*
Plant Biotechnology Center, Yangon, Myanmar
Email: moemoeoo022@gmail.com

MAY THAZIN HTUN
Plant Biotechnology Center, Yangon, Myanmar

MAY THU KYAW
Plant Biotechnology Center, Yangon, Myanmar

HNIN WAI LWIN
Plant Biotechnology Center, Yangon, Myanmar

LA MIN HTET HTET AUNG
Plant Biotechnology Center, Yangon, Myanmar

MOE ZAW AUNG
Plant Biotechnology Center, Yangon, Myanmar

HTAY HTAY AUNG
Plant Biotechnology Center, Yangon, Myanmar

PA PA AUNG
Plant Biotechnology Center, Yangon, Myanmar
Received 28 December 2017

Accepted 19 May 2018(*Corresponding Author)

Abstract Eighty eight Sein Ta Lone mango individuals from five Sein Ta Lone orchards(populations), namely, Mya Na De, Htone Bo, Se Bauk, Taik Kyi, and Nam Latt, and 100
years old plant as control, and a landrace of Yin Kwe individual, a total of 89 individuals,
were genotyped using 38 polymorphic markers showed 147 alleles with an average of 3.87
alleles per locus. Polymorphic information content varied from 0.02 to 0.83 and averaged of
0.37.Twenty four loci were Sein Ta Lone unique markers. Cluster analysis depicted thoseSP4 plant individuals from Se Bauk stand as an out-group from the rest of Sein Ta Lone
individuals which scattered independently from their locations. Principal coordinate
analysis confirmed grouping of Sein Ta Lone individuals closely to control Sein Ta Lone(100 years old) rather than Yin Kwe variety, and the first three principal coordinates
explained 62.05% of total variation. Analysis of molecular variance explained 75% ofgenetic variation occurred within populations and 25% among populations. The fixationindex (Fst) 0.63, pointed the high level of genetic differentiation among populations, it
might be due to Yin Kwe control rather than five Sein Ta Lone populations. Limited geneflow (Nm = 0.31) occurred among the populations. Almost the same value (0.32 to 0.35) of
the expected heterozygosity and the observed heterozgosity (0.16) were observed in Sein Ta
Lone populations. The number of effective alleles varied from 1.88 to 2.13, Shannon Index
ranged from 0.54 to 0.61, and same positive fixation index of (0.4), same value of thepercentage of polymorphic loci (52.63%). Pairwise Nei genetic identity showed higher
value to Sein Ta Lone control (100 years old), highest among five populations ranged from
0.916 to 0.957.

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Keywords genetic identification, Sein Ta Lone populations, Sein Ta Lone unique marker,
cluster analysis, analysis of molecular variance

INTRODUCTION
Mango (Mangifera indica) consisted of two distinct types such as monoembryonic andpolyembryonic type. Based upon taxonomic and molecular evidence, the mango (polyembryonictype) probably evolved in north-western Myanmar, Bangladesh and north-eastern India (Bompard,
1993; Mukherjee and Litz, 2009). Hirano et al. (2010) identified the uniqueness of Myanmar
mango individuals from Vegetable and Fruits Research and Development Center (VFRDC),
Yangon, using 11 SSR markers and results revealed that mango individuals from Myanmar weredistinguishable from those of Florida, India, and Southeast Asia.
Polyembryonic mango seeds contain several nucellar and zygotic embryo. Since adventitious
embryos develop from the nucellus, or can also originate by direct budding from the cotyledonsand hypocotyls of other nucellar, embryos seedlings are genetically identical to the maternal parent(Juiano, 1934). However, there was a little variation among seedlings derived from polyembryonicmangoes, due to somatic mutation (Mukherjee and Litz, 2009) and various biological and cultural
factors (Begum et al., 2013). Even, mango has cross-pollinated nature; Moore and Castle (1988)
recorded about 88-94% of self-pollinated zygotic seedlings using isozymes.
Nowadays, orchards are being used grafted plants (stock and scion), however, home yard
gardens are still using plants directly from seeds. Since Sein Ta Lone mango previously sown fromseed, and to date grafted Sein Ta Lone orchards were necessary to document their DNA profile forfingerprinting and genetic variability on farm.
OBJECTIVE
This study aims to analyze genetic diversity and population structure of Sein Ta Lone individuals,
using SSR markers.
METHODOLOGY
From May to July, 2017, the leaf samples from 88 Sein Ta Lone sample plants : 18, 15, 18, 18 and18 sample plants from Mya Na De, Htone Bo, Se Bauk, Taik Kyi, and Nam Latt, respectively, andone control Sein Ta Lone (assuming 100 years old according to villagers of Pae Gin Village,
Kyauk Se Township, Mandalay region), were collected. Mya Na De, Htone Bo and Se Baukorchards are from Mandalay region, Taik Kyi orchard from Yangon region, and Nam Latt orchard
from Shan region, respectively. In addition, Yin Kwe landrace, which widely used as rootstock forSein Ta Lone orchards, was also used as control. This experiment was carried out at the Plant
Biotechnology Center (PBC), Department of Agriculture (DOA), Myanmar.
Total genomic DNA was isolated from pale-brown young leaves using the hexadecyl
trimethyl ammonium-bromide (CTAB) method (Doyle and Doyle, 1987) with few modifications.
A total volume of 10 Pl of reaction mixture of PCR cocktails were performed in Labnet (Labnet
international Inc.) thermal cycler programmed for 5min at 94qC for initial Denaturation, 35 cyclesof 1 min at 94qC, min at 55qC - 60qC (marker dependent), 1 min at 72qC and final extension at
72qC for 5 minutes. PCR products were fractionated through 8% (w/v) polyacrylamide gel and theexpressions of gels were recorded on UV light using Cannon G12 digital camera.
SSR fragment sizing was performed with LabImage 2.7.1 Software and genetic distance andcluster analyses were evaluated for 89 individuals with 38 polymorphic loci using NTSYS-pc 2.01
program (Rohlf, 2000). Nei and Li’s genetic distance (1979) was conducted to calculate pair-wise
genetic distance among all individuals and the dendrogram was constructed by using a distancematrix using the unweighed pair group method with arithmetic average. The polymorphic
information content (PIC) was calculated by applying the formula provided by Anderson et al.
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IJERD – International Journal of Environmental and Rural Development (2018) 9-1(1993). To elucidate the genetic relationship among the populations, principal coordinate analysis
was conducted using Genalex6.2 (Peakall and Smouse, 2006).The Fst value was, in turn, used toestimate gene flow (Nm) as Nm = (1/Fst-1)/4 (Whitlock and McCauley, 1999).The number of alleles
per locus (Na), the effective number of alleles per locus (Ne), and the genetic heterozygosity (H)were used to detect the level of population genetic diversity.
RESULTS AND DISCUSSION
Genetic Diversity Analysis
The 89 mango individuals from five orchards (viz. Mya Na De, Htone Bo, Se Bauk, Taik Kyi, Nam
Latt), were assessed by using 64 SSR markers. A total of 58 markers were well amplified; 20 weremonomorphic and 38 were polymorphic markers (Table 1). Among the polymorphic markers, 24
were Sein Ta Lone unique markers; 18 homozygous loci (MiIIHR03a, MiIIHR05c, MiIIHR07a,
MiIIHR12a, MiIIHR16a, MiIIHR22a, MiIIHR25a, MiIIHR29a, MiIIHR31b, MiIIHR32a,
MiIIHR33a, MiIIHR34b, SSR-16, SSR-19, SSR-20, SSR-52, SSR-89 and MiMRD_l656), and 6
heterozyous loci (MiIIHR02c, MiIIHR04c, MiIIHR17b, MiKVR_a009, MiKVR_ao28 and
MiIIHR_f879).
A total of 147 alleles were detected, the average number of alleles per locus was 3.87, with arange from 2 to 8 (Table 1). Polymorphic Information Content (PIC) values ranged from 0.02 to
0.83 with 0.37 averaged, showing that a moderately low diversity exists in mango individuals
under study.
Cluster Analysis
A total of 89 mango individuals using 38 SSR were subjected into cluster analysis based on
UPGMA. In group I, Sein Ta Lone control (100 years old) clustered together with 12 Sein Ta Lone
individuals (viz. NL17, TK1, TK15, MND1, MND20, MND8, MND2, SP14, SP15, HB18,
MND16 and TK2) with genetic similarity of (0.86, 0.83, 0.8, 0.79, 0.83, 0.77, 0.75, 0.85, 0.75, 0.75,
0.8, and 0.76, respectively) (Fig. 1). Group II comprised of most of the individuals from fiveorchards were clustered independent of their locations. Group III consisted of the ten individuals(TK3, TK5, TK6, TK4, TK14, TK13, TK7, TK12, TK18, and TK20), from Taik Kyi orchard wereclustered together. Group IV depicted that Yin Kwe stand as separately from other. Interestingly,
Se Bauk (SP4) had distinct away the rest of Sein Ta Lone.
In dendrogram, individuals from Mya Na De orchard, MND1 and MND20 showed 0.89 and
MND10 and MND13 showed 0.86 genetic similarity and cluster together. In Htone Bo orchard,
HB6 and HB15 had 0.91, HB9 and HB10 had 0.88, HB5 and HB13 had 0.84, and HB1 and HB8
had 0.82, genetic similarity and cluster together. For Se Bauk orchard, SP10 and SP13 had 0.93,
both SP2 and SP3, and SP1 and SP5 had 0.86, genetic similarity, and clustered as a pair. Similarly,
individuals from Taik Kyi orchard, both pairs of TK1 and TK15, TK4 and TK14 had 0.90, TK18
and TK20 had 0.88, TK7 and TK12 had 0.87, TK3 and TK5 had 0.86, and TK10 and TK16 had
0.85, genetic similarity and clustered together. Likewise, individuals from Nam Latt orchard, NL9
and NL10 had 0.90, NL3 and NL6 had 0.86, NL7 and NL8 had 0.84, NL15 and NL16 had 0.78 and
NL18 and NL20 had 0.77, genetic similarity, and clustered together.
Principal Coordinate Analysis
In order to explore the distribution pattern of genetic diversity contained in individuals of each
population, a principal coordinate analysis was performed on 38 markers in 89 mango individuals(including Sein Ta Lone-100 years old and Yin Kwe controls). DNA scored of these two controls
was arbitrarily doubled in order to assume as separate populations. Therefore, a total of 91
individuals were subjected into multivariate analysis. Similar as cluster analysis, PCoA clearly
depicted all the individuals from five Sein Ta Lone orchards showed closely grouped to controlⒸ ISERD

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Sein Ta Lone (pop2) rather than Yin Kwe (pop1) (Fig. 2).The first three PC showed 62.05 % of
total variations, PC1 explained 29.75% of total variations, and PC2 and PC3 showed 17.90% and14.39%,respectively. Sein Ta Lone controls (100 years old sample, pop2) were close to most of
Sein Ta Lone individuals from Nam Latt orchard (Shan region), Se Bauk orchard (Mandalay
region), Mya Na De orchard (Mandalay region).
Table 1 Polymorphic markers with their product size, motif, no. of alleles amplified and
PIC valueNo.

Marker name

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38

MiIIHR09c
MiIIHR21b
MiIIHR24b
MiKVR_a394
MiKVR_d864
MiMRD_l744
MiKVR_a152
MiKVR_a965
MiKVR_c273
MiKVR_d656
MiKVR_n259
MiKVR_n613
MiKVR_u796
MiKVR_t130
MiIIHR02c
MiIIHR04c
MiIIHR03a
MiIIHR05c
MiIIHR07a
MiIIHR12a
MiIIHR16a
MiIIHR17b
MiIIHR22a
MiIIHR25a
MiIIHR29a
MiIIHR31b
MiIIHR32a
MiIIHR33a
MiIIHR34b
SSR-16
SSR-19SSR-20
SSR-52
SSR-89MiKVR_a009
MiKVR_ao28
MiMRD_l656
MiIIHR_f879

No. ofalleles

SSR motif(CT)3TTGC(CT)2GT(CT)4TC(GT)2(CT)2(GTTT)3(GT)2TTTTGTC(TG)4(AATGA)2(CA)9TACC(CATA)6(TG)7(ATC)4(AAAT)4(ATTAT)4(AAAAT)4(TAAAA)5(TAAAA)4(CACCCA)4(TGATGG)4(GGAAGG)5(GAAAAA)4(CA)2A(CA)7AG(CA)5(CA)11(CTT)6(CA)2(CT)8C(CT)2TTTT(CT)4(GA)11(GA)11(GA)10(GT)13GAGT(GA)10(GTCTC)2(TGTCTC)3T(CTC)2(GTTT)3ATTTG(ATT)2(GT)10(GAC)6(GA)12(GA)12(GGT)9(GAT)5(TA)2(CA)10TA(CA)3TA(CA)4(ACACACAT)3(ACACACACAT)3(AT)14(GT)18(GA)16
NA
(GT)8
(TA)
(ATTT)5(TTGGAC)4
AverageⒸ ISERD

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7
8
6
5
7
7
5
7
6
7
6
7
4
5
3
4
2
2
2
2
2
4
2
2
2
2
2
2
2
2
2
2
2
3
4
4
3
3
3.87

Size (bp)PIC

284-300
258-275242-252245-256282-300
283-303253-265278-300
239-255
252-277248-266244-264
208-216
255-267
176-239
170-202237-243207-213170-192
174-180
205-211248-284224-230
147-156
153-158
213-217194-206
171-173232-240
180-188170-254248-252
113-115105-119200-246221-259278-324116-131

0.79
0.76
0.78
0.78
0.78
0.8
0.73
0.82
0.8
0.81
0.71
0.83
0.67
0.72
0.51
0.51
0.02
0.02
0.02
0.02
0.02
0.51
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.04
0.51
0.51
0.04
0.51
0.37

IJERD – International Journal of Environmental and Rural Development (2018) 9-1

ST L
NL17
T K1
T K15
MND1
MND20
MND8
HB20
MND2
T K11
SP14
HB5
SP15
HB13
HB18
MND14
MND16
HB17
T K2
MND19
MND4
HB19
SP7
T K9
NL14
T K10
SP9
T K16
MND17
MND5
SP17
MND7
NL11
HB7
SP18
SP1
NL2
SP5
SP10
SP6
SP13
T K3
SP12
T K5
NL5
T K6
SP16
T K4
NL9
T K14
NL10
T K13
NL4
T K7
T K12
1.00
T K18
T K20
SP4
yk

ND10

D10
MND10

0.01

0.26

0.51

0.75

Coefficient

0.26

0.51

0.75

1.00

Coefficient

0.01

0.26

0.51

0.75

1.00

Coefficient

I

II

III
IV

Fig. 1 Dendrogram of 89 mango individuals on the basis of 38 SSR markers
using Nei and Li’s genetic distance (1979)
Principal Coordinates

Pop1
Pop2
Coord. 2

0.01

STL
NL17
TK1
TK15
MND1
MND20
MND8
MND2
SP14
SP15
HB18
MND16
TK2
MND4
SP7
NL14
SP9
MND17
SP17
NL11
SP18
NL2
SP10
SP13
SP12
NL5
SP16
NL9
NL10
NL4
TK19
NL7
NL8
NL15
NL16
NL18
NL20
SP8
NL19
NL3
NL6
MND9
MND12
SP20
MND15
MND6
HB3
SP2
SP3
HB1
HB8
HB9
HB10
NL13
MND10
MND13
MND11
HB6
HB15
HB12
HB20
TK11
HB5
HB13
MND14
HB17
MND19
HB19
TK9
TK10
TK16
MND5
MND7
HB7
SP1
SP5
SP6
TK3
TK5
TK6
TK4
TK14
TK13
TK7
TK12
TK18
TK20
SP4
yk

Pop3

Mandalay region

Pop4
Pop5
Pop6 Yangon region
Pop7 Shan region

Coord. 1

Fig. 2 Two dimensional axis of Principal coordinate analysis (axis 1 vs axis 2) of
91 mango individuals, using 38 markers
pop1-Yin Kwe , pop2 - Sein Ta Lone, pop3 - Mya Na De orchard, pop4 - Htone Bo orchard,
pop5- Se Bauk, pop6 - Taik Kyi orchard, and pop7 - Nam Latt orchard

Population Structure and Gene Flow
To determine the pattern of diversity between and within the mango orchards (pop), an analysis
molecular variance (AMOVA) in the 91 mango individuals was conducted and results reveal 7
main groups as the orchards which obtained DNA samples and two controls (Yin Kwe and 100
years old Sein Ta Lone). AMOVA using 7 populations suggested that the largest proportion ofgenetic diversity (75%) was found among plants belonging to the same population, whereas
variation present among populations was 25% and no divergence observed between regions, allsources variation were significant at P>0.01 (Table 2). Large variability detected within
populations could be explained by the occurrence of gene flow or most probably, by sowing thesame materials in different regions. The fixation index (Fst), showed the value of 0.63, confirming
the high level of genetic differentiation among the populations (Hartl and Clark, 1997). Thisdifferentiation might be mainly due to Yin Kwe individuals differ from Sein Ta Lone individuals.
Hirano et al. (2011) mentioned that Yin Kwe and Sein Ta Lone showed significantly varianceat DNA level (Fst= 0.44). Gene flow (Nm) was equal to 0.31 indicating that limited gene flow
among the populations (a local differentiation of populations). It may be due to the occurrence of
zygotic seedling (2% to 90%) in polyembryonic cultivars or various biological and cultural
practices like continuous grafting from one population to others (Begum et al., 2013). DiazMatallana et al. (2009) observed high gene flow value in analysis of diversity among sixⒸ ISERD

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Colombian mango populations, and they concluded that it is probably caused by both insect
pollinators and human intervention. Since pop1 and pop2, arbitrary population, used the same DNA
samples as controls in each pop, there had no consideration for population analysis. Expected
heterozygosity (He) values of populations from five orchards (i.e. pop3 to pop7) showed 0.31 to
0.34, which pointed that not much difference in population diversity (Table 3). Observed
heterozygosity (Ho) was the same value (0.16) in all populations. Observed heterozygosity of eachpop was smaller than the expected heterozygosity based on Hardy-Weinberg expectations.
Consequently the fixation index was positive over five pops (0.4). The number of effective alleles(Ne) ranged from 1.88 (pop7) to 2.13 (pop 5).The Shannon index (I) ranged from (0.54) in pop7 to(0.61) in pop3 and pop5. The percentage of polymorphic loci (P) across populations was the samevalue (52.63) %.
Table 2 Analysis of molecular variance for mango individuals
SourceAmong Regions
Among Pops
Within Pops
Total

df
2
4
84
90

SS
140.67348.56
1719.12208.3

MS
70.33
87.14
20.47

Est. Var.
0
6.84
20.47
27.31

%
0%
25%
75%
100%

Table 3 Parameters (Mean and SE) of genetic variability of mango populations
Pop*
Pop 1
Pop2
Pop3
Pop4
Pop5
Pop6
Pop7
Total

Na
1.16±0.06
1.16±0.06
2.55±0.31
2.37±0.26
2.47±0.28
2.42±0.29
2.26±0.25
2.06±0.10Ne
1.16±0.06
1.16±0.06
2.13±0.22
1.88±0.16
2.10±0.21
1.98±0.20
1.92±0.18
1.76±0.07I
0.11±0.04
0.11±0.04
0.61±0.11
0.56±0.1
0.61±0.10
0.57±0.10
0.54±0.09
0.44±0.04*For abbreviation see Figure 2
Na = no. of alleles
I = Information index = -1*sum (pi*ln(pi))
He = Expected Heterozygosity = 1- Sum pi^2
%P= Percentage of polymorphic lociHo
0.16±0.06
0.16±0.06
0.16±0.06
0.16±0.06
0.16±0.06
0.16±0.06
0.16±0.06
0.16±0.02

He
0.08±0.03
0.08±0.03
0.34±0.06
0.32±0.05
0.34±0.05
0.32±0.05
0.31±0.05
0.26±0.02

F-1±0.00-1±0.00
0.4±0.15
0.4±0.15
0.4±0.15
0.4±0.15
0.4±0.15
0.25±0.06

%P
15.79
15.79
52.63
52.63
52.63
52.63
52.63
42.11±6.79

Ne = no. of effective alleles = 1/(sum pi^2)
Ho = Observed Heterozygosity =no. of Hets/N
F = Fixation Index= (He- Ho) / He = 1- (Ho/ He)

All populations (pop3 to pop7) from five orchards showed high pairwise genetic identity tocontrol Sein Ta Lone (100 years old) (pop2) than Yin Kwe (pop1) (Table 4). Likewise, pairwise
genetic identity among five orchards were high, indicating quite similar mango clone, pop7 (Nam
Latt orchard) and pop3 (Mya Na De orchard) showed high value of genetic identity (0.831 and
0.822). Pop4 (Htone Bo orchard) showed the lowest value of 0.766. Pop 5 (Se Bauk orchard)
showed high genetic identity (0.957) to pop7 (Nam Latt orchard), and (0.951) to pop3 (Mya Na De
orchard). Five populations (grafted orchards) showed more similar to each other than Sein Ta Lone(100 years old control), it might be mainly due to cross pollinated nature of mango (gene flow).
Table 4 Pairwise population matrix of Nei genetic identity
Pop*
Pop1
Pop2
Pop3
Pop4
Pop5
Pop6
Pop7

Pop1
1.000
0.000
0.084
0.096
0.095
0.055
0.072

Pop2

Pop3

Pop4

Pop5

Pop6

Pop7

1.000
0.822
0.766
0.803
0.789
0.831

1.000
0.935
0.951
0.923
0.916

1.000
0.945
0.929
0.917

1.000
0.918
0.957

1.000
0.919

1.000

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CONCLUSIONAs Sein Ta Lone mango is an economically important crop in Myanmar, this study provides insight
into extent and genetic variability at DNA level of five Sein Ta Lone orchards to control Sein Ta
Lone (100 years old plant). Genotyping with 38 SSR markers confirmed differentiation of Yin Kwe
and Sein Ta Lone at DNA level which was consistent with Hirano et al. (2011). Moderately low
genetic diversity in PIC value indicated five Sein Ta Lone orchards(grafted trees) have highgenetic similarity to 100 years old Sein Ta Lone control (seeded tree). Moreover, variation within
populations (within orchards) were observed and next grafting procedure from present Sein Ta
Lone individuals should be carefully carried out for ensure to get true to type Sein Ta Lone mango.
Sein Ta Lone unique markers will be useful in varietal identification in mango varieties. This study
will provide valuable information to varietal identification and protection, and further varietal
improvement program at the national and global perspective.
ACKNOWLEDGEMENTS
This research was supported by DOA, Myanmar and this work was performed with thecollaboration of farm managers from Mya Na De, Htone Bo, Se Bauk, Nam Latt and Taik Kyi. We
wish to thanks to Dr. Ye Tint Htun, Director General, DOA, Mr. Thuya Soe, Director, Horticulture
and Plant Biotechnology Division, for facilitating this research and financial support. My extended
thanks and appreciation to Dr. Htay Htay Aung, Staff Officer ( PBC, Myanmar) for her kindcooperation in Analysis and invaluable suggestions and supporting for manuscript preparation.
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