Development and characterization of chromosome segment substitution lines derived from Oryza rufipogon in the genetic background of O. sativa spp. indica cultivar 9311
Qiao et al. BMC Genomics
Development and characterization of chromosome segment substitution lines derived from Oryza rufipogon in the genetic background of O. sativa spp. indica cultivar 9311
Weihua Qiao 2
Lan Qi 1 2
Zhijun Cheng 2
Long Su 2
Jing Li 2
Yan Sun 2
Junfang Ren 0
Xiaoming Zheng 2
Qingwen Yang 2
0 Institute of Tropical Horticulture, Hainan Academy of Agricultural Sciences , 14 Xingdan Road, Haikou, Hainan 571100 , China
1 Institute of Cereal Crop Science, Hainan Academy of Agricultural Sciences , 14 Xingdan Road, Haikou, Hainan 571100 , China
2 Institute of Crop Science, Chinese Academy of Agricultural Sciences , 12 Zhongguancun South Street, Beijing, Haidian 100081 , China
Background: Wild rice (Oryza rufipogon) constitutes a primary gene source for rice breed improvement. Chromosome segment substitution line (CSSL) for O. rufipogon is a powerful tool for fine mapping of quantitative traits, new gene discovery, and marker-assisted breeding. Thus, they provide a basis for a wide range of genomic and genetic studies. Results: In this study, a set of 198 CSSLs were developed from a cross between recurrent parent indica var. 9311 and an O. rufipogon donor parent; these were then genotyped using 313 polymorphic SSR markers evenly distributed across the 12 rice chromosomes. On average, each CSSL carried 2.16 introgressed segments, and the genetic distance of each segment was about 6 cM. The segments collectively covered 84.9 % of the wild rice genome. Based on these CSSLs, 25 QTLs involved in 10 agronomic traits were identified. Seven CSSLs were subjected to a whole-genome single nucleotide polymorphism chip assay and two QTLs, qSH4-1 and qDTH10-1, detected. In addition, a new QTL associated with the heading date was detected in a 78-Kb region on chromosome 10, thus proving the ability of these CSSLs to identify new QTLs and genes. Conclusions: The newly developed CSSL population proved a useful tool for both gene identification and whole-genome research of wild rice. These CSSL materials will provide a foundation for rice variety improvement. Abbreviations: cM, centimorgan; CSSL, chromosome segment substitution line; IL, introgression line; MAS, marker-assisted selection; NIL, near isogenic line; QTL, quantitative trait locus; SNP, single nucleotide polymorphism; SSR, simple sequence repeat
Chromosome segment substitution lines; Oryza rufipogon; Quantitative trait locus
A problem facing traditional rice breeding is that of yield
plateaus, these are caused by the narrow genetic basis of
parental materials [
]. The transfer of genes controlling
desirable traits from wild relatives to cultivated rice is an
important strategy in rice breeding. The Oryza family,
which includes cultivated rice and wild species, contains
highly diverse geographical, morphological, and
physiological characteristics [
]. Wild Oryza species with 2n =
24 or 48 chromosomes and genome constitutions AA,
BB, CC, BBCC, CCDD, EE, FF, GG, or HHJJ are
important reservoirs of genes with potential for use in rice
Common wild rice (Oryza rufipogon Griff.) has a
similar AA genome to cultivated rice, and is considered the
direct ancestor of cultivated rice (Oryza sativa L.) [
During the course of domestication to cultivated rice,
many desirable traits, such as resistance to diseases and
pests, and adaptation to unfavorable environments were
lost, profoundly decreasing genetic diversity [
genes controlling important agronomic traits in rice
domestication, such as days to heading, seed shattering,
and seed dormancy, have been found in wild rice relatives
]. Although the overall economical characters of
wild rice are inferior to those of cultivated rice, modern
molecular biology studies have revealed potential genes
hidden in wild rice are essential for yield-related trait
]. Therefore, it is important to
discover these useful genes and to apply their use in rice
Many important traits in rice, including heading date,
culm length, eating quality, and yield are controlled by
quantitative trait loci (QTL) and show continuous
phenotypic variation in progenies. Construction and use of a
suitable genetic population is pivotal for fine mapping
of QTLs and map-based cloning of target genes.
Temporary mapping populations such as F2 or BC1 [
and permanent primary mapping populations including
doubled-haploid and recombinant inbred lines have been
developed for genetic analysis of complex traits [
However, these mapping populations cannot be used to
estimate individual QTLs precisely owing to genetic
background noise [
]. Thus, they are not adequate for
fine mapping and characterization of target QTLs and
further analyses [
]. Furthermore, phenotypic effects of
QTLs are always influenced by genetic backgrounds and
environmental factors. Therefore, the development of
advanced mapping populations such as introgression lines
(ILs) and chromosome segment substitution lines (CSSLs)
to analyze QTLs has received great attention. A CSSL
population is generally developed through advanced
backcrossing, selfing, and marker-assisted selection (MAS). In
CSSL populations, each line carries a single or a few
chromosomal segments from the donor parent in the genetic
background of the recurrent parent. An ideal CSSL
population is composed of lines carrying only one different
chromosome segment from the donor parent, but with the whole
population carrying the entire genome of the donor.
CSSLs from interspecific hybridization represent a
powerful and useful genetic resource for genome research,
especially QTL mapping and gene cloning, and for
pyramiding target segments and breeding [
]. The first
complete set of substitution lines were constructed in
tomato by Eshed and Zamir [
]. These consisted of
near isogenic lines (NILs) carrying single Lycopersicon
pennellii chromosomal segments in an otherwise
homogeneous L. esculentum background; these represented the
entire genome of wild tomato [
]. At least 20 sets of
ILs and CSSLs have been constructed in rice [
many agronomic QTLs have been identified and some
cloned using map-based methods [
]. Tian, Tan,
and their co-workers constructed two series of ILs for
Yuanjiang and Dongxiang common wild rice in China,
]. Hirabayashi et al. developed ILs from
O. rufipogon and Oryza glumaepatula in a japonica
cultivated rice background . A difficulty with IL
populations is that they do not cover the entire wild rice genome,
making precise QTL mapping difficult. Recently, Furuta
et al. developed 33 CSSLs of O. rufipogon in an elite
japonica cultivar Koshihikari background using 149 single
nucleotide polymorphism (SNP) markers [
this small population was not large enough for a better
understanding of the wild rice genome, with long and
redundant introgressive segments blocking fine mapping
of new genes. Moreover, there are few CSSLs reported for
wild rice from the low-latitude areas of China that use the
sequenced indica cultivar background. Thus, to identify
and employ desirable genes, and to have a better
understanding of the genetic diversity of wild rice, it is necessary
to construct new O. rufipogon–O. sativa CSSLs.
In this study, a broad population of 198 CSSLs was
constructed from backcross progenies derived from a cross
between the commercial indica cultivar 9311 as the
recurrent parent and the wild rice CWR276 as the donor parent.
The CSSL population was genotyped using 313
polymorphic simple sequence repeat (SSR) markers distributed
evenly across the 12 rice chromosomes. The CSSL
population covered 84.9 % of the wild rice genome with an
average substituted segment length of 6 cM. Based on
evaluation of the phenotypic variation of quantitative trait
and identification of QTLs, seven CSSLs were selected for
whole-genome SNP chip assays. Finally, a new QTL
associated with the heading date was identified. We
demonstrated this CSSL population as a useful tool not only for
fine mapping of genes but also for wild rice genomic
research. In addition, these CSSL materials provide a
foundation for developing future rice cultivars for breeding
Identification of SSR markers for MAS
In all, 780 SSR markers distributed throughout the 12 rice
chromosomes were used to detect polymorphisms between
9311 and CWR276, among which 369 (52.7 %) were
polymorphic between these two parents. Finally, 313
polymorphic SSR markers were selected for analysis of the
CSSL genotypes. Information regarding the genetic distance
of these markers was downloaded from Gramene [
Sequence information of markers used in this study was
showed in S-Table 1. The average distance between two
adjacent markers on the rice linkage map was 5.7 cM, and
ranged from 0.1 cM to 25 cM (Table 1; Fig. 1). The
polymorphic markers were further used for MAS in the process
of developing CSSLs and genotyping of the CSSL
The procedure used for CSSL development is shown
schematically in Fig. 2. Following the initial cross between the
9311 ‘female’ and the CWR274 ‘male’, var. 9311 was used
as the recurrent parent to backcross the hybrid three
times, obtaining BC3F1. Over 1,000 BC3F1 plants were
investigated using 230 SSR markers distributed across the
12 chromosomes, and 236 plants were selected for further
backcrossing. In the BC4F1 generation, 376 individuals
were subjected to a whole-genome survey using 313 SSR
markers. Fifty-eight plants with less than three substituted
segments from wild rice were selected and successively
self-crossed to produce CSSLs. Similarly, 65, 43, and 32
CSSLs from BC5F1, BC6F1, and BC7F1, were obtained,
respectively. Thus, a total set of 198 CSSLs lines was
Distribution, number, and length of substituted chromosome segments in CSSLs
The 198 CSSLs carried 412 homozygous introgressed
chromosome segments and 72 heterozygous segments; on
average each CSSL contained 2.16 wild rice segments
(Table 2, Fig. 3). The CSSLs accumulatively covered
84.9 % (1531 cM) of the wild rice genome marker base.
There was an uneven distribution among the 12
chromosomes, with most introgressed segments (77) found on
chromosome 2, and chromosome 11 having the least (19).
Seventy-two CSSLs carried single introgressed segments,
these involved 37 heterozygous segments and 35
homozygous ones; these were considered NILs of the recurrent
parent. However, the transmission and recombination of
O. rufipogon substituted segments varied for each
individual chromosome, with coverage for chromosome 7 being
only 61.6 %, whereas chromosomes 2, 3, 5, and 10 had full
Among the 198 CSSLs, sizes of the 484 substituted
segments ranged from 0.7 cM (on Chr. 9 of CSSL157) to
34.4 cM (on Chr. 5 of CSSL 91), with an average of
6.03 cM. Forty percent of substituted segments were
smaller than 5 cM, 40 % of substituted segments were
from 5 to 10 cM, 18 % ranged from 10 to 20 cM, and
2 % of segments were over 20 cM (Fig. 4).
Evaluation of phenotypic variation of quantitative traits in the CSSLs
Morphometric measurements of agronomic traits in the
198 CSSLs are presented in Table 3. The values of the
10 agronomic traits showed a large range of variation.
All of the traits were observed to have phenotypic
transgressive variation. Among the 10 investigated traits, seed
shattering and number of grains per panicle were the
most represented in the CSSLs, whereas days to heading
was the least represented trait. The correlations among 10
agronomic traits were shown in Additional file 1: Table S2.
In most cases, the correlation coefficients are significant or
highly significant. Additionally, some domestication-related
traits, such as lazy growth habit, red and long awn, spread
panicle, and black hull were observed in the CSSLs (data
QTLs analysis for the 10 agronomic traits was carried out
separately at both Nanjing and Sanya sites using
IciMapping software [
]. Forty-five QTLs were detected in the
CSSLs (Additional file 2: Table S3), with 25 significant
QTLs identified at both sites (Table 4, Fig. 1).
Days to heading
Three QTLs, located near RM485 and RM535 on Chr.2,
and RM590 on Chr.10 were associated with days to
heading. These loci showed an increasing effect on days to
Fig. 1 Genetic location of 313 polymorphic markers and distribution of 25 QTLs for 10 agronomic traits. Molecular markers are shown to the right of
chromosomes, and genetic locations (cM) of each marker shown to the left of chromosomes. The regions of 25 detected QTLs are shown using a
heading. Phenotypic variation explained by these three
QTLs ranged from 7.89 to 17.3 %.
Three QTLs associated with seed shattering were detected
at both sites. Two QTLs associated with increasing seed
shattering were located near markers RM7288 on Chr.2
and RM349 on Chr.4. A QTL near RM289 on Chr.5
showed a decreasing effect on seed shattering. The
phenotypic variation explained by these three QTLs ranged from
5.04 to 8.55 %.
RM280 on Chr.3; these showed an increasing effect on
plant height. A further QTL, near RM6318 on Chr.2, had
a decreasing effect on plant height. The phenotypic
variation explained by these four QTLs ranged from 5.20 to
Number of panicles per plant
One QTL associated with panicles per plant was detected
at both sites. This QTL was located near RM17308 on
Chr.4 and contributed a small positive effect, increasing
panicles per plant.
Four QTLs associated with plant height were detected at
both sites. The directions of their effects at both sites were
the same. Three QTLs derived from the wild rice were
located near markers RM128 and RM473 on Chr.1 and
Number of grains per panicle
One QTL located near RM184 on Chr.7 was associated
with number of grains per panicle. This QTL conferred
a negative effect, decreasing the number of grains per
Number of Average length
Number of Average length
Three QTLs associated with grain length were detected.
QTLs near RM467 on Chr.10 and RM519 on Chr.12
contributed an increasing effect on grain length, while the
QTL near RM15382 on Chr. 3 displayed a decreasing
effect. The phenotypic variation explained by these three
QTLs ranged from 6.07 to 15.3 %.
Four QTLs associated with grain width were detected at
both sites. The phenotypic variation explained by these
QTLs ranged from 6.08 to 19.0 %. Three QTLs, located
near RM514, RM544, and RM128 on chromosomes 3, 8,
and 1, respectively contributed a negative effect, while the
QTL near RM3467 on Chr. 3 contributed a positive effect.
Two QTLs were detected as controlling grain weight;
these were located near markers RM105 on Chr.9 and
RM188 on Chr.5. Both had a decreasing effect on grain
weight and the phenotypic variation explained by these
QTLs ranged from 8.07 to 11.9 %.
Length of flag leaf
Two QTLs were associated with flag leaf length. They
were located near RM280 on Chr.4 and RM17954 on
Chr.5. Both showed an increasing effect. The individual
locus explained 4.50-18.6 % of phenotypic variation.
Width of flag leaf
Two QTLs, located near RM206 on Chr.11 and RM128
on Chr.1, were associated with flag leaf width. Both
showed a decreasing effect. The phenotypic variation
explained by these QTLs ranged from 6.48 to 8.58 %.
SNP detection for mapping of the heading date and seed shattering QTLs
To both confirm SSR marker genotyping and for fine
mapping of QTLs associated with the heading date and
seed shattering, seven CSSLs were selected for further
SNP detection by whole-genome SNP chip scanning.
SNP genotyping was performed using an Illumina SNP
chip RiceSNP containing 9858 SNPs. Approximately
1500 SNPs were detected as polymorphic between the
two parents (data not shown). CSSL85, 102, and 167
have significant seed shattering and were detected for
seed shattering QTLs, while CSSL 53, 57, 73, and 172
have a significantly delayed heading phenotype, and thus
were detected for heading date QTLs. One of the seed
shattering QTLs, closest to marker RM349 on Chr.4,
was identified in all three seed shattering CSSLs (Fig. 5a).
Furthermore, one of the heading date QTLs, closest to
marker RM590 on Chr.10, was identified in all four
CSSLs with delayed heading. According to the positions
of SNP markers near RM349, and information from a
previous study [
], a sh4 gene was found near RM349
on Chr.4 (Additional file 3: Figure S5). qDTH10-1 was
identified in a 78-kb region in Chr.10 according to the
SNP markers and RM590 position (Fig. 5b); no gene has
been reported in this region. Therefore, qDTH10-1 is a
new QTL associated with the heading date.
Wild rice CSSL development and genome coverage
An excellent population of genetic materials is important
for a comprehensive understanding of quantitative traits.
CSSL have the potential to construct genome-wide genetic
stocks, to identify QTLs for functional genomics research,
and to discovering novel genes from newfound wild rice
accessions. So far, CSSLs derived from wild speices of
Oryza family including O. meridionalis, O. glumepatula,
O. rufipogon, and O. glaberrima have been constructed
]. Although the development of CSSLs for O.
rufipogon has previously been reported [
accessions should be included in such a genetic survey to
retrieve genes lost through domestication or evolution. In
this study, we developed a set of CSSLs derived from O.
rufipogon in the genetic background of an elite cultivated
rice. The donor parent CWR276 comes from the lowest
latitudes area of China, and is considered to be the unique
tropical O. rufipogon population in China. It has been
characterized with representative morphological
characteristics of Chinese common wild rice, including
resistance or tolerance to multiple biotic and abiotic stresses.
The primary objective of this study was to develop a wide
population of genetic materials for rice breeding and
The density of polymorphic markers throughout the
entire genome plays a vitally important role in the
development of CSSLs, the higher the marker density, the
lower the number of donor chromosomal segments lost.
Construction of CSSLs using MAS is well documented.
Ebitani et al. constructed and genotyped a set of CSSLs
using 129 RFLP markers [
]; Tian et al. used 126 SSR
]; Takai et al. used 140 SSR and 5 EST markers
]; Zhu et al. used 132 SSR markers [
]; Zhu et al.
used 260 SSR and sequence tagged site markers [
and Xu et al. used 254 markers, including SSR and InDel
]. This study made use of 313 polymorphic markers
fairly evenly distributed across the 12 rice chromosomes,
representing a higher density than previously reported.
Compared with re-sequencing-based high-throughput
methods, the molecular marker-based genotyping approach
is tedious and labor intensive. However, the CSSL
population developed using high-density molecular markers
provides starting material for advanced secondary
population construction and QTL analysis. MAS was started at
the BC3 generation, as some small chromosome segments
were missed in advanced backcrosses the final coverage of
the wild rice genome was about 85 % in the 198 CSSLs
population. Forty percent of the substituted segments were
smaller than 5 cM, and about 36 % of CSSLs had only one
substituted segment that could be considered as NILs. Data
demonstrated these CSSLs were better than the
aforementioned wild rice CSSLs on both lengths of introgressed
segments and amount of NILs. The uncovered regions in
this set of CSSLs might have occurred because MAS was
not performed until the BC3 generation, some biological
factors such as gametophyte, hybrid sterility and
headingdate genes might also be considered the cause. Tracing
back to an earlier generation, such as BC2, to fill the gaps
should make it possible for this CSSL set to cover the entire
wild rice genome.
CSSLs provided a platform for both rice breeding and
Although molecular tools and sequencing technique have
rapidly been developed, phenotyping remains the most
crucial and challenging factor in genetic analysis of
complex traits. The two parents used in this study have a
strong potential for heterosis. Thus, the CSSLs showed a
very large variation in all agronomic traits. Although wild
rice is generally inferior to cultivated rice in terms of yield
traits, previous reports demonstrated there are many
high-yielding QTLs in low-yielding wild rice [
13, 47, 48
Transgressive segregation of all yield traits was observed
in these CSSLs, especially for the number of panicles per
plant and grain length, which on average exceeded that of
recurrent parent 9311 at the two sites (Table 3). Cultivar
9311 has been planted on a large scale in China as it is an
elite variety. Some CSSLs in this stduy had a similar
genetic background as 9311, but their comprehensive
Width of flag leaf qWFL11-1 RM206
The value of PVE (Phenotypic variation explained by the QTL) and Add
(Estimated additive effect of the QTL) were from Nanjing site
characteristics were better than 9311 under different
environmental conditions. Therefore, they could be directly
used to develop new varieties, or as a parent to produce
new superior hybrids. These CSSLs could provide a useful
material population for cultivated rice breeding.
CSSLs can ben used to facilitate detecting and fine
mapping of QTLs by eliminating genetic background noise,
this would simplify the data analysis process and increase
the accuracy of results. In this study, CSSLs were used for
QTLs mapping of 10 agronomic traits, and 25 QTLs were
identified as present at both Nanjing and Sanya sites.
Some QTLs were either reported or contained previously
reported genes (Additional file 2: Table S3). SNP chip
scanning should prove an efficient tool for QTL
confirmation. The SNP chip used in this study was selected
from two parents indica and japonica [
], thus only
about 15 % of SNP sites were detected as polymorphic
between 9311 and CWR276. Two traits, days to heading
and seed shattering, which are considered to be the most
important traits involved in rice domestication, were
selected for further fine mapping using whole-genome
SNP chip scanning. The QTL qSH4-1 associated with seed
shattering was identified in Chr. 4 and a previously
reported gene sh4 has been found in this location [
The location of another QTL, qDTH10-1, associated with
the heading date was narrowed down to a 78-kb region on
Chr.10 (Fig. 5b); no previously reported gene is found in
this region. This QTL could be used for new heading date
gene searching. Generically, heading date often influences
many other agronomic traits, however, the qDTH10-1 did
not show pleiotropic effects to other agronomic traits
(Fig. 1 and Additional file 2: Table S3), this QTL might be
useful to alter heading date without influences other
agronomic traits in rice breeding. The results suggest the CSSLs
in this study will prove an efficient population for QTL
identification, and the use of different wild rice accessions
would lead to discovery of novel genes.
Plant breeding requires the combination of art and
science to improve the genetic basis of new crop varieties
so as to incorporate better agronomic and yield traits.
Systems of plant breeding using molecular MAS to
combine phenotype and genotype have entered a new era. The
development of wild rice CSSLs has provided a broad
platform for both wild rice genomic research and QTL
mapping. Novel genes found in wild rice using these CSSLs
could provide a new genetic resource for breakthroughs in
We successfully developed a wide population containing
198 CSSLs from wild rice in the genetic background of
elite indica cultivar 9311. The whole CSSL population
covered approximately 85 % of the wild rice genome with
an average substituted segment length of 6 cM. Each
CSSL contained an average of two introgressed segments.
Abundant QTLs associated with agronomic traits were
identified based on an evaluation of phenotypic variation
and genotyping of 313 SSR markers. Combined with a
SNP chip assay, a novel small QTL associated with the
heading date was fine mapped in the selected CSSL. The
CSSLs described in this study could prove a powerful tool
for large-scale gene discovery and provide an important
germplasm resource for rice breeding.
Development of CSSLs made use of the commercial elite
restorer indica cultivar 9311 as the recipient. 9311 is
characterized by its high yield, eating quality, and resistance
to multiple diseases. Chinese common wild rice accession
CWR276 (O. rufipogon) was collected from Sanya, Hainan
Province, and used as the donor parent. The ratoon was
collected from its original habitat and conserved in our
wild rice germplasm garden. The photo of two parents
was showed in S-Figure1.
Construction of CSSLs
The F1 plant derived from a cross between 9311 and
CWR276 was backcrossed to 9311 to produce 176 BC1F1
plants. BC1 plants were then backcrossed to 9311 two
times, without MAS, to produce a BC3F1 generation.
Whole-genome genotyping was performed in the BC3F1
generation using SSR markers distributed across the 12
rice chromosomes. One thousand BC3F1 plants were
surveyed by 230 SSR markers, and 236 plants selected for
backcrossing with 9311. According to SSR genotypes, the
BC4 generation was selfed or backcrossed consecutively to
generate BC4F5, or BC5F4, BC6F4, and BC7F4. Finally, 198
lines were used to construct the CSSLs.
DNA isolation and PCR
Genomic DNA was extracted from freshly frozen leaves
of individuals using the CTAB method described by
Rogers and Bendich [
]. Extracted DNA was stored in
ddH2O at −20 °C.
SSR marker primers were selected from dense rice
microsatellite maps, and synthesized in accordance with
sequences published by Ware et al. [
] or Temnykh
et al. [
]. Some markers were designed according to
information available from Gramene. DNA amplification
was performed using PCR with the following conditions:
95 °C for 5 min; 33 cycles of 94 °C for 30 s, 55 °C for
30 s, and 72 °C for 30 s; and a final cycle of 72 °C for
10 min. Reactions were carried out in 96-well PCR plates
in 25-μL volumes containing 1 μmol/L of each primer,
200 μmol/L of dNTPs, 5 ng of DNA template, 2 mmol/L
MgCl2, 2.5 μL 10× buffer, and 1 U of Taq polymerase
(Dong-Sheng Limited, Beijing,China). PCR products were
separated on 8 % polyacrylamide denaturing gels, and
bands visualized using the silver-staining protocol
described by Panaud et al. [
]. Some amplification products
were analyzed on 3.5 % agarose gels stained with ethidium
bromide and photographed using a UVP system.
Determination of length of substituted segments in CSSLs
A genetic linkage map was built to estimate marker
distances with reference to Temnykh et al. and Ware
et al. [
]. The lengths of substituted chromosome
segments in CSSLs were determined based on graphical
]. Construction of graphical genotypes
and calculation of percentage of the total genome in
each CSSL line were performed using GGT software [
A chromosome segment flanked by two markers of donor
type (DD) was considered 100 % donor type; a
chromosome segment flanked by two markers of recipient type
(RR) was considered 0 % donor type; and a chromosome
segment flanked by one marker of donor type and one
marker of recipient type (DR) was considered 50 % donor
type. The length of DD plus the length of two half DRs
were considered the estimated length of a substituted
Measurement of agronomic traits
The phenotypic evaluation of 198 CSSLs was performed
under natural conditions at the experimental stations of
the Chinese Academy of Agricultural Science in summer
(Nanjing, China; N32°03′, E118°47′) and winter (Sanya,
China; N18°15′, E109°30′) 2014. The field experiment
was designed in randomized plots with two replications.
For each CSSL and the parents, 60 plants were planted in
five rows, with 20 cm between plants within each row,
and 30 cm between rows. Fifteen plants in the center of
each plot were selected for the collection of data. Days to
heading was deemed the number of days from sowing to
heading of plants. Seed shattering was evaluated using the
method described by Han and Wei [
]. Plant height,
number of panicles per plant, and length and width of the
flag leaf were the mean value of 20 randomly selected
individuals. Number of grains per panicle, 1000-grain
weight, and grain length and width were detected by an
automatic seed investigation machine (Wanshen, Shenzhen,
China). Differences between CSSLs and 9311 were
determined using a t-test.
CSSL-based QTL mapping and SNP assay
The association between phenotype and marker genotype
was investigated by single-point analysis using Map
Manager QTXb17 [
] and SPSS 13.0 (SPSS Inc., Chicago, IL,
USA). The statistical threshold for single-point analysis
was P < 0.01. Genotyping of the seven selected CSSLs and
the two parents was performed using an Illumina SNP
chip RiceSNP containing 9858 SNPs at the Shenzhen
Academy of Crop Molecular Breeding, China. The mean
distance between adjacent SNP markers was 54.5 kb.
Chromosomal positions of SNPs were determined
according to the 9311 reference genome.
Additional file 1: Table S2. Correlation coefficients among the ten
agronomic traits. (DOCX 16 kb)
Additional file 2 Table S3. QTLs for ten agronomic traits detected in
198 CSSLs at Nanjing and Sanya sites. Table S1: Sequence information of
SSR primers used in this study. Figure S1: Photographs of Oryza sativa
spp. indica cultivar 9311 and wild rice CWR276. Figure S2: Nucleotide (A)
and amino acid (B) alignments of the sh4 gene between wild rice and
9311. Wild rice alleles were cloned from CSSL85, 102, 167, and the donor
parent BC276. The red rectangle indicates a substitution (K in the protein
of the wild rice allele). (DOCX 36 kb)
This research was supported by the Agricultural Science and Technology
Innovation Program of the Chinese Academy of Agricultural Science, and by
a grant from the National Natural Science Foundation of China (No.
31471471) to Weihua Qiao. We are grateful to Dr. Peng Zhang (Plant Breeding
Institute, The University of Sydney, Australia) for valuable suggestions and a review
of this paper.
WQ performed the experiments and wrote the manuscript. LQ analysed
the phenotypic data. ZC provided valuable suggestions for the experiment. JL, YS,
JR, and LS all contributed to PCR genotyping. XZ performed QTL analysis. QY
designed the experiment. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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