KR19990069344A - Quantitative Genetic Mapping (QTL) and Low Temperature Resistant Rice Variety Involved in Rice Seedling Resistance Using RAPD MARKER - Google Patents

Abstract

The present invention relates to a quantitative genetic map (QTL) involved in seedling cold resistance of rice using RAPD Marker and a method for selecting low temperature resistant rice varieties.

The present invention has the effect of providing a gene association map with an average distance of 10 cM and a total of 1,419.7 cM by a RAPD marker, and the gene association map according to the present invention has a high number of markers located on chromosomes. It is possible to greatly improve the accuracy of gene search and genetic analysis efficiency involved in cultivation, and the use of RAPD marker for cold resistance breeding can reduce the time and effort required for breeding new varieties and maximize the efficiency of breeding. There is.

Description

Quantitative Genetic Mapping (RTL) and Selection of Low Temperature Resistant Rice Varieties Involved in Cold Seedling Resistance of Rice Using RAD PD Method

The present invention relates to a method for selecting low temperature resistant rice varieties. More specifically, the present invention relates to a quantitative genetic map (QTL) involved in cold seedling resistance of rice using RAPD Marker and a low temperature resistant rice variety selection method.

Rice is the world's second food crop after wheat, with more than half of the world's population as rice staple food. Rice is produced in more than 90% of Asia and consumed mainly in Asia. Genes involved in the cold resistance of rice is a quantitative trait that is heavily influenced by the environment, and its genetic form is known to be complicated. Genetic characteristics of quantitative traits can also be analyzed by statistical methods, but unlike qualitative traits, it is important to reveal the number of genes involved or their location on the chromosome because the effects of individual environmental factors on the activity of individual genes are not high. It was virtually impossible. However, with the recent development of molecular biology, gene mapping method using DNA marker has been specified in various plants, and researches on gene locus analysis and gene mapping involved in quantitative traits as well as qualitative traits have been conducted in some plants. Quantitative trait loci (QTL) analysis using DNA markers has the advantage of revealing the effect of genes involved in quantitative traits and their location on chromosomes.

Genetic analysis or mapping of DNA using DNA markers involves restriction fragment length polymorphism (RFLP) analysis using DNA fragment size polymorphism resulting from restriction enzyme digestion, and genomic DNA as a template. Polymerase chain reaction (PCR) assays that amplify are known. Initially, it mainly relies on the RFLP method, but recently, PCR technology is not complicated, and there is no risk by using no radiation, it requires less sample volume, and the variability of DNA amplified by PCR is a synthetic primer. By varying the nucleotide sequence of, it is possible to obtain a wide variety of DNA band pattern variations, attracting interest in this method.

Accordingly, an object of the present invention is to efficiently select and provide a high ability to withstand low temperatures among the cold-resistant gene source of rice using RAPD marker.

In order to achieve the above object, the present invention investigates the degree of cold resistance between rice varieties using RAPD markers related to low temperature, and conducts experiments on the degree of seedling length reduction between 18 ° C. and normal temperature (25 ° C.). It was.

Hereinafter, the specific configuration and operation of the present invention.

Figure 1 is a dot blot analysis showing the RNA level showing the result of 4 ℃ (24 hours) low temperature treatment of seedlings of 10 tap varieties.

Figure 2 is a photograph showing the results of Southern hybridization between G309 and genomic DNA, one of the RFLP Marker.

Figure 3 is a photograph showing the results of PCR analysis of genomic DNA and primers OPXO ₃ of the Dul / Toyohatamochi combination F ₂ population of the test variety.

4 shows the total genome size of 1419.7 cM and the average distance between markers using 84 RAPD markers with chromosomal number identified in 54 RFLP markers showing various bands in the Dular / Toyohatamochi combination F ₂ population. Shows a gene association map of 10 cM.

Figure 5 shows the frequency distribution of seedlings at 18 ℃ of the Dular / Toyohatamochi combination F ₂ population of the test variety.

6 is a graph showing the results of QTL analysis based on the LOD value of the MAPL-MQTL program.

FIG. 7 is a diagram showing a quantitative trait (QTL) map involved in seedling cold resistance at 18 ° C. of the Dular / Toyohatamochi combination F ₂ population.

FIG. 8 is a graph showing the frequency distribution (continuous variation) of the planting of 18 ° C. seedlings of the F ₃ 291 strain of the Indica type K-sen4 / Zavanica type Silewah combination.

The present invention discloses a rice varieties and hybrid populations having different cold resistance and analyzing the degree of cold resistance between varieties at 4 ℃ and 18 ℃; Analyzing the relationship between the degree of accumulation of electrolyte and cold resistance of the rice treated at low temperature; This study consists of mapping rice genes using RFLP and RAPD markers and analyzing quantitative trait loci related to cold resistance.

Hereinafter, the specific configuration and operation of the present invention will be described in detail with examples and experimental examples.

Experimental Example 1. Cold resistance assay

In order to investigate the degree of cold resistance by rice varieties at 4 ℃, seedlings of 63 grains were planted in plastic square boxes (40 × 60 × 10 cm) containing 9 varieties of “Flower Rice” and topsoil. After culturing for 2 weeks at 2 ℃ was treated in the growth phase (4,000lux, 100% humidity, 12 hours / day) adjusted to 4 ℃. After 1 week of growth in low temperature treated seedlings at 28 ℃, the low temperature susceptibility value was investigated by examining varieties of cultivars (0: no dead individuals, 1: 1 to 20%, 2: 21 ~ 40% test, 3: 41-60% test, 4: 61-80% test, 5: 81-100% test).

In the cold-resistant assay at 18 ℃, 11 varieties of "Flower Rice" and others were disclosed and sown in the same manner as in the 4 ℃ cold-resistant assay, and grown for 15 days in a greenhouse (4,000lux, 12 hours / day) at 18 ℃ and 25 ℃. The height reduction rate of 18 ℃ was obtained by examining the height of each variety between the following two temperature conditions. In order to investigate the segregation characteristics of cold resistance in hybrids in which two varieties with different cold resistance were hybridized, F ₂ population of "Dular / Toyohatamochi" combination was disclosed with their parents and sown in the same manner as in the previous experiment. After three weeks of cultivation in a greenhouse controlled at (12 hours / day), the frequency distribution of grass heights was investigated and the grass heights of both parent and F ₂ populations were used as QTL analysis data. In order to verify the RAPD markers that were found to be highly related to 18 ° C cold resistance in QTL analysis, later 291 strains (F ₃ ) with their parents were crossed with "K-sen4" with low cold resistance and "Silewah" with strong cold resistance. Seeds were sown in plastic boxes (40 × 60 × 10 cm) and grown for 1 week in a greenhouse controlled to 25 ° / 20 ° C. (day / night). The seedling boxes were then transferred to a cold water treatment box (50 × 70 × 30cm) where the water temperature was 18 ° C (10cm deep) and the temperature was maintained at 24 ° / 20 ° C (day / night). This study was used for variance distribution survey and QTL analysis.

As shown in Table 1, the low temperature susceptibility values of "Hayayuki", "Mitak", "Nipponbare", "Toyohatamochi", etc., which are cultivated in Japan, were 1.0-1.5. The low-temperature susceptibility values of "Ippm rice" and "盈 德 rice" ranged from 2.0 to 3.0, whereas indica varieties such as "Dular", "Er Jiu Qing", and "Kele" showed high susceptibility values of 4.0-5.0. At 4 ℃, japonica-type varieties showed higher cold resistance than indica-type varieties.

Varietal Differences in Cold Resistance of Seedlings of 10 Water Varieties kind origin Cold resistance (0 ~ 5) Exp. I Exp. Ⅱ Average Hayayuki Japan One One 1.0 Mitak Japan One 2 1.5 Nipponbare Japan 2 One 1.5 Toyohatamochi Japan One 2 1.5 Hwayeongbyeo Korea 3 3 3.0 Ilpoombyeo Korea 2 2 2.0 Yeongdeogbyeo Korea 3 2 2.5 Dular India 5 3 4.0 Er Jiu Qing China 5 5 5.0 Kele India 5 5 5.0 Note: The cold resistance was recorded 7 days after cold treatment (2 days at 4 ° C.) about 2 weeks.

On the other hand, "Flower Rice" and 11 varieties were disclosed, after cultivation for 15 days in a greenhouse controlled at 18 ℃ and 25 ℃, the experimental results comparing the height and height reduction rate of the varieties between the two treatments are shown in Table 2 and same. Among the cultivars, the shortening rate of "Silewah" and "Toyohatamochi" was 18% and 25%, respectively, lower than those of other varieties, and the other varieties showed a relatively high shortening rate of 38-59% regardless of the breed type.

Plant bioequipment bridges in cold and untreated in 10 seedlings Cultivars Plant height (mm) Rate of shoot reduction (b-a / b × 100) 18 ° C (a) 25 ° C (b) Hayayuki 42 79 47 Mitak 29 70 59 Nipponbare 36 67 46 Toyohatamochi 81 108 25 Hwayeongbyeo 40 71 44 Ilppombyeo 21 35 40 Yeongdeogbyeo 55 89 38 Dular 97 177 45 Er Jiu Qing 30 55 46 Kele 86 160 46 K-sen4 83 168 51 Silewah 225 311 18

Experimental Example 2. RNA extraction and dot blot analysis

In order to investigate the relationship between the accumulation of rice electrolytic material and cold resistance under low temperature treatment, the seeds of the published varieties (Table 1) were sown in the same manner as in the 4 ° C cold resistance test and grown for 2 weeks in a 25 ° C greenhouse, and then 4 ° C. After 24 hours at, RNA was extracted according to Pawlowski et al. For analysis of dot blot, the results of hybridization with RNA extracted from cold-treated rice leaves and blotted onto nylon membrane and c167 DNADNA probes of RC167 and RC235, which were already identified as low-temperature inducible genes, were analyzed using Bio Imagine Analyzer (Fujix, BAS 1000MAC). The degree of accumulation of the electrolytic material was investigated.

In order to test the accumulation of electrolytes in cold-treated rice at the RNA level, seeds of seed varieties were sown in pots, grown for 2 weeks in a greenhouse at 28 ° C, treated for 12 hours at 4 ° C growth, and then 1g of leaves of each plant were collected. After extracting the whole RNA, the dot blot analysis using the cDNA (RC167, RC235) as a probe is shown in Figure 1 as a result. In the case of RC167 probe, the japonica-type varieties showed almost the same distinct response, but the indica-type varieties did not. The response of all 10 varieties to the RC235 probe was more pronounced than that of the japonica type.

Experimental Example 3. DNA Extraction

For PCR reaction and Southern hybridization, the CTAB method was followed by cultivation of the parent of the "Dular / Toyohatamochi" combination, the F ₂ population and the F ₃ generation of the "K-sen4 / Silewah" combination at 18 ° C and 25 ° C for 3 weeks, respectively. According to the DNA was extracted as follows. 1 g of healthy leaves of each plant were put in a mortar and crushed into fine powder in a state of rapid freezing with liquid nitrogen, mixed with 1 ml of 2xCTAB buffer and 0.5 ml of 1xCTAB, mixed, and then transferred to a 50 ml cap tube for 30 minutes at 55 ° C. Mix gently 2 to 3 times while reacting. After adding the same amount of chloroform for 30 minutes and centrifugation (4,000rpm, 7 minutes, 15 ℃), the supernatant was transferred to a 15ml polypropylene tube, 325μl of 10% CTAB-NaCl and 3.5μl of chloroform The mixture was mixed well, centrifuged (4,500 rpm, 10 minutes, 15 ° C), and the supernatant was transferred to 15 ml of fresh polypropylene tube. 3.5 ml of ppt (precipitation) CTAB solution (1% CTAB, 50 mM Tris pH 8.0, 10 mM EDTA) was added and allowed to stand at room temperature for at least 12 hours. Then, 0.7 ml and 0.42 ml of isopropanol were entangled and recovered. The DNA thus recovered was washed with 80% ethanol, dried, diluted 100 times with TE buffer, followed by electrophoresis on 0.8% agarose gel, and DNA concentration was controlled by staining with Ethidium Bromide (EtBr) under 260 nm UV. It was used for Southern hybridization.

Experimental Example 4. Selection of RFLP marker

To select markers showing polymorphic bands between "Dular" and "Toyohatamochi" using RFLP markers, which are already known on each chromosome in rice, 2 µg of genomic DNA extracted from leaves of "Dular" and "Toyohatamochi" was extracted from BamH I. 4-12 restriction enzymes such as Bgl Ⅱ, EcoR V, and Hind Ⅲ for 6 to 12 hours, followed by electrophoresis on 0.8% agarose gel, followed by transfer to nylon membrane filters (Nytran, S & S), Sambrook et al. By the method, hybridization was performed with probe DNA labeled with ³² PdCTP to select a marker indicating a polymorphic band. The probe DNA used in the experiment was a total of 218 markers, including 72 of Set I, 95 of Set II, and 49 probes from genomic DNA provided by the Rice Genome Research Program (RPG). It was.

As a result, 54 markers showed polymorphism by Southern hybridization of genomic DNA of "Dular" and "Toyohatamochi" among 218 RFLP markers. As shown in Table 3, 45 of the 54 selected RFLP markers banded to both the parental ("Dular") and pollen ("Toyohatamochi") but nine markers (G165B, G1314B, G282, G124B, G260). , G122, G1314B, G2132, and G1125) showed bands only on either parent.

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Southern analysis results between G309 and genomic DNA, one of 54 RFLP markers, are shown in FIG. 2.

Experimental Example 5. RAPD marker selection

DNA amplification sample preparation for selection of RAPD markers required for gene mapping was performed using 500 ng PCR reaction tubes with 1 ng of genomic DNA, 25 ng, primers of "Dular" and "Toyohatamochi", dATP, dTTP, dGTP, and dCTP, respectively. 2.5mM, buffer (10mM Tris pH 8.3, 50mM KCl, 1.5mM MgCl ₂ ), Taq-polymerase 0.5units were mixed and the reaction solution was totaled 10µl and used for PCR analysis. The initial denaturation using a thermo cycler (Gene Amp PCR System 9600) was 5 minutes at 96 ° C, subsequent denaturation at 15 ° C for 15 seconds, annealing at 38 ° C for 15 seconds, and DNA synthesis at 72 ° C for 7 minutes. The synthesized DNA was electrophoresed with 1.2% agarose gel (50 volts, 2 hours) and stained with EtBr to identify the band in the UV lamp. In the PCR, a band showing polymorphism between two varieties was used as a RAPD marker. The 10-mer primers used in this experiment were 662 out of 800 purchased from Operon (Operon Technologies, Inc. Alameda, Calif.) In 1995 at the Hokkaido Agricultural Experiment Station, Ministry of Agriculture, Forestry and Fisheries. RFLP markers L442 and C609, which are closely related to the chlorosis reaction of rice at 18 ° C and have already been sequenced, were prepared with 20-mer primer sequences and named PL442 and PC609 and used as RAPD markers, respectively.

As a result of PCR amplification of genomic DNA and 664 primers of test varieties "Dular" and "Toyohatamochi" to select RAPD markers, primers containing polymorphic bands were shown in 11% of the total as shown in Table 4 below. The corresponding 74 appeared.

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The number of bands represented by these primers was amplified to a total of 289, the size of the band was varied from 180 to 4,500bp. Of these, only 157 dominant bands appeared in either "Dular" or "Toyohatamochi". The PCR analysis was performed three times consecutively on 157 bands selected first, and finally 85 of them were clearly selected as RAPD markers.

PCR analysis of OPX03, one of the primers used, and genomic DNA of the cultivars showed bands at 2,400bp and 750bp in "Dular" and only 2,400bp in "Toyohatamochi" as shown in FIG. From 1 to 94, the F2 population was divided into bands at 750bp and no bands.

Experimental Example 6. Gene Mapping

In order to create a genetic map with the RFLP and RAPD marker, "Dular / Toyohatamochi" of F ₂ the proposed formulas the plant DNA isolated from the group藤卷after performing the primers and PCR starting from previous experiments [χ ² = ^{(a-3 / 4n) 2} /3 / 4n + (b-1 / 4n) 2/1/4 = (a-3b) 2 / 3n; n: total number, a, b: number of individuals separated by a, b]], the suitability test was performed for the separation ratio of bands representing the "Dular" type and the "Toyohatamochi" type. The grouping and association analysis of RAPD markers were analyzed by Bernatzky and Tanksley method, and the test score was obtained by Allard's F ₂ maximum likelihood algorithm. In order to determine the sequence of chromosome alignment of several closely related markers, three-point test and multiple test were performed using the test value calculated from each RFLP and RAPD marker. Using this MAPL program, the molecular gene map was completed. The RFLP markers selected in this experiment were named according to the Japanese REG standard. The RAPD markers were named in Arabic numerals, and the RAPD markers obtained from the primers PL442 and PC609 derived from the RFLP markers were named PKL442 and PKC609, respectively. . The relationship between the RAPD markers and the heights examined from the F ₂ population of "Dular / Toyohatamochi" treated at 18 ° C was performed by the t-test.

Based on the LOD values (LOD = 1.3, χ ² <0.05) using the MAPL program, the correlation between 85 RAPD markers was analyzed. As shown in Table 5, 16 independent groups and 1 independent group were analyzed. Groups were classified into 6 groups, including 7 markers, 8 markers, 11 markers, 12 markers, 15 markers, and 16 markers.

Grouping of 85 RAPD markers based on LOD values Group No. RAPD marker name One 2, 1, 49, 43, 56, 44, 58 2 3, 24, 25, 26, 8, 62, 63, 9 3 5, 30, 31, 37, 34 4 75, 23, 64, 57, 78, 10, 82, 12, 13, 35, 14, 60, 11, 3 5 83, 15, PKC609, PKL442 6 17, 76, 59, 72, 19 7 18, 40 8 20, 29 9 36, 61, 21, 69, 38 10 22, 73, 53 11 27, 28 12 32, 45 13 47, 81, 77, 80 14 50, 51, 52, 74, 71 15 54, 55 16 6, 33 Solitary 4, 7, 16, 41, 42, 48, 65, 66, 67, 68, 69

As a result of analyzing the association between the RAPD markers grouped in Table 5 and the 54 RFLP markers, as shown in Table 6, 84 markers except “marker 67” among 85 RAPD markers are all associated with the RFLP marker. The number of RAPD markers on each chromosome was the highest with 19 on the 4th chromosome. There were 2 RAPD markers on the 8th and 11th and 1 on the 7th chromosome.

As a result of the experiment, as shown in FIG. 4 and Table 7, a gene association map with an average distance of 10 cM and an overall genome size of 1419.7 cM was prepared using 85 RAPD markers and 54 RFLP markers.

Experimental Example 7. QTL Analysis

MATL-MQTL program was used to search for QTL on rice chromosome, and LOD value (logarithm score) of 4.0 was selected to increase the reliability of QTL. One or more LOD peaks on one chromosome were examined to determine whether they had single or multiple QTL, respectively.

As shown in FIG. 6, the region having an LOD value of 4.0 or more appeared only in the range of 15-42 cM of chromosome 5. The relationship between the three RFLP markers (G292, G396 and G337) located adjacent to the RAPD markers 20 and 29 on chromosome 5 and the height of the two varieties was examined. Significance was observed, and no significance was found in other markers G292 and G337.

In addition, the frequency distribution of seedlings at 18 ° C. of the “Dular / Toyohatamochi” combination F ₂ group was examined. As shown in FIG. 5, the average length of “Dular” grown at 18 ° C. for 20 days was 126 ± 8 mm. , "Toyohatamochi" was average 117 ± 2mm, and the average value of F ₂ group was 140 ± 17mm, which was higher than that of parents.

Relationship between RFLP markers and cold resistance at rice seedling stages for transgenic fragments RFLPmarker Average value of plant height in each genotype (mm) t-value Degree of freedom Dular type Toyohatamochi type G292 133.6 (31) ^a 161.8 (6) 1.937 35 G396 138.0 (29) 163.9 (8) 3.095 ** 35 G337 141.6 (30) 152.3 (7) 1.097 35 Note: a; number of lines, *, **; Significant at 5% and 10% levels, respectively.

On the other hand, as shown in Table 9, the maximum LOD value of the cold resistance-related QTL (29) identified as being located between the RAPD marker 29 and the RFLP marker G396 was 5.71, the difference was 2.55, the phenotype correlation was 0.81.

Detected QTL Characteristics of Plant Growth at Low Temperature in F ₂ Population of "Dular / Toyohatamochi" DNA marker Marker bordering the QTL Total length (cM) Peak LOD Epistasis Phenotypecorrelation G396 G292-G396 13.5 2.62 0.67 0.443 29 G396 ~ 29 10.4 5.71 2.55 0.81. 20 29-20 21.2 4.34 4.28 0.772

The QTL (20) located between RAPD markers 20 and 29 also showed a relatively high phenotypic correlation (0.772). 7 shows the results of the QTL-related QTLs identified on the chromosome map.

The variation of the seedlings was investigated at 18 ° C of the F ₃ 291 strain of the combination of the Indica type "K-sen 4" and the Javanese type "Silewah". exhibited a distribution, and the "K-sen 4" average plant height of is 225 ± 12mm, the average value of the "Silewah" was 355 ± 17mm, F ₃ system the mean of is appeared to 228 ± 42mm, choebinsu (77 lines) are It was 216-249 mm. As shown in Figure 8, the frequency distribution for the height of the F ₃ lines showed a continuous variation distribution.

As described through the above experimental example, the present invention has an effect that can provide a genetic association map of the average distance between the markers by the DNA marker 10cM and a total of 1,419.7cM. In addition, since the gene association map according to the present invention has a large number of marker factors located on the chromosome, it is possible to greatly improve the accuracy of gene search or genetic analysis involved in quality and quantitative traits, and to provide RAPD markers for the development of cold-resistant varieties. When used, it is possible to reduce the time and effort required for the breeding of new varieties, thereby maximizing the efficiency of breeding, which is a very useful invention for the breeding industry.

Claims (3)

85 RAPD Markers and 54 RFLP Markers were selected from the Indica type Dular and the Japonica type Toyohatamochi. The genetic map of claim 1, wherein the genetic map has a mean distance between markers of 10 cM and a total of 1,419.7 cM. A method of selecting rice cultivars having low temperature resistance by analyzing the following quantitative genetic map (QTL) involved in seedling resistance of rice using RAPD Marker.