Nucleotide Polymorphisms and Haplotype Diversity of RTCS Gene in China Elite Maize Inbred Lines
et al. (2013) Nucleotide Polymorphisms and Haplotype Diversity of RTCS Gene in China Elite Maize Inbred
Lines. PLoS ONE 8(2): e56495. doi:10.1371/journal.pone.0056495
Nucleotide Polymorphisms and Haplotype Diversity of RTCS Gene in China Elite Maize Inbred Lines
Enying Zhang 0
Zefeng Yang 0
Yifan Wang 0
Yunyun Hu 0
Xiyun Song 0
Chenwu Xu 0
Gregory Tranah, San Francisco Coordinating Center, United States of America
0 1 Key Laboratory of Crop Genetics and Physiology of Jiangsu Province, Key Laboratory of Plant Functional Genomics of the Ministry of Education, College of Agriculture, Yangzhou University , Yangzhou , China , 2 College of Agronomy and Plant Protection, Qingdao Agricultural University , Qingdao , China
The maize RTCS gene, encoding a LOB domain transcription factor, plays important roles in the initiation of embryonic seminal and postembryonic shoot-borne root. In this study, the genomic sequences of this gene in 73 China elite inbred lines, including 63 lines from 5 temperate heteroric groups and 10 tropic germplasms, were obtained, and the nucleotide polymorphisms and haplotype diversity were detected. A total of 63 sequence variants, including 44 SNPs and 19 indels, were identified at this locus, and most of them were found to be located in the regions of UTR and intron. The coding region of this gene in all tested inbred lines carried 14 haplotypes, which encoding 7 deferring RTCS proteins. Analysis of the polymorphism sites revealed that at least 6 recombination events have occurred. Among all 6 groups tested, only the P heterotic group had a much lower nucleotide diversity than the whole set, and selection analysis also revealed that only this group was under strong negative selection. However, the set of Huangzaosi and its derived lines possessed a higher nucleotide diversity than the whole set, and no selection signal were identified.
. These authors contributed equally to this work.
In the past, fundamental researches on increasing shoot biomass
and seed yield attracted most attentions of the crop scientists, and
the relevance of the root system for food production has often been
overlooked [1,2]. However, a healthy and well-developed root
stock architecture is especially important for the developing of
plant, because it is the organ absorbing water and inorganic
nutrients, in addition to anchoring of the plant body to the ground
[1,3]. Maize (Zea mays L.), one of the most widely grown grain crop
in the world, possesses a unique and complex root stock
architecture composed of embryonic and postembryonic roots
[4,5]. The embryonic roots, defined by the primary root and
a variable number of seminal roots, play important roles for early
vigor of the maize seedlings. However, at the postembryonic stage,
shoot-borne system forms the major backbone of the adult stock
Recently, several genes controlling the development of maize
shoot-borne roots, lateral roots, and root hairs have been isolated
[7,8,9]. Among them, the gene RTCS (rootless concerning crown
and seminal roots) was demonstrated to play a central role in the
auxin-mediated initiation of seminal and shoot-borne roots in
maize [5,9] and the mutant of this gene was impaired in the
formation of these roots. Map-based cloning revealed that this
gene was located in the short arm of chromosome 1, and encoded
a LOB domain protein. Sequence analysis illustrated the maize
RTCS gene was composed of 2 exons, separated by a 96-bp intron,
and its protein product contained 244 amino acid residues. The
maize RTCS gene is preferentially expressed in root tissues  and
its protein product showed typical features of a transcription factor
including nuclear localization, DNA-binding and downstream
gene activation .
Although the favorable root architecture plays critically
important roles for the development of plant, root architecture
was rarely considered as a selection criterion or traits for maize
improvement, mainly because of the practical difficulties with
their evaluation under field conditions . Recent researches in
maize revealed that changes in root architecture can strongly
affect the yield . Because increasing crop yield through
improvement of plant type and growing use of fertilizer has
reached a maximum, much attention should be focused on
improving the root system . Researches on the sequence
polymorphisms of key genes are important not only for crop
improvement but also for efficient management and
conservation of plant genetic resources [11,12,13]. However, rare
researches in genetic variants in the DNA sequence have
focused on the genes controlling the development of plant roots.
In addition, the genetic diversity at the DNA level of maize
RTCS gene is not known at present. Therefore, we detected
nucleotide polymorphisms, haplotype diversity and evolutionary
factors of the gene RTCS by direct sequencing 73 China elite
inbred lines, including the lines from 5 temperate heterotic
groups and some tropic germplasms.
Materials and Methods Plant Materials
A total of 73 China maize elite inbred lines were used in this
study (Table 1). Among these inbred lines, 63 temperate and 10
tropic germplasms were used. The 63 temperate inbred lines were
from 5 heterotic groups, including 15 from Tangsipingtou, 9 from
Lvdahonggu, 11 from Lancaster, 13 from Reid, and 14 from P
DNA-extraction and Sequencing RTCS Gene
Genomic DNA of was extracted from young leaves of the tested
inbred lines at the seedling stage using CTAB (cetyl trimethyl
ammonium bromide) method based on the modified protocol .
The sequences of the RTCS gene in 73 inbred lines was sequenced
by BGI Life Tech Co., Ltd. using the target sequence capture
sequencing technology on the NimbleGen platform .
Multiple sequence alignment was performed using Clustal X
 and was further edited manually. The software DNASP 5.0
[17,18] was used to analyze sequence nucleotide polymorphism
and allelic diversities. Two parameters of nucleotide diversity, p
and h were estimated. Where p is the average number of
nucleotide differences per site between any two DNA sequences,
and h is derived from the total number of segregating sites and
corrected for sampling size. Tajimas D  and Fu and Lis 
statistical tests were used to test the evidence of neutral evolution
within each group and each defined region. The minimum
number of recombination events  was estimated in the period
of evolution of RTCS gene among these inbred lines.
Nucleotide Diversity and Selection of RTCS Gene in China
Elite Inbred Lines
Sequence polymorphisms were detected among 73 maize
inbred lines across 1279 bp of sequence, which covers a 167 bp
59 untranslated region (UTR), a 735 bp coding region, a 104 bp
intron region, and a 273 bp 39 UTR. Nucleotide substitutions and
indels at the RTCS locus were identified, and the results were
summarized in Table 2. From the putative genomic sequences of
the 73 maize inbred lines, a total of 44 SNP sites were identified,
and among them, 16 and 28 sites belonged to singleton variable
sites and parsimony informative sites, respectively. In addition,
a total of 19 indel events covering 90 sites were identified in the
genomic sequences (Table S1). For all the 73 inbred lines, the
overall nucleotide diversity (p) of RTCS locus was 0.00666. Among
4 regions of the gene RTCS, the coding region showed much lower
nucleotide polymorphism than others, while the intron region had
the highest frequency of all sequence variants. This might be
caused by the variant of indels, because this region had the highest
aThe inbred lines of Huangzaosi and its derived lines.
frequency of indels per bp. However, the frequency of nucleotide
substitutions in 59UTR was higher than other regions. When we
used the sliding window of 100 bp under a step size of 25 bp, the
result revealed each region of the RTCS sequence possessed high
frequency of polymorphic sites (Figure 1). The highest nucleotide
diversity was within 1159 bp in 59-UTR with p~0:02296, while
the lowest value (p~0) was found in regions of exon-1 and exon-2,
respectively. The observed distribution of SNP sites and indel sites
was found to be significantly different (for SNP, x2~11:147,
df ~3, Pv0:05; for indel, x2~19:923, Pv0:01) from an
expected even distribution across the four defined regions
(Table 3). The uneven distribution of polymorphisms might be
particularly due to the low frequency of variants in coding region.
The Tajimas D test is a widely used test to identify sequences
which do not fit the neutral theory model at equilibrium between
mutation and genetic drift . All the values of Tajimas D in the
present study were not statistically significant, illustrating no
significant selection existed in the entire RTCS sequences. In
addition, Fu and Lis D* and F* were also not significant in almost
all regions except for intron. Although these results could not reject
the hypothesis of mutation drift equilibrium, a lack of footprint of
positive selection in most regions of RTCS was suggested.
Total length of amplicons (bp)
Number of all sequence variants (SNPs and indels)
Frequency of all sequence variants
Number of nucleotide substitutions (bp)
Frequency of polymorphic sites per bp
Number of indels
Number of indel sites
Average indel length
Frequency of indels per bp
Table 3. Nucleotide and allele diversities of RTCS gene by
analyzing 73 maize inbred lines.
Entire region 0.01726 0.00436 0.00860 0.01547 0.00395 0.01342
0.31120 0.29566 20.85026
1.38655 21.63477 22.51473*
1.20720 21.13533 22.32168*
*indicates the significance at P,0.05 level.
Nucleotide Diversity and Selection in Each Heterotic
The inbred lines used in this study can be classified into 6
groups, including 5 temperate heterotic groups and the group of
tropic germplasms. We also tested the nucleotide diversity of both
entire region and coding region of RTCS sequences for each
group, and the result revealed that the nucleotide diversities of 5
groups were higher than or very near to the whole set (Table 4).
The tropic group possessed the highest value nucleotide diversity,
and its haplotype diversity (Hd) is 1 for the entire region of RTCS,
suggesting each inbred line carried a haplotype. Only the P
heterotic group had much lower nucleotide and haplotype
diversities than the whole set both for the entire region and
coding region. This result suggested that the P group was more
conserved in RTCS locus than other groups. In addition, we also
noticed that the statistics for Tajimas D, Fu and Lis D* and F*
were all statistically significant in P group. This result illustrated
that the RTCS gene in P group were not evolved neutrally, and
also suggesting that selection might only acted the evolution of
RTCS gene in this group.
Huangzaosi is believed to be the representative line of the
Tangsipingtou heterotic group and was used as a key maize inbred
line in China . Among all inbred lines used in this study, at
least 11 lines were Huangzaosi and its derived lines. We also tested
the sequence polymorphisms of RTCS gene in Huangzaosi and its
derived lines. The result revealed that the nucleotide diversity (p) is
higher than the whole set, illustrating that there were abundant
nucleotide variations in Huangzaosi and its derived lines. In
addition, none of the statistics for Tajimas D, Fu and Lis D* and
F* were statistically significant for Huangzaosi and its derived
lines, suggesting that selection was not included in the RTCS locus
of this population.
Based on the whole length of the RTCS gene sequenced in 73
maize inbred lines, a total of 34 haplotypes were detected with a Hd
equal to 0.8992 (Table S2). The inbred lines were unbalancedly
distributed in these haplotypes. Among the haplotypes identified in
this analysis, 26 contained only one inbred line. The most frequent
haplotype was Hap_8, which contained 21 inbred lines. It should
be mentioned that nearly all the inbred lines in P group belonged
to this haplotype except for 91158, which was assigned to the
In the coding region of the gene RTCS, 16 sequence variants,
including 2 indels and 14 SNPs, were detected. Both of the 2 indels
contained 3 nucleotide acids, respectively, and this can not result
in frameshift of the codons. When we used the coding sequences to
identify the hapotype diversity, a total of 14 haplotypes were
identified for these 73 inbred lines (Table S3), and the hapotype
diversity was 0.7705. Among the haplotypes identified according
to CDS, 9 contained only one inbred line. The most frequent CDS
haplotype was CDS_Hap_5, which contained 29 inbred lines from
all 6 groups. In addition, CDS_Hap_7 and CDS_Hap_8 were also
haplotypes with high frequency, and only no inbred lines in P
group and tropic lines carried them, respectively.
Among 14 SNPs in the coding region, 8 were synonymous sites,
and the other 6 were nonsynonymous sites. The nonsynonymous
sites and the indels will lead to the changes of protein sequences.
When we translated the CDS into amino acid sequences, 7 types of
RTCS protein sequences were found to be encoded by these inbred
lines (Figure 2). Haplotypes CDS_Hap_5/6/7/14 encoded the
most frequent type of RTCS protein, and contributed to more than
half of all the inbred lines (42 out of 73). The variation of RTCS
protein sequences was the result of combinations of 6
nonsynonymous mutations and 2 indels in the coding region. All of the variants
at the protein level were found to be outside the LOB domain region
(Figure 2), and in other words, the region of LOB domain of RTCS
protein showed 100% identify in all the tested inbred lines.
Evidence of Recombination
The polymorphic sites in the entire RTCS sequence were used to
detect the evidence of recombination. The patterns of the
polymorphisms identified in inbred lines surveyed in this study
indicated the history of recombination at RTCS gene, which
contributed to the haplotype diversity. Under the algorithm of
Hudson and Kaplan , at least 6 recombination events were
found to be responsible to the polymorphism of RTCS gene. The
recombination events were detected in the informative sites of every
region, and they were found in the positions between 59-UTR and
exon-1 (82353), between exon-1 and intron (398468), between
intron and exon-2 (499553), the exon-2 (597667), between exon-2
and 39-UTR (8791038), and the 39UTR (10381154), respectively.
The consequences of recombination events are evident in the
pattern of polymorphisms when compared the sequence of one
haplotype with others. For example, the 59 UTR sequence of the
Hap_1 was the same as that of Hap_2. However, across the coding
region and intron region, there were 4 variants between them,
including 3 SNP and 1 indel covering 3 sites. The 39UTR region of
Hap_1 was found to be again virtually identical to Hap_2. This
result suggested that the RTCS sequence in Hap_2 has resulted from
at least two recombination events in the past relative to Hap_1.
The abundant genetic variations are the foundation for crop
improvement. The analysis of the genetic diversity of plant
variants is critical for understanding the genetic background of
phenotypic variation, and in turn will provide great help for crop
improvement . In this study, we detected the nucleotide
polymorphisms and the haplotype diversity of the gene RTCS, an
important regulator for the developing of roots, in 73 China elite
maize inbred lines. The identification of nucleotide variations
exerting functional effects, especially those causing changes of
amino acid composition, is the primary focus of association
mapping . Although most variants were found to be located in
the non-coding region, the SNP sites and indels in the coding
region also classified the tested inbred lines into 14 haplotypes. In
addition, a total 7 deferring RTCS proteins were encoded by this
gene in all the tested inbred lines. The nucleotide polymorphisms
of RTCS gene in this study would be helpful in identifying alleles
for further genetic analysis, and might also provide foundation for
Heterotic groups are of primary importance in hybrid breeding.
Crosses between inbred lines from different heterotic groups
generally result in vigorous F1 hybrids with significantly more
heterosis than F1 hybrids from inbred lines within the same
heterotic group . Heterotic groups are created by plant
breeders to classify inbred lines, and can be progressively
improved by reciprocal recurrent selection . Although the
classification of inbred lines into heterotic groups was based on
their general combining ability (GCA) and specific combining
ability (SCA) effects, the inbred lines within one heterotic group
were generally believed to possess lower genetic divergence than
those between different groups. Thus, molecular data, especially
SSR molecular markers, was thought to be the efficient method in
assigning inbred lines to specific heterotic groups [24,26,27,28,29].
The nucleotide polymorphisms of the RTCS locus were
investigated in 73 elite inbred lines from different heterotic groups.
The results revealed that sequence variants within each group
were higher or very near to those of the whole set except for P
group for both the entire region and the coding sequences.
Because breeders mainly focused on increasing shoot biomass and
seed yield in maize improvement in the past, the relevance of the
root system for crop improvement has often overlooked [1,2]. The
abundant variants within one heterotic group might be the result
of overlook in the selection by breeders, although this gene plays
important roles in formation of seminal and shoot-borne roots.
The purpose of the selection test is to distinguish between
a DNA sequence evolving randomly (neutrally) and one evolving
under a non-random process, including directional selection or
balancing selection, demographic expansion or contraction,
genetic hitchhiking, or introgression . The randomly evolving
mutations are called neutral, while mutations under selection
are non-neutral. In this study, we performed selective analysis
for each heterotic group, and the results revealed that only P group
was influenced by strong negative selection. Other groups have not
influenced by selection, suggesting that a bottleneck for the usage
of this locus in breeding in these heterotic groups. In addition, the
haplotype detection also found that P group has a lower value of
haplotype diversity than others. This might be the result of that
this group was used in China for a short period after 1980s, and
most of the inbred lines of this group in China were selected from
the pioneer hybrid P78599 . The consistency of the genetic
background for the inbred lines in P group resulted in the low
frequency of nucleotide variants. Huangzaosi is the most used
maize inbred line in China, and more than 42 hybrids and 70
derived lines used this inbred line since it was first bred in 1971
. 11 inbred lines of Huangzaosi and its derived lines were used
to test the nucleotide polymorphisms. The results revealed that this
set has a higher nucleotide diversity than the whole set, and no
selection was identified in this set. These result suggested that the
RTCS locus was not adopted when the breeders used Huangzaosi
as a key inbred line.
Table S1 The positions of nucleotide polymorphism of
RTCS gene among 73 inbred lines.
Table S2 The distribution of haplotypes of RTCS gene
in 73 inbred lines using the entire sequences.
Table S3 The distribution of haplotypes of RTCS gene
in 73 inbred lines using the coding sequences.
Conceived and designed the experiments: ZY CX. Performed the
experiments: EZ YW YH. Analyzed the data: ZY EZ YW. Contributed
reagents/materials/analysis tools: EZ ZY XS. Wrote the paper: ZY EZ.
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