Genet Resour Crop Evol

DOI 10.1007/s10722-016-0392-1

RESEARCH ARTICLE

Genetic relationships among improved varieties of rice

(Oryza sativa L.) in Indonesia over the last 60 years
as revealed by morphological traits and DNA markers
Kadapi Muhamad . Kaworu Ebana .
Shuichi Fukuoka . Kazutoshi Okuno

Received: 20 October 2015 / Accepted: 15 March 2016

Ó Springer Science+Business Media Dordrecht 2016

Abstract Morphological traits and two kinds of
molecular markers were employed to study the genetic
relationships among improved rice (Oryza sativa)
varieties of Indonesia since 1943. Dendrograms based
on morphological traits and both molecular markers
(simple sequence repeats, SSR and single nucleotide
polymorphism, SNP) agreed in separating the varieties
into two primary groups. Based on the morphological
traits, a larger group ([60 %) contains varieties with
smaller sizes compared with those in the smaller group
(\40 %). SSR and SNP markers revealed that most of
the varieties belonged to indica (88; 89 %) and
japonica (9; 8 %) subspecies, and 3 % of varieties
Electronic supplementary material The online version of
this article (doi:10.1007/s10722-016-0392-1) contains supple-
mentary material, which is available to authorized users.
K. Muhamad
K. Okuno (&)
Laboratory of Plant Genetics and Breeding Science,
Graduate School of Life and Environmental Sciences,
University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
Ibaraki 305-8572, Japan
e-mail: okuno.kazutoshi2423@gmail.com
K. Ebana
Genetic Resources Center, National Institute of
Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba,
Ibaraki 305-8602, Japan
S. Fukuoka
QTL Genomics Research Center, National Institute of
Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba,
Ibaraki 305-8602, Japan
were not involved in two subspecies. The molecular
markers revealed that the genetic diversity (H) stag-
nated between stage II (1967–1985) and stage III
(1986–2003). However, during stage I (1943–1966),
H was higher than in the other stages as revealed by
SNP markers, while H in stage I was lower than in the
other stages as revealed by SSR markers. In this study,
the two molecular data sets were positively correlated
and positive correlations between the phenotypic and
molecular data depended on the kind of molecular
marker: SNP had higher Mantel r values than SSRs.
Besides, SSR markers seem to be appropriate for
pedigree studies, while SNP markers could be used to
reveal genomic relationships. These findings were
attributable to the different properties of these two
different markers. These results suggested that the
diversity and differentiation of both the phenotypic and
molecular marker variations were probably resulted
from the crossing and selection in rice breeding in
Indonesia. We suggest that Indonesia needs another
strategy to improve new varieties to avoid a reduction
in genetic diversity and similarity.
Keywords Breeding history
Genetic similarity

Oryza sativa
SNP
SSR
Introduction
Rice is a staple food in Indonesia. The average rice
consumption is 150 kg per capita in Indonesia

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(UNCTAD 2011). The demand of rice has rapidly
increased in accordance with population growth in
Indonesia, China, India and other Asian countries even
though the increase in rice yields is slow (Hossain
2007). Corresponding to the increasing demand, new
elite varieties with ideal plant architectures that can
produce higher grain yields are needed to be devel-
oped (Peng et al. 1999). One of the reasons for the slow
growth of rice yields is the continuous use of
genetically related elite germplasm in breeding pro-
grams (Tanksley and McCouch 1997).
The International Rice Research Institute (IRRI)
was established in 1960 in cooperation with the
government of the Philippines to improve rice pro-
duction and productivity. In 1966, IR8 was the first rice
variety released that had excellent yield potential in the
tropics (Khush 1997). A recessive semi-dwarf1 gene
(sd1) was used to develop IR8 (Sasaki et al. 2002).
This variety was introduced into many Asian countries
and it was used as breeding material to develop new
varieties. Indonesia is one of several Asian countries
that have collaborative breeding projects with the IRRI
to improve Indonesian rice varieties.
The history of rice breeding in Indonesia is divided
into at least three stages according to the collaboration
with IRRI (Susanto et al. 2003). (1) The first stage
(1943–1966); varieties of this stage were bred using the
China variety from China, the Latisail variety from
India, and the Bengawan variety from Indonesia
(before collaboration). (2) During the second stage
(1967–1985), Indonesia released two famous IR vari-
eties, PB8/IR8 in 1967 and PB5/IR5 in 1968, as well as
the IR5-related varieties, Pelita I-1 and Pelita I-2.
Several collaborative programs with the IRRI were
formed as part of the International Network for the
Genetic Evaluation of Rice (INGER), the International
Rice Tungro Nursery (IRTN), the International Rice
Blast Nursery (IRBN), the International Rice Brown
Plant Hopper Nursery (IRBPHN), and the International
Rice Bacterial Blight Nursery (IRBBN). (3) During the
third stage (1986 to the present), a representative
variety, IR64, which possesses a wider genetic back-
ground than IR8 and has insect- and disease-resistance
genes, was used as a parent to develop new varieties.
These stages probably faced with serious problems,
especially the genetic similarity of their germplasm,
due to the continuous use of elite varieties as breeding
materials (Tanskley and McCouch 1997). Two
approaches have been used to evaluate the genetic
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Genet Resour Crop Evol
relationships of rice across several countries based on
the analysis of morphological traits and molecular
markers. According to Caldo et al. (1996), uniform
morphological traits were selected during the breeding
programs and resulted in the reduction of phenotypic
divergence of improved rice varieties in the Philip-
pines. Similar results were reported for improved
varieties of Cuba (Fuentes et al. 2005), Africa
(Ogunbayo et al. 2005) and India (Kunusoth et al.
2015). However, the use of morphological traits to
evaluate the genetic relationships among rice varieties
has encountered difficulties in obtaining reproducible
results in different environments, times and manage-
ment strategies (Yue et al. 2008; Tanaka and Shiraiwa
2009). In contrast, since molecular markers to evaluate
genetic relationships is not affected by the environ-
ments and/or management practices, molecular mark-
ers to analyze genetic relationships in crops has
become increasingly popular. Lu et al. (2005) reported
an increase in the uniformity of improved USA
varieties in 1900–2000 using SSR markers due to
limited range of crossing and artificial selection, which
is in agreement with the results of Wei et al. (2009)
who reported decreasing diversity in Chinese varieties
developed in 1950–1990 using SSRs. SNP markers are
frequently used for genetic analysis instead of SSRs
(Jenkins and Gibson 2002). Although SNPs are less
polymorphic than SSRs because of their biallelic
nature, scientists and breeders have used SNPs
because each SNP is a potentially useful marker, and
SNPs are an efficient way to monitor genetic diversity
over an entire genome (Ganal et al. 2009). McNally
et al. (2009) reported that SNPs were effective on
understanding of the breeding history and relationship
among improved varieties from different countries. A
reduction in the diversity and similarity of improved
rice varieties has been reported in several countries.
The study on the genetic relationships among
improved Indonesian rice varieties is important for
future rice improvement strategies using morpholog-
ical traits and molecular markers (SSRs and SNPs).
Materials and methods
Morphological characterization
100 Indonesian improved rice varieties used in this
study were selected from the rice germplasm
Genet Resour Crop Evol
collection at the Indonesian Centre for Rice Research
(ICRR Subang, Indonesia) (Supp. Table 1). The plants
were grown in a paddy field at the University of
Tsukuba from May to November in 2009 and 2010.
The seeds were sown in a nursery box and 30-day-old
seedlings were transplanted, using a 15 9 30-cm
spacing, with 30 plants per variety. Of the 100
varieties, ten traits of these varieties were recorded
at different growth stages according to the IRRI
(IBPGR-IRRI 1980) recommendations for O. sativa L.
However, as 22 varieties headed later, they did not
normally grow and did not reach to grain filling.
Therefore, only 78 out of 100 varieties were used for
the analysis in this study (Supp. Table 6).
DNA extraction
The plants were grown in nursery boxes in a green-
house at the University of Tsukuba, Japan, and leaf
samples were harvested from individual 30-day-old
plants of 78 varieties (Supp. Table 6). The leaf
samples were crushed in micro-tubes (2 mL) using a
crusher with a zirconium ball, and DNA was isolated
using a modified cetyl trimethylammonium bromide
(CTAB) method (McCouch et al. 1988).
SSR genotyping
The SSRs in this study consisted of SSRs used in
previous studies on Indonesian rice (Muhamad et al.
2015; Thomson et al. 2007) and Chinese rice (Zhang
et al. 2011). These SSRs were reported to effectively
differentiate rice varieties into two subspecies, subsp.
indica Kato and subsp. japonica Kato. We screened
148 SSRs mapped across all 12 chromosomes and
selected 32 polymorphic markers, the sequences of
which were obtained from the Gramene database
(http://www.gramene.org/) (Suppl. Table 3). The
PCR solution in each tube consisted of 1 lL of 109
buffer, 1 lL of dNTPs, 0.1 lL of Taq polymerase
(TOYOBO, Japan), 1 lL of primer (created from R
and F SSR sequences) (Invitrogen, Carlsbad, CA,
USA), 1 lL (5 ng) of DNA and 5.9 lL of distilled
water. The PCR conditions were 5 min at 94 °C; 35
cycles of 30 s at 94 °C, 30 s at the primer-specific
annealing temperature, 90 s at 72 °C; and a final
extension at 72 °C for 10 min. These SSRs were
genotyped in 100 rice varieties following elec-
trophoresis in 10 % polyacrylamide gels (250 V,
500 mA); the genotypes were determined visually
from the gel images. Of the 100 DNA samples, 78
genotypes provided sufficient data (\15 % missing)
for analysis.
SNP genotyping
The total genomic DNA isolated from the fresh leaf
tissue of one plant per variety was quantified and
diluted to 50 ng/lL in sterile 10 mM Tris–HCl (pH
8.0) and 1.0 mM EDTA (pH 8.0) (TE). Allele-specific
oligonucleotide hybridization used 5 lL of single-use
template genomic DNA (50 ng/lL) of each variety. In
this study, we used 768 SNPs. These were designed by
Yonemaru et al. (2014), who used these SNPs to
successfully differentiate 76 rice varieties into four
groups: aus, indica, tropical japonica, and temperate
japonica and it was used in Indonesian traditional rice
by Muhamad et al. (2015). The four variety groups in
rice classification were followed by Glaszmann (1987)
classification of Asian rice varieties. In which,
subspecies japonica comprise temperature japonica
and tropical japonica, while subspecies indica con-
tains indica and aus (Garris et al. 2005). Hereinafter
indica and tropical japonica groups in this study were
referred to as indica and japonica. The sequences of
these SNPs as well as additional information can be
found in their study (doi:10.1093/pcp/pct188). Tem-
plate DNA and a negative control (water) were mixed
with 768 different SNPs. The PCR conditions were
10 min at 37 °C, then 3 min at 95 °C, followed by 34
cycles of 35 s at 95 °C, 35 s at 56 °C, and 2 min at
72 °C, followed by a final extension at 72 °C for
10 min. These SNPs were genotyped in 100 rice
varieties using the GoldenGate BeadArray technology
platform (Illumina, San Diego, CA) and we followed
the manufacturer’s instructions for all experimental
procedures involving SNP genotyping. Of the 100
DNA samples, 78 genotypes provided sufficient data
(\15 % missing). The missing allele was classified
into the third allele for the analysis in this study.
Data analysis of morphological traits
Morphological data were statistically analyzed using
three types of software: JMP 5.1 (JMP 2000), NTSYS-
pc 2.0 (Rohlf 2000), and SPSS 21 for Windows. JMP
5.1 was used to create clusters within the improved
varieties using Ward’s hierarchical methods.
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Furthermore, this software was also used to observe
the contribution of morphological traits with stan-
dardized data for clustering by principal component
analysis (PCA) and bi-plot analysis. Correlations
among morphological traits and molecular markers
were calculated using the Mantel test generated by
NTSYS-pc, and Euclidean distances within improved
varieties were calculated using SPSS 21.
Data analysis of molecular markers
Molecular markers were analyzed using DARwin5
(Perrier and Jacquemod-Collet 2006), GenAlEx 6.5
(Peakall and Peter 2006), and BayeScan 2.1 (Foll and
Gaggiotti 2008). Furthermore, DARwin5 was used to
calculate a neighbor-joining tree based on genetic
distance using 1000 bootstraps (CIRAD 2009), and
GenAlEx 6.5 was used to calculate the number of
alleles (Na), the gene diversity (H; Shannon 1948), the
number of effective alleles (Ne), the polymorphic
percentage (P), the differentiation among groups (Fst)
and the genetic distance (D; Nei 1972). Bayescan v.
2.1 was used to calculate the differentiation among
neutral loci (FST) and to detect candidate loci for
divergence based on a NJ tree, in which outlier loci
generated by this software indicate natural selection.
The evidence of selection in the range 0.5 \ log10
(PO) \ 1 is substantial, strong if 1 \ log10
(PO) \ 1.5 and very strong if 1.5 \ log10 (PO) \ 2,
as explained in manual for this software.
Results
Morphological traits
Ward’s hierarchical cluster analysis indicated little
variation among the improved Indonesian rice vari-
eties released from 1943 to 2003 based on ten
morphological traits. According to the dendrogram,
the improved Indonesian varieties were classified into
two major groups in 2009 and 2010 (Figs. 1, 2).
The smaller group (group A) consisted of four out
of the five varieties released in the first stage and some
of the varieties released after this stage. The larger
group (group B) involved 62 and 73 % of the total
improved varieties in 2009 and 2010, respectively.
IRRI varieties and most of their derived varieties
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Genet Resour Crop Evol
belonged to group B. However, we did not find any
sub-groups clustered based on bred materials and/or
stage of variety release. The Euclidean distances
among the improved varieties, which are estimates of
phenotypic distances, were obtained from ten mor-
phological traits. We found various ranges of Eucli-
dean distances between all pairs, 3.92 to 152.01 in
2009 and 1.66 to 107.63 in 2010.
These values were compared to the genetic dis-
tances among varieties, determined by molecular
markers using Mantel’s test (Supp. Figs 1). PCA and
bi-plot analysis revealed that the morphological traits
contributed to the differentiation of the improved
Indonesian varieties into two groups (Figs. 3, 4). The
sum of three PCAs based on 10 traits of the 78
improved Indonesian varieties used in 2009 and 2010
explained 64.71 and 67.16 % of the total variation,
respectively. PC 1 explained 32.97 and 43.05 % of the
variation, PC 2 explained 19.37 and 12.63 % of the
variations, and PC 3 explained 12.36 and 11.48 % of
the variation in 2009 and 2010, respectively (Tables 1,
2). Most of the eigenvectors of the PCs were positive
and responsible for the differentiation in each year
(Tables 1, 2). This was also shown by the bi-plot
analysis, in which most of the traits were in quadrants I
and IV in both years (Figs. 3, 4).
There was a significant negative correlation between
plant height (PH) and heading date (HD) in the two
groups in 2009. However, a significant correlation
between plant height (PH) and heading date (HD) was
not found in 2010. In group B, we found a significant
correlation between plant height (PH) and panicle
length (PL), while the correlation between these
variables was not significant in group A in 2009 and
2010. Additionally, plant height (PH) was significantly
correlated with most of the traits in group B (Supp.
Tables 4, 5). We also found a delay in the heading date
(HD) for most of the Indonesian varieties when we
compared with the heading date (HD) in Japan and the
maturity date in Indonesia (data not shown). For
example, PB5 needed 160–162 days for heading in
Japan, but needed only 135–145 days to reach maturity
in Indonesia. IR36 needed 113–120 days for heading in
Japan versus 110–120 days in Indonesia. The Ciherang
variety, which is one of the famous varieties in
Indonesia, needed 117–135 days for heading in Japan
versus 116–125 days in Indonesia. We obtained the
data for the maturity date in Indonesia based on
descriptions of each variety (http://puslittan.bogor.net/).
Genet Resour Crop Evol

Fig. 1 Dendrogram using

hierarchical method in 2009
based on ten morphological
traits. X is 1st stage, Y is 2nd
stage, Z is 3rd stage and
asterisk IRRI varieties

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 Genet Resour Crop Evol

Fig. 2 Dendrogram using hierarchical method in 2010 based on ten morphological traits. X is 1st stage, Y is 2nd stage, Z is 3rd stage and
asterisk IRRI varieties

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Genet Resour Crop Evol
Fig. 3 Principal components analysis (PCA) and bi-plot based on ten morphological traits in 2009
Fig. 4 Principal components analysis (PCA) and bi-plot based on ten morphological traits in 2010
Molecular markers
Neighbor-joining analyses using 32 SSRs and 768
SNPs divided the improved Indonesian rice varieties
into two subspecies, indica and japonica. Most of the
improved Indonesian rice varieties fell into the indica
subspecies, i.e., 88.5 % using SSRs and 89.7 % using
SNPs. The varieties fell into the japonica subspecies
when the Kartuna variety was used as a varietal check
for tropical japonica. The SSRs categorized 8.9 % of
the varieties as japonica, while the SNPs did 7.7 % as
japonica. However, we found two varieties (2.6 %)
located in the center of the neighbor-joining tree using
SSRs and SNPs, and we called these varieties as group
C (Figs. 5, 6).
Most of the varieties belonging to the japonica were
old varieties released in the first stage (Bengawan,
Kartuna, and Dewi Ratih varieties), and some of them
(Danau Atas, Gajah Mungkur, and Kalimutu) were
bred without IRRI varieties as breeding materials.
However, certain varieties (Banyuasin and Situ
patenggang) in this group were developed using IRRI
varieties. Regarding the neighbor-joining tree, six
groups such as IR5, IR36, IR48, IR64, japonica and
‘‘C’’, were identified based on the SSRs and SNPs
(Figs. 5, 6). In addition, we identified the IR66 type
and the ‘‘X’’ groups in the neighbor-joining tree using
the SNPs. The differentiation between groups (Fst)
was significant for four out of six groups using SSRs
and four out of eight groups using SNPs (Table 3), and
the Fst using the SNPs was higher than that using the
SSRs. These groups (four out of six groups using SSRs
and four groups out of eight groups using SNPs) are
IR5, IR36, IR64 and japonica. We selected these
groups because the IR5, IR36 and IR64 varieties have
had the most influence on the varieties in breeding
history of Indonesia. Moreover, there was no infor-
mation on pedigree (crossing history) that the
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 Genet Resour Crop Evol

Table 1 PCs and eigenvector based on ten morphological 59.4 %, respectively) and japonica (81. 3 and 64.1 %,
traits in 2009 respectively). The diversity index (Shannon’s H) of
Traits Eigenvectors the two groups was the same when using the SSRs,
while the H of japonica was higher than that of indica
PC 1 PC 2 PC 3
when using the SNPs. Furthermore, the number of
PH (cm) 0.475 -0.282 0.016 effective alleles (Ne) of japonica was higher than that
CL (cm) 0.437 -0.360 0.116 of indica (1.86 and 1.69, respectively) when using the
PL (cm) 0.144 0.226 -0.312 SSRs and the SNPs (1.41 and 1.27, respectively)
LoL (cm) 0.308 0.423 0.157 (Table 4).
WoL (cm) 0.326 0.025 -0.544 According to the breeding history stages in Indone-
LoSL (cm) 0.353 0.128 0.321 sia, there was a higher percentage of polymorphic loci
LoFL (cm) 0.178 0.488 0.128 (P) for the SSRs (75.0–96.9 %) compared with the
WoFL (cm) 0.360 0.207 -0.363 SNPs (60.9–78.3 %). The diversity index (Shannon’s
HD (day) -0.048 0.454 0.320 H) of stage I was lower (0.53) than that of stages II and
AoFL (°) 0.272 -0.237 0.463 III (0.61 and 0.62, respectively) according to the SSRs,
Eigenvalue 3.297 1.937 1.236 while the H of stage I was higher (0.37) than that of
Cumulative of variation (%) 32.974 52.346 64.710 stages II and III (0.30 and 0.29, respectively) accord-
ing to the SNPs (Table 5). The number of effective
PH plant height, CL culm length, PL panicle length, LoL length
of leaf, WoL width of leaf, LoSL length of sheath leaf, LoFL
alleles (Ne) in stage I was lower (1.69) than that in
length of flag leaf, WoFL width of flag leaf, HD heading date, stages II and III (1.73 and 1.73, respectively) accord-
and AoFL angel of flag leaf ing to the SSRs, while the Ne in stage I was the highest
(1.46) and that in stage III was the lowest (1.29) when
using the SNPs. The differentiation (FST) and genetic
Table 2 PCs and Eigenvector based on ten morphological distance (D; Nei) between the three stages showed that
traits in 2010 there were significant differences in the FST
Traits Eigenvectors
(P \ 0.05 for SSRs, P \ 0.05, P \ 0.01 and for
SNPs) between stages I and II and between stages I
PC 1 PC 2 PC 3 and III (0.098 and 0.087, respectively) using SSRs and
PH (cm) 0.394 -0.311 -0.101 between stage I and II and between stages I and III
CL (cm) 0.372 -0.334 -0.367 (0.122 and 0.135, respectively) using SNPs. In con-
PL (cm) 0.087 0.043 0.726 trast, there was no significant difference in the FST
LoL (cm) 0.405 0.196 0.157 between stages II and III (0.017 and 0.021, respec-
WoL (cm) 0.338 -0.126 -0.178 tively) according to the SSRs and SNPs (Table 6).
LoSL (cm) 0.366 0.222 -0.103 Mantel’s test indicated positive correlations (r) be-
LoFL (cm) 0.333 0.335 0.266
tween the 2009 and 2010 morphological traits
WoFL (cm) 0.365 -0.071 0.147
(r = 0.62), the morphological traits and SSRs in
HD (day) 0.126 0.695 -0.311
2009 (r = 0.28) and 2010 (r = 0.19), the morpholog-
ical traits and SNPs in 2009 (r = 0.30) and 2010
AoFL (°) 0.164 -0.292 0.269
(r = 0.21), and an intermediate positive correlation
Eigenvalue 4.305 1.263 1.148
between the SSRs and SNPs (r = 0.44) (Table 7 and
Cumulative of variation (%) 43.045 55.676 67.159
Supp. Fig 1).
For abbreviations see Table 1 In addition, we found that breeding materials
affected differentiation of Indonesian improved vari-
eties, 41.4 % of improved varieties were grouped
improved varieties used in this study had been based on breeding materials by SSRs and 31.7 % of
produced using other bred materials (Supp. Table 6). them were grouped by SNPs (Supp. Table 6). Fur-
Diversity analysis revealed an intermediate to high thermore, we investigated the differentiation between
percentage of polymorphisms in the two groups loci (FST) according to the NJ tree using four out of six
according to the SSRs and SNPs: indica (100 and groups that were identified by the SSRs and four out of

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Genet Resour Crop Evol

Oryza sava subsp. indica

C type

75
15 61 17
59
65
53
54 IR42
52
50 60
57 69 24
25 40 3
70 16
22 27
IR48 64 43 68
55 67 13 2063
73 23
19 35 21
4834 76 33 IR66
37 41 56
IR36 62 8
32 9 47
45 51 10 44
49 2
PB 5 Kartuna
31 58 42
5
66 IR65
1872
74
IR70
77 71 26
14 IR64
78 36

IR48 type
IR5 type IR64 type
IR36 type 46

O.sava subsp. japonica

Fig. 5 Neighbor-joining tree of Indonesian varieties based on 32 SSR markers

Fig. 6 Neighbor-joining Oryza sava subsp. indica

tree of Indonesian varieties IR66 type C type
based on 768 SNP markers

56 71 57
44 69
33
38 62 52 13
70 77
6141
64 59
2620 6649 32 55 54 78
76 25 53
1518 39 14
IR64
IR424667 65 IR66 8 35 50 IR65
5 27 58 34
60 16
PB5 75 31 68 42
73
21 48IR48
74 3 22
IR70 43 IR36 10
63
40 9 47 19
23 17 2
24 45

IR64 type IR36 type IR48 type IR5 type

X type

Kartuna

72

51
7
1

O. sava subsp. japonica

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 Genet Resour Crop Evol

Table 3 Differentiation between groups according to neigh- Table 6 Differentiation (FST) and genetic distance (D; Nei’s)
bor-joining tree among stage in Indonesian improved varieties
Group pairwise SSR markers SNP markers Pairwise FST D
FST FST
SSR markers
IR36 versus IR5 0.056** 0.196** Stage I versus stage II 0.098* 0.117
IR36 versus IR64 0.108* 0.132** Stage I versus stage III 0.087* 0.103
IR36 versus Tj 0.163** 0.420** Stage II versus stage III 0.017 0.018
IR5 versus IR64 0.101* 0.163** SNP markers
IR5 versus Tj 0.153** 0.402** Stage I versus stage II 0.122* 0.080
IR64 versus Tj 0.185* 0.405** Stage I versus stage III 0.135** 0.088
Tj tropical japonica Stage II versus stage III 0.021 0.010
** P \ 0.001; * P \ 0.01 ** P \ 0.01; * P \ 0.05

Table 7 Correlation (r) between genetic similarities using

Table 4 Allelic values across populations, indica and japon- different approach
ica based on SSR and SNP markers Pairwise of genetic similarity Mantel’s test (r)
Mean values Indica Japonica
Morphology versus morphology
SSR markers 2009 versus 2010 0.617
Number of allele (Na) 3.031 2.188 Morphology versus molecular
Number effective of allele (Ne) 1.688 1.862 2009
Diversity index (H) 0.600 0.600 Morphology versus SSR 0.275
Polymorphic loci (P) (%) 100 81.25 Morphology versus SNP 0.304
SNP markers Morphology versus molecular
Number of allele (Na) 1.680 1.685 2010
Number effective of allele (Ne) 1.269 1.413 Morphology versus SSR 0.193
Diversity index (H) 0.247 0.360 Morphology versus SNP 0.206
Polymorphic loci (P) (%) 59.38 64.06 Molecular versus molecular
SSR versus SNP marker 0.443

eight groups that were identified by the SNPs. We

Table 5 Allelic values across populations, stage I found the highest value of differentiation among
(1943–1966), stage II (1967–1985) and stage III (1986–2003)
based on SSR and SNP markers
neutral loci (FST) using SNPs (Supp. Fig. 2). The FST
values ranged from 0.307 to 0.498 between IR5 and
Mean values Stage I Stage II Stage III IR36, from 0.234 to 0.519 between IR36 and IR64,
SSR markers from 0.307 to 0.475 between IR36 and japonica, from
Number of allele (Na) 2.000 2.813 2.906 0.260 to 0.504 between IR5 and IR64, from 0.281 to
Number effective of allele (Ne) 1.691 1.730 1.731 0.498 between IR5 and japonica and from 0.229 to
Diversity index (H) 0.525 0.612 0.616 0.519 between IR64 and japonica using SNPs. While
Polymorphic loci (P) (%) 75.00 93.75 96.88 the FST values ranged from 0.079 to 0.095 between
SNP markers IR5 and IR36, from 0.129 to 0.163 between IR36 and
Number of allele (Na) 1.633 1.814 1.891
IR64, from 0.156 to 0.217 between IR36 and japonica,
Number effective of allele (Ne) 1.461 1.313 1.290
from 0.119 to 0.137 between IR5 and IR64, from
Diversity index (H) 0.373 0.300 0.291
0.151 to 0.178 between IR5 and japonica and from
0.182 to 0.199 between IR64 and japonica using
Polymorphic loci (P) (%) 60.94 72.53 78.26
SSRs. We did not find evidence of strong selection

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Genet Resour Crop Evol
among neutral loci (FST) in these groups, in which we
detected the value of Log10 (PO) \ 1 in all compar-
isons using the two types of markers. However, we
found similiar locus (OSJNOa199K18 (AP006848.1))
between the IR36 and IR64 groups and between IR64
and japonica in the range of substantial selection
(0.55; 0.57). The locus (AP006848) was associated
with hypothetical protein in Oryza sativa subsp.
japonica, which does not have a characterized homo-
logue in the protein database (http://www.shigen.nig.
ac.jp/).
Discussion
In the present study, most of the traits contributed to
the differentiation of improved Indonesian rice vari-
eties into two groups based on clustering and PCA.
This result indicated that most of the varieties were
small in size (62 % in 2009 and 73 % in 2010). These
varieties were released after the introduction of IRRI
varieties as breeding materials. It seems that a semi-
dwarf variety with high yield potential became a
common Indonesian rice variety. Reduced plant
stature was a target trait of improved rice cultivars,
and most of the released varieties had a semi-dwarf1
(sd1) gene that originated from the Dee-gee-woo-gen
(Hargrove et al. 1988). A recessive semi-dwarf1 (sd1)
gene was used to develop IR8 (Sasaki et al. 2002).
However, we still found some varieties that were large
in size (tall stature), even for those varieties using
IRRI varieties as breeding materials. The clustering
based on morphological data is failure on grouping the
Indonesian rice varieties according to bred materials
and time stages. This indicated that the selection for
morphological traits might be directed to a similar
plant type as we mentioned above that rice breeding
programs in Indonesia exclusively focused on reduced
plant stature using semi-dwarf varieties that were
released by IRRI.
Based on the correlation analysis, most of the
varieties showed that plant height was directly asso-
ciated with other traits and affected the plant archi-
tecture. This result supported the view that reduced
plant stature was the target for rice breeding in
Indonesia using IRRI varieties. These changes also
affected the yield, as shown by the positive correlation
between plant height and panicle length in most of the
varieties developed using IRRI varieties. The sd1 gene
almost equally reduced plant height, panicle length,
and upper internodes (Tsai 1998), and panicle archi-
tecture correlated with yield and grain quality (Crow-
ell et al. 2014). This aspect could be explained by a
possible pleiotropic effect of sd1 (Murai et al. 2002).
The negative correlation between plant height and
heading date indicated that most of the Indonesian
semi-dwarf varieties were characterized by late head-
ing. However, this phenomenon was found only in
2009, which indicated that there was an environmental
change in 2010 and it causing the phenomenon was not
occurring in 2010. An interaction between environ-
ment and heading date was reported by Li et al. (2003).
Additionally, Chunhai and Zongtan (1995) reported
that the sd1 gene did not affect heading date. The delay
in heading in Indonesian rice varieties cultivated in
Japan was caused by photoperiod sensitivity. Indone-
sia, which is located along the equator, has a relatively
constant day length of approximately 12 h. In contrast,
Japan is located above the equator, and the day length
in the rice season (May to September) ranges from 13
to over 14 h. Rice, as a short-day plant, is sensitive to
photoperiod and long-day treatment considerably
delays heading and flowering (Vergara and Chang
1985). No significant correlation (r = 0.62) between
2-year data suggested the difference in the intensity of
environmental effects on traits in 2009 and 2010.
The genetic similarity analysis using the neighbor-
joining tree revealed that 88.5 and 89.7 % of the
Indonesian varieties were classified into indica by
SSRs and SNPs, respectively, while 8.9 and 7.7 % of
the varieties were classified into japonica by SSRs and
SNPs, respectively. In addition, 2.6 % of the varieties
were classified into admixtures by both markers.
These results differed from previous report (Thomson
et al. 2007). They showed that all of the Indonesian
improved varieties were indica. However, our results
agreed with a previous study that the Indonesian
breeding program focused on high-yielding irrigated
rice varieties which are largely indica rather than
upland rice varieties belonging to japonica (Thomson
et al. 2007). The varieties that did not use IRRI
varieties as breeding materials after the first stage and
which were classified into japonica in this study
involved in upland rice (Banyuasin, Gajah Mungkur,
Kalimutu and Situ Patenggang), based on their
description (http://puslittan.bogor.net/).
The differentiation between indica and japonica
was significant and both markers indicated strong
123

selection between indica and japonica as well as
differentiation among japonica and within indica sub-
groups was also significant. However, there was no
evidence showing strong selection at neutral loci
between indica and japonica and among sub-group.
Bayesian methods indicated that the strong selection
was not detected in the particular region of the genome
and/or the markers in this study and that particular loci
were selected in rice breeding in Indonesia. However,
similar locus in the substantial selective range was
detected between the IR36 and IR64 and between
IR64 and japonica groups using SNPs and these loci
was a likely target of artificial selection in rice
breeding in Indonesia. This locus (OSJNOa199K18)
might encode protein which is not homologs to
proteins registered in protein databases. Further study
of this region is needed to clarify its function. In
addition, group type ‘‘X’’ identified based on SNPs
indicated that SNPs has captured the distinction of this
group, while SSRs could identify some of the varieties
in this group as the breeding material.
The lower diversity in indica compared with
japonica was revealed by SNPs, while SSRs showed
the same diversity in indica as japonica. This was also
supported by the fact that Indonesian breeders focused
on improving indica rather than japonica, and it was
also shown by a lower effective number of alleles in
indica compared with japonica, as revealed by both
molecular markers. Changes in allelic frequency occur
in response to selection (Vieira et al. 2013). The
breeder is primarily interested in capturing unique
phenotypes, in which causing the recovery of desired
alleles and the elimination of undesirable ones. When
a gene is subjected to selection pressure its frequency
changes from parent to progeny, thus allelic frequency
in the progeny is variable depending on the differential
effect of selection on parental allelic frequency (Staub
1994).
The trend in the genetic diversity of Indonesian
improved rice varieties, according to their breeding
history in Indonesia, suggested that stagnation of genetic
diversity occurred between stage II (1967–1985) and
stage III (1986–the present), as revealed by both
molecular markers. This result indicated that the con-
tinuous use of elite varieties as breeding materials
caused genetic similarities of rice germplasm in Indone-
sia and it could be explained by relationship between
breeding materials and sub-clustering of variety group,
particularly in indica group, i.e., 41.4 % was grouped
123
Genet Resour Crop Evol
according to breeding materials by SSRs and 31.7 % by
SNPs. For example, 12 out of 17 varieties that were
clustered in IR5 type groups such as Atomita 2,
Mahakam, Cisokan, Ciliwung, Lusi, Way Seputih,
Cenrane, Bengawan Solo, Banyuasin, Batanghari,
Punggur and Lambur were successfully identified to
match with IR5 by SSRs, even though these varieties
were derived from complex cross combinations in
breeding programs. Moreover, we found decreased
genetic diversity (18.9 %) and a lower number of
effective alleles (13.7 %) in stage II compared with
stage I, as well as in comparison of stage III with stage I
(1943–1966), using SNPs. Stagnation and decreased
genetic diversity were reported in Chinese rice germ-
plasm during 1950–2004 (Yuan et al. 2007) and during
1950–1990 (Wei et al. 2009). In contrast, higher genetic
diversity (13.1 %) in stages II and III than that in stage I
was found using SSRs. This result is in agreement with
that reported for Indian rice varieties during 1970–2000
(Choudhary et al. 2013) and for Italian rice varieties
during 1880–2001 (Mantegazza et al. 2008). This
finding suggested that SNPs used in this study captured
more effective alleles involved in older varieties. The
insignificant difference between stages II and III indi-
cated that the breeding effort was not adequate to
enhance diversity in Indonesian rice germplasm, even
though Indonesian rice breeders had used complex cross
combinations to develop new varieties. Nevertheless, we
revealed that breeding program in Indonesia used the
common breeding materials in stage II for developing
new varieties in stage III, particularly IR5 type varieties.
The differences between the SNPs and SSRs in
terms of the level of genetic diversity were resulted
from the mutational properties of these two types of
molecular markers. A smaller proportion of rare
(private) alleles was observed in the frequency distri-
bution of the SNP data compared with the SSR data
(data not shown). This result is in agreement with that
of Emanuelli et al. (2013) who reported that SSRs
captured more rare (private) alleles than SNPs which
affected the diversity index. Additionally, the different
properties of these two markers also affected the
differentiation among varieties as suggested by Ham-
blin et al. (2007) and Emanuelli et al. (2013).
The positive correlations between the phenotypic
and molecular data depended on the kinds of molec-
ular markers. SSRs had lower Mantel r values than
SNPs. This may be attributed in part to the fact that the
SNP structure is more similar to the phenotypic data
Genet Resour Crop Evol
than SSR markers. This fact could be also due to the
different properties of these two markers. Although we
changed the missing allele in the SNP data to the third
allele, the SNP data revealed lower variations among
varieties compared with SSRs (Figs. 5, 6), and lower
phenotypic distances among varieties were observed
based on ten traits (Figs. 1, 2). However, the correla-
tion between these two markers and the phenotypic
distance among varieties were slightly different. The
slightly positive correlation caused by morphological
traits in this study are associated with a relatively
small number of loci, thus the potential difference
could be lost in the analysis of large amounts of
molecular data as suggested by Diederichsen (2009).
We concluded that the current distribution of
variations in phenotypes and molecular markers is
probably the result of artificial selection in rice
breeding in Indonesia. Although clustering based on
morphological traits has failed to group Indonesian
varieties according to breeding materials and time
stage, the clustering could reveal the focus of rice
breeding programs in Indonesia, reduced plant stature.
Meanwhile, the clustering based on molecular markers
revealed that the differentiation of improved rice
varieties in Indonesia was related to breeding mate-
rials. These indicated that molecular markers were
powerful to clarify the genetic relationship among
improved rice varieties in Indonesia while morpho-
logical traits could explain the effect of artificial
selection for plant stature. Hence, the extensive use of
elite varieties as breeding materials might cause the
stagnation and decrease of genetic diversity between
stage II and stage III in Indonesia. Besides, this study
demonstrated that the varieties bred before 1966 in
stage I might be valuable sources for increased
variability as revealed by SNPs.
The slight correlation between the diversity ana-
lyzed using SSRs and SNPs suggested that these two
molecular markers have different application. SSRs
seem to be more effective than SNPs in pedigree
studies, while SNPs are useful to analyze genomic
relationships. Spada et al. (2004) reported that SSRs
were more effective in pedigree studies on Italian rice
varieties compared with amplified fragment length
polymorphism (AFLP) markers. They also reported a
low correlation between SSR and AFLP similarity
because the different genomic information was pro-
vided by the two kinds of markers. Furthermore, using
SNPs, we identified candidate genes between IR5 and
IR36 and between IR36 and IR64 groups that might
have been selected during the period of rice breeding
in Indonesia. This study indicated that SNPs are also
useful to exploit selection at particular neutral loci,
compared with SSRs. The weak correlation between
phenotypic relationships in 2 years indicated that
evaluation of genetic relationships using molecular
markers includes difficulties with respect to the effects
of different management practices and different envi-
ronments. This study also suggested that Indonesia
needs another strategy to improve new varieties to
avoid a reduction in genetic diversity and similarity.
Acknowledgments We are grateful for the Dikti Scholarship
and would like to thank the Government of the Indonesian
Republic, who funded the first author for a Ph.D. program at the
University of Tsukuba, and the ICRR for providing us seed
material for this study.
Authors’ contributions The first author contributes to sub-
stantive conception, laboratory and field work, design, analysis,
interpretation of data and drafts the manuscript; the second, the
third and the fourth authors contributed to revise it critically for
important intellectual content; and the fourth author also as a
final approval person of the version to be published.
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