This document describes a study that used simple sequence repeat (SSR) markers to identify two popular rice hybrids (KRH-2 and DRRH-2) and their parental lines. Thirty-five SSR markers were tested, and six were found to be polymorphic across the hybrids and parents, allowing unique fingerprints for each hybrid. Five markers (RM 206, RM 276, RM 204, RM 234 and RM 228) differentiated the two hybrids. Analysis of parental lines found residual heterozygosity at two loci, highlighting the importance of SSR markers for maintaining genetic purity. A 20x20 grow-out matrix trial validated that the identified SSR markers effectively detected contaminants in commercial seed lots, comparable to
Dioscorea rotundata is a staple food crop for millions of people in the tropical and subtropical regions. In vitro germplasm conservation is a very useful tool in yam improvement strategies but very little is known about the genetic integrity and stability of in-vitro conserved yam plants. In this study, 42 accessions from in vitro and field populations were genotyped using 11 microsatellite markers and 23 morphological descriptors to assess variability within and between accessions. Out of the 23 morphological variables used, 13 were identified as most discriminate and were used to cluster the accessions into 4 clusters using the unweighted pair group arithmetic mean average (UPGMA). Accession maintained in field as well as in in-vitro showed high genetic similarity (R2 = 0.91, p-value: 1e-04). Out of the 42 accessions analyzed, nine accessions maintained in the field and in-vitro displayed different genetic profiles. This study provided basic information on the possible somaclonal variation of yam accessions maintained through in-vitro. Further study with advanced tools such as next-generation sequencing is required to elucidate the nature of the observed variation within clones.
It is a presentation prepared to tell people more about male sterility in brief. I have also included one case study to explain and help you. Hope you like it. Thanks!
Dioscorea rotundata is a staple food crop for millions of people in the tropical and subtropical regions. In vitro germplasm conservation is a very useful tool in yam improvement strategies but very little is known about the genetic integrity and stability of in-vitro conserved yam plants. In this study, 42 accessions from in vitro and field populations were genotyped using 11 microsatellite markers and 23 morphological descriptors to assess variability within and between accessions. Out of the 23 morphological variables used, 13 were identified as most discriminate and were used to cluster the accessions into 4 clusters using the unweighted pair group arithmetic mean average (UPGMA). Accession maintained in field as well as in in-vitro showed high genetic similarity (R2 = 0.91, p-value: 1e-04). Out of the 42 accessions analyzed, nine accessions maintained in the field and in-vitro displayed different genetic profiles. This study provided basic information on the possible somaclonal variation of yam accessions maintained through in-vitro. Further study with advanced tools such as next-generation sequencing is required to elucidate the nature of the observed variation within clones.
It is a presentation prepared to tell people more about male sterility in brief. I have also included one case study to explain and help you. Hope you like it. Thanks!
Development of biotic stress resistance technologiesMamtaChoudhary75
This ppt is regarding the topic 'Development of biotic stress resistance technologies'. in it, I've discussed biotic stress, effects on crop production, disease and insect resistance mechanisms, case studies, etc.
Genetic diversity in pea germplasm using RAPD MarkersShujaul Mulk Khan
Selection of the genotypes using plasmid assisted technology provides an efficient and useful tool for elaborating genetic relationships among genotypes. In present study, 48 Pea (Pisum sativum var sativum L.) genotypes obtained from different sources were analyzed through 20 RAPD, DNA markers for assessment of intraspecific DNA variations. Results revealed that significant variations were present in minor bands. Major bands also showed significant diversity. Considerable variations were also recorded in density of some common bands. Maximum and minimum genetic diversity i.e., 80% and 20% was found among 08 and 23 comparisons, respectively from banding profile. These variations can be
used further for enhancing variability, a prerequisite for crop breeding. Phylogenetic clustering (through dendrogram analysis) of genotypes revealed that genetic diversity is independent of origin of genotypes. Forty eight genotypes of pea clustered in three main groups A, B and C comprising 23, 5 and 20 genotypes, respectively. Group A1 and C1 included the most distantly related genotypes and hence can be recommended for breeding to obtain genetically diverse segregating populations.
S M Masiul Azam, Md Shahidul Islam, Parvin Shahanaz, Md Shafiqur Rahman and Sarder Md Shahriar Alam. “Molecular Characterization of Brassica Cultivars through RAPD Markers” United International Journal for Research & Technology (UIJRT) 1.3 (2019): 41-45.
Development of biotic stress resistance technologiesMamtaChoudhary75
This ppt is regarding the topic 'Development of biotic stress resistance technologies'. in it, I've discussed biotic stress, effects on crop production, disease and insect resistance mechanisms, case studies, etc.
Genetic diversity in pea germplasm using RAPD MarkersShujaul Mulk Khan
Selection of the genotypes using plasmid assisted technology provides an efficient and useful tool for elaborating genetic relationships among genotypes. In present study, 48 Pea (Pisum sativum var sativum L.) genotypes obtained from different sources were analyzed through 20 RAPD, DNA markers for assessment of intraspecific DNA variations. Results revealed that significant variations were present in minor bands. Major bands also showed significant diversity. Considerable variations were also recorded in density of some common bands. Maximum and minimum genetic diversity i.e., 80% and 20% was found among 08 and 23 comparisons, respectively from banding profile. These variations can be
used further for enhancing variability, a prerequisite for crop breeding. Phylogenetic clustering (through dendrogram analysis) of genotypes revealed that genetic diversity is independent of origin of genotypes. Forty eight genotypes of pea clustered in three main groups A, B and C comprising 23, 5 and 20 genotypes, respectively. Group A1 and C1 included the most distantly related genotypes and hence can be recommended for breeding to obtain genetically diverse segregating populations.
S M Masiul Azam, Md Shahidul Islam, Parvin Shahanaz, Md Shafiqur Rahman and Sarder Md Shahriar Alam. “Molecular Characterization of Brassica Cultivars through RAPD Markers” United International Journal for Research & Technology (UIJRT) 1.3 (2019): 41-45.
25. comparative study of genetic variations as determined from marker systemsVishwanath Koti
Tomato (Solanum lycopersicum L.) is most important Solanacous vegetable grown worldwide for
its edible fruits. Various marker techniques have been successfully applied, either individually or in
combination to study the genetic diversity of this crop. A Study to assess the usefulness of different
markers system for analyzing the genetic diversity and relation between different varieties and to find out
correlation between marker systems revealed that all tested tomato cultivars could be differentiated from
each other based on either morphological/protein/RAPD markers individually, and can be applied for
grouping of cultivars, pedigree analysis and genetic diversity analysis. However, markers system used in
this study showed variations in understanding the genetic relation between studied varieties.
Maize (Zea mays) is cultivated all over the world including Pakistan for fodder and grain. Genetic diversity and environmental effects greatly affect the fodder yield and quality in maize. Therefore, it is imperative to assess the genetic diversity among 31 maize genotypes for fodder and quality related traits. Genotypes were collected from the Australian Grain Gene Bank and grown under field conditions with three replications following randomized complete block design. The morphological traits such as plant height (cm), leaf area (cm2), leaf-stem ratio (wt. basis), stem girth (cm), leaves plant-1, days to 50% silking, days to 50% silking, leaf moisture (%), dry fodder yield plant-1 (g), green fodder yield plant-1 (g) and quality traits such as crude protein (%), ether extract (%), ash content (%), crude fiber (%) and nitrogen-free extract (%) were recorded.
Analysis of variance, biplot analysis and genotypic and phenotypic correlation were performed. Analysis of variance revealed significant differences with days to 50% silking, days to 50% tasseling, plant height, stem girth, leaf area, leaves plant-1, moisture percentage, green and dry fodder yield, crude protein, crude fiber, ether extract, ash content and nitrogen free extract and non-significant differences with leaf stem ratio. At phenotypic and genotypic level, dry fodder yield plant-1, plant height, stem diameter, leaf moisture %, No. of leaves, days to 50% silking, crude fiber and ether extract revealed significant correlation with fodder yield plant-1.
Biplot analysis based on PCA for different quantitative parameters showed that first two principal components i.e F1,F2 are contributing 23.29% and 14.53 % to the total variations respectively. Based on all the results the best genotypes were DTMA-271, DTMA-15, DTMA-281 and DTMA-295 could be used in breeding programs.
Molecular Diversity Analysis of Some Local Ginger (Zingiber officinale) Genot...AI Publications
Ginger (Zingiber officinale) rhizomes have been widely used as a spice and flavoring agent in foods and beverages in Bangladesh as well as in all over the world for its economical and medicinal values. The present investigation was undertaken for the assessment of 13 local ginger genotypes collected from different region of Bangladesh through 7 RAPD primers. Genomic DNA was extracted from ginger genotypes using CTAB method. A total of 34 distinct and differential amplification bands ranging from 150-1200 bp were observed with an average of 1.14 polymorphic bands per primer. The overall gene diversity was detected 0.8052 and the value of PIC was detected 0.7532. The RAPD marker generate enough polymorphism for possible use in diversity studies through cluster analysis and principal component analysis (PCA). PCA classified 13 ginger genotypes into four groups and showed in two dimensional scatter plot. The genetic similarity coefficients among genotypes ranged from 0.103 to 0.654. Cluster analysis based on Jaccard’s similarity-coefficient using UPGMA grouped the genotypes into two clusters: Cluster A and Cluster B. The cluster ‘A’ had only one genotype Kaptai local and the second cluster ‘B’ had rest of twelve genotypes. The prevalence level of polymorphism in the local genotypes of ginger will help to breeders for ginger improvement program.
Principal Component Analysis for Evaluation of Guinea grass (Panicum maximum...Agriculture Journal IJOEAR
Abstract— The present study was conducted to study the variability among the genotypes by Principal Component Analysis (PCA) in order to select those that are most suitable for breeding programme. This study included ten quantitative traits. The result of principal component analysis showed that the first four principal components with Eigen value greater than 0.88 contributed about 76.10 per cent of total variation in the population. The variability of the genotypes was interpreted based on four principal components, the first principal component described the yield level, the second principal component described the productivity and quality and the last two principal components described the quality of the fodder which indicating that the identified traits within the axes exhibited great influence on the phenotype and this could be effectively used for selection among the tested entries for further development of Guinea grass varieties with improved fodder yield and quality.
Heritability and genetic advance in F5 segregating generation of Tomato (Sola...Open Access Research Paper
The present study investigated the yield and its contributing attributes among F5 segregating tomato lines so as to find degree of genetic variability, heritability, and genetic advance. This research study was conducted using a randomized complete block design (RCBD) during season 2018-2019 at Agricultural Research Station Swabi, Khyber Pakhtunkhwa. The experimental material (23 segregating lines and 2 parental genotypes) were characterized for morphological days to first flowering, days to fruiting, plant height, stem diameter, cluster per plant, flowers per cluster, fruits cluster-1, fruits per plant, yield hectare-1. Analysis of variance regarding morphological attributes showed highly significant differences (P≤ 0.01) among tomato F5 segregating lines. Minimum days to first flowering and days to fruiting were recorded for ST-12, ST-14, ST-17 with values of (50.00), (78.33) each, respectively. Maximum plant height, stem diameter, clusters per plant, flowers per cluster, fruit per cluster, fruits per plant, single fruit weight were observed for ST-20, ST-17, ST-12, ST-21, Roma, ST-12, ST-8, Roma with values of (105.38), (1.69), (29.33), (6.18), (6.00), (150.27), (81.41). Very little differences were observed between phenotypic coefficient of variation and genotypic coefficient of variation for all traits except cluster plant-1 and fruits plant-1 indicating that most of the traits were less influenced by environmental factors for their phenotypic expression. All traits had high h2 but only fruit plant-1 (0.37), single fruit weight (0.58), yield ha-1 (0.39) were found to be moderate and clusters plant-1(0.12) had low h2. Low genetic advance (20.0) was recorded for all traits except yield. Moderate to low genetic advance suggests the action of both additive and non-additive genes and favorable influence of environment in the expression. Desired morphological characterization on the basis of the yield attributing traits to fruit yield showed these lines ST-1, ST-2, ST-4, ST-5, ST-6, ST-7, ST-9, ST-11, ST-12, ST-14, ST-17, ST-18, ST-19, ST-21, could further be used for the development of improved varieties in future tomato breeding program.
Assessment of Genetic Diversity in 13 Local Banana (Musa Spp.) Cultivars Usin...paperpublications3
Abstract: A Study was conducted to investigate the genetic variability among 13 local banana cultivars using 3 SSR primers of Mb1-69, Mb1-113 and Mb1-134. All the primer pairs amplified a total of 29 different marker bands with an average of 9.6 bands per primer. Among the 29 bands only 4 bands were monomorphic and the rest 25 bands were polymorphic. The sizes of the amplified DNA bands in 13 local banana cultivars varied from 200 bp to 600 bp. The primer Mb1-113 amplified the highest (14) number of DNA bands and the primer Mb1-69 amplified the lowest (7) number of DNA bands whilst primer Mb1-134 amplified 8 DNA bands. The values of pair-wise genetic distances ranged from 1.00 to 9.00 indicating the presence of wide genetic diversity. The dendogram constructed based on phylogenetic relationship analysis revealed that the highest genetic diversity (9.00) found between the cultivars champa and jawayta and also the cultivars champa and jahazy whilst the lowest (1.00) between the cultivars doubled haploid and kathaly, doubled haploid and sorishafruity, doubled haploid and amritsagor and doubled haploid and ganasundory. The UPGMA dendogram has segregated the 13 local banana cultivars into two major clusters. Agnishwar and champa formed in cluster 1 and the rest of the cultivars like sobri jesore, sobri, anazy, kathaly, jawayta, sorishafruity, amritsagor, jahazy, bangle, ganasundory and doubled haploid have constituted the cluster 2.
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Genome wide association studies (GWAS) analysis of karnal bunt resistance in ...Innspub Net
Karnal bunt (KB) disease is one of the most important challenges posed on of wheat (Triticum aestivum L.) industry of Pakistan because of itsinclusionin quarantine list around the globe. This disease is caused by the fungus Tilletia indica M. (Neovossia indica). It affects the grain quality of wheat and hampers its movement in international market resulting in economic losses. Presence of >3% infected grains in wheat lot makes it unsuitable for human consumption. Eradication of this disease is very difficult as no resistant cultivar has been found against KB in Pakistan so far. Genome wide association study (GWAS) was conducted on a set of 199 wheat germplasm collected from Pakistan. In this study 31,000 single nucleotide polymorphism markers were developed by 90K SNP array technology. A linear mixed model in GWAS, accounting for population structure, was fitted to identify significant genomic regions [-log(P) ≥ 4.0] on 6 different chromosomes i.e. 1A, 1D, 2D, 3B, 4A, 5A with novel loci. Candidate genes, through wheat genome assembly, were identified as putative genes related to KB resistance including kinase like protein family. The results of this study can be useful in wheat breeding through marker assisted selection for KB resistant varieties.
Detection of Genetic variation in tissue culture clones of date palm using IS...IJSRD
Date palm is a plant having high nutritional value and long life (yielding up to 100 years). Phoenix dactylifera requires 2-5 males for pollination of 100 females’ plant depending up on genetic and environment factors. Therefore paternity variation expected to very low according to PCR based techniques, Even though we have tried to find out genetic variation among tissue culture cloned plant. Tissue culture technique can be used for genetic improvement of date palm. The main purpose of this study was to evaluate the genetic variation in the tissue culture clones of date palm by using ISSR primers among mother and it’s two clones. The plant DNA was extracted and subjected to detection of genetic variation in two groups of date palm using ISSR primers. In this study ISSR primers produced monomorphic bands within group-1 and group-2. Genetic variation in tissue culture clones of date palm was not detecte by UBC primer series.
Incidence and toxigenicity of fungi contaminating sorghum from NigeriaPremier Publishers
Each Agro ecological zone was transversely delineated into 5 districts and five villages (at least 20 Km from each other) called “locations” were selected in each district. In each district, Sorghum grains in stores, bunches in the field and sorghum grains in the market were sampled from five locations, each approximately 20 km from the previous sampling location. The mycological analytical procedures were performed under aseptic condition. Plates were counted for fungal colonies using a colony counter and the number of fungal colonies per gram of sample was calculated as CFU/g. The fungi species were isolated and subsequently identified using MEA/CYA media for Aspergillus and Penicillium species and PDA for the fusarium species Toxigenicity studies on strains representing species of Aspergillus, Penicillium, Fusarium was carried out to determine their ability to produce aflatoxin B1 (AFB1); aflatoxin B2 (AFB2); aflatoxin G1 (AFG1); aflatoxin G2 (AFG2); OTA, ZEN, DON and FB1. A total of 701 isolates were recorded which consist of 67 confirmed fungal strains. Aspergillus species formed the majority with 346(49.6%) followed by the Fusarium species with 186(26.7%) then Penicillium species with 102(14.6%) while others such as Cuvularia, Phoma, Alternaria, Rhizormucor constitutes 67 (9.0% )strains of the total population.
Evaluation of seed storage proteins in common bean by some biplot analysisINNS PUBNET
In order to study of seed storage proteins, proteins samples of common bean genotypes were prepared by 0.2 M
NaCl of extracting soluble. Genotypes were located in two groups by cluster analysis using Wilks’ lambda
statistic. Two groups were different for yield components (number of pods per plant, number of seeds per plant
and seed weight). Factor analysis showed that two factors described 61% of total proteins variation. Correlated
bands with yield components characters had the highest coefficients for the first factor. This factor was named
“yield components proteins”. Protein bands via RM 58 and 64 had relationship with days to flowering.
Therefore, the second factor was named “phenologic proteins”. Genotypes were located in four groups by these
factors. Length, angle and presence of protein bands were important characteristics to explain graphical
information in GGE biplot compared to factor analysis. Get the full articles at: http://www.innspub.net/volume-3-number-5-may-2013/
JMeter webinar - integration with InfluxDB and GrafanaRTTS
Watch this recorded webinar about real-time monitoring of application performance. See how to integrate Apache JMeter, the open-source leader in performance testing, with InfluxDB, the open-source time-series database, and Grafana, the open-source analytics and visualization application.
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Length: 30 minutes
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- What out-of-the-box solutions are available for real-time monitoring JMeter tests?
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GraphRAG is All You need? LLM & Knowledge GraphGuy Korland
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Your campaign sent to target colleagues for approval
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22. utilization of ssr markers for seed purity testing in popular rice hybrids
1. ISSN: 2287-688X
Annals of plant sciences
Issue 1; Volume 1(November);1-5
*Corresponding Author:
Dr. Vishwanath K,
Department of Seed Science and Technology,
University of Agricultural sciences,
GKVK, Bangalore-65 Page| 1
Utilization of SSR Markers for Seed Purity Testing in Popular Rice Hybrids
(Oryza sativa L.)
MR Chetan Kumar1
, Vishwanath K1
*, Shivakumar N2
, Rajendra Prasad S1
, Radha BN1
and Ramegowda1
1
Department of Seed Science and Technology, 2
Department of Genetics and Plant Breeding,
University of Agricultural sciences, GKVK, Bangalore-65
Received for publication: September 05, 2012; Accepted: October 28, 2012.
Abstract: Microsatellite markers were used for fingerprinting of hybrids, assessing variation
within parental lines and testing the genetic purity of hybrid seed lot in rice. In this study 35
simple sequence repeats (SSR) markers were employed for fingerprinting 2 popular rice hybrids
and their parental lines. Six SSR markers were found polymorphic across the hybrids and
produced unique fingerprint for the 2 hybrids. A set of five markers (RM 206, RM 276, RM 204, RM
234 and RM 228) differentiated the 2 hybrids from each other, which can be used as referral
markers for unambiguous identification and protection of these hybrids. The analysis of plant-to-
plant variation within the parental lines of the hybrid KRH-2 and DRRH-2, using informative
markers indicated residual heterozygosity at two marker loci. This highlights the importance of
SSR markers in maintaining the genetic purity of the parental lines. To utilize these SSR markers
effectively for detection of impurities in hybrids, a two dimensional DNA sampling strategy
involving a 20 × 20 grow-out matrix has been designed and used for detection of contaminants in
a seed-lot of the popular Hybrids viz., KRH-2 and DRRH-2. The validation of the identified markers
was done with conventional grow out test (GOT) and they were comparable with the GOT.
Key words: Hybrid Rice, Molecular Markers, Identification, Genetic purity
INTRODUCTION
Rice is the staple food for a large segment
of the Asian population. It has been estimated that
rice production in India as well as several other
Asian countries must double by the year 2025 to
meet the requirements of the increasing population
(Hossain, 1996; Paroda, 1998). A self-pollinated
crop like rice, one of the challenges is the
production and supply of adequate quantities of
pure seeds to the farmers. And maintenance of
high level genetic purity of hybrid seeds and it is to
exploit the moderate level of heterosis in this crop.
It is estimated that for every 1per cent impurity in
the hybrid seed, the yield reduction is 100 kg per
hectare. Thus, there is a need for an assay to
assess genetic purity of seeds that is both accurate
and faster, so seed produced in the dry season can
be released for commercial cultivation in the
ensuing wet season.
The genuineness of the variety is one of
the most important characteristics of good quality
seed. Genetic purity test is done to verify any
deviation from genuineness of the variety during
its multiplications. Genetic purity test is
compulsory for seed certification of all foundation
and certified hybrid seeds. Higher genetic purity is
an essential prerequisite for the commercialization
of any hybrid seeds. Besides, success of any hybrid
technology depends on the availability of quality
seed supplied in time at reasonable cost. The
genetic purity during multiplication stages is prone
to contaminate due to the presence of pollen
shedders, out crossing with foreign pollens etc.,
besides physical admixtures. Thus use of seeds
with low genetic purity results in segregation of the
traits, lower yields and genetic deterioration of
varieties.
Traditional GOT based on morphological
markers are time consuming and are
environmental dependence. To overcome this
disadvantage, the molecular markers are being
used in many of the crops. However, due to
repeatability of the results and accuracy of the
obtained results are under question. This made a
way for use of molecular markers particularly the
co-dominant markers. The SSR markers are of
great importance for rapid assessment of hybrid
and parental line seed purity (Yashitola et al.,
2002, Antonova et al., 2006 and Pallavi et al.,
2011).
The primary objective of the present study
was to identify the public sector bred Indian two
popular rice hybrids, viz., KRH-2 and DRRH-2
together with their parental lines developed
recently, using SSR markers, to provide the DNA
fingerprint for these rice hybrids and their parental
lines, and to establish the basis for identification
and monitoring of seed purity for these hybrid rice
combinations.
MATERIALS AND METHODS
Plant materials:
For the purpose of molecular identification,
2 public sector rice hybrids viz., KRH-2 and DRRH-
2 released for commercial cultivation in different
parts of India and their parental lines were
selected for this study. The F1 seeds of KRH-2 and
their parental lines IR-58025A (the sterile female),
IR-58025B (the maintainer), KMR-3R (the
restorer) were obtained from Division of hybrid
rice, Zonal Agricultural Research Station, VC Farm,
Mandya and DRRH-2 and their seeds of parental
lines IR-68897A (the sterile female), IR-68897B
2. Chetan Kumar et al.:
Annals of plant sciences, 2012, 01 (November), 1-5 Page | 2
(the maintainer), DR 714-1-2R (the restorer) were
obtained from the concerned breeder, Directorate
of Rice Research, Hyderabad. For the purpose of
molecular identification seeds of above mentioned
hybrids were germinated in aseptic condition
grown in greenhouse, NSP, UAS, Bangalore. A
random sample of 400 seeds of KRH-2 and
DRRH-2 representing the commercial F1 seed lot
was used for testing their genetic purity. Out of
400, 30 randomly drawn F1 seeds were used for
marker analysis and others were used for
Grow-out test (GOT). The GOT was conducted at
National Seed Project, University of Agricultural
Sciences, GKVK, Bangalore during Rabi-2009.
Molecular analysis:
Plant DNA was isolated from seedlings
using CTAB (Van der Beek et al., 1999) protocol as
follows: about 0.1 g of young leaf tissue for each
sample was homogenized in liquid nitrogen, and
incubated at 600
C for 30–45 min with 500 μL of
CTAB buffer (1.0M pH 8.0 Tris-HCL, 3ML NaCl, 0.5
EDTA, 1% PVP-360). Then 500μL 24:1 of
chloroform: isoamyl alcohol mixture was added
and blended thoroughly for 5min. After
centrifugation (5 min, 13 000 rpm), aqueous layer
was pipetted into a new eppendorf tube and an
approximately equal volume of cold ethanol was
added. After storage at -20 0
C for 30–60 min,
precipitated DNA was centrifuged, vacuum dried
and finally stored in TE buffer.
For fingerprinting DNA from the bulk leaf
samples of 2 to 5 individual plants was used.
Quantification of DNA was accomplished by
analyzing the DNA on Nano Photometer analyzer
and also on 0.8 % Agarose gel using diluted uncut
lamda DNA as standard. DNA was diluted in TE
buffer to a concentration of approximately 25ng/μL
for PCR analysis. The sequence information for the
primer pairs was obtained from the publications of
Wu & Tanksley (1993), and Temnykh et al. (2000)
from sequence information obtained from DNA
libraries and published sequence data
(www.gramene.org). A total of 35 hyper variable
SSR primer pairs distributed across the 12
chromosomes were used for PCR amplification.
PCR amplification:
Thirty Five SSR primer pairs were selected
for this study. PCR was performed in a volume of
the reaction mixture was 20 μL containing of 30ng
of template DNA, 1 x PCR buffer with 1.5mM of
MgCl2, 0.2 mM of each dNTPs, 10 pmol of each
primers and 1U of Taq DNA polymerase. PCR was
carried out in a Thermal Cycler was used and
programmed for 35 cycles of 950
C (5 min), 940
C (1
min) 560
C (30 Sec.), 720
C (1 min) then followed
by final-extension at 720
C for 5 min. PCR products
(10–15 μL) were used for electrophoresis and the
amplicons were resolved on 1.5 % agarose gel
stained with ethidium bromide at 1 μg/mL, and
visualized under UV in a gel documentation system
and impurities were identified based on deviations
in expected amplification pattern.
Grow-out trials:
The parental and commercial seeds of the
two F1 hybrid cultivars were grown as 20 × 20
Grow-out matrix in National Seed project research
plots during Rabi-2009 with all the agronomic and
plant protection measures were adopted as per
recommended package of practices for raising a
healthy crop. Genetic purity visual evaluation was
conducted based on the main important
morphological characters thought the growth
period.
RESULTS AND DISCUSSION
Characterization and identification of
cultivars are crucial to varietal improvement,
release and in seed production programme. It is
mandatory to maintain the genetic purity of hybrid
seed for the successful crop production.
Unambiguous characteristic pattern of hybrids can
be obtained using DNA markers and had been
termed as DNA fingerprinting. The use of DNA
markers to obtain genotype specific profiles had
distinct advantages over morphological and
biochemical methods. The morphological markers
are influenced by the environmental conditions,
labour intensive and time consuming. However,
the biochemical markers such as isozyme and
protein patterns are least influenced by the
environment but exhibit limited polymorphism and
often do not allow discrimination between closely
related inbred lines (Lucchese et al., 1999). DNA
markers overcome most of these disadvantages of
morphological and biochemical markers that can
be useful to distinguish varieties and off types. The
usefulness of DNA fingerprinting technique for
cultivar identification was demonstrated by Dallas
(1988) for the first time in rice.
The present study utilized the SSR marker
techniques for identification of two Rice hybrids
along with their parental lines, demonstrating that
this technique can be successfully applied to
distinguish and identify the hybrids from its
parental lines. SSR had much more polymorphism
than most of other DNA markers, and is co-
dominant and large in quantity. Therefore, the high
polymorphic information content (PIC) of SSR had
promoted the application of microsatellites as
molecular markers in fingerprinting (Ashikawa et
al., 1999).
In this study primer pairs of 35 SSR’s
primer pairs associated with each hybrid and
parental lines were assessed on 1.5 to 2.00 per
cent agarose. The PCR products of the DNA
samples on the agarose did appeared and showed
polymorphism among the hybrids and their
parental lines.
Among the 35 primers studied, ten primers
viz., RM 234, RM 206, RM 276, RM 219, RM 216,
RM 209, RM 204, RM 228, and RM 335 showed
polymorphism between the parental lines which
were used for the production of rice hybrids KRH-
3. Chetan Kumar et al.:
Annals of plant sciences, 2012, 01 (November), 1-5 Page | 3
2and DRRH-2 and rest of primers showed
monomorphic banding pattern (Fig. 1). The male
sterile line IR-58025A of hybrid KRH-2 and its
restore line KMR-3R amplified an allele of size
120bp-140bp, while DRRH-2 CMS line IR-68897A
and its restorer line DR 714-1-2RR did amplified
the allele of size 100 to 210bp.
Figure.1: SSR markers profile showing the monomorphic
banding pattern of rice hybrids and their parental lines.
Lane 1A: IR-58025A, Lane 1B: IR-58025B, Lane 1R:
KMR-3R, Lane 1H: KRH-2, lane 2A: IR68897A, Lane
2B:IR68897B, Lane 2R: DR 714-1-2R, Lane 2H: DRRH-2
The two rice hybrids studied viz., KRH-2
and DRRH-2 were able to distinguish from their
parental lines using a specific SSR marker. Based
on the complementary banding patterns between
the hybrids and their parents the SSR marker RM
206, RM 234 and RM 276 were identified as the
three specific markers to distinguish F1 hybrid
KRH-2 form their parental lines (fig.2 & 3).
(a)
(b)
Figure 2: Polymorphic SSR markers profiles confirming
hybridity of KRH-2 obtained with (a) RM 206 and RM 234
(b) RM 276 and RM 234. M=Marker 100bp ladder,
A=IR-58025A,
B= IR-58025B, R= KMR-3R, H= KRH-2.
The RM 206 amplified a specific allele of
size 130 bp in F1 KRH-2; seed parent IR-58025A
and in its maintainer line but not in pollen parent
(KMR-3R). While the allele size of 120 bp was
absent in pollen parent. The same SSR marker RM
206 had amplified allele of size 130bp in restorer
parent (KMR-3R) which had restored the fertility in
male sterile parent. The same allele size of 130 bp
has also appered in F1 hybrid but not in female
parent (IR-58025A). Thus, it confirmed that the
allele of size 130 bp is very specific to the pollen
parent.
Thus, presence of both female and male
parent alleles was observed as a resultant of
crossing between two parents (F1 hybrid). This
confirmed the crossing and hybridity between two
parents. The appeared banding pattern is highly
specific to the KRH-2 and not observed DRRH-2
hybrid. Similarly RM 234 maker had also resulted
in amplifying allele of size 120 bp in female parent
(IR-58025A) and maintainer line (IR-58025B),
which was absent in pollen parent (KMR-3R).
While, the pollen parent had an amplicon at 130bp
which was absent in female parent (IR-58025A).
However, the F1 hybrid exhibited both the alleles of
the parents confirming the heterozygosity
condition of the hybrid by having bands at 120 and
130bp. The identified SSRs in F1 hybrids showed
complementary banding pattern of both parents. It
was valuable to distinguish the F1 from their male
and female parents.
Similarly, the newly released hybrid rice
DRRH-2, could be identified and distinguished by
the SSR marker RM 204, RM 234 and RM 228. The
marker RM 234 had amplicon of size 120bp size in
its female parent (IR-68897A) and its maintainer
line (fig. 3). The same marker had another
amplicon of size 130bp in pollen parent (DR 714-1-
2R).
(a)
(b)
(c)
Figure 3. Polymorphic SSR markers profiles confirming
hybridity of DRRH-2 obtained with (a) RM 234 (b) RM
204 (c) RM 228 and RM 335. M=Marker 100bp ladder,
1A=IR-68897A, 1B= IR-68897B, 1R= DR 714-1-2R, 1H=
DRRH-2, 2A=IR-58025A, 2B= IR-58025B, 2R= KMR-3R,
2H= KRH-2.
The banding pattern of this hybrid showed
both the amplicon at 120bp and 130bp. Thus it is
4. Chetan Kumar et al.:
Annals of plant sciences, 2012, 01 (November), 1-5 Page | 4
confirmed the genuine crossing and heterozygotic
condition of the hybrid. The SSR markers identified
had both female and male specific bands and are
useful in genetic purity testing. These markers
have an advantage of co-dominance inheritance,
easy scoring of the alleles, reproducibility and
accessibility to laboratories (Paniego et al., 2002).
The use of SSR markers for genetic purity testing
has been demonstrated in maize (Wang et al.,
2002); in rice (Nandakumar et al., 2004); in
sunflower (Pallavi et al., 2011).
A cytoplasmic male-sterile (CMS) system is
desirable for use in hybrid seed production as it
eliminates the need for hand emasculation. CMS is
a maternally inherited plant trait characterized by
the inability of flowers to produce viable pollen but
without affecting the female fertility and it is often
associated with mitochondrial DNA rearrangement,
mutation, and editing. CMS lines were multiplied
with adequate isolation distance leaving no scope
for a biological contamination through foreign
pollens coming from nearby Rice fields. Under such
circumstances, the only impurity that can be
expected in CMS line seed lot that comes from its
maintainer line probably could be as mechanical
admixture during various stages of seed handling
of CMS lines.
An attempt was made to distinguish CMS
lines (male sterile) used for the hybrid seed
production from its maintainer line (male fertile)
using SSR markers. The DNA samples of CMS lines
and its maintainer lines were amplified with primer
pairs and resolved in agarose gel (2 %). The
primers pairs studied did not show good
polymorphism between A lines and B lines.
However, the banding pattern could not able to
distinguish CMS A line from its maintainer CMS B
line. In Rice the cytoplasmic male sterility is
caused by a mutation in the mitochondrial genome
(mtDNA) and male sterile and male fertile Rice
lines differ in a 17 kbp fragment only (Korell et al.,
1992). This had resulted in isogenic lines and
differs only in one gene for pollen fertility. The
gene responsible for pollen fertility is present in
the mitochondrial genome and need to isolate for
distinguishing male fertile form male sterile line
(Begona et al., 2005).
Field performance of the rice hybrid
individuals:
In the Grow-out trials, purity evaluation
was conducted based on morphological traits
including plant height and days to maturity, pollen
sterility, and presence of panicle awns, panicle
exertion, panicle length, nodal pigmentation and
flag leaf senescence. The characters of few
individuals shown deviation from the standard
characters were identified as off-type and they
were similar to those of the male parental type,
which was also supported by the molecular marker
testing.
The results of the field grow-out test (GOT)
and SSR marker test were comparable. For
comparison of GOT test results with molecular
GOT, leaf sample collected by random sampling
were evaluated by using primer ‘RM 206’ for KRH-2
hybrids and RM 234 for DRRH-2 hybrids revealed
100, 93.34 and 91.43 per cent of off-types which
were validated with the theoretical purity (Field
GOT) per cent of 100, 95 and 90 per cent,
respectively. In this study, the validation of the
identified markers was done with conventional
grow out test (GOT) and they were comparable
with the GOT.
Figure.4: Genetic purity testing of KRH-2 hybrid seeds
using the SSR marker RM 206 L= 100 bp ladder, Lane 1-
23 = individual F1 plants representing a random sample
from hybrid seed lot of KRH-2. Lane 7 and 8 (arrow)
represents an off-type, a contaminant.
Figure.5: Genetic purity testing of DRRH-2 hybrid seeds
using the SSR marker RM 234; L= 100 bp ladder, Lane
1-18 = individual F1 plants representing a random
sample from hybrid seed lot of DRRH-2.
The present study showed that SSR
markers are quick, effective and results are
generally consistent with morphological analysis in
the field study. Primers identified in the study
could be utilized for routine genetic purity testing
of KRH-2 and DRRH-2 hybrids. The SSR marker
information developed through this study will be of
immense help for hybrid rice seed industry to
select appropriate marker combinations and assess
genetic purity of the crop.
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Source of support: Nil
Conflict of interest: None Declared