Development of AhMITE1 markers through genome-wide analysis in peanut (Arachis hypogaea L.)
Gayathri et al. BMC Res Notes
Development of AhMITE1 markers through genome-wide analysis in peanut (Arachis hypogaea L.)
M. Gayathri 0
R. K. Varshney
M. K. Pandey
R. S. Bhat 0
0 Department of Biotechnology, University of Agricultural Sciences , Dharwad 580 005 , India
Objective: In peanut, the DNA polymorphism is very low despite enormous phenotypic variations. This limits the use of genomics-assisted breeding to enhance peanut productivity. This study aimed to develop and validate new AhMITE1 and cleaved amplified polymorphic sequences (CAPS) markers. Results: In total, 2957 new AhMITE1 markers were developed in addition to identifying 465 already reported markers from the whole genome re-sequencing data (WGRS) of 33 diverse genotypes of peanut. The B sub-genome (1620) showed more number of markers than the A sub-genome (1337). Distribution also varied among the chromosomes of both the sub-genomes. Further, 52.6% of the markers were from genic regions; where 31.0% were from intronic regions and 5.2% were from exonic regions. Of the 343 randomly selected markers, 82.2% showed amplification validation, with up to 35.5% polymorphism. From the SNPs on the A03, B01, B02 and B03 chromosomes, 11,730 snip-SNPs (potential CAPS sites) were identified, and 500 CAPS markers were developed from chromosome A03. Of these markers, 30.0% showed validation and high polymorphism. This study demonstrated the potential of the WGRS data to develop AhMITE1 and CAPS markers, which showed high level of validation and polymorphism. These marker resources will be useful for various genetic studies and mapping in peanut.
Peanut; Diverse genotypes; WGRS; AhMITE1 markers; CAPS markers; Validation and polymorphism
High resolution trait mapping in peanut (Arachis
hypogaea L. 2n = 4x = 40) demands a considerably large
number of evenly distributed genome-wide markers to
identify marker-trait associations. The fact that the
genotypic polymorphism is very limited despite enormous
phenotypic differences among peanut genotypes signifies
the requirement for a large number of markers.
Different types of markers have been developed and employed
for diversity analysis, DNA finger-printing, trait
mapping and genomics-assisted breeding (GAB). The
prominent markers were RFLP, AFLP, DAF, SSR, DArT etc.
These DNA markers and other protein markers showed
low polymorphism among the peanut genotypes [
Though single nucleotide polymorphism (SNP) markers
are abundant and highly polymorphic in different
systems, they showed low polymorphism (14.0%) in
cultivated peanut [
Transposons are widely distributed in genomes, and
their polymorphic insertions allowed development of
transposon-based markers [
]. Both class I and class II
transposon-based markers have been developed and used
for various genetic analysis and mapping [
peanut, use of DNA transposon markers was proposed [
and one such marker was developed to track the activity
of A. hypogaea miniature inverted-repeat transposable
element (AhMITE1) to associate its transposition with
high-frequency origin of late leaf spot disease resistant
], and differentiation of two subspecies [
Subsequently, 1039 AhMITE1 markers were developed
], and used for mapping [
Use of diverse genotypes including the genetically
unstable peanut mutants which show hyperactivity of
AhMITE1 for marker discovery might detect a large
number of AhMITE1 insertion polymorphic sites (AIPs),
which could be employed to develop new markers. In
the past, transposon markers were developed using
transposon display [
] and in silico analysis [
]. But, analysis
of whole genome re-sequencing (WGRS) data from a
large number of diverse genotypes is expected to
capture all AIPs when the short reads are analyzed using
the computational method polymorphic TEs and their
movement detection (PTEMD) [
] for the de novo
discovery of AIPs. This study reports the development of
new AhMITE1 markers using diverse genotypes, and
their validation using the parents of various mapping
populations and backcross populations. SNPs discovered
from the WGRS data were also used to develop cleaved
amplified polymorphic sequences (CAPS) markers from
selected chromosomes (A03, B01, B02 and B03),
harboring quantitative trait loci (QTL) for the important
agronomical and productivity traits [
15, 24, 25
A total of 33 genotypes were employed for AhMITE1
marker development. Details on the genotypes [
] used for WGRS are given in Additional file 1: Table
S1. WGRS reads were generated using Illumina HiSeq
2000 for six genotypes and obtained from public DNA
sequence databases for the remaining 27 genotypes
(DRA004503–DRA004506 and SRA459965) (https://
www.ncbi.nlm.nih.gov/pubmed/27902796 and https://
file 1: Table S1), and used to detect AIPs using PTEMD
] without replication since the results were subjected
for confirmation (validation) using wet-lab experiment
through PCR, and detection in multiple genotypes to
support the results. The sequences flanking the AIPs
were retrieved, and primers were designed using default
parameters of BatchPrimer3 [
]. AhMITE1 markers
were validated by checking the amplicons from DER,
VL 1, 110 and 110(S) for the expected size. The seeds of
these genotypes were collected from the Department of
Genetics and Plant Breeding, University of Agricultural
Sciences, Dharwad, India. DNA was isolated from the
young leaves of the plants following the modified cetyl
trimethyl ammonium bromide (CTAB) method [
cells were lysed with CTAB buffer, and the debris were
removed by centrifugation. Proteins were removed from
the extract by phenol:chloroform extraction and the
RNA was removed by RNase treatment. The DNA was
washed with ethanol and finally dissolved in Tris–EDTA
(TE) buffer. Polymerase chain reaction (PCR) for the
AhMITE1 markers was carried out in a reaction volume
of 10 µl with the standard ingredients and PCR
profile (Additional file 2: Table S2a and S2b) [
eppendorf Mastercycler® pro. The PCR products were
resolved on 2% agarose gel. The markers amplifying the
expected product, depending on the presence or absence
of AhMITE1 at those marker loci, were considered to be
validated. The validated markers were checked for
polymorphic information content (PIC) using PowerMarker
V3.25 . For this, additional ten genotypes constituting
the recombinant inbred line (RIL) populations [
backcross populations [
] were employed. The seeds
of these genotypes were collected from the Department
of Genetics and Plant Breeding, University of
Agricultural Sciences, Dharwad, India.
Single nucleotide polymorphism identification from
the WGRS data was performed as described earlier [
A 1001 bp sequence was obtained for each SNP (500 bp
on left and right), and they were analyzed for snip-SNPs
(SNP sites which modify restriction enzyme recognition
sites) using CLC Sequence Viewer 7 (CLC Bio: http://
www.clcbio.com) for 25 restriction enzymes.
Primers were designed for those sequences which contained
snip-SNP using GeneTool Lite [
]. Preference was
given to those primers which could amplify 200–900 bp
amplicons, and generate restriction fragments of at least
Cleaved amplified polymorphic sequences
markers were validated by PCR amplification and
restriction digestion with the respective restriction enzyme
(Additional file 2: Tables S2c–S2e and Additional file 7:
Table S6). A reaction volume of 12 µl containing
Emerald Amp® GT PCR Master Mix (Catalog No. RR310A,
Clontech), 5 pmol of each primer and 50 ng of genomic
DNA was amplified, and used for restriction digestion.
The restriction fragments were separated on 2% agarose
gel, and checked for the products of expected size
(Additional file 7: Table S6). Those markers producing the PCR
and restriction fragments of expected size were
considered to be validated. PIC was calculated for the CAPS
markers as described for AhMITE1 markers.
Results and discussion
Large copy number [
], enormous genome-wide
insertion variation  and association with genes to
alter the function makes AhMITE1 a target for marker
]. A total of 3546 AIPs were identified
from a total reads of 9.9 billion across 33 genotypes, of
which 3081 were new and 465 were already reported [
]. This high success rate of marker discovery could
be attributed to the diverse genotypes and the software
(PTEMD) used in this study. Primers could be designed
for 2957 AIPs to amplify 100–405 bp amplicons
depicting the presence or absence of AhMITE1 at each marker
locus. Genotype-specific alleles were observed for all
the genotypes at varying number of markers (Additional
file 3: Table S3). At least two genotypes showed the same
type of allele at 1342 marker loci.
B sub-genome (1620) had marginally more number of
AhMITE1 markers than the A sub-genome (1337). An
unequal distribution of markers was observed across
the chromosomes of both the sub-genomes (Table 1). A
general correlation was observed between the number of
markers and the length of the chromosome [
]. In the A
sub-genome, the number of markers varied from 84 (A02
chromosome) to 210 (A03 chromosome); while it ranged
from 124 (B02 chromosome) to 269 (B03 chromosome)
in the B sub-genome. The recent efforts on sequencing
of the diploid progenitors of peanut, Arachis
duranensis (A genome) and Arachis ipaensis (B genome) showed
that transposable elements occupy larger space (68.5%) in
the B genome than in the A genome (61.7%), and DNA
transposons make about 10% of both A and B genome
]. Unequal distribution of DNA transposons was also
observed in rice [
], Brassica [
] and foxtail millet [
Analyzing the genomic location of these 2957
markers revealed that 1555 were genic and 1402 were
intergenic. A maximum of 562 (36.1%) marker loci had
AhMITE1 insertion at upstream regions (within 1 kb)
followed by 482 (31.0%) in intronic, 250 in downstream
regions (within 1 kb), 180 (11.6%) in UTRs and 81 (5.1%)
in exonic regions. Insertion of MITE in the genic region
as well as intergenic region is known to affect the gene
]. Thus, the AhMITE1 markers developed
in this study could have functional role as well. A sample
of 343 markers (Additional file 4: Table S4) was employed
for validation, and as high as 282 markers produced the
amplicons of expected size (Fig. 1) in all the four
genotypes (DER, VL 1, 110 and 110(S), indicating 82.2%
The validated markers (282) were tested for their PIC
using additional ten genotypes of cultivated peanut.
The PIC ranged from zero to 0.375 with an average of
0.155. In total, 221 and 61 markers were classified as low
(≤ 0.25) and moderate (0.26–0.50) for PIC. A maximum
of 35.5% polymorphism was observed between VL 1 and
110 (Table 2, Additional file 5: Figure S1), followed by
26.2% (TMV 2 and TMV 2-NLM), 23.1% (TMV 2 and
ICGV 86699), 22.3% (TMV 2 and ICGV 99005; TG 26
and GPBD 4) and 16.7% (TAG 24 and GPBD 4). High rate
of TE marker validation was also reported from foxtail
millet (Setaria italica) [
] and Caenorhabditis elegans
In total, 5,36,072 SNPs were identified when the WGRS
data from four peanut genotypes [DER, VL 1, 110 and
110(s)] were compared to the reference genomes [
Considering the mapped QTL for resistance to bacterial
wilt, late leaf spot and rust, and other important
productivity traits [
15, 24, 25
], the SNPs on A03, B01, B02 and
B03 were selected for identifying the snip-SNPs.
Screening of 64,416 SNPs from these four chromosomes
identified 11,730 (potential CAPS sites) for 25 restriction
enzymes (Additional file 6: Table S5). No significant
differences were found between the chromosomes for the
snip-SNPs. Further, 500 snip-SNPs on A03 chromosome
were used to develop CAPS markers. Currently, only two
CAPS markers are available in peanut [
]. They were
developed to detect specific mutations in AhFAD2A and
AhFAD2B leading to high oleic acid content [
Of the 500 CAPS markers identified, 30 were checked
for PCR amplification and restriction digestion
a out of 282 markers screened
(Additional file 7: Table S6). Twenty markers showed
PCR amplification, and 10 amplicons showed restriction
digestion; of which nine showed restriction fragments
of expected size (Additional file 8: Figure S2), and one
(CAPS0100) failed to show the fragments of expected
size with the enzyme BstKTI, indicating 30.0% validation
for CAPS markers. The CAPS sites for AluI and BamHI
showed maximum validation (100%) followed by AseI
with 57.1% validation. On the other hand, CAPS for AclI,
BglII and BstKTI showed no validation.
Polymorphic information content was calculated for
the nine markers showing the expected PCR product and
restriction products. PIC ranged from 0.195 (CAPS0072)
to 0.305 (CAPS0002, CAPS0043, CAPS0047, CAPS0050,
CAPS0053, CAPS0054, CAPS0058 and CAPS0059) with
a mean PIC of 0.293 (Additional file 7: Table S6). Parents
of the RILs and backcross populations like TAG 24 versus
GPBD 4, JL 24 versus GPBD 4, TMV 2 versus GPBD 4,
TMV 2 versus ICGV 86699, TMV 2 versus ICGV 99005,
TMV 2 versus IL 1 and TMV 2 versus IL 2 showed the
same level of polymorphism (88.9%), whereas VL 1 versus
110 failed to show any polymorphism for these markers.
The number of CAPS markers was more as compared
to AhMITE1 markers in peanut genome. However,
different families of class I and class II elements can be
considered to develop more AhMITE1 markers. Handling of
AhMITE1 markers in the laboratory is easy and requires
fewer resources. The transposition of the element from a
“donor” site can be validated by sequencing the
emptysite-related PCR, and searching for footprints (duplicated
regions). Thus, AhMITE1 markers can give an indication
not only of the genetic divergence that was caused by
AhMITE1 transposition, but also of the history of
transposition in each species [
]. Further, AhMITE1 markers
offer a DNA tag (AhMITE1) for gene discovery and
In this study, a large number of AhMITE1 and CAPS
markers were developed in peanut, where marker
polymorphism is a major limitation. In future,
retrotransposons and other DNA transposons can also be
considered for marker development. Similarly,
snipSNPs can be identified for the whole genome for the
development of CAPS markers. The major limitation
could be the fact that these markers are applicable only
for peanut. Currently, the new AhMITE1 markers are
being extensively used for trait mapping [
backcross breeding [
] to develop foliar disease
resistant genotypes in our laboratory. It is necessary to
test more number of CAPS markers to assess their true
rate of polymorphism.
Additional file 1: Table S1. Details on the genotypes used for marker
Additional file 2: Table S2a. PCR components for AhMITE1 marker assay.
Table S2b. PCR temperature profile used for AhMITE1 markers. Table S2c.
PCR components for CAPS marker assay. Table S2d. PCR temperature
profile used for CAPS markers. Table S2e. Restriction digestion
components for CAPS assay.
Additional file 3: Table S3. Genotype-specific AhMITE1 markers.
Additional file 4. Details on the newly developed AhMITE1 markers.
Additional file 5: Figure S1. Polymorphism survey for AhTE1131 among
the parents of RIL and backcross populations of peanut. [M: 100 bp ladder,
1: DER, 2: VL 1, 3: 110, 4: 110(S), 5: TAG 24, 6: GPBD 4, 7: JL 24, 8: TMV 2, 9:
ICGV 86699, 10: ICGV 99005, 11: IL 1 and 12: IL 2].
Additional file 6. Details on the potential CAPS sites for 25 restriction
enzymes on A03, B01, B02 and B03 chromosomes of peanut.
Additional file 7. Details on the newly developed CAPS markers.
Additional file 8: Figure S2. Validation of selected CAPS markers in
peanut. [M: 100 bp ladder, 1: DER, 2: VL 1, 3: 110 and 4: 110(S)].
AhMITE1: Arachis hypogaea miniature inverted-repeat transposable element
1; CAPS: cleaved amplified polymorphic sequences; WGRS: whole genome
re-sequencing; SNP: single nucleotide polymorphism; GAB: genomics-assisted
breeding; RFLP: restriction fragment length polymorphism; AFLP: amplified
fragment length polymorphism; DAF: DNA amplification fingerprinting; SSR:
simple sequence repeat; DArT: diversity arrays technology; AIP: AhMITE1
insertion polymorphism; PTEMD: polymorphic TEs and their movement detection;
QTL: quantitative trait loci; CTAB: cetyl trimethyl ammonium bromide; PCR:
polymerase chain reaction; PIC: polymorphic information content; RIL:
recombinant inbred line.
MG: Carried out bench work (DNA isolation, PCR, restriction digestion etc.).
KS: Helped in developing the AhMITE1 and CAPS markers. RKV: Generated the
WGRS data of GPBD 4 and TAG 24. MKP: Generated the WGRS data of GPBD 4
and TAG 24. RSB: Conceptualized the idea and prepared the manuscript. All
authors read and approved the final manuscript.
1 Department of Biotechnology, University of Agricultural Sciences,
Dharwad 580 005, India. 2 Department of Frontier Research, Kazusa DNA Research
Institute, Chiba 292-0818, Japan. 3 Center of Excellence in Genomics (CEG),
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT),
Hyderabad 502 324, India.
We acknowledge the receipt of genotypes DER, VL 1, 110 and 110(S) from Dr.
M. V. C. Gowda, Professor, Department of Genetics and Plant Breeding,
University of Agricultural Sciences, Dharwad 580 005, India. We thank S. Nakayama
(Kazusa DNA Research Institute) for technical assistance.
The authors declare that they have no competing interests.
Availability of data and materials
The sequences have been submitted and the GenBank accession numbers
DRA005803 (WGRS of DER and VL 1), DRA005805 [WGRS of 110 and 110(S)],
and DRA006239 (WGRS of ICGV 86855 and VG 9514) have been obtained. The
newly developed markers have been given under Additional file 1: Table S1,
Additional file 2: Table S2, Additional file 3: Table S3, Additional file 4: Table S4,
Additional file 6: Table S5, Additional file 7: Table S6. The genotypes used in this
study are the released varieties or the breeding lines.
Consent to publish
Ethics approval and consent to participate
This study involves peanut varieties that are publicly available and released to
the farmers for cultivation. Other genotypes are the breeding lines developed
and maintained at our university. Consent to participate: Not applicable.
Financial support received from the DST-JSPS Bilateral Program is gratefully
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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