CN108811869B - Large-scale accurate identification method for resistance to brown planthopper of rice in adult-plant stage - Google Patents
Large-scale accurate identification method for resistance to brown planthopper of rice in adult-plant stage Download PDFInfo
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- 230000006378 damage Effects 0.000 claims description 15
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Abstract
The invention discloses a large-scale accurate identification method for the resistance of rice brown planthopper resistance in a rice plant growing period, and belongs to the field of rice genetic breeding science. The invention discloses a large-scale accurate identification method for the resistance of rice against brown planthopper in the adult stage of rice, which comprises the following steps: (1) preparing a rice straw sample; (2) planting a rice straw sample; (3) excessive inoculation identification; (4) managing after inoculation; (5) and (5) evaluating the identification result. The invention establishes a rice stump identification method and provides a high-efficiency resistance identification technology for the rice brown planthopper resistance breeding of various scales. Generally, the resistance breeding materials with large, medium and small scales can be identified once or repeatedly for many times, and the identification result of the seedling stage identification can be repeatedly identified in the adult plant stage, so that the identification result is accurate, and the progress of resistance genetic breeding projects is effectively promoted.
Description
Technical Field
The invention belongs to the field of rice genetic breeding science, and particularly relates to a large-scale accurate identification method for the resistance of rice brown planthopper resistance in a rice adult plant stage.
Background
Rice is one of the main grain crops in China, and the rice production process faces serious threat of brown planthopper. The brown planthopper is a rice pest with outbreak and great harm, the generation area of the rice planthopper (mainly brown planthopper) in each year in rice areas in China is more than 2000 million hectares at present, and the direct rice loss caused by the damage of the rice planthopper reaches more than 280 million tons. For a long time, the prevention and control of the brown planthopper mainly depend on the application of chemical insecticides, and the application of a large amount of insecticides often causes the destruction of the ecological environment, the killing of a large amount of natural enemies of the brown planthopper and the improvement of the drug resistance of pests, thereby causing the 'rampant' of the brown planthopper and bringing greater difficulty to the prevention and control. Practice proves that the cultivation of the resistant rice variety is the most economic, effective, safe and ecological method in the comprehensive control of the brown planthopper. Resistance breeding mainly comprises the steps of introducing resistance genes into excellent rice breeding materials, wherein resistance identification is a key step in resistance gene mining and resistance breeding, stable and reliable resistance progeny are obtained through resistance identification of breeding progeny, and resistant rice varieties are bred from the stable and reliable resistance progeny; by identifying the resistance of the genetic segregation population, the genetic analysis and the molecular marker positioning of the resistance gene can be carried out. The brown planthopper is harmful in production, but the resistance identification of the brown planthopper in the rice plant forming period always has the problems of low identification efficiency, poor identification result reliability and the like, and the application in resistance breeding, construction of genetic analysis groups and the like is not large, so that the method has important practical significance in solving the technical problems of high efficiency, rapidness and accurate identification of the rice plant forming period.
Disclosure of Invention
The invention aims to establish a high-efficiency and accurate identification technical system for the resistance of the rice to the brown planthopper in the adult stage of the rice and application of the technology in breeding of the rice resistant to the brown planthopper and establishment of genetic analysis groups.
The purpose of the invention is realized by the following technical scheme:
a large-scale accurate identification method for the resistance of rice against brown planthoppers in the adult stage of rice comprises the following steps:
(1) preparing a rice straw sample;
planting the rice group to be identified in a field, and performing field management on the rice by adopting a conventional planting method; stripping one rooted tillering from each rice plant of a group to be identified in a plant growing period, and cutting a lower rice stump by using scissors to serve as an identification sample;
(2) planting a rice straw sample;
planting rice piles in rows in a tray pot filled with shallow soil, and using pest-sensitive varieties as comparison, wherein 1 row of comparison varieties is planted in each tray pot;
(3) excessive inoculation identification;
inoculating and identifying within 3 days after planting rice stakes, and inoculating more than 20 heads of 2-3 instar brown planthopper nymphs to each rice stake;
(4) managing after inoculation;
i, after the insects are inoculated, keeping the insect prevention net cover sealed to prevent the insects from escaping;
II, keeping a water layer with the depth of not more than 1cm in the basin; water body isolation is needed outside the basin to prevent ants from being harmful;
III, placing the tray in an identification room with a sunshade net or a place with weak sunlight for culturing, and preventing the high temperature of more than 32 ℃;
IV, moving to a shady and cool place in time when encountering high temperature and direct strong sunlight;
reducing airflow flowing in an identification room or a place for placing an identification basin outdoors, closing a windshield window door on the windward side of the identification room when obvious airflow occurs, and arranging a wind shield for outdoor identification;
(5) evaluating the identification result;
recording resistance identification results 1-2 days after all the pest-susceptible control varieties die, carrying out evaluation recording according to 9-grade standard, grading each seedling, and calculating the average damage grade of each material:
grade 1 (1.0-1.9) is High Resistance (HR), and the damage is not obvious;
grade 3 (2.0-3.9) is anti (R), and the first leaf sheath on the periphery turns yellow;
grade 5 (4.0-5.9) is Medium Resistance (MR), the second leaf sheath on the periphery turns yellow, and the growth potential of plants is obviously weakened;
grade 7 (6.0-7.9) is a medium feeling (MS), and the plant begins to wither;
grade 9 (8.0-9.0) is high-sensitivity (HS), and the plant dies;
(6) repeatedly identifying;
repeated identification can be carried out according to actual needs;
(1) performing instant repeated identification, namely taking repeated rice stakes according to the repeated times during sampling, and performing repeated identification for more than 2 times according to the experimental design;
(2) and (4) once repeated identification, after one-time identification is finished, referring to the identification result, and if necessary, flexibly sampling before heading stage to perform repeated identification.
Preferably, in the step (1), each tiller is used for cutting a lower rice stump with scissors to serve as an identification sample, and the cutting length is 10-20 cm.
Preferably, in the step (1), the tillers stripped from different plants are required to be consistent in size, strong in growth and free of plant diseases and insect pests.
Preferably, in the step (2), the pest susceptible variety is TN1, 9311 or nipponica.
Preferably, in the step (2), the spacing between the rows of the plants is 4-8 cm.
Preferably, in the step (3), within 3 days after inoculation, the inoculation source is added to the tray with possibly insufficient insect source to achieve sufficient insect population density.
Preferably, in the step (3), after the insects are inoculated, an insect-proof net cover is covered on the rice stakes on the supporting pot.
Preferably, the identification method can be carried out at any time during the adult stage from 20 days after rice transplantation to heading stage.
The invention discloses a large-scale accurate identification method for the resistance of brown planthopper resistance of rice in the adult stage, which is applied to brown planthopper resistant rice genetic breeding, genetic research or wild rice resistance resource screening.
The invention has the following beneficial effects:
1. the invention establishes a rice stump identification method and provides a high-efficiency resistance identification technology for the rice brown planthopper resistance breeding of various scales. Generally, the resistance breeding materials with large, medium and small scales can be identified once or repeatedly for many times, and the identification result of the seedling stage identification can be repeatedly identified in the adult plant stage, so that the identification result is accurate, and the progress of the resistance breeding project is effectively promoted.
2. The rice stub identification method is suitable for construction of mapping populations for genetic analysis research and gene marker positioning research of genes resisting brown planthoppers of rice, and particularly can more effectively provide an accurate identification technology for construction and family analysis of large-scale mapping populations when the conventional adult-plant-stage identification method is not suitable for construction and accurate analysis of large-scale mapping populations.
3. In the breeding process of brown planthopper resistance of rice, when the source of resistance genes is unclear, the genetic background is complex, and resistance characters do not have applicable molecular markers, or when marker-assisted selection is difficult to accurately identify resistant plants, the technical method provides an effective screening alternative method for resistant plants. Through detecting the breeding material, whether the detected material has the resistance of the brown planthopper can be judged, and then the insect-resistant variety or strain is rapidly screened for rice breeding.
4. The invention can carry out resistance identification at any time in the adult rice plant stage, namely, the optimal time or the optimal conditions can be selected for identification according to the actual conditions from 20 days after rice transplantation to heading stage.
5. Compared with plants, the rice stakes have greatly reduced biomass, can realize excessive inoculation of pests, lead the sensitive materials to die quickly, lead the identification period to be synchronous with the pest development period, improve the accuracy of the identification result and obtain the identification result in a shorter time.
6. According to the invention, multiple rice stakes can be taken from each plant for repeated identification, and 1 rice stake represents 1 repetition, so that the accuracy of the identification result is improved.
7. If the seedling stage identification is carried out, the obtained resistant plants can be repeatedly identified and verified in the adult stage in the same season, so that the accuracy and the timeliness of the identification result are improved.
Drawings
Fig. 1 is a graph of the growth condition of rice stakes 4 days after resistance identification and insect inoculation in the rice stake forming stage, the viability of sensitive plants is obviously weakened and begins to turn yellow [ 1 row for each material, the left 2 nd row is respectively: ptb33 resistance, candidate Bph3 near isogenic line 1, TN1 (sensory control), candidate Bph3 near isogenic line 2, 9311 (recurrent parent), candidate Bph3 near isogenic line 3 ].
Fig. 2 is a diagram of the growth condition of rice stumps after resistance identification and identification in the plant-growing period by the rice stump method, wherein all the sensitive comparison materials and the sensitive identification materials die [ 1 row of each material, the left part is as follows: ptb33 resistance, candidate Bph3 near isogenic line 1, TN1 (sensory control), candidate Bph3 near isogenic line 2, 9311 (recurrent parent), candidate Bph3 near isogenic line 3 ].
FIG. 3 is a graph showing the growth of rice plants before the identification of plant resistance (control) and inoculation by a conventional method.
FIG. 4 is a diagram showing the growth of rice plants after the identification of plant resistance by the conventional method (control) is completed, [ two lines per material, left: ptb33 resistance, candidate Bph3 near isogenic line 1, TN1 (sensory control), candidate Bph3 near isogenic line 2, 9311 (recurrent parent), candidate Bph3 near isogenic line 3 ].
The results of the rice stump method resistance identification (figure 2) and the conventional plant resistance identification (contrast) (figure 4) are completely the same, the Ptb33 resistance source is expressed as high resistance, the candidate Bph3 near isogenic line 3 is expressed as medium resistance, the other materials are expressed as high sensitivity, and the resistance grading difference is obvious, which indicates that the rice stump method resistance identification result is reliable, simple and efficient.
Detailed Description
The present invention is further illustrated by the following specific examples in order that the advantages and features of the invention may be more readily understood, it being understood that the following examples are only preferred versions of the invention, and are not intended to limit the invention.
Example 1
A large-scale accurate identification method for the resistance of rice against brown planthoppers in the adult stage of rice comprises the following steps:
(1) preparing a rice straw sample;
planting the rice group to be identified in a field, and performing field management on the rice by adopting a conventional planting method; stripping one rooted tillering from each rice plant of a group to be identified in a plant growing period, wherein the tillering stripped from different plants is required to be consistent in size, strong in growth and free of diseases and insect pests, each tillering uses scissors to cut a lower rice stump as an identification sample, and the cutting length is 20 cm;
(2) planting a rice straw sample;
planting rice piles in rows in a tray pot filled with shallow soil, and using pest-susceptible variety TN1 as a contrast, and planting 1 row of contrast varieties in each tray pot; when planting, the spacing between the rows and the plants is 4-6 cm;
(3) excessive inoculation identification;
inoculating and identifying insects within 3 days after the rice stakes are planted, inoculating more than 20 nymphs of 2-3 instar brown planthoppers to each rice stake, and covering an insect prevention mesh enclosure on the rice stakes on the supporting basin after the insects are inoculated; within 3 days after inoculation, adding an inoculation source to a tray with possibly insufficient insect source to achieve sufficient insect population density;
(4) managing after inoculation;
i, after the insects are inoculated, keeping the insect prevention net cover sealed to prevent the insects from escaping;
II, keeping a water layer with the depth of not more than 1cm in the basin; water body isolation is needed outside the basin to prevent ants from being harmful;
III, placing the tray in an identification room with a sunshade net or a place with weak sunlight for culturing, and preventing the high temperature of more than 32 ℃;
IV, moving to a shady and cool place in time when encountering high temperature and direct strong sunlight;
reducing airflow flowing in an identification room or a place for placing an identification basin outdoors, closing a windshield window door on the windward side of the identification room when obvious airflow occurs, and arranging a wind shield for outdoor identification;
(5) evaluating the identification result;
recording resistance identification results 1-2 days after all the pest-susceptible control varieties die, carrying out evaluation recording according to 9-grade standard, grading each seedling, and calculating the average damage grade of each material:
grade 1 (1.0-1.9) is High Resistance (HR), and the damage is not obvious;
grade 3 (2.0-3.9) is anti (R), and the first leaf sheath on the periphery turns yellow;
grade 5 (4.0-5.9) is Medium Resistance (MR), the second leaf sheath on the periphery turns yellow, and the growth potential of plants is obviously weakened;
grade 7 (6.0-7.9) is a medium feeling (MS), and the plant begins to wither;
grade 9 (8.0-9.0) is high-sensitivity (HS), and the plant dies;
(6) repeatedly identifying;
repeated identification can be carried out according to actual needs;
(1) performing instant repeated identification, namely taking repeated rice stakes according to the repeated times during sampling, and performing repeated identification for more than 2 times according to the experimental design;
(2) and (4) once repeated identification, after one-time identification is finished, referring to the identification result, and if necessary, flexibly sampling before heading stage to perform repeated identification.
Technical application verification one: identification and screening of Bph3 near isogenic line
The test material comprises a rice planthopper resistance source Ptb33, a sensitive control variety TN1, a sensitive parent 9311 and 3 to-be-identified Bph3 near isogenic lines, wherein the to-be-identified Bph3 near isogenic lines are bred by the hybridization of Ptb33 and 9311 and the continuous multi-generation backcross of offspring and 9311. After the materials are sowed and cultured, the materials are transplanted into a planting pond of a brown planthopper identification room, 2 rows of materials are planted, 8 families are planted in each row, and normal cultivation management is carried out after planting. In 25 days after transplantation, 10 families of materials in each plant stage are randomly selected, 1 rice stump in each family is planted in a supporting pot, and 1 row of rice stumps are planted in each material.
The selected material was then identified using the identification method described in this example. The plant identification material is infected with the insect control TN1 plant and completely dies at the 10 th day after the inoculation, and the rice stump identification material is infected with the insect control TN1 plant and completely dies at the 6 th day. Both treatments were investigated for resistance results after total death of TN1 plants, and samples of each material were taken for Bph3 gene marker detection, with the results shown in FIG. 1 and Table 1.
The test shows that (1) the plant resistance identification result is completely consistent with the rice stump resistance identification result; (2) the resistance source Ptb33 contains a plurality of resistance genes, and the resistance of the resistance source Ptb33 to the brown planthopper is strongest, so that the resistance level 1 is reached; (3) the susceptible control TN1, the recurrent parent 9311 and the candidate Bph3 near isogenic lines 1 and 2 do not carry resistance genes, and the resistance is 9-grade high susceptibility; (4) the resistance of a candidate Bph3 near isogenic line 3 carrying the resistance gene Bph3 is detected to reach the level of resistance in grade 5. The results show that the rice stump identification method has reliable results, short identification time, less insect consumption and high efficiency.
TABLE 1 identification of the resistance of plants and stakes to Nilaparvata lugens in the adult stage of rice materials and the detection of the molecular markers of the resistance gene Bph3
Note: "+" indicates the presence of the Bph3 gene marker and "-" indicates the absence of the Bph3 gene marker.
Example 2
A large-scale accurate identification method for the resistance of rice against brown planthoppers in the adult stage of rice comprises the following steps:
(1) preparing a rice straw sample;
planting the rice group to be identified in a field, and performing field management on the rice by adopting a conventional planting method; stripping one rooted tillering from each rice plant of a group to be identified in a plant growing period, wherein the tillering stripped from different plants is required to be consistent in size, strong in growth and free of diseases and insect pests, each tillering uses scissors to cut a lower rice stump as an identification sample, and the cutting length is 10 cm;
(2) planting a rice straw sample;
planting rice stakes in rows in a tray pot filled with shallow soil, and using pest-sensitive variety 9311 as a control, and planting 1 row of control variety in each tray pot; when planting, the spacing between the rows and the plants is 6-8 cm;
(3) excessive inoculation identification;
inoculating and identifying insects within 3 days after the rice stakes are planted, inoculating more than 25-head nymphs of 2-3 instar brown planthoppers to each rice stake, and covering an insect prevention mesh enclosure on the rice stakes on the supporting basin after the insects are inoculated; within 3 days after inoculation, adding an inoculation source to a tray with possibly insufficient insect source to achieve sufficient insect population density;
(4) managing after inoculation;
i, after the insects are inoculated, keeping the insect prevention net cover sealed to prevent the insects from escaping;
II, keeping a water layer with the depth of not more than 1cm in the basin; water body isolation is needed outside the basin to prevent ants from being harmful;
III, placing the tray in an identification room with a sunshade net or a place with weak sunlight for culturing, and preventing the high temperature of more than 32 ℃;
IV, moving to a shady and cool place in time when encountering high temperature and direct strong sunlight;
reducing airflow flowing in an identification room or a place for placing an identification basin outdoors, closing a windshield window door on the windward side of the identification room when obvious airflow occurs, and arranging a wind shield for outdoor identification;
(5) evaluating the identification result;
recording resistance identification results 1-2 days after all the pest-susceptible control varieties die, carrying out evaluation recording according to 9-grade standard, grading each seedling, and calculating the average damage grade of each material:
grade 1 (1.0-1.9) is High Resistance (HR), and the damage is not obvious;
grade 3 (2.0-3.9) is anti (R), and the first leaf sheath on the periphery turns yellow;
grade 5 (4.0-5.9) is Medium Resistance (MR), the second leaf sheath on the periphery turns yellow, and the growth potential of plants is obviously weakened;
grade 7 (6.0-7.9) is a medium feeling (MS), and the plant begins to wither;
grade 9 (8.0-9.0) is high-sensitivity (HS), and the plant dies;
(6) repeatedly identifying;
repeated identification can be carried out according to actual needs;
(1) performing instant repeated identification, namely taking repeated rice stakes according to the repeated times during sampling, and performing repeated identification for more than 2 times according to the experimental design;
(2) and (4) once repeated identification, after one-time identification is finished, referring to the identification result, and if necessary, flexibly sampling before heading stage to perform repeated identification.
And (5) technical application verification: large-scale application of' rice stake identification method
Respectively hybridizing the rice resistance source 05BPH16 (containing a resistance gene qBph30(t)) and the rice resistance source 08BPH327 (containing a resistance gene qBph29(t)) with a rice hair restorer line 9311, a Guanghui 998, a Gui 99, a test 253 and the like, the progeny F2 is separated to obtain 500 plants with excellent personality, F3 is used for planting the plants in rows, the resistance identification of the selected rice is carried out according to the method of the embodiment in the adult stage, 1 family is selected for each plant, 3 rice stakes are selected for each family for 3 times of repeated identification, and simultaneously, the rice leaf samples of the offspring of the two hybridization combinations are taken to extract DNA for gene marker analysis and verification, a special molecular marker QY14 (primer sequence 5'-AGCCCAAATGGAACAAACAA-3', 5'-GCTCACGGTTAAGCAATGGT-3') is used for detecting qBph30(t), and a closely linked molecular marker YM54 (primer sequence 5'-CTCTCCCCTCCAATCCTTTT-3', 5'-AGCACCCTGGAAAGCAGTAG-3') is used for detecting qBph29 (t). The results of identification and detection are shown in table 2, and the identified partial resistant plants contain homozygous or heterozygous resistant genes through detection, and the results show that the rice stump identification method is very suitable for large-scale resistance identification, and the identification results are completely accurate.
TABLE 2 identification of Nilaparvata lugens resistance to rice progeny by Rice stump identification method
Example 3
And (3) technical verification: wild rice resistance resource screening
Wild rice is an important resource in China, and contains a plurality of resistance genes, wherein the resistance genes comprise brown planthopper resistance genes with important utilization values. Resistant resources are screened from wild rice, and research materials can be provided for finding new genes resisting brown planthopper. However, it is difficult to harvest seeds from wild rice, and it is very inconvenient and inefficient to use seeds for identifying the resistance to brown planthopper. The test uses the rice stump identification method to identify the brown planthopper of the wild rice resource, and obtains beneficial effect.
A large-scale accurate identification method for the resistance of rice against brown planthoppers in the adult stage of rice comprises the following steps:
(1) preparing a rice straw sample;
sampling and identifying from a common wild rice storage garden of Guangxi university, taking 3 rooted tender tillers from each wild rice resource as 3 times of repeated identification materials, cutting a lower rice stub with the length of about 15cm from each tillerer by using scissors to serve as an identification sample, and taking 800 parts of materials in the batch of identification;
(2) planting a rice straw sample;
planting rice piles in rows in a tray pot filled with shallow soil, and using pest-susceptible variety TN1 as a contrast, and planting 1 row of contrast varieties in each tray pot; when planting, the spacing between the rows and the plants is 5-8 cm; 80 parts of materials to be identified are planted in 8 rows. Repeating the planting of 800 parts of materials for 3 times for 30 pots;
(3) excessive inoculation identification;
inoculating and identifying insects within 3 days after planting rice stakes, inoculating more than 30 heads of nymphs of 2-3 instar brown planthoppers of rice to each rice stake, and covering an insect prevention mesh enclosure on the rice stakes on the supporting basin after inoculating the insects; within 3 days after inoculation, adding an inoculation source to a tray with possibly insufficient insect source to achieve sufficient insect population density;
(4) managing after inoculation;
i, after the insects are inoculated, keeping the insect prevention net cover sealed to prevent the insects from escaping;
II, keeping a water layer with the depth of not more than 1cm in the basin; water body isolation is needed outside the basin to prevent ants from being harmful;
III, placing the tray in an identification room with a sunshade net or a place with weak sunlight for culturing, and preventing the high temperature of more than 32 ℃;
IV, moving to a shady and cool place in time when encountering high temperature and direct strong sunlight;
reducing airflow flowing in an identification room or a place for placing an identification basin outdoors, closing a windshield window door on the windward side of the identification room when obvious airflow occurs, and arranging a wind shield for outdoor identification;
(5) evaluating the identification result;
recording resistance identification results 1-2 days after all the pest-susceptible control varieties die, carrying out evaluation recording according to 9-grade standard, grading each seedling, and calculating the average damage grade of each material:
grade 1 (1.0-1.9) is High Resistance (HR), and the damage is not obvious;
grade 3 (2.0-3.9) is anti (R), and the first leaf sheath on the periphery turns yellow;
grade 5 (4.0-5.9) is Medium Resistance (MR), the second leaf sheath on the periphery turns yellow, and the growth potential of plants is obviously weakened;
grade 7 (6.0-7.9) is a medium feeling (MS), and the plant begins to wither;
grade 9 (8.0-9.0) is high-sensitivity (HS), and the plant dies;
(6) repeatedly identifying;
the resources with the resistance to brown planthopper of 1 grade and 3 grades obtained in the primary screening identification are repeatedly screened according to the methods (1) to (5) above.
And (3) test results:
among 800 parts of the common wild rice resources, 12 parts of the grade 1 resistant resource and 16 parts of the grade 3 resistant resource are obtained, and the ratio of the resistant resources is low (as shown in Table 3). And (3) re-screening the resources of the 1 level and the 3 level, wherein the re-screening of 12 resources of which the primary screening is the 1 level totally has the resistance of the 1 level, and when the re-screening of 16 resources of which the primary screening is the 3 level is carried out, 1 resource of which the primary screening is the 1 level and the rest is the 3 level. The result shows that the accuracy of the initially screened resistance identification result is very high, and the obtained grade 1 resistance resource can be used as a resistance source.
TABLE 3 Primary screening and identification results of resistance of common wild rice seed stem rice stump to Nilaparvata lugens
Example 4
A large-scale accurate identification method for the resistance of rice against brown planthoppers in the adult stage of rice comprises the following steps:
(1) preparing a rice straw sample;
planting the rice group to be identified in a field, and performing field management on the rice by adopting a conventional planting method; stripping one rooted tillering from each rice plant of a group to be identified in a plant growing period, cutting a lower rice stump as an identification sample by using scissors, wherein the cutting length is 18 cm;
(2) planting a rice straw sample;
rice stakes are planted in rows in a tray pot filled with shallow soil, insect-susceptible Nipponbare is used as a comparison, and 1 row of comparison varieties are planted in each tray pot;
(3) excessive inoculation identification;
inoculating and identifying within 3 days after planting rice stakes, and inoculating more than 30 heads of nymphs of 2-3 instar brown planthoppers to each rice stake;
(4) managing after inoculation;
i, after the insects are inoculated, keeping the insect prevention net cover sealed to prevent the insects from escaping;
II, keeping a water layer with the depth of not more than 1cm in the basin; water body isolation is needed outside the basin to prevent ants from being harmful;
III, placing the tray in an identification room with a sunshade net or a place with weak sunlight for culturing, and preventing the high temperature of more than 32 ℃;
IV, moving to a shady and cool place in time when encountering high temperature and direct strong sunlight;
reducing airflow flowing in an identification room or a place for placing an identification basin outdoors, closing a windshield window door on the windward side of the identification room when obvious airflow occurs, and arranging a wind shield for outdoor identification;
(5) evaluating the identification result;
recording resistance identification results 1-2 days after all the pest-susceptible control varieties die, carrying out evaluation recording according to 9-grade standard, grading each seedling, and calculating the average damage grade of each material:
grade 1 (1.0-1.9) is High Resistance (HR), and the damage is not obvious;
grade 3 (2.0-3.9) is anti (R), and the first leaf sheath on the periphery turns yellow;
grade 5 (4.0-5.9) is Medium Resistance (MR), the second leaf sheath on the periphery turns yellow, and the growth potential of plants is obviously weakened;
grade 7 (6.0-7.9) is a medium feeling (MS), and the plant begins to wither;
grade 9 (8.0-9.0) is high-sensitivity (HS), and the plant dies;
(6) repeatedly identifying;
repeated identification can be carried out according to actual needs;
(1) performing instant repeated identification, namely taking repeated rice stakes according to the repeated times during sampling, and performing repeated identification for more than 2 times according to the experimental design;
(2) and (4) once repeated identification, after one-time identification is finished, referring to the identification result, and if necessary, flexibly sampling before heading stage to perform repeated identification.
The invention establishes a rice stump identification method and provides a high-efficiency resistance identification technology for the rice brown planthopper resistance breeding of various scales. Generally, the resistance breeding materials with large, medium and small scales can be identified once or repeatedly for many times, and the identification result of the seedling stage identification can be repeatedly identified in the adult plant stage, so that the identification result is accurate, and the progress of the resistance breeding project is effectively promoted.
The method of the present invention is used for identification, and the following identification figures 1 to 4 are obtained. Fig. 1 is a graph of the growth condition of rice stakes 4 days after resistance identification and insect inoculation in the rice stake forming stage, the viability of sensitive plants is obviously weakened and begins to turn yellow [ 1 row for each material, the left 2 nd row is respectively: ptb33 resistance, candidate Bph3 near isogenic line 1, TN1 (sensory control), candidate Bph3 near isogenic line 2, 9311 (recurrent parent), candidate Bph3 near isogenic line 3 ].
Fig. 2 is a diagram of the growth condition of rice stumps after resistance identification and identification in the plant-growing period by the rice stump method, wherein all the sensitive comparison materials and the sensitive identification materials die [ 1 row of each material, the left part is as follows: ptb33 resistance, candidate Bph3 near isogenic line 1, TN1 (sensory control), candidate Bph3 near isogenic line 2, 9311 (recurrent parent), candidate Bph3 near isogenic line 3 ].
FIG. 3 is a graph showing the growth of rice plants before the identification of plant resistance (control) and inoculation by a conventional method. FIG. 4 is a diagram showing the growth of rice plants after the identification of plant resistance by the conventional method (control) is completed, [ two lines per material, left: ptb33 resistance, candidate Bph3 near isogenic line 1, TN1 (sensory control), candidate Bph3 near isogenic line 2, 9311 (recurrent parent), candidate Bph3 near isogenic line 3 ].
The results of the rice stump method resistance identification (figure 2) and the conventional plant resistance identification (contrast) (figure 4) are completely the same, the Ptb33 resistance source is expressed as high resistance, the candidate Bph3 near isogenic line 3 is expressed as medium resistance, the other materials are expressed as high sensitivity, and the resistance grading difference is obvious, which indicates that the rice stump method resistance identification result is reliable, simple and efficient.
Claims (7)
1. A large-scale accurate identification method for the resistance of rice against brown planthopper in the adult stage of rice is characterized by comprising the following steps: the method comprises the following steps:
(1) preparing a rice straw sample;
planting the rice group to be identified in a field, and performing field management on the rice by adopting a conventional planting method; stripping one rooted tillering from each rice plant of a group to be identified in a plant growing period, cutting a lower rice stump as an identification sample by using scissors, wherein the cutting length is 10-20 cm;
(2) planting a rice straw sample;
planting rice piles in rows in a tray pot filled with shallow soil, and using pest-sensitive varieties as comparison, wherein 1 row of comparison varieties is planted in each tray pot;
(3) excessive inoculation identification;
inoculating and identifying within 3 days after planting rice stakes, and inoculating more than 20 heads of 2-3 instar brown planthopper nymphs to each rice stake;
(4) managing after inoculation;
i, after the insects are inoculated, keeping the insect prevention net cover sealed to prevent the insects from escaping;
II, keeping a water layer with the depth of not more than 1cm in the basin; water body isolation is needed outside the basin to prevent ants from being harmful;
III, placing the tray in an identification room with a sunshade net or a place with weak sunlight for culturing, and preventing the high temperature of more than 32 ℃;
IV, moving to a shady and cool place in time when encountering high temperature and direct strong sunlight;
reducing airflow flowing in an identification room or a place for placing an identification basin outdoors, closing a windshield window door on the windward side of the identification room when obvious airflow occurs, and arranging a wind shield for outdoor identification;
(5) evaluating the identification result;
recording resistance identification results 1-2 days after all the pest-susceptible control varieties die, carrying out evaluation recording according to 9-grade standard, grading each seedling, and calculating the average damage grade of each material:
grade 1 is High Resistance (HR), with insignificant damage;
grade 3 is anti (R), the first sheath on the periphery turns yellow;
grade 5 is Medium Resistance (MR), the second leaf sheath on the periphery turns yellow, and the growth potential of plants is obviously weakened;
grade 7 is a Medium (MS) and the plants begin to wither;
grade 9 is high-sensitivity (HS), and the plant dies;
(6) repeated identification is specifically as follows:
i, immediate repeated identification, namely, taking repeated rice piles according to the repeated times during sampling, and performing repeated identification for more than 2 times according to the experimental design;
and II, once repeated identification, wherein after one identification is finished, the identification result is referred, and if necessary, the sample can be flexibly sampled before the heading stage for repeated identification.
2. The large-scale accurate identification method for the resistance of the rice to the brown planthopper in the adult rice plant period according to claim 1, which is characterized in that: in the step (1), the tillers stripped from different plants are required to be consistent in size, strong in growth and free of plant diseases and insect pests.
3. The large-scale accurate identification method for the resistance of the rice to the brown planthopper in the adult rice plant period according to claim 1, which is characterized in that: the plant-row spacing of the rice pile row-by-row planting in the step (2) is 4-8 cm; the insect susceptible variety is TN1, 9311 or Nipponbare.
4. The large-scale accurate identification method for the resistance of the rice to the brown planthopper in the adult rice plant period according to claim 1, which is characterized in that: in the step (3), within 3 days after inoculation, the inoculation source is added to the tray with possibly insufficient insect source to reach sufficient insect population density.
5. The large-scale accurate identification method for the resistance of the rice to the brown planthopper in the adult rice plant period according to claim 1, which is characterized in that: and (4) covering an insect prevention net cover on the rice stake on the supporting basin after the insects are inoculated in the step (3).
6. The large-scale accurate identification method for the resistance of the rice to the brown planthopper in the adult rice plant period according to claim 1, which is characterized in that: the identification method identifies the rice at any time in the adult plant period from 20 days after rice transplantation to heading.
7. The application of the large-scale accurate identification method for the resistance of the rice to the brown planthopper in the adult rice plant period as claimed in claim 1, is characterized in that: the application of the gene is in the aspects of brown planthopper resistant rice genetic breeding and genetic research or wild rice resistance resource screening.
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