RU2294965C1 - Method for evaluation of inbred line uniformity and hybridism level of sunflower f1 seeds - Google Patents

Abstract

FIELD: agriculture, in particular determination of plant material genotype and sunflower breeding.

SUBSTANCE: claimed method includes isolation of DNA from each sprout in seed group of sunflower plant followed by amplification thereof using PCR. Amplification is carried out on HA 432, HA 514, HA 1442, and ORS 5 microsatellite locuses by using primers corresponding to flanking regions of such locuses. Amplification products are separated by electrophoresis in colored, e.g. agarose gel. Visual comparison of electrephoresis spectra, e.g., in ultraviolet beam makes in possible to evaluate inbred line uniformity and hybridism level in tested sample based on number of single-type spectra.

EFFECT: method of increased accuracy.

5 cl, 3 dwg, 2 ex

Description

The invention relates to agriculture and can be used in breeding and seed production of sunflower.

Sunflower is the most important oilseed crop in Russia. Along with the selection of varieties of populations, the selection of hybrids of this culture has acquired great importance. To maintain the high efficiency of linear hybrid selection, monitoring of the genetic purity of inbred lines and the level of seed hybridity F _{1 is} necessary. To solve these problems, it is possible to use the traditional method of soil control, which requires a significant investment of time and sown area. In addition, morphological characterization is highly dependent on environmental conditions. To some extent, these problems are solved by analyzing the polymorphism of storage proteins and isoenzymes. But the limited number of detected protein markers and the high cost of a complete isoenzyme analysis for sunflower seeds do not satisfy the requirement of a complete assessment of the typicality and hybridity of seed material. It is generally recognized that the most promising systems for labeling seed and selection material are based on the assessment of DNA polymorphism. In particular, microsatellite DNA repeats (SSRs) located randomly in the genome exhibit a high level of polymorphism and are codominant. The use of SSR markers makes it possible to increase the level of identification of genotypes, assessment of the typicality and hybridity of seeds.

The well-known "Method for marking breeding achievements of meadow clover based on RAPD-markers" (RF Patent No. 2244416 by application No. 2002102791/13 of 02/06/2002, A 01 H 1/04, publ. 20.01.2005, Bull. No. 2), consisting in the assessment of DNA polymorphism by the RAPD method using a set of 10-membered primers. According to this method, based on the assessment of DNA amplification products, profiles that are characteristic of only this variety are selected and serve as markers of selection achievement.

The disadvantage of this method is the poor reproducibility of the results and the resulting impossibility of accurately determining the typicality of inbred lines and hybridity of seeds F ₁ sunflower.

Closest to the claimed invention is a "Method for establishing the typicality and level of hybridity of sunflower genotypes" (Patent of Ukraine No. 63 265 A according to application No. 2003032413 of 03.20.2003, publ. 15.01.2004 Bull. No. 1. Authors: Sivolap Yu .M. And Solodenko A.E.). In accordance with the known method, a sample is taken consisting of 100 pieces of the seeds of the studied batch thereof, germinated for 3 days, DNA is extracted from each seedling, DNA amplification is carried out at selected microsatellite loci ORS 78, ORS 509, ORS 595, ORS 815 using the corresponding flanking sequences of the forward and reverse primers using the polymerase chain reaction, the amplification products are separated into fractions by electrophoresis in 10% polyacrylamide gel stained by a specific method using iem nitric acid silver, and DNA fragments were visualized (gel was placed for 5 minutes in 10% ethanol, transferred to 1% HNO ₃ solution for 3 minutes, washed several times with distilled water, soaked 20 minutes in AgNO ₃ in the dark , washed several times with distilled water, incubated the gel in a solution (0.28 M NaCO ₃ (anhydrous), 0.019% formalin), replacing the solution each time it was darkened until amplification fragments appeared, washed several times with bidistilled water), and typicality and level I establish the hybridity of sunflower genotypes by counting the number of plants with the same allelic composition, i.e. by the number of similar spectra.

The disadvantage of the prototype is not a wide range of loci used in the analysis of the typical lines and seed hybridity F _{1 of} sunflower seeds, which limits the number of analyzed genotypes, in addition, the carcinogenicity of the polyacrylamide gel used in the prototype, the complexity of its preparation reduces the effectiveness of the method.

The problem solved by the claimed invention is to increase the level of identification of genotypes, assess the typicality and hybridity of seeds F ₁ sunflower by increasing the number of microsellite loci used, polymorphic for lines and hybrids of sunflower, as well as to increase the efficiency of the method by simplifying the operation of separating amplification products and Improving the work of staff during the analysis.

The purpose of the invention is the expansion of the arsenal of loci recommended for assessing the typicality of inbred lines and the level of seed hybridity F _{1 of} sunflower, as well as reducing the carcinogenicity of the gel used for electrophoresis, and simplifying its staining.

The technical result is achieved by the fact that in the known method of establishing the typicality and level of hybridity of sunflower genotypes, including sampling, consisting of 100 pieces of seeds of the studied batch thereof, their germination for at least 3 days, DNA extraction from each seedling, DNA amplification at selected microsatellite loci using flanking primers using polymerase chain reaction, separation of amplification products into fractions by electrophoresis in a stained gel, visual comparison of ele spectrophoretic spectra and the assessment of typical or hybrid samples by the number of spectra of the same type, according to the invention, DNA amplification is carried out according to the following set of microsatellite loci using primers flanking them:

Microsatellite locus Forward and reverse primer sequences AT 432 P STT TAT SSS SSS SSS SST SSS O GGG TTT AGT GGC CAG TAG TTG TC AT 514 P GGT CAA CGG ATT TAG AGT C O GTA TTG ATT SSA ASA TCC AG AT 1442 P GCT TAT GTG STT ACG TGT TCC TG O STA AAC AGT TGG GCG AGT GTA GC ORS 5 P ATS TGC TGG AGG AAA TTS AG O CTG CTG CCC ACC ATA CTG

moreover, to separate the amplification products, an agarose gel is used, which is stained with ethidium bromide by directly adding the latter to it, and a visual comparison of the electrophoretic spectra is carried out in ultraviolet light.

A comparative analysis of the proposed technical solution with the prototype allows us to conclude that the claimed method for assessing the typicality of inbred lines and the level of seed hybridity F _{1 of} sunflower differs from the known conditions for the implementation of actions, namely, the substances used. So, in the known method during PCR, the following microsatellite loci are used: ORS 78, ORS 509, ORS 595, ORS 815, as well as “their” primers flanking them (loci). Thus, the claimed method meets the criteria of patentability NOVELTY.

Studying the state of the art in the process of conducting a patent search on all types of information publicly available in the press, we did not find a hallmark that differs from the prototype regarding a set of microsatellite loci and primers flanking them during DNA amplification. The inventive method, including the claimed combination of features, for a specialist in the field of selection and seed production of sunflower does not explicitly follow from the current prior art. Our studies have revealed that the claimed primer sequences flank the corresponding loci that have a sufficient level of polymorphism in different sunflower genotypes for their identification and subsequent assessment of typicality. The loci of this set have codominant inheritance, which allows them to be used in assessing the level of hybridity using primers flanking them that correspond to the declared ones (direct and reverse).

Based on this, it can be concluded that the claimed invention meets the patentability criterion. INVENTIVE LEVEL.

The claimed technical solution meets the patentability criterion INDUSTRIAL APPLICABILITY, because It can be used in agriculture. And, in addition, the description of the invention provides means and methods by which it is possible to implement a technical solution in the form as described in the independent claim.

The method is as follows.

To assess the typicality of seeds of the inbred line or the level of hybridity of seeds F _1, sunflower seeds are taken by a standard method from the studied seed lot of the inbred line or hybrid F _{1 a} sample consisting of 100 pieces of seeds, they are germinated for at least 3 days and DNA is extracted from each seedling by any known method, According to a known method, DNA of selected seeds is amplified according to the declared microsatellite loci using primers flanking them using PCR.

The obtained amplification products are separated into fractions by electrophoresis in a stained gel according to standard methods. The obtained electrophoretic spectra are visually compared and the number of spectra of the same type is used to evaluate the typicality of the line or the hybridity of the seeds of F ₁ sunflower.

Examples of the method.

Example No. 1. Assessment of the typology of the inbred line VK 678.

To assess the typical seed lot of the inbred line VK 678, a sample consisting of 100 seeds was taken in a standard way. Seeds were germinated for 5 days in rolls of filter paper in a thermostat at a temperature of + 24 ° C.

DNA was extracted from each seedling obtained by the standard method. To do this, weighed samples weighing 0.2 g from each seedling and homogenized in a porcelain mortar with the addition of 0.9 ml of lysis buffer containing 20 mm Na EDTA; 01M Tris-HCl pH 8.5; 14M NaCl, 20% CTAB. The resulting mass was transferred into a clean plastic tube and incubated at a temperature of 65 ° C for 60 minutes, periodically mixing. Next, 0.45 ml of a mixture of chloroform-isoamyl alcohol (in a ratio of 24: 1 by volume) was added, stirred for 10 minutes. Then centrifuged for 5 min at 10,000 rpm. The aqueous phase was transferred to clean tubes. The procedure was repeated twice. To the resulting subnatant, 7 μl of RNAse solution was added and incubated at 37 ° C for 60 min to disrupt the RNA molecules. DNA was besieged by adding an equal volume of chilled isopropyl alcohol, followed by centrifugation at 10,000 rpm for 5 minutes. The subnatant was discarded. The precipitate was washed with 80% ethanol. The purified DNA was dried at room temperature. The isolated DNA of each seedling was dissolved in 100 μl of sterile water.

For each seedling analyzed, DNA amplification was carried out at microsatellite loci: AT 432, AT 514; AT 1442; ORS 5 by polymerase chain reaction. The reactions were carried out in 25 μl of a standard reaction mixture containing 67 mM Tris HCl pH 8.8; 16.6 mM ammonium sulfate; 3 mM MgCl ₂ ; 0.01% Tween 20; 0.2 mm each of dATP, dGTP, dCTP; dTTP; 10 ng of DNA; 1 unit of recombinant thermostable DNA polymerase and 10 pM primers flanking the amplified locus. Amplification was carried out in the following temperature-time regime: initial denaturation at 96 ° C for 2 min, 30 cycles; denaturation at 94 ° C for 30 sec; firing at 60 ° C for 40 sec; elongation at 70 ° C for 1 min; final elongation 2 min.

The amplification products of DNA according to the claimed microsatellite loci were separated into fractions by a known method of electrophoresis in a stained agarose gel. For this, a 2% agarose gel was prepared in TAE buffer containing 0.004 M Tris-acetate and 0.002 M EDTA. As the electrode buffer used 1 TAE buffer. To prepare it, 1 g of agarose was added to 50 ml of a single TAE buffer, the mixture was brought to a boil. Then the hot gel flask was cooled under running water to a temperature of 50-60 ° C, after which 2-3 μl of a 1% solution of ethidium bromide was added. Electrophoresis was carried out in a chamber for horizontal electrophoresis for 1 h 30 min at a current strength of 50 mA.

A visual comparison of the electrophoretic spectra was carried out in ultraviolet rays. The fractions of the spectra differ in different lengths of amplified DNA fragments. Fragment lengths were set using a standard marker. Earlier, our studies established a typical picture (spectra) of the amplified locus for the estimated inbred line. Among the estimated 100 seedlings, 100 were typical at the HA 514 loci (see FIG. 2) and AT 1442, one seedling is atypical at the HA 432 locus, and two at the ORS 5 locus (see FIG. 1). Thus, 97 seedlings had spectra of amplification products typical for the VK 678 line at all four declared loci, i.e. the level of typicality of the seed lot of the inbred line VK 678 was 97%.

Example No. 2. Assessment of the level of seed hybridity F ₁ Triumph sunflower hybrid.

To assess the level of hybridity of the batch of seeds F _{1 of the} Triumph hybrid, sampling of seeds, their germination, and DNA isolation were carried out in the same way as in example No. 1. Amplification of DNA was carried out alternately with four pairs of primers flanking the claimed loci using the polymerase chain reaction in the standard reaction mixture shown in example No. 1. The VK 678 and VK 580 lines, which are parental for the Triumph sunflower hybrid, have clearly different spectra of amplification products at the HA 1442 locus.

The maternal line VK 678 gives the allele A, the paternal line VK 580 is characterized by the allele B. The co-dominant type of inheritance of microsatellite loci makes it possible to distinguish between the analyzed seeds hybrid seeds, seeds with a spectrum of the maternal or paternal form and seeds with an atypical foreign spectrum.

The amplification products of DNA at the HA 1442 locus isolated from the seedlings were separated by gel electrophoresis in the same manner as in Example 1.

A visual comparison of the spectra of PCR products showed that 87 seeds in the spectra of amplified DNA had allele A and allele B, i.e. turned out to be hybrid (Fig. 3), in 13 seedlings, the spectra of amplification products contained only the allele of the maternal line, which indicated the possibility of their belonging to the maternal line of VK 678.

Thus, the level of seed hybridity F ₁ of the Triumph hybrid batch was 87%, the possible level of maternal form impurity was 13%.

So, the claimed set of microsatellite loci using primers flanking them expands the arsenal of loci recommended for assessing the typicality of inbred lines and the level of seed hybridity F _{1 of} sunflower, i.e. achievement of the goal. In addition, the use, in particular, of an agarose gel, and for its staining - ethidium bromide, allows to reduce the carcinogenicity of the gel and simplify its staining, i.e. achievement and additional purpose of the invention. The inventive method allows to solve the problem, which consists in increasing the level of identification of genotypes, assessing the typicality and hybridity of seeds F ₁ sunflower, as well as the effectiveness of the method.

Claims (5)

1. A method for assessing the typicality of inbred lines and the level of seed hybridity F _{1 of} sunflower seeds, including sampling consisting of 100 seeds of the studied lot thereof, germinating them for at least 3 days, isolating DNA from each seedling, amplifying DNA at selected microsatellite loci using flanking primers using polymerase chain reaction, separation of amplification products into fractions by electrophoresis in a stained gel, visual comparison of electrophoretic spectra and assessment of t pichnosti or hybridity sample by the number of similar spectra, characterized in that the DNA amplification is performed on the next set of microsatellite loci with their flanking primers: Microsatellite locus Forward and reverse primer sequences AT 432 P STT TAT SSS SSS SSS SST SSS O GGG TTT AGT GGC CAG TAG TTG TC HA514 P GGT CAA CGG ATT TAG AGT C O GTA TTG ATT SSA ASA TCC AG AT 1442 P GCT TAT GTG CTT ACG TGT TCC TG O STA AAC AGT TGG GCG AGT GTA GC ORS 5 P PBX TGC TGG AGG AAA TTC AG O CTG CTG CCC ACC ATA CTG 2. The method according to claim 1, characterized in that the colored gel used to separate the amplification products into fractions is agarose. 3. The method according to claim 2, characterized in that they use a gel stained with ethidium bromide. 4. The method according to claim 3, characterized in that the staining of the gel with ethidium bromide is carried out by directly adding the latter to the gel. 5. The method according to claim 1, characterized in that the visual comparison of the electrophoretic spectra is carried out in ultraviolet light.

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