CN110468229B - Coseparation molecular marker Hxjy-1 of rice broad-spectrum high-resistance bacterial leaf blight gene Xa45(t) - Google Patents
Coseparation molecular marker Hxjy-1 of rice broad-spectrum high-resistance bacterial leaf blight gene Xa45(t) Download PDFInfo
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Abstract
The invention discloses a coseparation molecular marker Hxjy-1 of rice broad-spectrum high-resistance bacterial leaf blight gene Xa45(t), wherein the nucleotide sequence of the coseparation molecular marker is shown as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The coseparation molecular marker Hxjy-1 can accurately screen out materials containing broad-spectrum high-resistance bacterial blight gene Xa45(t), can effectively predict whether rice plants have resistance to bacterial blight, and can greatly accelerate the screening progress of the bacterial blight-resistant rice materials.
Description
Technical Field
The invention belongs to the technical field of crop molecular genetics research, and particularly relates to a coseparation molecular marker Hxjy-1 of a rice broad-spectrum high-resistance bacterial blight gene Xa45(t), which is suitable for auxiliary selective breeding of the gene Xa45(t) by using the Hxjy-1.
Background
Rice is one of the most important food crops in the world, more than half of the population in the world takes rice as staple food, and the great challenge facing the world is to ensure the food safety, however, diseases and pests are the main factors threatening the rice production, and the control of various rice diseases and pests is a serious problem to be solved urgently. Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae is a major bacterial disease, which frequently outbreaks into disasters in southern China and southeast Asia rice areas and is one of the main limiting factors for high and stable yield of rice. The most economical, effective, environment-friendly and sustainable disease control method is to cultivate and popularize rice varieties with wide resistance to bacterial blight. However, because rice varieties carrying single disease-resistant genes are popularized in a large area in part of rice regions, a synergistic evolution relationship exists between rice and the bacterial blight bacterium, and bacterial strains which are virulent to the disease-resistant genes Xa4 and Xa21 mainly promoted at home and abroad have been sequentially appeared in China and parts of rice regions in southeast Asia, the new genes for bacterial blight resistance are continuously identified, and the method has important significance for culturing rice varieties with broad-spectrum and lasting resistance through new gene polymerization.
At present, genetic variation in excellent germplasm resources is introduced into main cultivars, and new genes are explored and utilized by combining with offspring-related traits, so that not only can target trait introgression line groups of different recurrent parents be cultivated, but also beneficial genes can be effectively explored, and the strategy is relatively effective for exploring the beneficial genes from wild rice.
Yuanjiang common wild rice (Yuanjiang Oryza rufipogon Griff) is common wild rice with highest distribution altitude (750m) found in China so far, and is considered as the common wild rice with the best originality because no cultivated rice is planted around the habitat of the rice because of unique climatic ecological environmentConstruction of a wild rice leaf cDNA library and analysis of partial gene fragments, inheritance HEREDITAS(Beijing)2008, 6.2008, 30(6): 776-780). The applicant has developed a BC with a syngeneic 35 (syngeneic 35, a high-yield and high-quality japonica rice variety bred in China) as a recurrent parent and Yuanjiang common wild rice as a donor parent2F16And (3) artificially inoculating 11 strong pathogenic bacteria in a infiltration line group, namely PXO99, T7147, YN18, YN1, GD414, HEN11, ScYc-b, YN7, FuJ, YN24 and Y8 collected from Japan, Philippines and China, in the plant booting stage, and identifying the bacterial leaf blight resistance of each infiltration line and parents. The applicant subject group found that the strain G252 and the donor parent Yuanjiang common wild rice were highly resistant to the above 11 strains, and the acceptor parent syngeneic line No. 35 was highly sensitive to the above 11 strains, indicating that G252 had the same resistance reaction to bacterial blight as Yuanjiang common wild rice. Further indicates that the resistance gene carried by G252 may be from Yuanjiang common wild rice. G252 is hybridized with japonica rice 02428 which is susceptible to bacterial leaf blight to construct F2Locate the population, pair F2Plants were inoculated with the 11 strains described above. Genetic analysis found that F2The number of disease-resistant plants and the number of susceptible plants of each strain of the plants accord with 3:1 segregation ratio, indicating that the resistance of G252 to bacterial blight is controlled by a dominant gene. The group of the applicant obtained 1 broad-spectrum high-resistance genes having resistance to all of the above 11 strains by gene mapping, and was tentatively named Xa45 (t).
Since Xa45(t) is a broad-spectrum dominant gene with high resistance to bacterial blight, the gene has wide application prospect in breeding new resistant varieties. Therefore, it is necessary to find a cosegregation marker of Xa45(t), anchor the cosegregation marker on chromosome, facilitate the research on the function of the gene Xa45(t) on chromosome through the cosegregation marker, and have great significance for cloning and utilizing the gene Xa45(t) to develop new rice varieties resisting bacterial blight.
Disclosure of Invention
Against the background of the above research, the present invention aims at providing coseparation molecular marker Hxjy-1 of rice broad-spectrum highly-resistant bacterial blight gene Xa45(t), which can anchor gene Xa45(t) on chromosome accurately. Through detecting the molecular marker Hxjy-1 which is co-separated from the Xa45(t), the bacterial leaf blight resistance of the rice Xa45(t) monogenic line progeny material can be detected, the operation is simple, the accuracy rate reaches 100%, and the breeding progress of the bacterial leaf blight resistant rice variety can be accelerated.
In order to achieve the aim, the coseparation molecular marker Hxjy-1 of the rice broad-spectrum high-resistance bacterial blight gene Xa45(t) consists of Hxjy-1-F primer and Hxjy-1-R primer, the length of a target fragment is 167bp, and the nucleotide sequence of the Hxjy-1-F primer is shown as SEQ ID NO: 1, the nucleotide sequence of the Hxjy-1-R primer is shown as SEQ ID NO: 2, respectively.
The physical distance of the coseparation molecular marker Hxjy-1 of the rice broad-spectrum high-resistance bacterial blight gene Xa45(t) on a rice chromosome is 27.978Mb, the gene Xa45(t) can be marked on the tail end of the long arm of the rice chromosome 11, and the coseparation molecular marker can also be used for screening single plants containing the target gene.
The invention also provides application of the coseparation molecular marker Hxjy-1 of the rice broad-spectrum high-resistance bacterial leaf blight gene Xa45(t) in auxiliary selective breeding of rice molecular markers for resisting bacterial leaf blight.
In the application, the co-separation molecular marker Hxjy-1 can amplify an amplified fragment of 167bp, so that the target rice material contains a site of a gene Xa45(t) resisting bacterial blight.
The amplification adopts a 15 mu L PCR reaction system to carry out PCR amplification; the 15 μ L PCR reaction system was: 5U/. mu.LTaq enzyme 0.1. mu.L, Mg 2+10 XPCR Buffer 1.5 uL, 2.5mmol/L dNTP1.2 uL, 10 umol/L upstream and downstream primers 0.4 uL, 20 ng/uL template DNA 2 uL, ddH2O9.4 μ L; the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30sec, renaturation at 55 ℃ for 30sec, and extension at 72 ℃ for 1min for 35 cycles; extending for 10min at 72 ℃; mu.L of the PCR amplification product was electrophoresed on a 4% agarose gel at a constant voltage of 5V/cm, then imaged by a gel imaging system and stored.
In order to prevent the evaporation of the 15. mu.L of PCR reaction system liquid, 1 drop of paraffin oil is finally added into the 15. mu.L of PCR reaction system.
The invention also provides application of the coseparation molecular marker Hxjy-1 of the rice broad-spectrum high-resistance bacterial blight gene Xa45(t) in molecular marker-assisted selection.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention utilizes Yuanjiang common wild rice and a molecular marking method to discover a broad-spectrum high-resistance bacterial leaf blight gene Xa45(t) for the first time.
(2) The co-separation molecular marker Hxjy-1 of the gene Xa45(t) developed by the invention is verified on the solid genetic experiment result, the marker specificity is high, the difference between parents is obvious, the operability is strong, the amplified target fragment is single, the detection is easy, the target gene can be accurately positioned only by a simple PCR amplification technology and an agarose gel electrophoresis detection technology, the polyacrylamide gel electrophoresis detection with fussy, high toxicity and long time consumption is not needed, and the detection is environment-friendly, convenient, rapid and efficient.
(3) The Xa45(t) gene is finely anchored at the tail end of the long arm of the No. 11 rice chromosome, and the coseparation molecular marker Hxjy-1 provided by the invention can be used for accurately and effectively screening the candidate gene of Xa45(t), so that the screening efficiency of the candidate gene is improved, and a good theoretical basis is laid for subsequent cloning, utilization of the Xa45(t) gene and research of the function of the Xa 3526 (t) gene.
(4) The auxiliary breeding target is clear, and the auxiliary breeding efficiency of conventional breeding is effectively improved. Conventional breeding often selects individuals for subsequent crossing or backcross transformation by phenotype. However, the identification procedure of the bacterial leaf blight resistance of rice is complex, long in period and easy to be influenced by environmental factors and human factors. For example, the identification of bacterial leaf blight of rice generally requires that the plant grows to the booting stage for inoculation, so that the culture period of the plant is long and the plant is labor-consuming before resistance identification; in addition, a large amount of manpower and material resources are needed for culturing strains, artificial inoculation, field investigation and the like, and plum rain weather needs to be avoided. Therefore, the resistance breeding by using the conventional means has higher difficulty, low efficiency and high cost. However, the molecular marker detects the bacterial leaf blight resistance gene locus in a target plant, so that a single plant with high resistance homozygous genotype can be quickly identified only in the seedling stage, a single plant with susceptible genotype or a single plant with heterozygous genotype is eliminated in time, the production cost is saved, the selection efficiency of a resistant material is greatly improved, and the breeding period of a rice variety is greatly shortened.
SEQ ID NO: 1 shows the nucleotide sequence of Hxjy-1-F primer.
SEQ ID NO: 2 shows the nucleotide sequence of Hxjy-1-R primer.
SEQ ID NO: 3 shows the nucleotide sequence of the R13I14-F primer.
SEQ ID NO: 4 shows the nucleotide sequence of the R13I14-R primer.
SEQ ID NO: 5 shows the nucleotide sequence of Hxjy-14-F primer.
SEQ ID NO: 6 shows the nucleotide sequence of Hxjy-14-R primer.
SEQ ID NO: 7 shows the nucleotide sequence of RM224-F primer.
SEQ ID NO: shown in FIG. 8 is the nucleotide sequence of RM224-R primer.
SEQ ID NO: shown in FIG. 9 is the nucleotide sequence of RM27322-F primer.
SEQ ID NO: 10 shows the nucleotide sequence of the RM27322-R primer.
Drawings
FIG. 1: identification of 20 shares of F from G252/02428 using the coseparation molecular marker Hxjy-12Electrophorogram for separating the colony susceptible offspring gene Xa45 (t). M represents a standard molecular weight Marker (DL1000 Marker), G252 is a disease-resistant parent, 02428 is a susceptible parent, Nipponbare is susceptible cultivated rice, F1Numbers 1-20 represent 20F randomly drawn from the G252/02428 population for the first generation resulting from the cross between the disease-resistant parent G252 and the susceptible parent 024282And (4) infected individual plants. The disease grade corresponding to each individual is marked above the number of each individual, S represents infection (the length of the disease spot is more than 6cm), and R represents disease resistance (the length of the disease spot is less than or equal to 6 cm).
FIG. 2: identification of 22 shares of F from G252/02428 using the coseparation molecular marker Hxjy-12Electrophorogram for separating disease-resistant progeny gene Xa45 (t). M represents a standard molecular weight Marker (DL1000 Marker), G252 is a disease-resistant parent, 02428 is a susceptible parent, and the number is 1-2A number of 2 represents 22F's randomly drawn from the G252/02428 population2Disease-resistant single plants. The disease grade corresponding to each individual is marked above the number of each individual, and R represents disease resistance (the length of the lesion is less than or equal to 6 cm).
Detailed Description
The present invention will be described with reference to examples, which are not specifically illustrated and are conventional methods. The reagents or apparatus used are commercially available.
Example 1 the method for obtaining the co-segregation molecular marker Hxjy-1 of the rice broad-spectrum high-resistance bacterial blight gene Xa45(t) comprises the following steps:
(1) F2Population construction and phenotypic identification
(1) Performing bacterial leaf blight resistance identification on a transferred progeny of Yuanjiang common wild rice (collected from Yuanjiang county in Yunnan province), wherein the transferred progeny of the Yuanjiang common wild rice is obtained by hybridizing Yuanjiang common wild rice with a zygosity 35, taking the zygosity 35 as a recurrent parent and breeding an introgression line BC which takes the zygosity 35 as a genetic background and carries a gene resisting bacterial leaf blight2F16A material.
The following 11 strong pathogenic bacteria of bacterial blight collected from Philippines, Japan, China were used to pair the introgression lines BC2F16Inoculating the material to identify bacterial leaf blight resistance, and inoculating to introgression line BC2F16And (3) carrying out artificial inoculation on the plant of the material in the booting stage, and identifying the bacterial leaf blight resistance of each introgression line and the parents. The strain G252 (i.e. Yuanjiang common wild rice introgression line G252) and the donor parent Yuanjiang common wild rice are found to have high resistance to the 11 strains, and the acceptor parent syngeneic line No. 35 is highly sensitive to the 11 strains, so that the result shows that the G252 has the same resistance reaction to bacterial leaf blight as the Yuanjiang common wild rice, and the result shows that the bacterial leaf blight resistance gene carried by the G252 can come from Yuanjiang common wild rice, namely the resistance source G252 with broad spectrum and high resistance to the 11 bacterial leaf blight strong pathogenic bacteria is screened.
G252 as disease resistant parent, japonica rice 02428 as susceptible parent, and performing conventional hybridization, bagging and single plant collection on G252 and 02428 when the G252 and 02428 bloom to obtain F1Generation of seed, F1After the seeds germinate and grow into seedlings, DNA is respectively extractedUsing CTAB method), using SSR marker to detect F1Authenticity of the plant will be authentic F1Plant selfing, bagging and individual plant collection to obtain F2Generation of seed, F2Form F after the seeds germinate and become seedlings2Isolating the population, separating F2The separated groups are used as gene positioning group materials and are all planted in bases of Yuanjiang county in Yunnan province, single plants are planted and inserted, 10 plants are planted in each row, the row spacing of the plants is 12cm multiplied by 24cm, conventional water and fertilizer management is carried out, and protective rows are arranged around the plants.
The 11 bacterial blight strong pathogenic bacteria are PXO99, T7147, YN18, YN1, GD414, HEN11, ScYc-b, YN7, FuJ, YN24 and Y8.
The above-mentioned strains and related materials are disclosed in the following non-patent documents, and the applicants have stored the strains and can provide them within 20 years from the date of the present patent application.
PXO99, philippine standard strain 6, collected from philippines, published in the literature "zheng wei et al, china, japan and philippine rice xanthophylls germ genetic diversity comparison analysis, microbiology report, 2008, 35 (4): 519-523'.
T7147, Japanese Standard Strain No. 2, collected from Japan, published in the literature "Zhengwei et al, China, Japan, and Philippine Rice Blackspot bacterial blight genetic diversity comparative analysis. microbiological reports, 2008, 35 (4): 519 to 523 "
YN18 (Chinese standard strain No. 1), YN1 (Chinese standard strain No. 2), GD414 (Chinese standard strain No. 3), HEN11 (Chinese standard strain No. 4), ScYc-b (Chinese standard strain No. 5), YN7 (Chinese standard strain No. 6), FuJ (Chinese standard strain No. 8) and YN24 (Chinese standard strain No. 9) are collected from the northeast China rice district and published in the literature "Wuxi et al. 290 to 295 ".
Y8, a strong pathotype physiological race in Yunnan, collected from Yunnan of China, published in the literature "Yinji et al. evaluation of bacterial leaf blight resistance of filial generation of cultivated rice of ordinary wild rice. Jiangxi agricultural science, 2010, 22 (8): 81-84'
The zygosity 35 (sold in the market), cultivated rice and the high-yield and high-quality japonica rice variety bred in China are published in the literature, "Yinji et al.,. evaluation of bacterial leaf blight resistance of filial generation of the cultivated rice of the common wild rice, Jiangxi agricultural science, 2010, 22 (8): 81 to 84 ".
02428 japonica rice, a dwarf and wide-compatible japonica rice (cultivated rice) bred in China, published in the literature "xie warong wu et al. 02428 mutant of japonica rice, recombinant inbred line brown rice functional component content and related analysis of the same with agronomic traits. southwest agro-journal, 2011 24 vol 5: 1620 to 1624'.
(2) Inoculating the 11 bacterial blight strong pathogenic bacteria to the parent strain F in the booting stage of the plant2And (5) carrying out disease resistance identification on the families. Before inoculation, the strain is inoculated on an NA culture medium (the formula of the NA culture medium is that beef extract 3g, yeast extract 1g, peptone 5g, cane sugar 10g and agar 17g are added with water to reach the constant volume of 1000ml and then are mixedPH is 6.8 to 7.0. ) Culturing at 28 +/-2 deg.c for 48-72 hr. The strain is eluted with sterile distilled water during inoculation, suspended uniformly, and prepared into 3 × 108cfu/mL(OD6000.5). In the booting stage of plants (about 40 days after seedling transplanting), after sterilization by surgical scissors, dipping the prepared germ suspension, selecting sword leaves, horizontally placing the scissors with the tool tip upward, cutting off the leaf tips by 1-3 cm, inoculating 1 strain to each strain, changing the scissors for each inoculated 1 strain, marking, and observing and recording under natural conditions after inoculation.
When the disease development of the reference material tends to be stable after inoculation for about 15 days, 3 harmless leaves (free of insect pest and other diseases except bacterial blight and mechanical damage) with the longest lesion are selected from each plant, and the length of the lesion is measured. The disease resistance is achieved by taking 6cm as the anti-infection boundary, namely the length of the disease spot is less than or equal to 6cm, and the disease is affected by more than 6 cm.
(II) resistance Pattern analysis
F2The resistance identification of the plant can judge the gene type contained in the disease-resistant parent, namely: if F2The theoretical isolation ratio of the plant to the influenza is 3:1, and the plant is controlled by 1 dominant gene; 1:3 is controlled by 1 recessive gene; 15:1 is controlled by 2 dominant genes; the 1:15 is the control of 2 recessive genes, and the like, so that the disease-resistant characteristics of the disease-resistant parent can be known to be the control of the recessive genes. The theoretical isolation ratio of resistance to infection can be determined byChi square test for analysis, i.e.: the deviation degree between the actual observed value and the theoretical estimated value of the statistical sample is expressed as [ X2 ═ Σ (actual observed value-theoretical estimated value) 2/theoretical estimated value]Calculating a chi-square value by a formula, calculating a P value by a CHIDIST function according to the chi-square value, if the P value is larger than 0.05, the difference is not obvious, accepting the hypothesis, and if the P value is smaller than 0.05, the difference is obvious, and not accepting the hypothesis.
(III) extraction of genomic DNA and PCR reaction
(1) Extracting parent and F respectively by CTAB method2DNA from individual leaves of the population was purified and examined on a 0.8% agarose gel to determine the quality. The DNA concentration was adjusted to 20 ng/. mu.L by means of an ultraviolet spectrophotometer and stored in a refrigerator at-20 ℃ for further use.
(2) PCR amplification and electrophoresis
The PCR reaction was performed on a PCR amplification apparatus. The PCR reaction system (15. mu.L) was: 5U/. mu.L Taq enzyme 0.1. mu.L containing Mg 2+10 XPCR Buffer 1.5 uL, 2.5mmol/L dNTP1.2 uL, 10 umol/L upstream and downstream primers 0.4 uL, 20 ng/uL template DNA 2 uL, ddH2O9.4 mu L, and finally adding 1 drop of paraffin oil to prevent evaporation; the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30sec, renaturation at 55 ℃ for 30sec, and extension at 72 ℃ for 1min for 35 cycles; extending for 10min at 72 ℃; mu.L of the PCR amplification product was electrophoresed on a 4% agarose gel at a constant voltage of 5V/cm, then imaged by a gel imaging system and stored.
The forward primer refers to a primer located at the 5' end of the entire amplicon (i.e., the sequence of interest being amplified). The downstream primer refers to the primer located at the 3' end of the entire amplicon (i.e., the sequence of interest being amplified).
(IV) Gene mapping
(1) Polymorphic marker screening
309 markers are selected according to SSR molecular markers published by a Gramene website (https:// www.gramene.org /) according to relatively uniform physical distances, PCR amplification and 4% agarose gel electrophoresis detection are carried out on parents (G252 and 02428) according to the PCR reaction system and the PCR reaction conditions of the step (three) in the example 1, and SSR markers which are good in polymorphism and easy to determine in band type are screened out.
(2) Initial gene mapping
Gene mapping population Material (F) described in step (one) of example 1 above2Segregating population), 10 extreme disease-resistant plants and 10 extreme susceptible plants are selected, nuclear genome DNA is extracted by a CTAB method respectively, the disease-resistant plants and the susceptible plants are mixed in equal amount respectively to form resistant and susceptible DNA pools, the resistant and susceptible DNA pools of a positioning population are subjected to molecular detection respectively by using polymorphic SSR markers screened by the step (IV) (1) of the embodiment 1, G252 and 02428 parents are used as controls, markers linked with genes resistant to bacterial blight are preliminarily screened, and then linked markers are verified by using partial susceptible single plants. When a polymorphism is detected between the two parents and the pool of resistant and sensitive DNA, it is indicated that the gene of interest is located between the two markers. The two markers are used for carrying out PCR amplification on the single plants with the positioned population diseases, according to the PCR analysis result (carrying out PCR according to the step (three) of the embodiment 1, namely genomic DNA extraction and PCR reaction), the banding pattern of the disease-resistant parent is marked as 'A', the banding pattern of the disease-sensitive parent is marked as 'B', the heterozygous banding pattern is marked as 'H', and the PCR banding pattern amplified by each SSR marker is combined with the parents and the F2The phenotype of the infected individual plant is detected whether the two markers are linked with the target gene, thereby initially positioning the gene on the chromosome. The extreme disease resistance refers to the disease resistance in all F2In the family, the plant with the shortest lesion and the strongest disease resistance; the extreme susceptibility refers to all F2In the family, the disease spot is the longest and the disease resistance is the weakest.
(3) Fine localization of genes
According to the initial positioning result, the Japanese fine genome of the rice among the markers linked with the target gene is downloaded by referring to the Japanese fine genome, the sequences are uploaded to an NCBI database (https:// www.ncbi.nlm.nih.gov /), simple repeated sequences are searched, and the SSR marker is designed by using a Primer premier 5.0. In NCBI database, through Blast program, the downloaded sequence is compared with rice 9311 genome sequence, the insertion deletion area is searched, and InDel marker is designed. Polymorphism screening is carried out on the designed SSR marker and the InDel marker, linkage analysis is carried out on the screened polymorphism markers among the individual susceptible strains of the positioning population, and finally the target gene is finely positioned on the chromosome.
(V) results and analysis
(1) Genetic analysis of resistance of Yuanjiang common wild rice introgression line G252
F is to be2The plants were divided into 11 small groups (G1-G11), each of which was inoculated with 1 strain out of the 11 strains described in the step (one) of example 1 above, while G252 was used as a positive control and japonica rice 02428 was used as a negative control, and F was investigated and counted when the lesions of G252 and japonica rice 02428 were stable2The disease condition and the result show that the resistance difference of each small population to the corresponding strain is obvious. According to the inoculation identification condition, counting the number of disease-resistant plants and susceptible plants of each small group, and finding out that theoretical deduction values of each small group to corresponding strains are 3:1 isolation of infection, it is speculated that G252 may carry 1 broad-spectrum dominant gene against bacterial blight or that multiple resistance genes interact to promote broad-spectrum resistance of G252 against bacterial blight.
(2) Initial localization of Gene Xa45(t)
Selecting 10 extreme disease-resistant plants and 10 extreme disease-sensitive plants from each small population, respectively extracting DNA by a CTAB method, respectively mixing the equivalent resistant plants to respectively form resistant and sensitive DNA pools, performing molecular detection on the resistant pool of each small population by using the screened 86 pair SSR polymorphic markers, and taking G252 and 02428 parents as controls, so that the result shows that the SSR marker of the No. 11 chromosome can detect polymorphism between an amphipathic sample and the resistant pool of each small population, and the target gene can be located on the chromosome. Selecting F of each small group2Linkage analysis is carried out on susceptible single strains, the target genes of each small population are located between RM224 and RM27322, the fact that G252 carries 1 resistance gene which is consistent in genetic effect on each test strain is shown, the genes are tentatively named as Xa45(t), the fact that the genes Xa45(t) are resistant to bacterial blight in a broad spectrum is further shown, the fact that 11 constructed small populations are not different in resistance reaction of each test strain is also shown, therefore, 11 small populations can be combined into 1 large population, and follow-up experiments are all researched by using the large populations.
The linkage marker RM224 consists of an RM224-F primer and an RM224-R primer, and the nucleotide sequence of the RM224-F primer is shown in SEQ ID NO: 7, the nucleotide sequence of the RM224-R primer is shown as SEQ ID NO: shown in fig. 8.
The linkage marker RM27322 consists of a RM27322-F primer and a RM27322-R primer, wherein the nucleotide sequence of the RM27322-F primer is shown as SEQ ID NO: 9, the nucleotide sequence of the RM27322-R primer is shown as SEQ ID NO: shown at 10.
(3) Fine localization of Gene Xa45(t)
In order to further locate the Xa45(t) gene, new 131 pairs of SSRs and 136 pairs of InDel markers are developed, 5 pairs of SSRs and 9 pairs of InDel polymorphic markers are screened out, the obtained polymorphic markers are subjected to high-precision linkage analysis among susceptible individuals of a located population, finally, the gene Xa45(t) is finely located in a 26kb physical distance range between the two markers of the R13I14 and Hxjy-14 at the tail end of the long arm of the No. 11 chromosome of rice, and the genotype of the InDel marker Hxjy-1 amplified among the susceptible individuals is found to be consistent with the genotype of a susceptible parent 02428, which indicates that the Hxjy-1 and the gene Xa45(t) are co-separated (see Table 1).
TABLE 1 linkage markers of rice broad-spectrum highly-resistant bacterial blight gene Xa45(t)
a: is the physical distance on the japanese clear genome.
Example 2
Verification of coseparation molecular marker Hxjy-1
Materials and methods
(1) Material
Negative varieties: 299 parts of disease-resistant material for a bacterial leaf blight-sensitive variety 02428 (as a negative control), Nipponbare and G252/02428 filial generation.
Positive variety: and 63 parts of disease-resistant materials of G252 (used as a positive control) and G252/02428 filial generation, and 64 parts in total.
Co-separation molecular marking: hxjy-1
Nipponbare, Japanese bred japonica type conventional rice, published in the literature "Panxiawu et al. the differential regulation and control mechanism of CBF regulon in Nipponbare and 93-11 low-temperature domestication process of rice variety Chinese Rice science, 2012, 26 (5): 521 ~ 528 ".
(2) Method of producing a composite material
The method for extracting genomic DNA of each rice and performing PCR amplification of each genomic DNA using the coseparation molecular marker Hxjy-1 was the same as in example 1.
(II) results
The DNAs of 363 negative and positive samples, such as 02428 rice sample, were amplified by PCR. The results show that: the coseparation molecular marker Hxjy-1 can amplify corresponding target fragments (the length of the target fragment is 167bp) in a positive sample, does not amplify fragments with the same size in a negative sample, and has the detection rate of 100 percent (363/363). The result shows that the coseparation molecular marker Hxjy-1 and the detection method thereof provided by the invention can accurately screen the material containing the gene Xa45(t), can effectively predict whether the rice plant has resistance to bacterial blight, and can greatly accelerate the screening progress of the rice material resistant to bacterial blight.
Sequence listing
<110> institute of biotechnology and germplasm resources of academy of agricultural sciences of Yunnan province
<120> coseparation molecular marker Hxjy-1 of rice broad-spectrum high resistance bacterial blight gene Xa45(t)
<160>10
<170>SIPOSequenceListing 1.0
<210>1
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
<210>2
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
<210>3
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
<210>4
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
gtccatcaaa caccaagatc 20
<210>5
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
<210>6
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
<210>7
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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<212>DNA
<213> Artificial Sequence (Artificial Sequence)
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agagcccatg tagctacgcc ttcg 24
<210>10
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
aatcatgccg gctgaaattg tacc 24
Claims (6)
1. A co-separation molecular marker Hxjy-1 of a rice broad-spectrum highly-resistant bacterial blight gene Xa45(t) is characterized in that the co-separation molecular marker Hxjy-1 is a fragment amplified by Hxjy-1-F primers and Hxjy-1-R primers, the length of a target fragment is 167bp, and the nucleotide sequence of the Hxjy-1-F primers is shown as SEQ ID NO: 1, the nucleotide sequence of the Hxjy-1-R primer is shown as SEQ ID NO: 2, respectively.
2. The co-segregating molecular marker Hxjy-1 according to claim 1, wherein the physical distance between the co-segregating molecular marker Hxjy-1 and the rice chromosome is 27.978 Mb.
3. Use of the co-segregation molecular marker Hxjy-1 of claim 1 or 2 in rice bacterial blight-resistant molecular marker assisted selective breeding.
4. The use of claim 3, wherein the coseparation molecular marker Hxjy-1 is obtained by amplifying a 167bp fragment in the target rice material through Hxjy-1-F primer and Hxjy-1-R primer, and the target rice material contains the site Xa45(t) of the gene resisting bacterial blight.
5. The use of claim 4, wherein the amplification is performed using a 15 μ L PCR reaction: 5U/. mu.LTaq enzyme 0.1. mu.L, Mg2+10 XPCR Buffer 1.5. mu.L, 2.5mmol/L dNTP 1.2. mu.L, 10. mu. mol/L upstream and downstream primers 0.4. mu.L each, 20 ng/. mu.L template DNA 2. mu.L, ddH2O9.4 μ L; the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30sec, renaturation at 55 ℃ for 30sec, and extension at 72 ℃ for 1min for 35 cycles; extending for 10min at 72 ℃; mu.L of the PCR amplification product was electrophoresed on a 4% agarose gel at a constant voltage of 5V/cm, then imaged by a gel imaging system and stored.
6. The use according to claim 5, wherein 1 drop of paraffin oil is added to a 15 μ L PCR reaction system.
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| CN111349632B (en) * | 2020-03-04 | 2021-01-22 | 上海交通大学 | Rice bacterial leaf blight resistant genes Xa2, Xa14 and Xa45(t) and application thereof |
| CN114438100B (en) * | 2022-03-01 | 2023-11-10 | 云南省农业科学院生物技术与种质资源研究所 | Method for efficiently separating bacterial leaf blight-resistant gene with wild rice blood margin and family members thereof |
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| CN101892244A (en) * | 2010-03-17 | 2010-11-24 | 华中农业大学 | Oryza officinalis anti-Xanthomonas oryzae major gene Xa3/Xa26-2 and application for improving disease resistance of rice thereof |
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| CN102888399B (en) * | 2012-09-30 | 2014-02-05 | 浙江师范大学 | STS molecular marker for identifying high rice bacterial blight resistant genes and application thereof |
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| CN110066883A (en) * | 2018-12-03 | 2019-07-30 | 中国农业科学院深圳生物育种创新研究院 | With the molecular labeling R112146 of Rice Bacterial Blight Xa23 close linkage |
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