CN111719011B - Method for determining genetic differentiation between rice hybrid intercropping varieties by SSR (simple sequence repeat) markers - Google Patents
Method for determining genetic differentiation between rice hybrid intercropping varieties by SSR (simple sequence repeat) markers Download PDFInfo
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Abstract
The utility model relates to a method for determining genetic differentiation between rice hybrid intercropping varieties by SSR (simple sequence repeat) markers, belonging to the technical field of molecular markers; comprising the following steps: extracting total DNA from rice to be detected, amplifying by using 48 pairs of SSR specific primers, separating PCR products by a capillary electrophoresis method, reading the molecular weight of the PCR products, processing SSR molecular fingerprints by using data, and calculating genetic differentiation index (genetic divergence index, GDI) according to a genetic differentiation formula; with the increase of the genetic differentiation index GDI, the better the rice blast prevention and control effect of the variety collocation combination in the mixed planting. The utility model can rapidly and accurately identify the genetic differentiation level among varieties by using a trace amount of DNA sample, predicts the control effect of the variety mixed interplanting composition on rice blast, and has important significance in the research of the collocation selection of varieties in the variety mixed interplanting.
Description
Technical Field
The utility model relates to the technical field of molecular markers, in particular to a method for determining genetic differentiation between rice hybrid intercropping varieties by using SSR markers.
Background
The rice blast is one of main diseases threatening the rice production, and is an important factor restricting the high and stable yield of the rice. With the mechanization of rice and the large-area popularization of high-yield varieties, the rice varieties are singly planted in a large area for a long time, so that the rice blast is continuously mutated, the dominant species are rapidly appeared and proliferated, the occurrence of rice blast is gradually serious, the epidemic period is shorter and shorter, the resistance of the rice blast of the popularized rice varieties is finally rapidly reduced, and the large-scale use of pesticides and fertilizers causes the frailty and degradation of a farmland ecological system, and the serious problems such as biodiversity loss, ecological environment degradation and the like are caused, so that the control of the rice blast by using the rice diversity hybrid intercropping technology becomes key. The mixed interplanting of rice varieties provides an ecological rice blast control method, which not only controls the effective means of rice blast, but also reduces the application of pesticides, thereby achieving the purpose of promoting sustainable production of rice. However, in actual production, a large number of rice variety combinations need to be screened first. The traditional variety combination screening method is to select varieties with large differences of physiological race resistance, genetic background, plant height and the like of rice blast for variety collocation, and then determine the combined field control effect through field experiments. Therefore, the traditional method is often low in efficiency, and a large amount of field test work is performed in the early stage, so that time and labor are wasted.
Disclosure of Invention
In order to solve the problems, the utility model provides a simple and stable method for determining the rice mixed interplanting variety combination, and the genetic differentiation index among rice varieties is calculated to predict the rice blast control effect of the related variety combination in the field, so that a large amount of time required for screening the combination is reduced.
In order to achieve the above purpose, the utility model provides an SSR marker for determining 48 SSR sites evenly distributed on 12 chromosomes of rice, wherein 48 pairs are shown in table 1:
TABLE 1 SSR primer sequence listing
The above sequences are sequentially noted as: SEQ ID NO.1-SEQ ID NO.48
A method for determining genetic differentiation between rice hybrid intercropping varieties by SSR markers comprises the following specific steps:
(1) Extracting rice genome DNA;
(2) Detecting the concentration of the rice genome DNA extracted in the step (1): detecting the concentration of the genome DNA by adopting an ethidium bromide fluorescence staining method;
(3) Performing PCR amplification on the extracted rice genomic DNA sample by using 48 pairs of specific primers according to claim 1;
(4) Separating the PCR product by utilizing a capillary electrophoresis method, and reading the molecular weight of the PCR product;
(5) Calculation of genetic differentiation index: the genetic differentiation index is calculated by utilizing the datamation processing SSR molecular fingerprint, and the calculation formula of the genetic differentiation index of the hybrid intercropped rice variety pair is as follows:
wherein A is i1 And A i2 Representing polymorphism assignment (0-n) of A sample allele at ith SSR site, B i1 And B i2 Representing polymorphism assignment (0-n) of B sample allele at ith SSR site, max i Assigning a maximum value (N) to all allelic polymorphism bands contained in the ith SSR locus, wherein N represents the total number of SSR primer pairs (48 at present);
(6) Linear regression analysis: and carrying out correlation analysis on the GDI and the effect value of the related parameters for preventing and treating rice blast by using a statistical linear correlation analysis method.
Further, the step (1) specifically includes:
step (1) putting rice seeds into a 96-well plate one by one, putting the 96-well plate on a water absorbing disc to absorb water, transferring to an illumination incubator, culturing until seedlings grow to 10-20cm, taking a rice seedling, adding liquid nitrogen, grinding into powder, adding 700 mu L of 2% CTAB buffer solution, carrying out water bath, adding chloroform/isoamyl alcohol, mixing uniformly, and centrifuging;
step (2) taking 650 mu L of supernatant, transferring into a sterilized centrifuge tube, adding 650 mu L of chloroform/isoamyl alcohol, fully and uniformly mixing, centrifuging, taking 600 mu L of supernatant, adding 600 mu L of isopropanol, uniformly mixing, refrigerating and storing for more than 2 hours, and precipitating DNA;
and (3) centrifuging, discarding supernatant, reserving precipitate, adding 70% ethanol for rinsing twice to remove impurities, centrifuging, collecting precipitate, performing sterile air drying on water, adding 100 mu LTE buffer solution or sterilized ultrapure water to dissolve DNA, shaking, mixing uniformly, and storing in a refrigerator at-20 ℃ for later use.
Further, the condition of the illumination culture in the step (1) is 28 ℃, the humidity is 90 percent, and the illumination is carried out for 12 hours; CTAB buffer was 100mM Tris-HCl, pH8.0, 20mM EDTA, pH8.0,1.4mol/LNaCl,2% cetyltrimethylammonium bromide.
Further, the CTAB buffer solution in step (1) is preheated to 65 ℃ before being added, and the water bath condition is 65 ℃ water bath for 30min-1h.
Further, the volume ratio of chloroform/isoamyl alcohol in the step (1) is 24:1.
further, the isopropanol in step (1) in (2) is precooled at-20 ℃.
Further, the PCR amplification system comprises: :1 mu L of 10 Xbuffer containing 2mmol/LMgCl 2 0.8. Mu.L of 10mmol/LdNTP, 0.2. Mu.L of 10. Mu. Mol/L forward primer, 0.2. Mu.L of 10. Mu. Mol/L reverse primer, 0.05. Mu.L of LTaqDNA polymerase, 50ng of 1. Mu.L of template DNA, 10. Mu.L of double distilled water.
Further, the conditions for PCR amplification are: pre-denaturation at 94℃for 4min … … … … … 1cycle94℃30s,55℃30s,72℃30s … … … cycles72℃7min as final extension stage … … … … 1cycle
The product was stored at 4 ℃.
Compared with the prior art, the utility model has the beneficial effects that: the utility model establishes a formula and a method for detecting and determining the genetic differentiation index (genetic divergence index, GDI) between rice mixed intercropping varieties by utilizing a microsatellite SSR molecular marker technology for the first time. The method is an algorithm which is designed and researched by itself according to the fragment size of the allele. The application of the algorithm can maximally solve the problem of optimum variety pairs in the planting among various mixtures when a plurality of varieties are selected, and the genetic differentiation index provides a simple evaluation method for acquiring information, so that the problems of high complexity and selection optimization can be rapidly solved. The development and application of the method have important significance in the research of collocation selection of varieties in the diversity mixed planting.
The utility model establishes a formula and a method for detecting and determining the genetic differentiation index between rice mixed intercropping varieties by utilizing a microsatellite SSR molecular marker technology for the first time. The method is an algorithm which is designed and researched by itself according to the fragment size of the allele. Along with the increase of the genetic differentiation index, the better the control effect of the whole combined variety in the mixed intercropping planting is on rice blast, and the higher the corresponding genetic blocking effect is, the higher the genetic differentiation index is. The application of the algorithm helps us predict the field rice blast resistance level of different mixed interplanting variety combinations, and provides theoretical guidance and technical parameters for reasonably utilizing the matching of the rice varieties planted in the biodiversity mixing interplanting to control rice blast.
Drawings
FIG. 1 is a diagram showing the electrophoresis separation of amplification products of a rice sample at the same microsatellite locus (RM 263), wherein A is genotype AA, B is genotype BB, C is genotype CC, and D is genotype DD;
FIG. 2 is a correlation between control effects of main cultivars and control effects of interplanted cultivars in a variety of interplanting modes and Genetic Differentiation Index (GDI) between rice cultivars; the diamond shape in the figure is an intercropping variety, and the square shape is a main cultivation variety;
FIG. 3 is a plot of test cell, wherein the numbers are variety numbers and I, II, III represent the number of repetitions;
FIG. 4 is a schematic diagram of a plot, wherein A is a cross-sectional view of a diversity interplanted plot, B is a cross-sectional view of a net planted plot, P is a protective row variety, X is a main cultivated variety, mainly conventional rice and hybrid rice, O is an interplanted variety, mainly a conventional local variety, C is a seedling field photograph, D is a seedling photograph, and EF is a disease investigation photograph;
FIG. 5 is a graph showing the average difference of disease indexes (C and D) of rice blast morbidity (A and B) of a 12-pair variety combination in field experiments in 2013 and 2014, wherein T represents a traditional variety, M represents a modern variety, mon is net planting, and Mix is diversity inter planting;
FIG. 6 shows the relationship between the Genetic Differentiation (GDI) between rice varieties and the rice blast control effect in 2013 (A) and 2014 (B);
fig. 7 shows the rice blast control effect in the diversity inter-planting mode for the main and inter-cultivated varieties in 2013 and 2014, M is the main cultivated variety, and T is the inter-cultivated variety.
Detailed Description
The technical scheme of the present utility model will be further described with reference to specific examples, but the scope of the present utility model is not limited thereto.
Example 1
1. A method for determining genetic differentiation between rice hybrid interplanting varieties by SSR (simple sequence repeat) markers comprises the following steps:
(1) Extraction of rice genomic DNA
(1) The method comprises the steps of reducing the lower opening of a washed 96-well plate, putting seeds into the 96-well plate one by one, placing the 96-well plate on a water absorbing plate for absorbing water, moving the 96-well plate to an illumination incubator (28 ℃, 90% of humidity) for 12 hours of illumination, germinating after three days, and taking the seeds as a material for extracting DNA when the seeds grow to 10-20 cm.
(2) A seedling of rice is taken, sheared and placed in a 1.5ML centrifuge tube, added into 1/3 volume of the centrifuge tube, added with liquid nitrogen, and repeatedly ground into powder by a grinding rod.
(3) The powder-filled centrifuge tube was added with 700. Mu.L of 2% CTAB buffer [100mM Tris-HCl (pH 8.0), 20mM EDTA (pH 8.0), 1.4mol/LNaCl,2% cetyltrimethylammonium bromide (CTAB) ] and the CTAB buffer was preheated to 65℃before addition, the tube lid was closed and inverted up and down several times, and after this, it was incubated in a water bath at 65℃for 30min to 1h, and inverted up and down several times every 15 minutes.
(4) The sample was removed from the water bath, chloroform/isoamyl alcohol (24:1) was added, and the volume was equal to 700. Mu.L of the extract, and the tube was gently shaken for 5-10 minutes to thoroughly mix and centrifuged at 12,000rpm for 15min at room temperature.
(5) The supernatant was pipetted in 650. Mu.L, transferred to another sterilized 1.5mL centrifuge tube, the lower organic phase was discarded, and chloroform/isoamyl alcohol (24:1) was added in 650. Mu.L equal volume to the supernatant, and the centrifuge tube was inverted upside down to thoroughly mix, and centrifuged at 12,000rpm for 15min.
(6) The supernatant 600. Mu.L was pipetted into a sterilized 1.5mL centrifuge tube, the same volume of 600. Mu.L of pre-chilled isopropyl alcohol at-20℃was added, the centrifuge tube was gently inverted to mix, and the centrifuge tube was kept in a refrigerator at-20℃for 2 hours or more to precipitate DNA.
(7) Centrifuge at 10,000rpm for 10min, pour off supernatant and leave the pellet. The DNA with good quality should be colorless transparent gel-like object.
(8) The mixture was rinsed twice with 400. Mu.L of 70% ethanol to remove impurities, centrifuged at 10,000rpm for 5min, and the precipitate was collected. Air-drying in a sterile workbench until there is no water drop at the bottom of the tube, at which time the DNA should be invisible to naked eyes.
(9) 100. Mu. LTE buffer (10 mM Tris-HCl (pH 8.0), 1mM EDTA (pH 8.0)) or sterilized ultrapure water was added to dissolve the DNA, and the mixture was shaken and mixed, and stored in a refrigerator at-20℃for use.
(2) Detection of Rice genomic DNA concentration
The genomic DNA concentration was detected by ethidium bromide fluorescent staining. A2. Mu.L sample of LDNA was mixed with 2. Mu.L loading buffer (98% formamide, 10mM EDTA, 0.25% bromophenol blue, 0.25% xylene blue) (with anthocyanin addition at a ratio of 39:1), and the sample was added to a 0.8% agarose gel well, while the prepared lambda DNA solutions (25 ng/. Mu.L, 50 ng/. Mu.L, 100 ng/. Mu.L, respectively) were added as controls. And (3) electrophoresis at 220V for 15min, taking out, placing on an ultraviolet imaging system for observation and shooting, and judging the concentration of the sample DNA according to the contrast between the brightness of the DNA band and the standard Marker DNA.
(3) The SSR primers are evenly distributed on 48 SSR sites on 12 chromosomes of rice (each chromosome contains 4 SSR sites on the long and short arms of the 12 chromosomes respectively) for molecular fingerprint experiments, 48 pairs of fluorescent primers capable of effectively distinguishing genetic differentiation of rice varieties (fluorescent tags are added to all the primers), the molecular weight of amplified target fragments is moderate (about 80 bp-350 bp), the 5' ends of the 48 pairs of microsatellite SRR primers are respectively connected with fluorescent groups with different colors to serve as fluorescent detection markers, and the numbers, the chromosome positions and the sequences of the SSR primers are shown in Table 1 in detail.
(4) PCR amplification and electrophoresis
All PCR reactions were performed using a Applied Biosystem2720 thermocycler (company Applied Biosystems, usa). The volume of each reaction system was 10. Mu.l, and the components were as follows: mu.L 10 Xbuffer (containing 2 mmol/lMgCl) 2 ) 0.8. Mu.L dNTP (10 mmol/L), 0.2. Mu.L forward primer (10. Mu. Mol/L), 0.2. Mu.L reverse primer (10. Mu. Mol/L), 0.05. Mu.L TaqDNA polymerase, 50ng (1. Mu.L) template DNA, about 10. Mu.L double distilled water;
the PCR reaction was performed under the following conditions:
pre-denatured at 94℃for 4min … … … … … … … … … 1cycle
94℃30s，55℃30s，72℃30s………30cycles
72 ℃ for 7min as final extension stage … … … … 1cycle
The product is stored at 4deg.C
The PCR products were isolated and analyzed by Genotyper (ABI 3130xl;Applied Biosystems) fluorescent capillary electrophoresis system.
(5) Electrophoresis detection and electrophoresis result reading
The SSR primers are 48 SSR sites which are evenly distributed on 12 chromosomes of rice, 48 pairs of fluorescent primers (all of which are added with fluorescent labels) capable of effectively distinguishing genetic differentiation of rice varieties are selected, the molecular weight of amplified target fragments is moderate (about 80 bp-350 bp), 5' ends of the 48 pairs of microsatellite SRR primers are respectively connected with fluorescent groups with different colors to serve as fluorescent detection markers, and the numbers, the chromosome positions and the sequences of the SSR primers are shown in Table 1 in detail.
The amplified microsatellite fragments show different colors, red, blue or green fluorescent marking groups, 3-5 pairs of SSR primer amplified fragments are mixed according to the colors of the fluorescent marking groups and the molecular weight of the amplified fragments, 1 mu L of mixed products are extracted, 9 mu L of Hi-Di formamide and GeneSca-nTM-500LIZ internal standard (U.S. Applied Biosystems Inc, the internal standard lengths are 35bp, 50bp, 75bp, 100bp, 139bp, 150bp, 160bp, 200bp, 300bp, 340bp, 350bp and 400bp in sequence) are added, and the mixed uniformly. The above mixture was denatured at 94℃for 5min, cooled to 4℃and subjected to capillary electrophoresis separation above ABI 3130X 1 (U.S. Applied Biosystems Inc.) sequencing. After separation of the amplified products by capillary electrophoresis, the molecular weight was read using the biological software genemap river.3.7 (us Applied Biosystems inc.). Based on the co-dominant nature of the SSR primers, two equal amounts of molecular weight (bp) are read if the amplified fragment appears as a single band, and two different molecular weights (bp) are read if the amplified fragment appears as a double band.
(6) Calculation of Genetic Differentiation Index (GDI)
The genetic differentiation index was calculated using a data-based processed SSR molecular fingerprint. According to the SSR fingerprint, all samples are arranged and assigned from small to large in molecular weight at the band of any one SSR site (labeled as the i-th primer, i=1, 2,3 … … N, n=48), and the band with the smallest molecular weight is assigned to 0 and is increased from small to large, for example: 100bp,102bp,108bp and 110bp … … are assigned 0,1,4,5 … … respectively. After the assignment is completed, an assignment data matrix covering all rice samples is formed. And selecting any two samples (such as A and B) in the assignment matrix, namely all SSR locus allelic polymorphism, and calculating the Genetic Differentiation Index (GDI) of the two varieties (variety pairs) by using the locus polymorphism.
The calculation formula of the seed Genetic Differentiation Index (GDI) of the hybrid rice variety pair is as follows:
wherein A is i1 And A i2 Representing polymorphism assignment (0-n) of A sample allele at ith SSR site, B i1 And B i2 Representing the polymorphism assignment (0-n) of the B sample allele at the ith SSR site. Ma xi Assigning a band comprising all allelic polymorphisms at the ith SSR siteIs (n) the maximum value of (c). N represents the total number of SSR primer pairs (currently 48).
(7) Linear regression analysis
The experiment uses a statistical linear correlation analysis method to perform correlation analysis on the effect value of the correlation parameters of the GDI of the rice diversity mixed-planting combined variety and the rice blast prevention and control and the yield. R is a correlation coefficient, and represents the degree of correlation (P >0.05 represents uncorrelation, P <0.05 represents significant correlation, and P <0.01 represents extremely significant) and properties (positive correlation or negative correlation) of two variables (X, Y) with a straight line. Regression equations are used to represent the form of the relationship between the two variables.
2. In order to establish a standardized procedure for detecting genetic differentiation levels between any rice varieties, a neutral molecular marker SSR is selected, 13 rice varieties which are collected for diversity interplanting in Yunnan province are analyzed, and the result shows that 48 pairs of SSR primers show polymorphism in 13 rice varieties, the molecular weight variation range is 100-500 bp, FIG. 1 is an amplification product diagram of a primer RM236 on partial materials, and 4 alleles of a primer RM263 in a population are known from FIG. 1, so that the selected SSR primers have polymorphism in an experimental population and polymorphic sites can be well distinguished.
And reading electrophoresis bands of SSR sites at 48 according to 13 varieties, and arranging and assigning the molecular weights of the amplified bands from small to large to form an assignment data matrix covering all rice samples. The Genetic Differentiation (GDI) value between the combined varieties is calculated by the genetic differentiation formula between the main variety and the intercropping variety, and the variation range of the Genetic Differentiation Index (GDI) is between 0.00 and 1.00. 0.00 shows that there is no genetic differentiation among varieties, 1.00 shows that there is extremely high level of genetic differentiation among varieties, 13 varieties are matched with other varieties respectively, the result shows that the Genetic Differentiation Index (GDI) among different rice matched combination variety pairs has great change, the change range is 0.00-0.41 (see table 2), the different rice varieties have great difference based on the genetic basis determined by SSR molecular markers, the quantitative description is carried out on the genetic differentiation level among varieties by using the SSR molecular markers and the method for calculating the GDI, and the description result is effective and reliable.
TABLE 2 Paddy rice variety Paddy differentiation index (GDI)
3. Genetic Differentiation (GDI) between rice varieties in previous experiments
Selecting a variety of rice mixed intercropping popularization demonstration combination varieties in Yunnan province in 2001-2002, selecting 16 pairs of variety collocation combination pairs in total, wherein the variety collocation combination pairs are shown in a table 3, the variety collocation combination pairs are shown in a table 4, and determining the genetic differentiation condition of the rice intercropping varieties by utilizing SSR molecular markers.
Polymorphism analysis was performed on 20 rice varieties among 16 variety combination pairs by using 48 pairs of SSR primers, an SSR matrix chart was formed, and Genetic Differentiation Index (GDI) analysis was performed on the 16 pairs of variety combination pairs by band reading and assignment (Table 4). The results show that the Genetic Differentiation Index (GDI) between different rice collocation combination variety pairs is different, wherein the combined genetic differentiation index value of HuangKN/II Y838 and DBN-2/GY527 is 0.36 at the highest, the genetic differentiation index value of HuangKN/DX4 is 0.11 at the smallest, the genetic differentiation indexes of other 13 pairs of combinations are distributed between 0.11 and 0.36, and the average value of the two groups of genetic differentiation indexes is compared, so that the average value of the GDI value in the I high-efficiency collocation combination is 0.34 and is obviously higher than the GDI value of the III low-efficiency collocation combination by 0.17 (P < 0.001).
TABLE 3 Rice sample information Table
TABLE 4 genetic differentiation values (GDI) and control Effect of the combination variety pairs
Further analyzing the correlation between the control effect and the Genetic Differentiation Index (GDI) of 16 pairs of main cultivars and intercropped cultivars, the result shows that the control effect and the Genetic Differentiation Index (GDI) of the intercropped cultivars show a remarkable positive correlation (R 2 ＝0.72，R＝0.85，P<0.01, FIG. 2), the control effect of the main cultivar has the same trend as that of the interplanted cultivar, i.e., the control effect of the main cultivar also has a significant positive correlation with the Genetic Differentiation Index (GDI) (R 2 ＝0.68，R＝0.82，P<0.01, FIG. 2), that is, the better the control effect of rice blast in the diversified intercropping system, the higher the GDI value of the genetic differentiation index, and the better the genetic blocking effect caused by the higher the corresponding genetic differentiation index.
Example 2
1.1 materials and methods
1.1.1 Experimental materials
According to rice varieties which are used for large-area planting in a diversity mixed planting mode in 10 years of Yunnan province, 22 rice varieties representing different ecological planting areas are selected, wherein the 22 rice varieties comprise traditional varieties and modern improved varieties. The names, types and sources of varieties are shown in Table 5. The method of variety DNA extraction was the same as in example 1. Based on the Genetic Differentiation Index (GDI) value obtained between varieties, a variety combination pair for field test was selected in combination with a combination example of main-cultivated and interplanted rice varieties widely used for diversity mixed-cultivation within 10 years (see Table 6).
TABLE 5 Rice sample information Table for this study
1.1.2 Genetic Differentiation Index (GDI)
The calculation of the genetic differentiation index is based on the result of the amplified product of the SSR molecular marker, the SSR primer used in the application is identical to that in example 1, PCR amplification and band reading are identical to those in example 1, and the calculation of the Genetic Differentiation Index (GDI) is identical to that in example 1.
1.1.3 field controlled Experimental design
1.1.3.1 test site
The test was conducted in 4 to 10 months in 2013 in the baoshan county of the indica-japonica mixed rice district of Yunnan province and in Jianshan county of red river, and in 4 to 10 months in 2014 in the baoshan county of the indica-japonica mixed rice district of Yunnan province and in the rogowski county of the triangu. The region is a main rice planting yield region and a rice blast perennial region in province.
1.1.3.2 test treatment
The test sets 3 treatments, namely (1) diversity mixed intercropping planting, namely rice main planting variety and intercropping variety mixed planting, wherein the planting mode adopts a double-dragon water outlet planting mode, each two rows of main planting variety are combined, the row spacing in the group is 15cm, the row spacing between the groups is 30cm, the plant spacing is 15cm, each two groups of middle row planting intercropping variety, the cell length and width are (400 cm multiplied by 235 cm) (see fig. 4, wherein A is diversity intercropping cell section view, B is net planting cell section view, "P" is protection row variety, "X" is main planting variety, mainly uses conventional rice and hybrid rice as main variety, "O" is intercropping variety, mainly uses traditional local variety as main variety, C is seedling field photo, D is seedling, EF is disease photo investigation, (2) the main planting variety is single-work, the single-work mode is that each two rows of single-work variety are combined, the row spacing between the group is 15cm, the cell spacing is 30cm, the plant spacing is 15cm, and the cell length is (200 cm multiplied by 3.5 cm) (see fig. 4). The single-cropping mode is identical to that of the main cultivated variety, and the length and width of the cell are (200 cm multiplied by 117.5 cm). The experiment design of the complete random block is repeated for 3 times, 1 row of protection rows are arranged among the cells, 4 rows of protection rows are arranged around the whole field, and the protection rows are used for planting rice blast-sensing varieties, so that the rice blast is facilitated.
1.1.3.3 field management
Sowing the seeds in 20 days of 3 months each year, sterilizing the seeds by using 0.1% carbendazim seed dressing, raising the seedlings in a water seedling raising mode, scattering each experimental material into the seedling bed to raise the seedlings, separating each variety by 30cm, transplanting the seedlings in about 4 days of 5 months, transplanting the seedlings in a transplanting cell distribution diagram (figure 3, wherein the numbers are variety numbers, I, II and III represent the repeated times), performing water and fertilizer management in a test area according to local measures, and applying pesticides for preventing and controlling pests, namely high-chlorine toxicity and imidacloprid for preventing and controlling borers and rice planthoppers according to the occurrence condition of insect pests, and not applying any pesticides for preventing and controlling rice blast. Spraying and injecting spore suspension in the initial spike period to ensure rice blast. The field management of each treatment is consistent in the whole experimental process.
1.1.4 Rice blast disease investigation method
The rice blast investigation standard is implemented according to a grade 5 standard of national standard of the people's republic of China (GB/T1579021995), the spike stalk blast is investigated in the yellow ripe stage of rice, a 5-point sampling method is adopted for each cell, and 10-point investigation is carried out. And counting the investigation result and calculating the morbidity and the disease index. The severity of the panicle blast was rated as shown in Table 6.
TABLE 6 ear blast disease score index (in ear units)
Calculating morbidity, disease index and rice blast prevention and treatment effect according to investigation results:
(1) The morbidity formula is: i (%) = ×100, wherein: i is the incidence (%); p is the number of pathogenic plants (leaves, ears); z is the number of total plants (leaves, ears) investigated.
(2) The disease index formula is: r= ×100, where: r is disease index; pi is the number of incidences at each level; di is the representative value of each level; p is the total plant (leaf) number of investigation; DM is the highest level representative value.
(3) The disease control effect is calculated by the following formula: e (%) = ×100, wherein: e is the prevention and treatment effect; r is disease index.
1.1.5 data analysis
And (3) sorting the data obtained by investigation, and respectively calculating the incidence rate, disease index and disease control effect of rice blast in the field of each cell. The differences of the morbidity, disease index and disease control effect of each pair of variety combinations in single cropping and diversity intercropping of rice are analyzed by adopting a paired sample T test.
And carrying out correlation analysis on the correlation parameters of the GDI of the rice diversity mixed-planting combined variety and the rice blast control and the effect value of the yield by utilizing a statistical linear correlation analysis method. R is a correlation coefficient, and represents the degree of correlation (P >0.05 represents uncorrelation, P <0.05 represents significant correlation, and P <0.01 represents extremely significant) and properties (positive correlation or negative correlation) of two variables (X, Y) with a straight line. Regression equations are used to represent the form of the relationship between two variables.
The difference among disease indexes, prevention and treatment effects and morbidity of two groups with GDI more than or equal to 0.3 and GDI less than or equal to 0.22 is compared, and analysis and comparison analysis are carried out by adopting single-factor ANOVA. All the statistical analyses were performed using SPSS19.0 statistical software. All plots were made in the microsoft toffever.2010 software platform.
2.1 results and analysis
2.1.1 field controlled laboratory Combined variety Magnaporthe grisea investigation results
According to rice varieties which are used for large-area utilization in a biodiversity mixed interplanting mode in 10 years of Yunnan province, 22 varieties representing different rice ecological planting areas are selected in 2013 and 2014 respectively, wherein the 22 varieties comprise traditional (farmyard) varieties and modern improved varieties, and 12 varieties are combined together according to the genetic differentiation index GDI between the rice varieties to carry out field controlled experiments. According to the rice blast disease condition of each tested rice combination in the diversity mixed planting mode, calculating the rice blast disease index and the control effect of different varieties in different planting modes (see Table 7).
The rice blast investigation of the whole experimental area is carried out in the beginning of 9 to 10 months, the rice blast happens successively in the experimental area, the disease-sensitive variety planted in the surrounding protection row is purple glutinous, the incidence rate reaches 82.86%, and the disease index reaches 46.45. The degree of rice blast in 2013 was slightly higher than that in 2014, but there was no obvious difference. As can be seen from FIG. 5, where T represents a traditional variety, M represents a modern variety, mon is a net planting, mix is a diversity interval, the average of the combined morbidity and disease index is lower than that of the net planting, and the same trend is observed in two years of experiments.
From the selected combination, the incidence rate, disease index and control effect of each combination are greatly different in two years, the control effect of the intermediate cultivar is obviously higher than that of the main cultivar, and the improvement of the efficiency of the diversity mixed intercropping mode on the rice blast resistance of the traditional cultivar is more obvious (see Table 7).
TABLE 7 Genetic Differentiation Index (GDI) of the combination variety pairs, disease index and control Effect (E) in the pure planting (S1) and intercropping (S2) planting modes
Note that: * The numbers represent the degree of significant difference, "+" represents P <0.05, "+" represents P <0.01
4.3.2 relationship between genetic differentiation index and disease control Effect
By analyzing the results of combined field experiments of different rice varieties and analyzing the correlation of GDI between different combined varieties, we found that in the experiments in 2013, the Genetic Differentiation Index (GDI) and the control effect (R 2 ＝0.85，R＝0.92，P<0.01 Control effect (R) of main cultivar 2 ＝0.92，R＝0.96，P<0.01 All show very remarkable positive correlation (figure 6A), and the same result is obtained in the experiment of 2014, the Genetic Differentiation Index (GDI) and the control effect (R) of the intercropped variety 2 ＝0.77，R＝0.88，P<0.01 Control effect (R) of main cultivar 2 ＝0.76，R＝0.87，P<0.01 All show very significant positive correlationAs the GDI value between the combination varieties increases, the rice blast control effect increases (FIG. 6B).
4.3.3 genetic differentiation index the difference between the GDI.gtoreq.0.3 group and the GDI.gtoreq.0.22 group
In the group according to the Genetic Differentiation Index (GDI), the average value of the incidence rate and the disease index of the rice net planting in the combination that the GDI is more than or equal to 0.3 is obviously higher than that of the diversity interplanting (P < 0.05). The average incidence rate of the two-year traditional variety net planting is 38.38%; the average incidence of the interplanting was 23.52%; the average disease index of the net planting is 17.60; the average disease index of the intercropping is 7.65; the average incidence rate of the two-year modern breed net planting is 20.57%; the average incidence rate of the intercropping is 12.03%; the average disease index of the net planting is 9.34; the average disease index of the interplanting was 4.84.
In the combination that GDI is less than or equal to 0.22, the average value of the incidence and the disease index of the rice net planting treatment and the diversified intercropping treatment are not obvious (P > 0.05), the average value of the incidence and the disease index of the traditional variety for two years is expressed as that the net planting treatment is higher than the intercropping treatment (P > 0.05), and the average value of the incidence and the disease index of the modern variety for two years is expressed as that the net planting treatment is lower than the intercropping treatment (P > 0.05). The average incidence rate of the pure planting of the traditional variety is 20.23%; the average incidence of the interplanting was 17.70%; the average disease index of the net planting is 8.79; the average disease index of the intercropping is 7.39; the average incidence rate of the two-year modern breed net planting is 37.57%; the average incidence of the interplanting was 39.41%; the average disease index of the net planting is 20.45; the average disease index of the interplanting was 21.11.
As shown in FIG. 7, M is the main cultivar and T is the intercropping cultivar, the control effect of the combination with GDI not less than 0.3 is obviously higher than that of the combination with GDI not less than 0.22 (P < 0.01), the control effect in 2013 is better than that in 2014, and the average control effect of the traditional cultivar in two years is 55.47% in the combination with GDI not less than 0.3; the average control effect of modern varieties for two years is 45.88%; the average control effect of the traditional variety in the combination with GDI less than or equal to 0.22 is 15.36 percent for two years; the average control effect of modern varieties is 4.49 percent in two years.
According to the results of two-year field controlled experiments and the data analysis results obtained by previous large-scale rice diversity field production, we propose to determine GDI values 0.22 and 0.3 obtained by SSR molecular marker detection analysis calculation as the threshold value of the selected variety combination, that is, when the GDI value obtained by SSR molecular marker detection and calculation of the rice hybrid variety is higher than 0.3, the mixed interplanted rice variety combination shows lower incidence of rice blast and good rice blast control effect; when the GDI value obtained by using SSR molecular marker detection and calculation to detect and calculate the rice hybrid cultivars is lower than 0.2, the rice hybrid interplanted rice variety combination is characterized by higher incidence of rice blast and poorer or invalid control effect. The variety combination is screened through the reference threshold value of the Genetic Differentiation Index (GDI), so that the variety combination of the variety mixed planting can be simply, conveniently and reasonably determined, and the principle and parameters for optimizing the planting combination are provided for predicting the reduction of the incidence rate of rice blast and the improvement of the control efficiency of the rice blast.
The above study results show that: the SSR molecular markers established by us can be used for detecting and calculating the genetic differentiation indexes of rice varieties to be subjected to diversity mixed intercropping, and predicting the rice blast resistance after intercropping by using the degree of the genetic differentiation indexes of matched combined varieties. The method not only provides genetic explanation for the variety assembly principle of the rice variety mixed interplanting, but also provides theoretical basis and parameter index for the implementation of reasonable assembly of the variety of the rice variety mixed interplanting.
The rice blast is the most important disease threatening the rice production, is also the limiting factor of the Yunnan rice production in southwest China, and the use of pesticides and chemical fertilizers can not effectively control the rice blast, but also leads to the deterioration of a rice ecological system, so that the further improvement of the rice productivity is limited. The mode of planting between the various hybrid rice varieties plays a positive role in reducing the epidemic and hazard of rice blast, and provides an example of preventing and controlling farmland diseases and insect pests by utilizing biological diversity. However, with the updating of varieties, more varieties are required to be assembled, and the efficient and rapid variety mixing-planting variety assembling method becomes key. The utility model screens a method for determining genetic differentiation between a main cultivar and an intercropped cultivar by utilizing an SSR molecular marker technology, reads the molecular weight of the main cultivar and the intercropped cultivar after PCR amplification and capillary fluorescent electrophoresis separation, and forms a valuation data matrix covering all rice samples. And selecting all SSR locus allelic polymorphism of any two varieties in the assignment matrix, and calculating Genetic Differentiation Index (GDI) of the two varieties (variety pairs) by utilizing the locus polymorphism. The Genetic Differentiation Index (GDI) varies from 0.00 to 1.00. 0.00 shows no genetic differentiation among varieties, 1.00 shows extremely high level genetic differentiation among varieties, 13 varieties selected through the research are matched with each other, the result shows that different rice matched combination variety pairs have great change, the change range is between 0.00 and 0.41, different rice varieties have great difference based on the genetic basis determined by SSR molecular markers, the method for calculating GDI can effectively and accurately quantitatively describe the genetic differentiation level among combination varieties by utilizing SSR molecular markers, and the establishment of the method solves the problem that the genetic differentiation detection of rice variety combinations lacks quantitative analysis tools in the mixed interplanting process. Lays a foundation for further verifying the relation between the genetic differentiation of the combined varieties under the diversity mixed planting and the rice blast resistance performance.
SSR calculation to establish a standardized procedure for detecting genetic differentiation levels between arbitrary rice varieties, we selected neutral molecular markers SSR to establish SSR fingerprints of rice varieties, and calculated genetic differentiation index (genetic divergence index, GDI) between every two rice varieties according to each rice SSR fingerprint. And through the research of disease related parameters, the positive correlation between the genetic differentiation index and the control of rice blast is found. This result strongly explains the phenomenon that different combinations have different control effects on rice blast in field data, namely, the greater the genetic differentiation level between rice combination varieties, the higher the efficiency of rice blast resistance in fields in hybrid planting. Therefore, by utilizing the genetic differentiation index, the field rice blast resistance level of different mixed varieties can be predicted, and theoretical guidance and technical parameters are provided for reasonably utilizing the matching of the rice varieties planted in the biological diversity mixed varieties to control rice blast.
The foregoing description is only of specific embodiments of the present utility model and is not intended to limit the scope of the present utility model, and any changes and substitutions without inventive effort are intended to be included in the scope of the present utility model. Therefore, the protection scope of the present utility model should be subject to the protection scope defined by the claims.
Sequence listing
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Claims (9)
1. The SSR marker-determined rice hybrid interplanting variety genetic differentiation specific primer pair is characterized in that the SSR primers are 48 SSR sites evenly distributed on 12 chromosomes of rice, 48 pairs are total, and the list is as follows:
2. a method for determining genetic differentiation between rice hybrid interplanting varieties by SSR (simple sequence repeat) marking, which is characterized by comprising the following steps:
(1) Extracting rice genome DNA;
(2) Performing PCR amplification on the extracted rice genomic DNA sample by using 48 pairs of specific primers according to claim 1;
(3) Separating the PCR product by utilizing a capillary electrophoresis method, and reading the molecular weight of the PCR product;
(4) Calculation of genetic differentiation index: the genetic differentiation index is calculated by utilizing the datamation processing SSR molecular fingerprint, and the calculation formula of the genetic differentiation index among the mixed intercropped rice varieties is as follows:
wherein A is i1 And A i2 Representing polymorphism assignment (0-n) of A sample allele at ith SSR site, B i1 And B i2 Representing polymorphism assignment (0-n) of B sample allele at ith SSR site, max i Assigning a maximum value (N) to all allele polymorphism bands contained in the ith SSR locus, wherein N represents the total number of SSR primer pairs;
(5) Linear regression analysis: and carrying out correlation analysis on the GDI and the effect value of the related parameters for preventing and treating rice blast by using a statistical linear correlation analysis method.
3. The method according to claim 2, characterized in that: the step (1) specifically comprises the following steps:
step (1) putting rice seeds into a 96-well plate one by one, putting the 96-well plate on a water absorbing disc to absorb water, transferring to an illumination incubator, culturing until seedlings grow to 10-20cm, taking a rice seedling, adding liquid nitrogen, grinding into powder, adding 700 mu L of 2% CTAB buffer solution, carrying out water bath, adding chloroform/isoamyl alcohol, mixing uniformly, and centrifuging;
step (2) taking 650 mu L of supernatant, transferring into a sterilized centrifuge tube, adding 650 mu L of chloroform/isoamyl alcohol, fully and uniformly mixing, centrifuging, taking 600 mu L of supernatant, adding 600 mu L of isopropanol, uniformly mixing, refrigerating and storing for more than 2 hours, and precipitating DNA;
and (3) centrifuging, discarding supernatant, reserving precipitate, adding 70% ethanol for rinsing twice to remove impurities, centrifuging, collecting precipitate, performing sterile air drying on water, adding 100 mu LTE buffer solution or sterilized ultrapure water to dissolve DNA, shaking, mixing uniformly, and storing in a refrigerator at-20 ℃ for later use.
4. A method according to claim 3, characterized in that: the condition of illumination culture in the step (1) is 28 ℃, the humidity is 90 percent, and illumination is carried out for 12 hours; CTAB buffer was 100mM Tris-HCl, pH8.0, 20mM EDTA,pH8.0,1.4mol/L NaCl,2% cetyltrimethylammonium bromide.
5. A method according to claim 3, characterized in that: the CTAB buffer solution in the step (1) is preheated to 65 ℃ before being added, and the water bath condition is that the water bath is carried out for 30min-1h at 65 ℃.
6. A method according to claim 3, characterized in that: the volume ratio of chloroform to isoamyl alcohol in the step (1) is 24:1.
7. A method according to claim 3, characterized in that: the isopropanol in the step (2) is precooled under the condition of-20 ℃.
8. A method according to claim 3, characterized in that: the PCR amplification in step (3)The system comprises: 1 mu L of 10 Xbuffer containing 2mmol/L MgCl 2 0.8. Mu.L of 10mmol/LdNTP, 0.2. Mu.L of 10. Mu. Mol/L forward primer, 0.2. Mu.L of 10. Mu. Mol/L reverse primer, 0.05. Mu.L Taq DNA polymerase, 50ng of 1. Mu.L template DNA, 10. Mu.L double distilled water.
9. A method according to claim 3, characterized in that: the conditions for PCR amplification were: pre-denatured at 94℃for 4min … … 1cycle
94℃30s，55℃30s，72℃30s……30cycles
72 ℃ for 7min as final extension stage … … 1cycle
The product was stored at 4 ℃.
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| WO2003085133A2 (en) * | 2002-04-08 | 2003-10-16 | Centre For Dna Fingerprinting And Diagnostics | Novel fissr-pcr primers and method of genotyping diverse genomes of plant and animal systems including rice varieties |
| CN103789308A (en) * | 2014-01-23 | 2014-05-14 | 黑龙江省农业科学院耕作栽培研究所 | Molecular marker for japonica rice genetic diversity analysis and authentication method for molecular marker |
| CN103911441A (en) * | 2014-03-13 | 2014-07-09 | 南宁益谱检测技术有限公司 | Genetic analysis method based on capillary electrophoresis and SSR fluorescence labeling for rice |
| CN108374054A (en) * | 2018-05-21 | 2018-08-07 | 黑龙江省农业科学院农产品质量安全研究所 | Suitable for one group of rice SSR molecular marker of capillary electrophoresis detection technology and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003085133A2 (en) * | 2002-04-08 | 2003-10-16 | Centre For Dna Fingerprinting And Diagnostics | Novel fissr-pcr primers and method of genotyping diverse genomes of plant and animal systems including rice varieties |
| CN103789308A (en) * | 2014-01-23 | 2014-05-14 | 黑龙江省农业科学院耕作栽培研究所 | Molecular marker for japonica rice genetic diversity analysis and authentication method for molecular marker |
| CN103911441A (en) * | 2014-03-13 | 2014-07-09 | 南宁益谱检测技术有限公司 | Genetic analysis method based on capillary electrophoresis and SSR fluorescence labeling for rice |
| CN108374054A (en) * | 2018-05-21 | 2018-08-07 | 黑龙江省农业科学院农产品质量安全研究所 | Suitable for one group of rice SSR molecular marker of capillary electrophoresis detection technology and its application |
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