DISCUSSION

DISCUSSION
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 hybrids.
In present study aimed at confirmation of cotton hybrids G.cot.DH-7 ( Sujay G.27)
and G.cot.DH-9 ( 4011 824) using morphological markers as well as DNA markers.
Cotton is the kind of fibre crop and is the most important commerical crop of India. In
India desi hybrids have been released in 1974 and 1976 respectively and their number is
expected to increase in future. The varieties and hybrids attain acceptance when the farmer
gets genetically pure seeds of high standards. For this purpose, each cultivar should be
properly defined with suitable descriptors, so as to maintain its identifying during seed
production through field inspection and certification. Apart from this, characterization of

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cultivars is also required for their protection under PPV & FR Act 2001. In India, while
certain diagnostic features for released and notified crop varieties and hybrids are known and
used in seed certification, but by and large descriptors are incomplete. Thus present study was
undertaken to characterize three hybrids and their parental lines that are inactive seed
multiplication chain on the basis of qualitative morphological characters.
The hybrid G.cot.DH-7, G.27, and 4011 exhibited erect type of growth habit, while
Sujay G.cot.DH-9 and 824 had semi-errect type of growth. The variation exhibited by the
genotypes for growth habit is due to genetic character of the genotypes. The genotypes
exhibited variation for stem hairiness and grouped as sparsely hairy (G.cot.DH-7, G.cot.DH9, 4011, and 824) and glabrous (Sujay). Similarly, Sethi et al. (1960) reported that stem
hairiness is a genetically controlled and it varies from genotype to genotype. However, the
five lobs per leaves ware observed in both the two hybrids and their parents. The leaf foliage
colour of G.cot.DH-7 and Sujay was light green and dark green leaf was observed in G.27.
Whereas in case of hybrid G.cot.DH-9 and female parent 4011 were green colour leaf foliage
and male 824 dark green in colour. The genotypic variation in the colour of the leaf may be
due to genetic character of the parents and may be due to edaphic and environmental
conditions such as nutritional factors and light intensity during crop growth.
All the genotypes except 824 have medium hairs on leaves. However, the hairs on leaf
behaves like a simple mendelian trait giving 1:2:1 ratio in crosses between sparsely hairy and
medium hairy leaves (Harland, 1944).
The petal colour of the flower is one of the important characters for characterization
on flower petal colour, the genotypes were categorized as cream (G.27), yellow (G.cot.DH-9,
G.cot.DH-7, Sujay and 824) and deep yellow (4011). Similarly, petal spot is used as marker
for varietal identification, which is present in G.cot.DH-9, G.cot.DH-7, Sujay and 824 and
absent in 4011 parent. The variation in petal spot is due to genetic constituent of the
genotypes which has cumulative effect and is governed by dominant gene as it was reported
by Ahuja et al. (2006) in cotton. The variation in the anther colour was noticed and the
genotypes were grouped as dark yellow in both the female parent as well as hybrids and light
yellow in male parents G.27 and 824, which is a heritable character and it is genetically
controlled. The cylindrical boll shape observed both the hybrid and their parent except 824 in
varied and it is genetically controlled by the genotype.

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The polymorphic data obtained in RAPD fingerprint pattern were utilized as marker
for hybrid identification. The RAPD profile of parents and their hybrids were correlated to
find out direct introduction of character/ genes in to the hybrids through male or female
parent by possessing male and female parent specific bands among RAPD profiles of two
hybrids. Nevertheless, it was exploited to document polymorphism and relationship between
two hybrids and their parents. RAPD analysis was performed with 25 decamers from the
series of RPI1 to RPI25. Good amplification was obtained with all the primers. Three
decamers viz., RPI5, RPI7, RPI8 showed polymorphism among the hybrids and their parents.
Further, RPI7 was polymorphic for parents of G.cot.DH-7 and G.cot.DH-9 hybrid. RPI7
generated female (Sujay) specific amplicon RPI7-500bp and RPI7-450bp in hybrid
G.cot.DH-7. RPI7 identified female (4011) specific amplicons 450bp and 500bp in
G.cot.DH-9 (figure 3). RPI 8 identified male (824) specific amplicon 550bp in G.cot.DH-9
hybrid (figure 3).
Polymorphic molecular markers produced unique banding and not only discriminated
the two cotton parents, but also identified their true hybrids. Polymorphism revealed by
RAPDs is based on the position and orientation of primers annealing sitesand the interval
they span. Polymorphism between individuals can arise through nucleotide substitutions and
insertions or deletions (Williams et al., 1990). This technique can be adopted for large scale
screening of hybrids in cotton (Mehetre et al. 2004; Dongre and Parkhi 2005).RAPD analysis
has also been utilized for hybrid identification and assessment of genetic diversity in wheat
(Awan et al., 2008), rice (Haiyuan et al., 1998), maize (Iva et al., 2005), leucadendron (Lui et
al., 2007), muskmelon (Park and rosby 2004), cyrtandra (Gesneriaceae) (Smith et al., 1996)
and theobroma (Wilde et al., 1992).
Out of 19 ISSR primers, two primers ISSR9 and ISSR12 showed polymorphism in
parents of hybrid G.cot.DH-7. Out of which only one primer ISSR9 was found to be
heteroallelic for parents. Primer ISSR9 produced two distinguishable alleles, in which allele3
was common in both parents, allele one was specific to male (Sujay) about 200bp and allele
two was specific to female (G.27) parent about 550bp.Where as ISSR12 primer resulted in
amplification of 700bp male (Sujay) specific amplicon that was present in hybrid. The ISSR9
primer was genereted female specific amplicon of 200bp and 900bp in G.cot.DH-7hybird
(figure 2). ISSR 12 has identified male specific amplicon of

ISSR12 (700bp) in the

G.cot.DH-7.

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Our results confirm that ISSR markers are efficient tools for the discrimination of F1
hybrids from the self-pollinated progeny of female parents in controlled crosses. They can
also be effectively used to fingerprint and differentiate plants with highly similar
morphological characteristics. The hybridity status of F1 hybrids can be easily verified by
comparing amplified polymorphic bands between F1 hybrids and female parents (bands
specific to the male parents). In addition, ISSR markers allow the easy, fast, inexpensive,
accurate, reliable, and simultaneous detection of polymorphisms at multiple loci in the
genome using low quantities of DNA. These properties have made the markers useful for the
genetic analysis of various plants (Reddy et al., 2002). Our results agreed with those of Desai
(2014), who reported the use of 19 ISSR primers to identify the parentage of 5 hybrids of
cotton. Similarly, Liu et al., (2007) have used 54 ISSR primers to confirm the hybrids of
three crosses of the cotton and found that two ISSR primers gave polymorphic DNA bands
between male and female parents in each cross and could verify their interspecific hybrids.
Eight ISSR primers have also been used successfully in the identification of cotton hybrids
(Dongre et al., (2005, 2010 and 2012)).
Microsatellite repeats used as primers in ISSR markers, amplify DNA segment
present at an amplifiable distance in between two identical microsatellite repeat relatively
more stable and repeatable. The polymorphic ISSR markers produced female specific
amplicon. Thus ISSRs can be used to identify female parent of the respective hybrid.
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).

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It is concluded from this study that it is possible to differentiate the cotton hybrids
more accurately and efficiently from its parental lines using molecular markers. It also
indicates that RAPD and ISSR banding pattern of the parents compared with their respective
hybrid clearly recognize true hybrid.

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