Title of Invention	A NOVEL DNA MARKER FOR DROUGHT TOLERANCE IN PLANTS
Abstract	A novel DNA marker for drought tolerance in tea plants comprises of an amplified fragment 1400bp generated by using primer AP OPAHO2.

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Field of Invention This invention relates to a novel DNA marker for drought tolerance in plants Beckground of the Invention In plants DNA markers are conventionally developed using homogenous mapping population. This is impractical where the repreductive phase cycle is long- Developmant of trail-ralated DNA marker in planta have been done in homogenous breeding populations (real leoganic lines-RIL/near isoganic Iines- NIL) focucing on trait-related physiological parameters. This ia a time consuming process, taking many years for development of RILs and NILs. Further correlation with any one phenotypic parameters often would not give a DNA marker that could ba directly correlated with a quantitative trail. Most of tha markers for drought tolerance known till data exhibit correlation with one or another physiolosical parameter associated with the trail. The different phenotypes focused on are ueually ecosystem spacific and thus would not be useful for rapid and precise assessment/screening gamplasm in laboratory plants. Objects of the Invention An object of this invention is to establish a novel DNA marker for drought tolerance in plants. A further object of this invention it to propose a DNA marker for precise selection in the laboratory of plants for ecosystem specific cultivation. A still further object of this invention is to propose a DNA marker for developing improved varieties through plant breeding for drought tolerance. Another object of this invention is to propose a DNA marker for rapid diagnosis of drought tolerance trail in plants. Still another object of this invention is to propose a DNA marker useful in identification of genes for plant transformation in developing improved varieties. Brief description of the invention According to this invention there is provided a novel DNA marker for drought tolerance in plants comprising of an amplified fragment of 1400bp. In accordance to this invention there is provided a method for developing a DNA marker for drought tolerance comprising extracting ganomic DNA from frozen young leaves, subjecting the DNA thus obtained to the step of calibration, subjecting tha calibrated DNA to the step of analysis. Clening tha analyzed DNA fragment sequencing tha DNA fragment to obtain tha DNA marker. Detailed description of the invention: Tha process undertaken to establish this DNA marker to novel in that it developed through correlation with trail-specific precise and rapidly accessed biochemical marker. With the use of this novel, non-obvious, inventive process, a DNA marker for drought tolerance has been established. GENOMIC DNA EXTRACTION: 200mg of frozan young leaves were used for DNA isolation. Tha CTAB (cetyltrimethylemmonium bromide) method was employed to extract DNA and on an average 20μg of DNA was obtained. After agarose gel electrophoresis, DNA was calibrated on tha basis of ethidium bromide fluorescence using defined amount of DNA at standard RAPD STUDIES For Randomly amplified polymorphic DNA (RAPD) analysis, DNA of young leaves from each clone was used as template in the PCR reaction nature of total volume 25μl, conteining 26 μm dNTPs, 40ng primer and 0.3μl Taq DNA polymerase. The reaction was allowed for 45 cycles under 36° C annealing temperature. PCR products obtaimed using eleven random decamer primers such at OPAB06, OPAB18, OPAH18, OPAH02, OPAB17, OPAC19, OPAH12, OPAL04, OPAA02, OPAG13 and OPAL08, were separated on 1.2% agarose gel and visuallized over UV Iight in pretence of ethidium bromide. RAPD analysis of randomly selected plants within each clonal propagation confirmed the genetic uniformly of plants within each clonal plot (data not pretented). For use in the present study a comparative analysis obtained short listed (on the basis of drought tolerance / susceptibility) clones were undertaken. For use in the present study a comparative analysis of 10 short-listed (on the basis of drought tolerance / susceptibility) clones was undertaken. This RAPO study was repeated (for each set of 10 clones), with leaves collected from randomly selected plants within each clonal plot. Representative gel images of RAPO pattern to shown in Figure 1a. Figure 1b shows the RAPD pattern with the tame primer on genomes of 4 clones (that are also present in Figure 1a) that exhibit drought tolerance in the field (Figrue 1b was not used for scoring RAPD bands). Primers used In the RAPD study, total number of RAPD bands observed through all the 10 clones, total number of monomophic / polymorphic bands present and percentage of polymorphic bands obtained are given in table 1. Stress related bozyme activity: Detached (water stressed), young tea leaves were extracted in extraction buffer and the extracts were run on non-denaturing gets. Activity of enzymes was studied by allowing the reaction of iso-enzyme in gets by immersing the gel in appropriate reaction mixture, followed by stopping enzyme activity by immensing gel in appropriate staining solution. SOD and APX activity: Isolated leaves were homogenized in 50mM Pottasium phosphate (pH-7.8) buffers for SOD (EC 1.15.1.1) and in 100mM Sodium Phosphate (pH. 7.8) buffer for APX (EC 1.11.1.11). Enzyme activities in the extract was visualizad on non- denaturing activity gel (12% polyacrylamide) after electrophoretic separation folowed by immesing the gel in appropriate staining solution. Elecrophoretically separated SOD isozymes were visualized by reduction of Nitro Blue Tetrezolium (NBT) by O2 generated in reaction between N,N,N1,N1-tetra methylethylem diamine (TEMED) and Riboflavin. Bands representing the isozymes remain colorless due to absence of O2 that had been scavenged by isozyme activity; rest of the gel was stained blue due to reduced NBT. Doneltometric scant (at 992.8 nm) of negatively stained isozyme activity bands is shown in Figure 2a. The APX activity gal staining was similar accepting that insteed of ribeflavin, H2O2 and Atcorbate were used to ganarate O2. Negative staining of isozyme bands is documantad in a gal doc apparatus (image Master VDS, Phannacis Biotech). The negatively stained isozoma activity bands scanned at 632.8 nm; a rapresentative picture of the denstiometric scans is shown in Figura 2b. Ragrassion analysis was conducted using data of isozyme study (of SOD and APX) and RAPD bands. Association of RAPD markers with 800 activity and APX activity were investigated using multiple ragrassion analysis approach. Enzyme activities viz. of appropriate Suboxide dismuiase (800) and APX isozyme (peak area) were treated as dapandant variables and the various RAPD bands (Scored as 1 for presence of band and 0 for absence) were treated as independent variables. The regression analysis was based on the modal Where Y to the enzymatic tralt, in the RAPD marker, b the partial regression coefficient, d the between accesaion residual which is left after regression. Extant of association batween tha RAPD bands and copperzine superoxide diamutase (Cu-Zn SOD) and Atcorbate Poroxidase (APX) (band II) activites are shown in Table2. Tha sequence for this marker is being submitted for patenting. The marker could be utilized for selection of appropriate genotypes (a) for ecosystem specific cultivation (b) for developing improved varieties through plant breeding/plant transformation endeavours. EXAMPLE Taa clones, reported to be tolerant /sensetive to drought (on tha basis of field performance) where selected for this study • Tea clones of Darjeeling used in establishing (through RAPO and isozyme studies correlated with information on field studies) a precise DNA marker in the present study use: RR17/144, T246, CPI, TV26, TV 25, B777, AV2, K1/1, B688, T78 (all Darjeeling tea clones). • Tea clones used for validating (through RAPD studies correlated with field studies) that the established DNA marker is associated with drought tolerance in other tea clones viz the Assam tea clones: TV1, TV2, TV14, TV17, TV20, TV30 (All Assam tea clones) Leaves from plants of each of the clones listed above were collected from Ging Tea Estate, Darjeeling, and from Tea Research Association (TRA), Assam. For enzyme studies, freshly detached leaves (from the Darjeeling clones) were used for extraction within 24h. Tea plants art perennial and have long reproductive cycles. Thus for rapid avaiblebility of uniform raw material (i.e. fresh tea leaf) vegetatively propagated tea clones constitute the plant population for use in the tea industry. In such populations, linkage study through final generations is time consuming. Under such circumstances, developing DNA markers through association studies supported by regression analysis between DNA fingerprinting pattern and precise biochemical parameters of the desired phenotypic tralt in plants belonging to a narrow gone pool, could provide an useful allernative. Cell protective enzymes, specifically Cu-Zn cytosolic superoxide dismutase (SOD) and ascordate peroxedase (APX), have been reported to be correlated with drought tolerance in plants. In this study, an attempt has boon mode to establish DNA markers associated with drought tolerance in tea plants through use of regression analysis involving RAPD pattern and drought-specific isozyme activity. Fig 1a and Figure 1b shows RAPD patterns of toa clones used in establishing the DNA marker. Reproductibility of data was confirmed after observing identical fingarprinting patterns in 3 report experiments. A similar approach in demonstrating reproductibility in RAPD studies have also been reported earlier. Use of eleven primers in RAPD studies on 10 clanes revealed 180 PCR bands; among these 131 bands (i.e. 69.87% of total 180 amplified fragments) were polymorphic (table 1). This shows that the RAPD method it capable of revesting appraciable levels of polymorphism in tea plant germplesm. Bearing in mind that polymorphic markers generated from tha fingarprinting pattern of plants belonging to a narrow gana pool rapresent independent characters for specific trails, an attempt was made to develop drought tolerance- associated molecular (DMA) marker(s) using correlative studies between RAPD (ganoma) analysis and activity of drought specific isozymes. For this a study was made between easily scorable RAPD (dominant) markers and biochemical parameters (viz SOD and APX activity) of drought tolerance in pients. Activity of stress-related cell-protective enzymes has been reviewed at length. Cytosolic Cu-Zn SOD and APX are known to be associated with drought stress in plants. Isozyme marker based assessment for drought tolerance in the clones under study was mada by assessing activity of SOD and APX isozymes. Densitiomatric scan of the activity gel of SOD (Figure 2a) shows 4 major peaks; tha peak at about 29kD (band IV) represents the Cu-Zn SOD. This peak was found to show higher activity (peak area above 100 mm2) in clones RR17/144, CP1, TV26 and AV2 compared to tha area under peak IV in T24S, TV26, B777, K1/1, B688, T78 (peek araa below 70 mm3). The suggests that clones RR17/144, CP1, TV26 and AV2 possess the drought tolerant tralt. Cultivars of Nicotiena tabacum with high drought tolerance show highest aecorbate peroxidase activity. My data of APX activity fig. (2b) in drought stressed leaf (of tea) from non-daneturing polyacryamide gel electrophoresis study ravealed by NBT staining 2 isozyme bands, ona of these, namely APX II, was found to be a monomer with molacutar weight of about 29kD and the other at about 40 Kd (APX band 1). In densilometric scane of the activity gels (Fig. 2b), the 2 bands are represented as 2 peaks at about 40 KD (APX band I) and at about 29 kD (APX band II). The isozyme peak at about 29kD (APX II) exhibited particularly high activity in clones RR17/144, CP1, TV26, AV2 (peak area above 90 mm2); T246, TV25, B777, K1/1, B688, T78 exhibited lower activity (peak area below 50 mm2). It may be noted that the clones that showed high APXII activity (Figure 2b) also showed high Cu-Zn SOD activity (Figure 2a). These clones also appear drought tolerant from field performance data. Association studies between RAPD bands and trails of interest have been reported. For precision in establishment of trait-related DNA markers, have used RAPD analysis of highly diverse accessions of rice in muliple regression analysis to determine associations between the DMA merker(s) and quantitative traits, in the present study regression analysis (Table 2) in represent was undertaken to determine the association of specific isozyme activity peak areas as dependent variables and RAPD band (s) (Figure 1a) score as independent variables. Step- wise regression showed that the RAPO band (at 1400 bp) obtained with OPAH02 primer has a highly significant regression coefficient for Cu/Zn SOD activity (b=0.970) and for APX II activity (b-0.968). Using Fisher's exact test (F-test) the association between Cu/Zn SOD and APX II and tha RAPO band of 1400 bp wat found to be significant at 99.9% confidence. Being associated win clones exhibiting high activity of drought tolarance-specific iaozymes this DNA band (marker) could be used in germplasm screening for drought tolarance in tea piants. This brand at 1400 bp is marked win an arrow in Fig 1a and 1b. Validation studias in date shown in RAPD patterns reveals that the same primer, viz AP0H02,)amplified a DNA fragment of 1400bp also in some Assam tea plants (marked with an arrow -Fig 3a and 3b) This band at 1400 bp is now being further characterized The DNA fragment has been further checked for its validity in other randomly selected tea plants including some Assam tea clones. Velidation studies till date shown in RAPO patterns reveals that the same primer, viz AP0H02, amplified a DNA fragment of 1400bp also in some Assam tea plants (marked win an arrow -Fig 3a and 3b) Table: 1 Sequence or 11 random primers with the number of scprable amplified and polymorphic bands We Claim: 1. A novel DNA marker for drought tolerance in tea plants comprises of an amplified fragment 1400bp generated by using primer OPAHO2. 2. A method for developing a DNA marker for drought tolerance comprising extracting genomic DNA from frozen young leaves, Subjecting the DNA thus obtained to the step of calibration, Subjecting the calibrated DNA to the step of analysis, Cloning the drought specific DNA fragment for obtaining the DNA sequence of the marker brand using the primer such as OPAB06, OPAB18, OPAH18, OPAH02, OPAB17, OPAC19, OPAH12, OPAL04, OPAA02, OPAG13 and OPAL08. 3. The method as claimed in claim 2 wherein the step of analysis is done by regression analysis approach using (RAPD study) correlated with activity of drought specific isozyme of superoxide dismutase (SOD) and ascorbate proxidase (APX) in drought stressed detached leaves. A novel DNA marker for drought tolerance in tea plants comprises of an amplified fragment 1400bp generated by using primer AP OPAHO2.

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77-kol-2004-granted-abstract.pdf

77-kol-2004-granted-claims.pdf

77-kol-2004-granted-correspondence.pdf

77-kol-2004-granted-description (complete).pdf

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77-kol-2004-granted-examination report.pdf

77-kol-2004-granted-form 1.pdf

77-kol-2004-granted-form 18.pdf

77-kol-2004-granted-form 2.pdf

77-kol-2004-granted-form 26.pdf

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