CN106566888A - Method for selecting and breeding variety with diversified resistance by molecular marker-assisted selection - Google Patents
Method for selecting and breeding variety with diversified resistance by molecular marker-assisted selection Download PDFInfo
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Abstract
The invention provides a method for selecting and breeding a new rice variety resistant to Pyricularia oryzae and Xanthomonas oryzae, and belongs to the technical field of rice disease resistance breeding. The method comprises the steps of selecting and using a two-line restoring line 9311 with a maximum promotion area in production as a to-be-improved parent, and performing hybridization with a Pyricularia oryzae and Xanthomonas oryzae resistant parent, backcrossing and selfing by utilizing a method of combining molecular marker-assisted selection with field agronomic selection; performing resistance identification on an obtained target gene homozygous line, and selecting and breeding three lines with constant agronomic traits and improved target resistance traits; and finally mixing the three lines by different ratios according to actual conditions of different planting areas to obtain the new variety with the diversified resistance, namely, "a super-resistant variety 9311". According to the method, the Pyricularia oryzae and Xanthomonas oryzae resistance of the rice variety with the diversified resistance is hugely improved on the basis of ensuring agronomic traits of an original background material, namely, the production increment and stabilization are facilitated, and the environmental protection is facilitated; and the method is of great significance for ensuring grain sustainable development.
Description
Technical field
The invention belongs to paddy disease-resistant breeding technical field, is especially a kind of many using molecular marking supplementary breeding resistance
The method of sample kind.
Background technology
Now studies have reported that, by the rice blast of rice blast fungus (Pyricutaria oryzae Cav.) initiation and by bacterial leaf spot
The bacterial leaf-blight that bacterium (Xanthomonas campestris pv.oryzae) causes is that Rice Production is endangered in world wide
The fungal disease and bacterial disease of most serious.The stable yields volume increase of both disease feedwater rice brings serious harm, one
As can make the paddy rice underproduction 20% or so in the popular time, total crop failure can be even caused when serious.In recent years, as hybrid rice parentses are lost
Pass basis to narrow, and the unreasonable of nitrogenous fertilizer uses in cultivation, rice blast and bacterial leaf spot disease are in cumulative year after year trend, rice of feeding water
Production brings very big threat.Gimmick of preventing and treating main at present is spraying agent and popularization and application disease-resistant varieties.Medicament is prevented
Although controlling play during paddy disease-resistant very important effect, it brings the increase of labour cost and environmental pollution
Aggravation, it has also become the outstanding problem of current agricultural production.Therefore, cultivate and plant disease-resistant variety be preventing and treating rice blast it is most economical,
Effectively, environmentally friendly measure and method.
But, it is at present conventional to cultivate and using the strategy of resistant rice cultivars, there is breeding cycle length, to germ plasm resource
Utilization rate is low, and educated varietal resistance loses defect and the problem such as fast.Therefore, system research and the letter using disease-resistant gene
Breath, sets up new application strategy and technical method, improves the utilization of antagonism genetic resources, is the trend of paddy disease-resistant breeding.
There are some researches show, because pathogen frequently occurs pathogenicity variation, cause common disease-resistant variety often plant 3～
Resistance is gradually lost after 5 years；It is used in mixed way different cultivars and combines the heterogeneous environment to be formed, is conducive to enhancing paddy rice to hold germ
Long property resistance.Resistant variety, binding molecule marker assisted selection (Molecular marker- are cultivated using paddy disease-resistant gene
Assisted selection, MAS), can quickly realize that resistant gene, to the transfer of purpose material, extends its genetic diversity
Property, anti-spectrum is widened, strengthen resistance, so as to reach the purpose of control disease.Can be realized using molecular marker assisted selection various
The transformation of resistant gene, for further breeding for disease resistance germ plasm resource is provided.
In the rice blast resistance gene and bacterial leaf spot disease-resistant gene reported at present, rice blast disease-resistant gene Pi2, Pi9
It is one of gene that wherein resistance is best, practical application is best with bacterial leaf spot disease-resistant gene Xa23.The anti-spectrums of Pi2 are quite wide, to
Overwhelming majority performance resistance in 792 rice blast fungus biological strains that China collects, only 7.55% microspecies can infect carrying
The parent C101A51 of Pi2, additionally, C101A51 is to 36 performance resistances in 43 rice blast bacterial strains from 13 countries.
Equally, Pi9 shows resistance to 43 rice blast bacterial strains from 13 countries.Xa23 has been reported to bacterial leaf-blight
One of best gene of resistance, it is little from Filipine bacterial leaf spot bacterial strain biological strain, 3 physiology from Japan to 10
Plant, and seven biological strains from China all show good resistance.In recent years, numerous researchs both domestic and external are also indicated that
These genes are used to improve the breed resistance aspect with good effect.
Therefore, in view of rice blast and bacterial leaf spot disease are selected to blast resisting to the harm produced by Rice Production amount
And bacterial leaf-blight show resistance new rice variety/strain method it is extremely urgent.
The content of the invention
For the deficiencies in the prior art, the invention discloses a kind of utilize molecular marking supplementary breeding resistance diversity kind
Method, in resistance diversity rice varieties on the basis of original background material economical character is ensured, by the disease-resistant base of rice blast
Because of Pi2, Pi9 and bacterial leaf spot disease-resistant gene Xa23 import to paddy rice and treat modified parent, improve the anti-of rice blast and bacterial leaf-blight
Property, that is, be conducive to volume increase stable yields to be conducive to environmental protection again, to guaranteeing that Grain Sustainable development is significant.
What the present invention was realized in：A kind of method of utilization molecular marking supplementary breeding resistance diversity kind, comprising
Following steps：
(1) be that strong advantage restorer 9311 treats modified parent from two, respectively with containing rice blast resistance gene Pi2's
Rice material, the rice material containing rice blast resistance gene Pi9 and the paddy rice material containing bacterial leaf spot resistance gene Xa23
Material is hybridized, is returned and selfing；Molecular Detection is the genomic DNA by extracting heterozygosis progeny population, using each gene
Specific molecular marker primer enters performing PCR amplification and electrophoretic analysis；
(2) to respectively obtaining the BC containing Pi2, Pi9, Xa23 difference resistant gene homozygosis3F4For seed, BC is planted3F4Dai Cai
Material, sowing, and Resistance Identification is carried out respectively, select economical character and be maintained, and target disease resistance trait obtains the three of improvement
Plant strain；
(3) finally regional concrete condition is planted according to each, by a certain percentage to these three materials containing different resistant genes
Mixing, obtains the multifarious new varieties of resistance " super anti-9311 ".
Further, described Molecular Detection is the genomic DNA by extracting each filial generation colony, using each gene
Specific molecular marker primer enters performing PCR amplification and electrophoretic analysis；PCR amplifications molecular labeling primer used sequence as follows：
The sequence of Pi2-InDel-F is：5’GCAGCGGCTAGGGTTTATC3’
The sequence of Pi2-InDel-R is:5’CACCCAGCAACTGATTTGTCA 3’
The sequence of Pi9-195-F is：5’TTGCTCCATCTCCTCTGTT 3’
The sequence of Pi9-195-R is：5’ATGGTCCTTTATCTTTATTG 3’
The sequence of Xa23-C189-F is：5’TAAGTTCTACATCGACCCCA 3’
The sequence of Xa23-C189-R is：5’CACATGAAGAGCTGGAAACG 3’.
Further, in described step (1) it is the rice material containing rice blast resistance gene Pi9, anti-containing rice blast
The rice material and the rice material containing bacterial leaf spot resistance gene Xa23 of property gene Pi2 refers to respectively containing rice blast resistance
The rice material China of gene Pi2 accounts for, the rice material 75-1-127 containing rice blast resistance gene Pi9 and contain bacterial leaf-blight
The rice material CBB23 of resistant gene Xa23.
Further, described step (1) is referred specifically to：
1.1,9311 treat improved materials, respectively with blast resisting, bacterial blight-resisting donor parents 75-1-127 (Pi9),
China accounts for (Pi2) and CBB23 (Xa23) hybridization；
1.2, offspring plants using Pi9, Pi2 and Xa23 genetic marker 195, Pi2-Indel and C189 are individual to filial generation
Strain is selected, and while is partial to 9311 offspring individuals and recurrent parent backcrossing 4 times, then selfing one with Agronomic characteristic
It is secondary, finally give the BC containing different resistant genes4F2Strain.
Further, pcr amplification reaction system is as follows in described step (1):
2x Reaction Mix:12.5μL；
Primers F l (10 μ Μ):1μL；
Primer Rl (10 μ Μ):1μL；
Golden DNA Polymerase:0.2μL；
DNA profiling (20-50ng/ μ L):1μL；
ddH2O:Complement to 25 μ L；
PCR Thermal cycling conditions are as follows:94 DEG C 5 minutes；94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；72
DEG C 7 minutes；10 DEG C of preservations.
Further, described step (3) is referred specifically to by 1：1：1 mixing three types improved materials, obtain anti-containing three
New varieties " super anti-9311 " of ospc gene.
The present invention is compared to the beneficial effect of prior art：1. the high efficiency of Breeding Process：It is disease-resistant from rice blast
The rice material difference modified parent 9311 of gene Pi2, Pi9 and bacterial leaf spot disease-resistant gene Xa23, can quickly obtain each single base
Because of the homozygous lines for improveing；2. the applicability for producing：Each homozygous lines that seed selection is obtained, can occur according to different regions disease
Difference, targetedly allocates the ratio of different strains, to reach the combination of optimum efficiency；The method of the present invention combines field agriculture
Skill proterties and molecular biological variety identification method, selection has efficient and agility.
Description of the drawings
Fig. 1 is that molecular labeling Pi2-InDel of the present invention is accounted in the resistance parent China of rice blast resistance gene Pi2 and waited to improve
The polyacrylamide gel electrophoresis figure of the amplified production of parent 9311 and homozygosis offspring individuals；
Fig. 2 is molecular labeling of the present invention 195 in the resistance parent 75-1-127 of rice blast resistance gene Pi9 and treats improvement parent
The agarose gel electrophoresis figure of the amplified production of sheet 9311 and homozygosis offspring individuals；
Fig. 3 is molecular labeling C189 of the present invention in the resistance parent CBB23 of bacterial leaf spot resistance gene Xa23 and waits to improve
The agarose gel electrophoresis figure of the amplified production of parent 9311 and homozygosis offspring individuals；
Fig. 4：Super anti-9311 seed selection flow chart.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
As shown in figure 4, seed selection of the present invention super anti-9311 is concretely comprised the following steps, will be respectively from paddy rice China account for, 75-1-
127th, the disease-resistant material and receptor parent 9311 of CBB23 is hybridized and three backcrossings, and by molecular marker assisted selection and
Phenotypic Selection obtains BC3F1, 3 selfings are subsequently carried out, obtain three kinds of BC3F4Improved materials；Then according to variant growing area disease
The actual conditions that evil occurs, mix by a certain percentage three types improved materials, obtain containing three disease-resistant genes, agronomic shape
Keep excellent new rice variety " super anti-9311 " consistent with recurrent parent 9311.Specific embodiment is as follows：
Embodiment 1
Resistance parent Hua Zhanyu containing rice blast resistance gene Pi2 treats that modified parent 9311 hybridizes, is returned and inbreeding population
Evaluation and screening：
It is rice using the rice material Hua Zhanyu high-quality containing rice blast resistance gene Pi2, the middle Xian kinds 9311 of high yield
Seasonal febrile diseases and bacterial leaf spot resistance treat that improved materials hybridize, and obtain F1 generation material, and seedling stage selected positive single using Pi2-InDel
Strain, pulls out negative individual plant；F1 generation material carries out the identification of seedling stage rice blast resistance gene Pi2, retains the individual plant containing target gene；
Field phenotypic character is instituted an inquiry from seedling stage, the economical character individual plant consistent with 9311 is then filtered out, is taken in the appropriate hybridization phase
The pollen mixture of these individual plants and 9311 backcrossings.BC1F1 is also to select positive individual plant in seedling stage mark for material, filters out agronomy
The proterties individual plant consistent with 9311, in the appropriate hybridization phase pollen mixture of these individual plants and 9311 backcrossings are taken.By that analogy, until
Obtain BC4F1 seeds.Plantation BC4F1 seeds, the positive individual plant for marking secondary combined field Phenotypic Selection consistent with 9311 phenotypes
Selfing.Genes of interest is pure and mild, the individual plant selfing that economical character is consistent with 9311 for selecting for BC4F2.BC4F2 is carried out for selfed seed
Indoor and outdoor blast resistance identification, selects the excellent strain of general performance.Genomic DNA is extracted to filial generation individual plant, to examine
The primer for surveying each gene enters performing PCR amplification.
As shown in figure 1, with Pi2-InDel-F, the primer pair of sequence shown in R, DNA to be detected is expanded, in detection paddy DNA
With the presence or absence of rice blast resistance gene Pi2；
The sequence of Pi2-InDel-F is：5’GCAGCGGCTAGGGTTTATC 3’
The sequence of Pi2-InDel-R is:5’CACCCAGCAACTGATTTGTCA3’
As shown in figure 1, Hua Zhanzhong target gene Pi2 gene identification methods, band shown in 110bp is containing Pi2 gene specifics
Band, and stripe size is 108bp in control material 9311.
Specific detection method is as follows：
1.DNA is extracted
Take 1-50g fresh plant materials to be fitted in 2mlEP pipes, plus steel ball shakes into powder in liquid nitrogen.Add 600 μ l (w/
V) 2 × CTAB Extraction buffers of preheating, 65 DEG C of insulation 10-20 minutes, shake therebetween frequently.Add the chloroform/isoamyl of 500 μ l
Alcohol, light and slow reverse centrifuge tube is mixed, and under room temperature, 12000r/min is centrifuged 10 minutes.Supernatant is proceeded to into a clean 1.5mlEP
Guan Zhong, adds isopyknic chloroform/isoamyl alcohol, overturns centrifuge tube and mixes, and room temperature, 12000r/min are centrifuged 10 minutes.By supernatant
Liquid is proceeded in another new 1.5mlEP pipes, adds the isopropanol of 0.6-1 times of volume, is mixed, and is placed 30 minutes under room temperature.3500-
4000r/min is centrifuged 5-10 minutes, goes supernatant, 70% ethanol rinse, precipitation to dry up.It is subsequently adding the ddH of 40ul2O dissolves
DNA, takes the detection of 2ul solution electrophoresis, and 4 DEG C save backup.
2.PCR is expanded, and amplification reaction system is as follows:
2x Reaction Mix:12.5μL
Primers F l (10 μ Μ):1μL
Primer Rl (10 μ Μ):1μL
Golden DNA Polymerase:0.2μL
DNA profiling (20-50ng/ μ L):1μL
ddH2O:Complement to 25 μ L.
PCR Thermal cycling conditions are as follows:94 DEG C 5 minutes；94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；72
DEG C 7 minutes；10 DEG C of preservations.
3. electrophoretic analysis
After the reaction of Pi2-InDel primer pairs PCR terminates, take appropriate amount of sample carries out electricity on 10% polyacrylamide gel
Swimming detection, deposition condition is 180V, and electrophoresis time is 1:30 hours.
Embodiment 2
Resistance parent 75-1-127 containing rice blast resistance gene Pi9 with treat modified parent 9311 and hybridize, be returned and selfing
The evaluation and screening of colony：
It is rice using the rice material Hua Zhanyu high-quality containing rice blast resistance gene Pi9, the middle Xian kinds 9311 of high yield
Seasonal febrile diseases and bacterial leaf spot resistance treat that improved materials hybridize, F1The identification of seedling stage rice blast resistance gene Pi9 is carried out for material, is protected
The individual plant containing target gene is stayed, the economical character individual plant consistent with 9311 is then filtered out, in the appropriate hybridization phase these individual plants are taken
Pollen mixture and 9311 backcrossing.BC1F1Be also to select positive individual plant in seedling stage mark for material, filter out economical character with
9311 consistent individual plants, in the appropriate hybridization phase pollen mixture of these individual plants and 9311 backcrossings are taken.By that analogy, until obtaining
BC4F1Seed.Plantation BC4F1Seed, the positive individual plant selfing for marking secondary combined field Phenotypic Selection consistent with 9311 phenotypes.
Genes of interest is pure and mild, the individual plant selfing that economical character is consistent with 9311 for selecting for BC4F2.BC4F2Indoor and outdoor is carried out for selfed seed
Blast resistance identification, selects the excellent strain of general performance.Genomic DNA is extracted to filial generation individual plant, to detect each base
The primer of cause enters performing PCR amplification.
As shown in Fig. 2 with 195-F, the primer pair of sequence shown in 195-R, expanding DNA to be detected, it is in detection paddy DNA
It is no to there is rice blast resistance gene Pi9；
The sequence of 195-F is：5’TTGCTCCATCTCCTCTGTT 3’
The sequence of 195-R is：5’ATGGTCCTTTATCTTTATTG 3’
As shown in Fig. 2 target gene Pi9 gene identification methods in 75-1-27, band shown in 2031bp is containing Pi9 genes
Specific band, and without this band in control material 9311.
Specific detection method is as follows：
1.DNA is extracted：
Take 1-50g fresh plant materials to be fitted in 2mlEP pipes, plus steel ball shakes into powder in liquid nitrogen.Add 600 μ l (w/
V) 2 × CTAB Extraction buffers of preheating, 65 DEG C of insulation 10-20 minutes, shake therebetween frequently.Add the chloroform/isoamyl of 500 μ l
Alcohol, light and slow reverse centrifuge tube is mixed, and under room temperature, 12000r/min is centrifuged 10 minutes.Supernatant is proceeded to another clean
In 1.5mlEP pipes, isopyknic chloroform/isoamyl alcohol is added, overturn centrifuge tube and mix, room temperature, 12000r/min are centrifuged 10 minutes.
Supernatant is proceeded in new 1.5mlEP pipes, the isopropanol of 0.6-1 times of volume is added, is mixed, placed 30 minutes under room temperature.
3500-4000r/min is centrifuged 5-10 minutes, goes supernatant, 70% ethanol rinse, precipitation to dry up.It is subsequently adding the ddH of 40ul2O
Dissolving DNA, takes the detection of 2ul solution electrophoresis, and 4 DEG C save backup.
2.PCR is expanded：
Amplification reaction system is as follows:
2x Reaction Mix:12.5μL
Primers F l (10 μ Μ):1μL
Primer Rl (10 μ Μ):1μL
Golden DNA Polymerase:0.2μL
DNA profiling (20-50ng/ μ L):1μL
ddH2O:Complement to 25 μ L.
PCR Thermal cycling conditions are as follows:94 DEG C 5 minutes；94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；72
DEG C 7 minutes；10 DEG C of preservations.
3. electrophoretic analysis：
After the reaction of 195 primer pairs PCR terminates, take appropriate amount of sample carries out electrophoresis detection on the Ago-Gel of 1-2%, electricity
Swimming condition be 80V, 0:30 hours.
Embodiment 3
The resistance parent CBB23 of the Xa23 of gene containing bacterial leaf spot resistance with treat modified parent 9311 and hybridize, be returned and selfing
The evaluation and screening of colony：
Using the rice material CBB23 containing bacterial leaf spot resistance gene Xa23 and high-quality, the middle Xian kinds 9311 of high yield
Treat that improved materials hybridize for rice blast and bacterial leaf spot resistance, F1 generation material carries out seedling stage bacterial leaf spot resistance gene Xa23's
Identification, retains the individual plant containing target gene, then filters out the economical character individual plant consistent with 9311, and in the appropriate hybridization phase this is taken
The pollen mixture of a little individual plants and 9311 backcrossings.BC1F1 is also to select positive individual plant in seedling stage mark for material, filters out agronomy
The shape individual plant consistent with 9311, in the appropriate hybridization phase pollen mixture of these individual plants and 9311 backcrossings are taken.By that analogy, until obtaining
To BC4F1 seeds.Plantation BC4F1 seeds, mark the secondary combined field Phenotypic Selection positive individual plant consistent with 9311 phenotypes certainly
Hand over.Genes of interest is pure and mild, the individual plant selfing that economical character is consistent with 9311 for selecting for BC4F2.BC4F2 is connect for selfed seed
The strong pathogenic bacteria P6 identifications of bacterial leaf-blight are planted, the excellent strain of general performance is selected.Genomic DNA is extracted to filial generation individual plant,
To detect that the primer of each gene enters performing PCR amplification.
As shown in figure 3, with C189-F, the primer pair of sequence shown in C189-R, DNA to be detected is expanded, in detection paddy DNA
With the presence or absence of rice blast resistance gene Xa23；
The sequence of C189-F is：5’TAAGTTCTACATCGACCCCA 3’
The sequence of C189-R is：5’CACATGAAGAGCTGGAAACG 3’
As shown in figure 3, target gene Xa23 identified for genes figures in CBB23, band shown in 750bp is containing Xa23 gene specifics
Band, and stripe size is 800bp in control material 9311.
Specific detection method is as follows：
1.DNA is extracted：
Take 1-50g fresh plant materials to be fitted in 2mlEP pipes, plus steel ball shakes into powder in liquid nitrogen.Add 600 μ l (w/
V) 2 × CTAB Extraction buffers of preheating, 65 DEG C of insulation 10-20 minutes, shake therebetween frequently.Add the chloroform/isoamyl of 500 μ l
Alcohol, light and slow reverse centrifuge tube is mixed, and under room temperature, 12000r/min is centrifuged 10 minutes.Supernatant is proceeded in another 1.5mlEP,
Isopyknic chloroform/isoamyl alcohol is added, centrifuge tube is overturned and is mixed, room temperature, 12000r/min are centrifuged 10 minutes.Supernatant is proceeded to
In new 1.5mlEP pipes, the isopropanol of 0.6-1 times of volume is added, mixed, placed 30 minutes under room temperature.3500-4000r/min
Centrifugation 5-10 minutes, supernatant, 70% ethanol rinse, precipitation is gone to dry up.The ddH2O dissolving DNAs of 40ul are subsequently adding, 2ul is taken
Solution electrophoresis detect that 4 DEG C save backup.
2.PCR is expanded：
Amplification reaction system is as follows:
2x Reaction Mix:12.5μL
Primers F l (10 μ Μ):1μL
Primer Rl (10 μ Μ):1μL
Golden DNA Polymerase:0.2μL
DNA profiling (20-50ng/ μ L):1μL
ddH2O:Complement to 25 μ L.
PCR Thermal cycling conditions are as follows:94 DEG C 5 minutes；94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；72
DEG C 7 minutes；10 DEG C of preservations.
3. electrophoretic analysis：
After the reaction of C189 primer pairs PCR terminates, take appropriate amount of sample carries out electrophoresis detection on the Ago-Gel of 1-2%,
Deposition condition is 80V, 0:30 hours.
Embodiment 4
The rice blast of Pi2, Pi9 and Xa23 homozygous lines, the identification of bacterial leaf spot resistance indoor and outdoor；Wherein for rice blast is anti-
Property grade scale, is shown in Table 1；The grade scale of bacterial leaf-blight identification, is shown in Table 2.
The grade scale of the blast resistance identification of table 1.
The grade scale of the bacterial leaf-blight of table 2. identification
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any Spirit Essences without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (6)
1. a kind of method of utilization molecular marking supplementary breeding resistance diversity kind, it is characterised in that comprise the steps of：
(1) it is that strong advantage restorer 9311 treats modified parent from two, respectively with the paddy rice containing rice blast resistance gene Pi2
Material, the rice material containing rice blast resistance gene Pi9 and the rice material containing bacterial leaf spot resistance gene Xa23 enter
Row hybridization, backcrossing and selfing, after Molecular Detection is carried out to each filial generation colony；
(2) to respectively obtaining the BC containing Pi2, Pi9, Xa23 difference resistant gene homozygosis3F4For seed, BC is planted3F4For material, receive
Kind, and Resistance Identification is carried out respectively, select economical character and be maintained, and target disease resistance trait obtains three kinds of strains of improvement
System；
(3) finally the multifarious new varieties of resistance " super anti-9311 " are obtained to these three material mixing containing different resistant genes.
2. the method for utilization molecular marking supplementary breeding resistance diversity kind according to claim 1, it is characterised in that
Described Molecular Detection is the genomic DNA by extracting each filial generation colony, is drawn using the specific molecular marker of each gene
Thing enters performing PCR amplification and electrophoretic analysis.
3. the method for utilization molecular marking supplementary breeding resistance diversity kind according to claim 1, it is characterised in that
The rice material containing rice blast resistance gene Pi9, the paddy rice containing rice blast resistance gene Pi2 in described step (1)
Material and the rice material containing bacterial leaf spot resistance gene Xa23 refer to respectively the paddy rice material containing rice blast resistance gene Pi2
Material China accounts for, the rice material 75-1-127 containing rice blast resistance gene Pi9 and containing bacterial leaf spot resistance gene Xa23's
Rice material CBB23.
4. the method for utilization molecular marking supplementary breeding resistance diversity kind according to claim 3, it is characterised in that
Described step (1) is referred specifically to：
1.1,9311 treat improved materials, respectively account for blast resisting, bacterial blight-resisting donor parents 75-1-127 (Pi9), China
(Pi2) hybridize with CBB23 (Xa23)；
1.2, offspring is entered using Pi9, Pi2 and Xa23 genetic marker 195, Pi2-Indel and C189 to filial generation individuality plant
Row is selected, and is returned four times with recurrent parent while being partial to 9311 offspring individuals with Agronomic characteristic, then selfing is once,
Finally give the BC containing different resistant genes3F4Strain.
5. the method for utilization molecular marking supplementary breeding resistance diversity kind according to claim 1, it is characterised in that
Pcr amplification reaction system is as follows in described step (1):
2x Reaction Mix:12.5μL；
Primers F l (10 μ Μ):1μL；
Primer Rl (10 μ Μ):1μL；
Golden DNA Polymerase:0.2μL；
DNA profiling (20-50ng/ μ L):1μL；
ddH2O:Complement to 25 μ L；
PCR Thermal cycling conditions are as follows:94 DEG C 5 minutes；94 DEG C 30 seconds, 58 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulation；
72 DEG C 7 minutes；10 DEG C of preservations.
6. the method for utilization molecular marking supplementary breeding resistance diversity kind according to claim 1, it is characterised in that
Described step (3) is referred specifically to by 1：1：1 mixing three types improved materials, obtain the new varieties containing three disease-resistant genes
" super anti-9311 ".
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