Morphological and Molecular Variability in Weedy Rices of Haryana
Weedy rices commonly known as red rices, infest rice fields worldwide. They compete with rice for nutrients, deteriorate quality and are difficult to eradicate. Understanding their genetic diversity and the origin may prove helpful in designing an effective way for managing them. A collection of weedy rices from Haryana were evaluated along with cultivated and wild rices for variability and diversity at morphological and molecular level using 35 ISSR primers. Significant variation was observed at morphological and genetic level in weedy rices of Haryana. Weedy rices were found to be distinct from white and red type cultivated rices and wild strains. Twenty one ISSR markers gave amplification of DNA and a total of 91 polymorphic bands were found. With 9 ISSR markers 20 unique bands were observed in 17 genotypes. Markers such as Pr814, Pr836 and Pr858 produced unique bands in weedy rices only. Alleles unique to weedy rices observed by ISSR markers could be converted into SCAR markers for identification. Weedy rices were placed alone as well as with other groups in cluster analysis. Both short and long grain weedy rices were found in collection though at present only long grain types are cultivated indicating that leftover progenies of local cultivars segregated into various plant types giving rise to the weedy rice.
Received: August 18, 2011;
Accepted: November 02, 2011;
Published: December 16, 2011
Rice is the most important cereal crop. The plant has wild, cultivated and
weedy forms. Off type rice plants with red seeds are considered weedy (Oka,
1988) and color of bran layer of red rices varies from light to dark
red. Weedy and cultivated rice are taxonomically classified as same species
as (O. sativa L.). Weedy rice shares traits with both cultivated and
wild types of rice. It is thought to have evolved either by hybridization between
wild and cultivated rice, or as a transitional form changing from wild to cultivated
types, a relic or primitive cultivar, or an off type derived from the present
cultivars (Tang and Morishima, 1988). Earlier to advent
of high yielding white varieties, red rices formed an important group in rice
growing Asian countries because of their tolerance to adverse conditions and
high Zn and iron content (Ahuja et al., 2007)
but weedy rice competes with rice for space, nutrients and has higher N use
efficiency for biomass production and poses a number of problems (Burgos
et al., 2006).
The close similarity between weedy rice and cultivated rice has prevented use
of selective herbicides (Hoagland and Paul, 1978). Also
concern exists for the danger of development of more aggressive weed through
crop allele escape (Chen et al., 2004). In field,
U. S. growers spend much energy and money in fighting red rices termed as Red
Menace. Costly Color sorters are installed to separate them from milled
product (Rood, 2000). The Asians are somewhat tolerant
towards red weedy rices as they eat and assign special names for them e.g.,
Sharei in Korea and Ludao in Japan (Ahuja
et al., 2007).
In recent past, weedy rices have been used for various types of studies as
variability, evolution of rice, gene flow, gene cloning, introgression and QTL
analysis of low temperature tolerance, inheritance of seed dormancy, shedding
and pollen sterility (Suh and Ha, 1987; Tang
and Morishima, 1988; Vaughan et al., 1995;
Suh et al., 1999; Ahn et
al., 2002; Jing et al., 2003; Gealy
et al., 2009). Understanding the origin of weedy rice may be helpful
in designing an effective way for their manage (Pyon et
al., 2000) and they have been utilized in rice varietal improvement
programmes to impart resistance against many diseases and pests. Haryana state
is known for cultivation of scented and fine grain rices. At present white,
medium to long grain varieties are grown and no red cultivated or wild variety
is known. Rice fields in Haryana are often contaminated by red colored weedy
rice called choba. Color sorters are used to separate red grains from
milled product in quality rices (Ahuja et al., 2007).
A collection of weedy rices from Haryana were evaluated for variability at morphological
and molecular level along with cultivated and wild rices to investigate its
MATERIALS AND METHODS
Plant material: As many as 15 weedy entries were collected from Kaul and Sonipat areas of Haryana. Weedy plants were distinct from cultivated rice by height, husk color and awns. Two cultivated white strains were obtained from Rice Research Station, Kaul whereas 11 red cultivated and 2 wild from P.A.U Ludhiana.
Morphological variability: Wild entries were grown in pots and the rest
28 rice genotypes were sown at Rice Research Station, Kaul in a dry bed nursery.
Seedlings were transplanted after 30 days in randomized block design with three
replications following standard package and practices. At maturity data were
recorded for plant height, panicle length, grain yield per plant, 1000-grain
weight, hull color, grain color, awning and grain dimensions (paddy length,
breadth and thickness) on 10 plants per replication, selected randomly following
Standard Evaluation System of International Rice Research Institute (IRRI,
Molecular variability: Genomic DNA was isolated from leaf samples of
60 day old rice plants using CTAB method as described by (Saghai-Maroof
et al., 1984). The quantity and quality of DNA samples were checked
by 0.8% agarose gel electrophoresis (Sambrook et al.,
1989) using a standard containing 100 ng μL-1 genomic λ
A total of 35 ISSR primers (UBC primer set # 9, John Hobbs, NAPS Unit, University of British Columbia, Vancouver, V6T 1Z3 Canada) were used in present study. The PCR reaction was carried out using a single primer at a time in 10 μL reaction mix containing 12.5 ng of template DNA, 1xPCR buffer, 2.5 mM MgCl2, 500 μM dNTP mix, 0.3 μM primer and 1.5 unit Taq DNA polymerase. PCR amplifications were performed in 96-well plates on a PTC100TM 96V thermocycler (MJ Research, Watertown, MA, U.S.A.) with initial denaturation at 94°C for 4 min; followed by 45 amplification cycles of denaturing at 94°C for 1 min, annealing at 50°C for few primers and 55°C for rest for 1 min; extension at 72°C for 2 min and final extension at 72°C for 15 min. The PCR products were resolved by electrophoresis on 1.5% (w/v) agarose gel. They were viewed under UV light (350 nm) and photographed using Vilber Lourmat gel documentation system. Molecular weight of bands was estimated using a wide range ladder (100 bp) from Fermentas, India.
Statistical analysis: Based on morphological data mean performance and variance was analyzed among all genotypes and grouping was done using Wards minimum variance method using software Windostat version 7.0. Intra and inter-cluster Euclidean distances generated were used to describe the relationships among the rice genotypes. Euclidean distance [d (i,j)] between two individuals i and j, having observations on characters (p) denoted by x1, x2,
,xp and y1, y2
., yp for i and j, respectively, can be calculated as under:
d (i,j) = [(x1 y1)2+(x2-y2)2+
Based on presence/absence of band, genetic similarity was evaluated by calculating
the Jaccard similarity coefficient to estimate all pair wise differences in
the amplification products produced by the primers for all rice genotypes. The
0/1 matrix was used to calculate the similarity/genetic distance using simqual
sub-program of NTSYS-PC version 2.02 software package (Rohlf,
1990). The similarity matrix thus obtained was subjected to cluster tree
and principal component analyses using the Un-weighted Pair-group Method with
an Arithmetic Average (UPGMA) sub-program of NTSYS-PC.
RESULTS AND DISCUSSION
Variation for morphological traits: Analysis of variance showed significant differences among genotypes for all the characters under study (Table 1).
Agronomic traits: The plant height ranged from 101 cm (PR111) to 222.67 cm (IC334112). All wild, red cultivated and weedy rices were tall whereas both cultivated white entries were semi-dwarf in stature. The panicle length ranged from 27 cm (IC334112)-17.16 cm (O. nivara). The yield per plant ranged from 22.16 (HKR47)-3.72 g (K1). Yield per plant was highest in cultivated white followed by red cultivated, weedy rice and wild types. Grain weight ranged from 32.69 (IC334141)-8.16 g (W1) (Table 2).
Cultivated white and cultivated red genotypes were awn less. Both wild entries possessed awns however, awns of different length were found in weedy rice. Hull color varied from straw to black. Both the wild possessed black hulls and both cultivated white entries possessed straw hulls. However various shades of hull colors were observed in cultivated red and weedy rices. In weedy rice husk color ranged from straw, brown spots, brown furrows, reddish to light purple and purple spots (scale 0-9). Grain color ranged from white to red. Weedy rices showed different shades from white to light brown, brown, speckled red and red (scale 0-7). Interestingly in entry W3, red, pink and white grains coexisted in a single panicle (Table 2).
Cluster analysis for morphological data: Dendrogram was constructed
based on data collected from all the rice genotypes for all characters mentioned
above using Ward's Minimum Variance method.
|| Analysis of variance for morphological traits of wild, red
and weedy rices
|*Mean values are significant at 5% level of significance
|| Mean performance of different rice genotypes for different
|Red cultivated 1-11, weedy Kaul, 12-17, Sonipat 18-26, Wild
27-28, White cultivated 29-30, *Mean values are significant at 5% level
Five clusters were obtained. Number 1-30 correspond to serial number of rice
genotypes as mentioned in (Table 3). Cluster I included 10
entries (8 cultivated red, 2 wild). Cluster II included 2 (both of cultivated
white). Cluster IV included 7(weedy red). Cluster V included 9 (one cultivated
red, 8 weedy red).
Euclidean intra-cluster distances showed a range of 1.14 to 3.01. Minimum intra-cluster distance (1.14) was within cluster III and maximum (3.01) was within cluster I. Euclidean inter-cluster distances showed a range of 2.99 to 6.18 (Fig. 1, 2).
ISSR markers based polymorphism among rice genotypes: Out of 35, only
21 ISSR markers gave amplification. A total of 95 bands were detected out of
which 4 were monomorphic and 91 were polymorphic.
|| Cluster information using wards minimum variance
|| Ward's Minimum Variance Dendrogram based on agronomic data
of all accessions
|| Inter and intra cluster distance among all accessions
ISSR DNA bands varied between 1 (primer IS21) and 14 (UBC primer No. 845) with
an average of 4.5 bands per primer. Maximum number of polymorphic bands (14)
was obtained using UBC primer No. 845; the average number of polymorphic bands
was 4.3 per primer. The polymorphism percentage ranged from 0-100% with an average
polymorphism of 88.9% across all the varieties. The size of PCR amplified products
ranged between 200 and 1300 bp. UBC No. 854, IS21, IS99 and IS121 gave monomorphic
bands whereas UBC No. 809, 811 814, 822, 835, 836, 845, 852, 856, 858, 873 and
IS29, IS43, IS69, IS88 IS90 and IS116 gave polymorphic bands.
Genetic diversity analysis: ISSR marker analysis generated high levels
of molecular polymorphism within this selection of cultivated red and weedy
rices (Fig. 3). Dendrogram obtained using the ISSR allelic
diversity data shows that at 0.46 similarity coefficient the 30 rice genotypes
could be divided into five groups (Table 4). All the cultivated
and weedy rice selections were found to be genetically distinct from one another
except W5 and W6 which has a high similarity coefficient of 0.77. Though number
of indica rice varieties included in this study were low but the study
do suggest huge diversity among the indica and cultivated red and weedy
rice genotypes. Notably, indica rice variety, HKR47, was relatively closer
to the wild ancestors, O. rufipogon and O. nivara, compared to
cultivated red and weedy rice genotypes.
Unique alleles: A total of 19 unique alleles were detected in a total
of 17 genotypes with 9 ISSR primers. These unique bands ranged from 1-4 with
an average of 2.22.
|| Dendrogram based on ISSR Polymorphisms of all 30 genotypes
by UPGMA cluster analysis
||Unique alleles obtained using 9 ISSR primers in 17 rice genotypes
The size of unique bands ranged from 250-1300 bp (Table 5).
UBC primer No. 814, 836 and 858 produced unique bands in weedy rices only.
A collection of weedy rices of Haryana showed wide variation in morphological
traits. Weedy rices were tall and poor yielded. Two weedy entries (K5 and W8)
possessed grain weight near cultivated rices whereas, the seven entries possessed
lowest 1000 grain weight (8-12 g) among all the entries under test. The lighter
grain weight in weedy in comparison with cultivated rice is in accordance with
earlier reports (Tang and Morishima, 1989). Weedy entry,
W8 possessed grains longer (10.02 mm) than local Basmati rice (9.81 mm). Four
weedy rices could be classified under short grain category. At present in Haryana
only long grain types are cultivated while short grain types were grown some
50 years back (Ahuja et al., 1995). Therefore,
short grain weedy rices seem to be progeny of volunteer plants of old cultivars
(Zhang et al., 2008). Weedy rices possessed thin
grains and the grain thickness was lower than that of cultivated rices. Paddy
grain dimensions (length, breadth and thickness) in Indianrices ranges from
5.15-11.27, 1.97-3.73 and 1.61-2.57 mm (Hector et al.,
1933; Ramaiah and Rao, 1953).
The husk color of weedy rices was found to be between cultivated rices and
wild i.e., varied from straw color (4 out of 15) to various shades of brown.
Eight out of 15 weedy rices possessed awns and 14 out of 15 red grains. Red,
pink and white grains in a single panicle of W3 might be the result of crossing
between weedy and cultivated or between weedy and weedy. Gealy
et al. (2009) showed evidence of hybridization of weedy rice with
cultivated rice by DNA studies. Different hull colors were reported by (Shivrain
et al., 2010) in weedy rices of Arkansas. Federici
et al. (2001) reported weedy rices with straw hull and no awn mimicking
cultivated as well as wild types. It is observed that weedy rices are distinct
from cultivated rices (both red and white) in awning, husk color, grain color,
grain weight and thickness. Similar variation in grain traits was reported by
Suh and Ha (1987) and Vaughan et
In the present study, both the cultivated white rice genotypes are placed in extremes groups by molecular marker analysis though were placed in same cluster by Euclidean square analysis.
Similar is the case with wild rice and other groups. Similar results i.e.,
variability at morphological level did not coincided with diversity at molecular
level were reported by Vanaja et al. (2007) in
rice, Bandyopadhyay et al. (2007) in finger millets
and Devanshi et al. (2007) in Ber. In the present
study Oryza nivara and rufipogon are placed in different cluster. Similar results
were reported by Sujatha et al. (2004). Ryza
rufipogon is clustered along with f our cultivated red rices in cluster 4. It
is thought to be the progenitor species of the cultivated rice, O. sativa
L. (Oka, 1988).
The genetic clustering of weedy rice accessions from Haryana was found to be
random with respect to location as samples from one location did not belong
to the same genotypic cluster. Most of the weedy rices were placed with cultivated
white, red and wild rices except K1 which was placed in separate cluster. UBC
primers No. 814, 836 and 858 produced unique bands in weedy rices which indicate
that these ISSR primers may be used to distinguish cultivated and weedy rice
varieties at molecular level. However, this will require converting them into
SCAR markers. Gealy et al. (2009) reported SSR
marker loci which could distinguish between U.S. weedy red rice and white cultivars.
Chen et al. (2004) reported frequent occurrence
of gene flow from cultivated rice to weedy rices and according to Yu
et al. (2005) unique alleles in weedy rices are through gene flow.
Possible origin of weedy rices have been proposed as segregating progenies
of cultivated rice (Suh et al., 1999), intervarietal
hybridization or mutation (Cao et al., 2006),
hybridization of discrete types of rice cultivars generating partially sterile
plants resulting into weedy strains (Ishikawa et al.,
2005) or parallel evolution, hybridization, gene flow etc (Kane
and Baack, 2007). Watanabe et al. (2000) reported
that different rice growing locations show different patterns of genetic diversity,
depending on specific combinations of germplasm from which weedy rices originated.
Present material comprises weedy strains genetically close to red and white
cultivated strains, wild strains and distinct from all three groups indicates
that the weedy rices of Haryana might have originated by local cultivars leaving
volunteer plants through the succeeding cropping seasons, hybridization of discrete
types, segregating progenies of cultivated rice and gene flow as proposed by
From the above study it is concluded that weedy rices possess genes useful under adverse environmental conditions are genetically more similar to cultivated rice than wild rices but with the help of some ISSR primers weedy rices are isolated from cultivated and wild rice and they are easily eliminated from the rice field. This was a small sample collected from two districts of Haryana and variability obtained shows that there is need to collect and evaluate more weedy rices having important genes.
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