P. SELVAKUMAR, R. RAVIKESAVAN, A. GOPIKRISHNAN, K. THIYAGU, S. PREETHA AND N.

MANIKANDA BOOPATHI

Selvakumar, P., Ravikesavan, R., Gopikrishnan, A., Thiyagu, K., Preetha, S. and Manikanda Boopathi, N.
(2010), Seed Sci. & Technol., 38, 358-366

Genetic purity analysis of cotton (Gossypium spp.) hybrids using

SSR markers
P. SELVAKUMAR1, R. RAVIKESAVAN1, A. GOPIKRISHNAN2, K. THIYAGU1, S. PREETHA1
AND N. MANIKANDA BOOPATHI2*

1
Department of Cotton, Centre for Plant Breeding and Genetics
2
Department of Plant Molecular Biology and Biotechnology, CPMB, Tamil Nadu Agricultural University,
Coimbatore 641003, India (E-mail: nmboopathi@tnau.ac.in; biotechboopathi@yahoo.com)

(Accepted December 2009)

Summary

Use of morphological differences, between true hybrids and off types in grow out test (GOT) for genetic purity
analysis, are not always apparent and cannot be recognised easily. Further, morphological traits are costly, tedious
to score and environment sensitive. Alternatively, it is suggested that recent breakthrough in molecular markers
can be employed in genetic purity analysis. The genetic purity of three cotton hybrids (TCHB 4510, TCHB 2310
and TCHB 213) that are widely cultivated in Tamil Nadu, India were assessed by GOT and molecular markers.
A total of 400 individuals from each one of the three hybrids were raised in the field and morphological traits
were recorded. Results of this GOT have shown that TCHB 2310 had lowest genetic purity (62.5%) followed
by TCHB 4510 (78.2%) and TCHB 213 (95.2%). Simple sequence repeats (SSR) marker analysis of parents that
were involved in the production of all the three hybrids have shown that 45 out of 150 SSRs were polymorphic
among the parents. From this set of polymorphic SSRs, BNL686, BNL1679, BNL3971, BNL3955, CIR407 and
CIR413 were selected to test the genetic purity of hybrid seeds since they have produced clear, scorable and
unambiguous polymorphic bands among the parents. All the three hybrids were clearly distinguished from their
selfed females and off types using these six SSRs. Hence, it is proposed that these SSR markers can be used in
efficient analysis of hybrid seed purity since this technique is simple to use, more accurate and not affected by
environment when compared with GOT.

Introduction

Cotton is an important commercial crop in India and contributes a major share to the
national economy. Increasing lint production and fiber quality has long been a major
breeding objective to meet out the demands of growing population and modernized textile
mills. Heterosis breeding is a key genetic tool that facilitates yield enhancement and helps
in enrichment of desirable quantitative and qualitative traits in cotton (Dongre and Parkhi,
2005). India ranks second in cotton production (22% of the global production) with the
average productivity of 526 kgha-1. Cotton is cultivated in ~ 9 million hectares and 70%
of this area is occupied by hybrids (Mehetre et al., 2007). The first hybrid H-4 has been

* Author for correspondence

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released in 1971 and henceforth large core of improved hybrids gifted with high yield
potential, early maturity, better fibre quality and tolerance to key pests and diseases have
been developed during the last five decades. This has brought about a sea change in the
cotton scenario of the country by causing a quantum jump in yield.
Hybrid seed production in cotton is usually taken up by hand emasculation and
pollination. Being often cross pollinated crop, the genetic purity of cotton hybrid seeds
is adversely affected by the foreign pollen. Hence, in order to get better returns from
the hybrids, greater seed purity and quality are emphasized elsewhere. Traditionally, it
has been the practice to carry out GOT to analyze the genetic purity of hybrid seeds
using morphological traits (Tatineti et al., 1996; Gumber, 2003; Ankaiah et al., 2005
and Patel et al., 2005). However, morphological differences between true hybrids and
off types are not always apparent and cannot be recognised easily, especially when the
parents are genetically similar. The sensitivity of morphological traits to the environment
further limits the application of GOT. It has also been reported that GOT is an expensive
and time consuming procedure which may delay planting and hence lead to loss of seed
viability (Ali et al., 2008). Alternatively, biochemical markers such as isozymes and seed
storage proteins have been suggested for genetic purity determination (Dadlani et al.,
1997; Mehetre and Dahat, 2001; Borle et al., 2007 and Rakshit et al., 2008). Nevertheless,
the main weaknesses of biochemical marker are its low abundance and sensitivity to
environmental and experimental conditions. Therefore, it is necessary to develop a rapid,
reliable and reproducible technique to assess the genetic purity of cotton hybrids. With
the advent of the molecular marker technology, it is now possible to test the purity of the
hybrid seed immediately after harvesting and processing by DNA markers. DNA markers
such as RFLP (Pendse et al., 2001; Dongre and Parkhi, 2005) RAPD (Geng et al., 1995;
Venu, 2001; Rao et al., 2002; Mehetre et al., 2007), AFLP (Rana and Bhat, 2004), SSR
(Rana, 2003; Dongre and Parkhi, 2005; Saravanan et al., 2007) and ISSR (Dongre and
Parkhi, 2005; Rana et al., 2006) have been used to rapidly screen genetic purity of hybrid
seed lots.
Though the usefulness and effectiveness of different type of molecular markers were
reported, the selection of marker type is of great importance for successful genetic purity
testing. In case of dominant markers such as RAPD and ISSR, only those primers which
amplify bands specific to the male parent might reveal a proper pattern of a true hybrid
as opposed to that of a selfed seed of the female parent. On the other hand, co-dominant
markers (e.g., SSR) produce polymorphic markers from each of the parental line of the
hybrid and hence it would be useful for hybridity and purity testing (Dongre and Parkhi,
2005). Further, SSRs can be easily automated using florescent labeled primers on an
automated sequencer and it is possible to multiplex (combine) several markers with non
overlapping size ranges on a single electrophoresis run (Edwards and McCouch, 2007).
It has also been particularly indicated that the SSRs are more discriminative, reliable
and repeatable and they could potentially be standardized more easily for Distinctness,
Uniformity and Stability (DUS) testing (UPOV, 1997).
Among the hybrids released by Tamil Nadu Agricultural University (TNAU), TCHB
4510, TCHB2310 and TCHB 213 are having significant area in Tamil Nadu, India and
hybrid seeds are routinely being produced to meet out the demands. Usually the genetic
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 P. SELVAKUMAR, R. RAVIKESAVAN, A. GOPIKRISHNAN, K. THIYAGU, S. PREETHA AND N. MANIKANDA BOOPATHI

purity of the hybrid seeds are tested by GOT; but because of the above said reasons the
University is looking for alternative method. Hence SSR markers were employed in this
study for genetic purity analysis of cotton hybrids.

Materials and methods

Plant materials
The present study was carried out at TNAU, Coimbatore, India during 2007 - 2009.
The plant material for this study comprised of two G. hirsutum (H23 and H45), two G.
barbadense (B1 and B10) and their three inter-specific hybrids (TCHB 4510 (derived
from H45 and B10), TCHB 2310 (derived from H23 and B10) and TCHB 213 (derived
from H23 and B1). The crossing programme was done by conventional hand emasculation
and pollination method developed by Doak (1934). To get sufficient amount of seeds, the
hybridization programme was continued for 45 days.

Grow Out Test (GOT)

Four hundred seeds from each hybrid seed lot were sown in 5 m row with 10 cm plant to
plant and 45 cm row to row spacing in August 2008. The details on morphological traits
that have been recorded to distinguish the true hybrids from off types for all the three
hybrids along with their parents are furnished in table 1. The genetic purity of hybrids
was calculated as:
(Total number of plants – number of off types)
Genetic purity % = × 100
Total number of plants

Isolation of genomic DNA

Hundred seeds from each one of the three hybrid seed lots were used for SSR marker based
genetic purity testing. Genomic DNA was isolated from every seed as described by Krishna
and Jawali (1997), were stored at –20°C and used as template for PCR amplification. The
isolated genomic DNA was quantified by NanoDropTM 1000 Spectrophotometer. After
quantification, the final concentration of DNA was adjusted to15 ng/µl.

SSR analysis
A total of 150 SSR markers were used to identify polymorphic marker among the parents.
The forward and reverse primers of these SSR markers were obtained from www.
cottonmarker.org. From the 45 markers that were found to be polymorphic, six markers
were selected to test the genetic purity of hybrid seeds since they have produced clear,
scorable and unambiguous polymorphic bands among the parents. Among the six SSR
markers, BNL686, BNL1679, BNL3971 and BNL3955 were utilized to test the hybrids
TCHB 4510 and TCHB 213. The hybrid TCHB 2310 was tested using the BNL686,
BNL1679, CIR407 and CIR413. The cocktail (15 µl) for PCR amplification was prepared
as 40 ng of DNA, 200 µM of each dNTPs, 0.4 µM of each forward and reverse primers,
1X Taq buffer and 0.03 units of Taq DNA polymerase. The amplification reaction was
carried out in thermo cycler (MyCycler®, Bio-Rad Laboratories, California) and the
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 Table 1. Distinguishable morphological characters of parents and hybrids.

Parent Hybrid Parent Parent Hybrid Parent Parent Hybrid Parent

Characters
H 45 TCHB 4510 B 10 H 23 TCHB 2310 B 10 H 23 TCHB 213 B1

Medium
Leaf colour Green Green Green Green Green Green Green Dark Green
green

Leaf texture Glabrous Glabrous Glabrous Glabrous Glabrous Glabrous Glabrous Glabrous Hairy
GENETIC PURITY ANALYSIS USING SSRs

Light to
Light Light
Petal colour Cream Yellow Cream Yellow Cream sulphur Yellow
yellow yellow
yellow

Light Yellow to
Anther colour Cream Yellow Buff Yellow Yellow Buff Yellow
Yellow deep yellow

Present Present
Petal spot Absent Present Present Absent Present Present Absent
(light colour) (dark colour)

Round Round Round Oblong Conical

Boll shape Oblong Oblong Oval Oblong
to oblong to ovoid to ovoid to conical to oval

Sparse Sparse
Slightly Profusely and
Boll surface Smooth pitted Pitted and slightly Pitted and slightly Pitted
pitted deeply pitted
pitted pitted

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 P. SELVAKUMAR, R. RAVIKESAVAN, A. GOPIKRISHNAN, K. THIYAGU, S. PREETHA AND N. MANIKANDA BOOPATHI

programme of reaction was set as 93°C for initial denaturation, 72°C for final extension
and 40 cycles of 95°C for denaturation, 50°C for annealing and 72°C for extension. After
adding 5 µl of loading buffer (40% sucrose and 0.25% bromophenol blue), the PCR
products were analyzed using 3% Metaphor agarose gel electrophoresis. A 100 bp DNA
ladder was used to calculate PCR product size. The bands were visualized under UV
transillumination after staining with ethidium bromide and gel images were documented
using Alpha Imager TM 1200 (Alpha Innotech Corporation, USA). SSR gel images were
manually scored for number of alleles produced by each SSR marker. The bands were
counted as allele 1, 2, 3 and so on, from top of the lanes to their bottom.

Results

Grow out test

Observations were recorded on four hundred individuals of each hybrid for a number of
characters. The parents and F1 plants were carefully observed on the basis of morphology
to see if they were true hybrids. The details on number of off types and analysis of genetic
purity using GOT results are given in table 2. It has been recorded that the hybrid TCHB
2310 had lowest genetic purity (62.5%) followed by TCHB 4510 (78.2%) and TCHB 213
(95.2%).
Table 2. Efficiency of GOT and molecular markers in testing genetic purity of cotton hybrids.

Primers Total seeds tested Number of off types Genetic purity (%)

TCHB 4510
BNL1679 99 20 79.8
BNL686 100 20 80.0
BNL3955 98 18 82.0
BNL3971 100 16 84.0
GOT 400 87 78.2

TCHB 2310
CIR407 99 28 71.7
CIR413 98 33 66.3
BNL686 100 32 68.0
BNL1679 97 29 70.1
GOT 400 150 62.5

TCHB 213
BNL1679 99 9 91.9
BNL686 100 7 93.0
BNL3955 97 7 92.8
BNL3971 100 8 92.0
GOT 400 19 95.2

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Genetic purity testing using SSR markers

The polymorphisms observed between the parents were used as markers for hybrid
identification. The SSR marker, BNL1679 produced three distinguishable alleles, in
which allele 3 was common in both parents, allele 1 was specific to male and allele
2 was specific to female parent. All the three alleles were present in hybrid (figure 1).
Similarly, BNL686 produced two polymorphic alleles and one common allele (Allele 1).
Allele 2 and 3 were specific to female and male parent, respectively. On the other hand,
two distinguishable alleles were produced by BNL3955. The female parent had allele 2
and male parent had allele 1.

M P1 P2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
200bp

100bp

M P1 P2 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

200bp

100bp

M P1 P2 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

200bp

100bp

M P1 P2 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

200bp

100bp

M P1 P2 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100

200bp

100bp

Figure 1. Genetic purity analysis of TCHB 2310 using SSR primer BNL 1679. M – DNA ladder; P1- Parent
H23; P2- Parent B10; lane number 1 to 100 – hybrid progenies. Allele 1, 2 and 3 are marked with different
arrow viz., , and respectively.

The SSR marker, BNL3971 had shown polymorphism between two parents by
producing two alleles in which allele 1 was specific to female and 2 was specific to
male parent. Interestingly, CIR407 had produced one male specific allele (allele 1) and
two common alleles (allele 2 and 3). Two different alleles were produced by this primer
in which allele 1 was female specific and allele 2 was male specific. The hybrids were
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 P. SELVAKUMAR, R. RAVIKESAVAN, A. GOPIKRISHNAN, K. THIYAGU, S. PREETHA AND N. MANIKANDA BOOPATHI

distinguished from off types by checking the presence of both female and male specific
alleles. The details on number of off types and genetic purity tested by different SSR
markers along with GOT results are given in table 2.

Discussion

Assessment of seed purity is one of the most important quality control components in
hybrid seed production. Traditionally, GOT has been employed to assess the purity of
hybrid seeds using morphological traits. The traits such as petal colour, petal spot, anther
colour, boll shape, boll surface, leaf colour and leaf texture were studied to distinguish
the interspecific hybrids from off types. TCHB 4510 had yellow anthers and its parents
H45 and B10 had cream and yellow anthers, respectively. This hybrid was identified
by yellow anthers which was male specific. The usefulness of anther colour to identify
genotypes was already reported (Ankiah et al., 2005). The petal spot was also served as a
morphological marker to assess the genetic purity of hybrids. The petal spot was absent in
female parent H23 but present as deep red in male parent B10, and diluted red in hybrid
TCHB 2310. The significance of petal spot as a marker trait for testing the genetic purity
of hybrids had been established (Gumber, 2003). The genetic purity of hybrid TCHB
213 was confirmed using leaf shapes and the same trait was also used by Venu (2001) to
distinguish the hybrid with that of off types. Though GOT is used to determine genetic
purity of cotton hybrids, it is tedious, space demanding and time consuming and often
does not allow the unequivocal identification of genotypes.
Hence, a recent development in molecular markers has been suggested for genetic
purity testing since they are used to assess precisely the genotype and not the phenotype
(Sundaram et al., 2008). Among different types of molecular markers, SSR markers found
to be very useful for testing genetic purity since they are co-dominant and using SSR
the heterozygous state can be easily discerned from the homozygous state. It is possible
to differentiate the hybrids from its female selfed individuals alone in GOT. However,
molecular markers can be employed to differentiate the hybrids from all kinds of off types
(Pendse et al., 2001).
The hybrid TCHB 2310 recorded low genetic purity as per GOT and it was also
supported by the results of SSR markers. Interestingly, the SSR marker, CIR413 has
produced a non parental banding pattern in few individuals of hybrid TCHB 2310.
This non parental banding pattern may be due to pollen contamination during crossing
programme or use of impure parents. Hence this type of markers could be useful in
differentiating hybrid from off types other than selfed seeds of female. Similar kind of
result was reported by Pendse et al. (2001), where off types (not an identical to either the
female or male parent) were noticed in cotton hybrid NHH-44 using RAPD markers.
Hybrid TCHB 213 was found to be genetically pure for a random sample of 100 seeds
as tested by SSR markers BNL1679, BNL686, BNL3971 and BNL3955. These findings
were in accordance with GOT. Apart from regular hybrid allelic pattern, an unusual allelic
pattern was also observed. For example, in TCHB 4510, some of the individuals have
shown hybrid allelic pattern for primer BNL1679 whereas the same individuals have shown
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GENETIC PURITY ANALYSIS USING SSRs

female parent specific allele for primer BNL686. One possible reason for this discrepancy
is that parental individuals are advanced inbred lines and they may have residual amounts
of heterozygosity that can be detected only at molecular level (Nandakumar et al., 2004).
Therefore, hybrid genotypes occasionally exhibit slight variation, which could account for
the differences observed with these molecular markers. Furthermore, cotton is an often
cross pollinated crop and hence, parental individual may not be genetically identical and
could be heterozygous at one or more loci. Consequently, few loci in hybrids may be non
heterozygous.
In summary, it is concluded from this study that it is possible to differentiate the
cotton hybrids more accurately and efficiently from its parental lines and off types using
molecular markers. These molecular markers would be more efficient than GOT, since
DNA markers would be more accurate for determining hybrid seed purity. Further, marker
analysis will also result in considerable savings for the seed industry as this technique may
avoid the cost of storage for a whole season and cost of acquiring land and cultivation.
In addition to that the SSRs used in the present investigation, may offer efficient genetic
purity testing of other hybrids provided their parental polymorphism has already been
established with these markers.

Acknowledgements

This work was funded by Department of Biotechnology, Ministry of Science and

Technology, Government of India under Program Support for Research and Development
in Agricultural Biotechnology at TNAU.

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