KR20070043925A - A novel dna maker for drought tolerance in plants - Google Patents

Abstract

Drought-tolerant new DNA markers in plants consist of amplified 1400 bp fragments obtained with the use of primer APOH02.

Primer, DNA Fragment, DNA Sequence

Description

A novel DNA maker for drought tolerance in plants

The present invention relates to the development of new DNA markers resistant to drought in plants.

DNA markers in plants have typically been developed by using homogenous mapping populations. This is not practical at long regeneration cycle stages. The development of trait-related DNA markers in plants has led to homologous breeding populations (actual homogenous genotype lines -RIL / near genotypes) focusing on physiological parameters associated with traits. NIL). This is a time consuming process that takes years to develop RILs and NILs. Moreover, interactions with any one phenotypic parameter often do not give DNA markers that can be directly related to quantitative traits.

Most of the drought resistant markers known to date represent interactions with one or other physiological parameters associated with the trait. Other phenotypes are usually focused on ecosystem features and are not useful for the rapid and accurate evaluation and testing of germplasm in experimental plants.

An object of the present invention is to establish a new DNA marker resistant to drought in plants.

It is a further object of the present invention to present DNA markers for accurate selection in the laboratory of plants for ecosystem specific cultivation.

Another object of the present invention is to provide a DNA marker for developing improved varieties through drought resistant plant breeding.

Another object of the present invention is to provide a DNA marker for rapid diagnosis of traits resistant to drought in plants.

It is a still further object of the present invention to present a DNA marker useful in the identification of genes for plant transformation in the development of improved varieties.

According to the present invention a new DNA marker resistant to drought is provided in a plant composed of 1400 bp amplified fragments.

According to the present invention, there is provided a method of extracting genomic DNA from frozen young leaves, measuring the obtained DNA, analyzing the measured DNA, cloning the analyzed DNA fragment sequencing the DNA fragment to obtain a DNA marker. A method is provided for developing a drought resistant DNA marker comprising a step.

The process started to establish these DNA markers is new in that they are developed through interactions with trait specific, accurate and rapidly evaluated biochemical markers. With the use of this new, unclear, and ingenious process, drought resistant DNA markers have been established.

Genome DNA Extraction Method:

200 mg of frozen young leaves were used for DNA isolation. CTAB (cetyltrimethylammonium bromide) method was used for DNA extraction, and an average of 20 µg of DNA was obtained. After electrophoresis through an agarose gel, DNA is measured based on ethidium bromide fluorescence using a standard amount of DNA.

RAPD Research Method:

For randomly amplified polymerphic DNA (RAPD) studies, the young leaf DNA from each clone was subjected to 25 μl of dNTPs, 40 ng primers and 25 ul of PCR reaction mixture containing 0.3 ul of Taq DNA polymerase. It was used as a template. The reaction was carried out in 45 cycles at annealing temperature of 36 ° C. PCR products obtained by the use of 11 optional decanter primers such as OPAB06, OPAB18, OPAH18, OPAH02, OPAB17, OPAC19, OPAH12, OPAL04, OPAA02, OPAG13 and OPAL08 were obtained with 1.2% agarose gel. ) And is ethidium bromide and visualized on UV light. RAPD analysis of randomly selected plants from each clonal propagation confirms the genetic uniformity of the plants within each clonal plot (data is Not shown). A comparative analysis of the short-lived clones (based on drought tolerance / sensitivity to drought) obtained for use in this study began. A comparative analysis of 10 short-lived clones (based on drought tolerance / sensitivity to drought) obtained for use in this study was initiated. This RAPD study is repeated with leaves collected from randomly selected plants within each clonal plot (in sets of 10 clones each). Gel images specimens of the RAPD pattern are shown in FIG. 1A. FIG. 1B shows a RAPD pattern (also shown in FIG. 1A) with the same primers for the genomes of four clones showing drought resistance in nature (FIG. 1B is not used to count RAPD bands).

The primers used in the RAPD study, the total number of RAPD bands observed through 10 clones, the total number of monomorphic / polymorphic bands present and the percentage of polymorphic bands obtained are given in Table 1.

Stimuli associated with isoenzyme activity:

Separated young tea leaves (in water-deficient) were extracted in extraction buffer, which was run on a non-denaturing gel. The activity of the enzyme was studied by immersing the gel in an appropriate reaction mixture to induce the reaction of iso-enzymes in the gel and then stopping the enzyme activity by immersing the gel in an appropriate staining solution.

Active SOD and APX:

The separated leaves were 50 mM potassium phosphate (pH-7.8) buffer for SOD (EC 1.15.1.1) and 100 mM sodium phosphate (pH-7.8) for APX (EC 1.11.1.11). Homogenized in buffer. Enzyme activity in the extract was visualized on an unmodified active gel (12% polyacrylamide) after electrophoretic separation following the deposition of the gel in a suitable colorant.

The electrophoretically isolated SOD isozyme was nitro blue tetrazolum (Ntro) by O ₂ ^- produced by the reaction between N, N, N ¹ , N ¹ -tetramethyletylem diamine (TEMED) and riboflavin. Blue Tetrazolium: hereafter visualized by reduction of NBT). Bands representing isozymes remain colorless due to lack of O ₂ ⁻ removed by the activity of the isoenzyme and the remaining gel remains blue colored due to reduced NBT. Densitometric scans (632.8 nm) of the active band of negatively colored activators are shown in FIG. 2A.

APX active gel staining is also similar except that ascorbate is used to produce O ₂ ⁻ instead of riboflavin and hydrogen peroxide (H ₂ O ₂ ). Negative staining of the isoenzyme bands is detailed in the gel doc apparatus (Image Master VDS, Pharmacia Biotech).

The negatively colored isoenzyme activity bands were scanned at 632.8 nm; Representative diagrams of densitometric scans are shown in FIG. 2B.

Correlation studies between isoenzyme activity and DNA fringerprint to develop specific DNA markers resistant to drought using regression analysis:

Regression analysis is performed using RAPD band and isoenzyme (SOD and APX) study information. The association of SOD and APX activity with RAPD markers was studied through several regression approaches. In other words, the enzyme activity of the appropriate Suboxide dismutase (SOD) and APX isoenzyme is treated as a dependent variable at the peak, with various RAPD bands (zero without bands and one with bands). Are treated as independent variables. The regression analysis is based on the following model.

Y = a + b _i m _i , + b ₂ m ₂ + ------ b _j m _j + ---- b _n m _n + d

Where Y is the trait of the enzyme, m is the RAPD marker, b is the partial regression coefficient, and d is the residual residual after regression. The degree of association between the RAPD band and the Asaryri superoxide dimutase (Cu-Zn SOD) and ascorbate poroxidase (APX) (band II) activity is shown in Table 2.

The sequence of these markers was submitted for patent. This marker can be used for the selection of the appropriate phenotype for (a) ecosystem specific cultures and (b) development of improved varieties through plant breeding / plant transformation efforts.

Clones of tea reported to be drought resistant / sensitive based on field practice were selected for this study.

Darjeeling's Tea clone was used to establish DNA markers (through studies of RAPD and isoenzymes related to field research information). Ticlones used in this study were RR17 / 144, T246, CPI, TV26, TV25, B777, AV2, K1 / 1. B688, T78 (both Darjeeling Ticlones).

Other Tea clones have different established DNA markers (DNA markers established through field research-related RAPD studies) that differ from other Ti clones, the Assam Ticlones (TV1, TV2, TV14, TV17, TV20, TV30). ) Is also used to demonstrate that it is associated with drought tolerance.

The plant leaves of each of the clones listed above were collected from Ging Tea Estate, Darjeeling, and Tea Research Association (TRA), Asam.

For enzyme studies, freshly separated leaves from Darjeeling Ticlones were used for extraction within 24 hours.

Tea plants are perennial plants and have a long regeneration cycle. Thus, for the rapid use of homogeneous raw materials (ie fresh tea leaves), lushly grown tea clones constitute a plant population for use in the tea industry. In such populations, linkage studies through fidelity generation are time consuming. Under such circumstances, DNA markers through association studies supported by regression analysis between the exact biochemical parameters of the desired phenotype of plants belonging to a narrow gene pool and DNA fingerprinting Development can be a useful alternative.

Cytoprotective enzymes, in particular zinc-copper (Cu-Zn) cytosolic Subside dismutase (SOD) and ascorbate Poroxidase (APX), are reported to be associated with drought tolerance in plants It became. In this study, experiments were conducted to establish DNA markers related to drought resistance in tea plants through regression analysis involving RAPD patterns and drought specific isoenzyme activity.

1A and 1B show the RAPD pattern of ticlones used to establish DNA markers. The reproducibility of the information was confirmed after observing the same fingerprinting in three repeated experiments. Similar approaches to demonstrating reproducibility in RAPD studies have already been reported. 180 PCR bands were identified using 11 primers used for RAPD studies on 10 clones. Here 131 bands (ie 69.87% of the total 180 amplified fragments) are polymorphic bands (Table 1). This shows that the RAPD method can reveal significant levels of polymorphism in the germ cells of tea plants.

Keeping in mind that polymorphic markers arising from the fingerprinting patterns of plants in a narrow gene pool exhibit independent characteristics for specific traits, this experiment is based on RAPD (genome) analysis and Interaction studies between the activity of drought-specific isoenzymes have been done to develop drought tolerance associated with molecular (DNA) markers. So this study was done between easily countable RAPD markers and biochemical parameters of drought tolerance in plants (ie SOD and APX activity).

The activity of cytoprotective enzymes associated with stress has been examined in detail. Cytosolic archery SOD and APX are known to be associated with drought stress in plants. Under this study, isoenzyme markers based on the assessment of drought resistance in clones were made by assessing the activity of SOD and APX isoenzymes. Densitometric scans of gel activity of SOD (FIG. 2A) show four major peaks. The highest point at 29 kD (Band IV) represents the Ag-Zn SOD. These peaks were found to show higher activity in the RR17 / 144, CP1, TV26 and AV2 clones compared to the area below the peak IV of the T246, TV25, B777, K1 / 1, B688, and T78 (peak area below 70 mm²). Peak area above 100 mm²). This indicates that clones RR17 / 144, CP1, TV26 and AV2 have drought resistant traits.

Nicotiana tabacum varieties, which are highly resistant to drought, have the highest ascorbate peroxidase activity. The data in FIG. 2 (b) of APX activity on drought stressed leaf of tea from unmodified polyacryamide gel electrophoresis study is shown by two isoenzyme bands colored with NBT One of them, APX II, is a monomer having a molecular weight of about 29 kD, and the other is identified as 40 kD (APX band I). In a densitometric scan of the active gel (FIG. 2B), the two bands peaked at about 40 kD (APX band I) and about 29 kD (APX band II). At about 29 kD (APX band II), the isoenzyme peak was particularly high in the RR17 / 144, CP1, TV26 and AV2 clones (highest 90 mm 2 peak region). T246, TV25, B777, K1 / 1, B688, and T78 showed lower activity (peak area below 50 mm²). It is noted that clones exhibiting high APX II activity (FIG. 2B) also exhibited high Cu-Zn SOD activity (FIG. 2A). These clones also appear to be drought resistant in field practice data.

Association studies between RAPD bands and traits of interest have been reported. For precision in the establishment of trait related DNA markers, a highly diverse accessions of rice RAPD analysis was used in multiple regression analysis to determine the association between DNA markers and quantitative traits. The regression analysis in this study, shown in Table 2, was performed to determine the association of the number of RAPD bands (FIG. 1A) as the independent variable with the highest region of specific isozyme activity as dependent variable. Sequential regression analysis indicated that the 1,400 bp RAPD band obtained with the OPAH02 primer was very significant for the Cu-Zn SOD activity (regression coefficient: b = 0.970) and APX II activity (regression coefficient: b = 0.968). It has been shown that it has a regression coefficient. A Fisher's exact test (F-test) for the association between the Cu-Zn SOD and APX II and the RAPD band of 1400bp was found to be significant with a confidence of 99.9%. Because of its association with clones that exhibit high activity of specific isotopes that are resistant to drought, this DNA band (marker) can be used to screen germ cells that are resistant to drought in tea plants. This band of 1400 bp is indicated by arrows in FIGS. 1A and 1B.

The confirmation studies so far seen in the RAPD pattern show that the same primer, APOH02, also amplifies DNA fragments of 1400 bp in Assam tea plants (indicated by arrows in FIGS. 3A and 3B).

This band at 1400bp will now be further characterized.

This DNA fragment has further confirmed its usefulness in other randomly selected Tea plants, including several Asam Ticlones.

Table 1: Amplified Number of 11 Random Primer Sequences and Polymorphic Bands

Primer Sequence of primers (5'-3 ') Number of bands amplified Number of polymorphic bands Percentage of Polymorphic Bands (%) OPAA02 GAGACCAGAC 11 5 45.45 OPAB06 GTGGCTTGGA 15 10 66.66 OPAB17 TCGCATCCAG 16 8 50.00 OPAB18 CTGGCGTGTC 16 10 62.50 OPAC19 AGTCCGCCTG 21 20 95.24 OPAH02 CACTTCCGCT 15 14 93.33 OPAH12 TCCAACGGCT 14 11 78.57 OPAH18 GGGCTAGTCA 24 22 91.66 OPAG13 GGCTTGGCGA 14 5 35.71 OPAL08 GTCGCCCTCA 19 17 89.47 OPAL04 ACAACGGTCC 15 9 60.00 Total 180 131 69.87

Table 2: Association between Cu-Zn SOD, APX II and RAPD bands of 1400bp

Tea clone Peak area of Cu-Zn activity (mm ² ) Peak area of APX II activity (mm ² ) Association with isoenzyme activity associated with drought RR17 / 144 104 91 RAPD band observed at 1400 bp using OPAH02 primer (significant, P <0.001) CP1 121 110 TV26 122 93 AV2 120 97 T246 74 33 No band with specific significance observed

Claims (7)

A new DNA marker resistant to drought in plants consisting of amplified 1400 bp fragments generated by primer APOHO2. The DNA marker of claim 1, wherein the amplified fragment is present in clones RR17 / 144, CP1, TV26 and AV2. The DNA marker according to claim 1, having a sequence list as follows. Tea Sequence

In a method for developing a DNA marker resistant to drought, Extracting genomic DNA from frozen young leaves, Measuring the obtained DNA, Analyzing the measured DNA, Developing a drought resistant DNA marker comprising cloning a dreg-specific DNA fragment (identified by regression as shown in claim 6) to obtain a DNA sequence of the marker band Way. The method of claim 4, wherein the marker identification step comprises a DNA marker resistant to drought, characterized in that using a primer such as OPAB06, OPAB18, OPAH18, OPAH02, OPAB17, OPAC19, OPAH12, OPAL04, OPAA02, OPAG13 and OPAL08. How to develop. 5. The method of claim 4, wherein the analyzing step comprises a drought specific isozyme of suboxide dismutase (SOD) and ascorbate poroxidase (APX) in isolated leaves subjected to drought stress. A method for developing a drought resistant DNA marker, characterized by a regression approach using RAPD research methods related to 5. The method of claim 4, wherein the assay step is based on an association study between RAPD bands (with asgnotypes) as an independent variable and biochemical parameters resistant to drought in plants as an independent variable. A method for developing a DNA marker resistant to drought.

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