CN110358862A - With the molecular labeling Hxjy-14 of rice wide spectrum high resistance to hoja blanca gene Xa45 (t) close linkage - Google Patents
With the molecular labeling Hxjy-14 of rice wide spectrum high resistance to hoja blanca gene Xa45 (t) close linkage Download PDFInfo
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Abstract
The present invention discloses a kind of molecular labeling Hxjy-14 with rice wide spectrum high resistance to hoja blanca gene Xa45 (t) close linkage, and nucleotide sequence is as shown in SEQ ID NO:1 and SEQ ID NO:2.Physical distance of the molecular labeling Hxjy-14 on rice chromosome is 27.983Mb, the material of the gene Xa45 (t) containing wide spectrum high resistance to hoja blanca can accurately be filtered out, whether resistance of the rice plant to bacterial leaf-blight is effectively predicted, the rice material screening progress of bacterial blight-resisting can be greatly speeded up.
Description
Technical field
The invention belongs to crop molecule genetics research technical fields, and in particular to rice wide spectrum high resistance to hoja blanca base
Because of the molecular labeling Hxjy-14 of Xa45 (t) close linkage, it is suitable for carrying out assisted Selection to gene Xa45 (t) using the label
Breeding.
Background technique
Rice is one of the cereal crops that world's populations more than half are staple food, and rice safety production is always tie society
The key subjects that agricultural development and the people's livelihood are stabilized.Pest and disease damage is the principal element for threatening Rice Production, controls various rice disease and pests
Evil is serious problem urgently to be resolved.Caused by rice Xanthomonas campestris (Xanthomonas oryzae pv.oryzae, Xoo)
Bacterial blight of rice is a kind of main bacteriosis, which in south China and the frequent population outbreak of Southeast Asia rice region, is
One of rice high yield, key constraints of stable yields.The most economical effective and sustainable Disease management method of environmental protection is cultivation pair
Bacterial leaf-blight has the rice varieties of extensive resistance and is promoted.However, single due to being widely applied carrying in part rice region
The rice varieties of one disease-resistant gene, there are the relationship of coevolution, China and the southeast between rice and leaf spot bacteria
Has there is the bacterial strain for having virulence to featured both at home and abroad disease-resistant gene Xa4 and Xa21 in succession in sub-part rice region, therefore, constantly identifies
Bacterial leaf spot resistance new gene has important meaning to the rice varieties with broad spectrum durable resistance are cultivated by new gene polymerization
Justice.
Currently, the hereditary variation in quality germplasm is imported into main breed, carried out in conjunction with offspring's correlated traits
New gene discovers and uses, and can not only cultivate the objective trait introgression line group of different recurrent parents, and can effectively excavate advantageous
Gene, such strategy from wild rice for excavating beneficial gene relative efficiency.
Yuanjiang River common wild-rice (Yuanjiang Oryza rufipogon Griff.) is that China finds Distribution Sea so far
The common wild-rice for pulling out highest (750m) is planted without cultivated rice around habitat because of climatic ecological environment uniqueness, it is considered to be
The best common wild-rice of primitiveness (building of the Yuanjiang River common wild-rice Leaf cDNA Library such as history winter swallow and portion gene piece
Piecewise analysis heredity HEREDITAS (Beijing) in June, 2008,30 (6): 776~780).The applicant seminar cultivated with
Collaboration 35 is recurrent parent, and Yuanjiang River common wild-rice is the BC of donor parents2F16Introgression line group, with collected from Japan, Fei Lv
Guest, PXO99, T7147, YN18, YN1, GD414, HEN11, ScYc-b, YN7, FuJ, YN24 and Y8 of China are totally 11 strong pathogenic
Bacterium carries out artificial infection in plant boot stage, identifies each introgression line and the bacterial leaf spot resistance of parents.The discovery of applicant seminar
Strain G252 and above-mentioned 11 bacterial strains of donor parents Yuanjiang River common wild-rice highly resistance, receptor parent collaboration 35 above-mentioned 11 of high senses
Bacterial strain shows that G252 has Resistant reaction identical with Yuanjiang River common wild-rice to bacterial leaf-blight.Further illustrate that G252 is carried
Resistant gene may be from Yuanjiang River common wild-rice.G252 is hybridized with the japonica rice 02428 of sense bacterial leaf-blight, constructs F2Positioning
Group, to F2Plant is inoculated with above-mentioned 11 bacterial strains.Genetic analysis discovery, F2The disease-resistant plant number and sense of each bacterial strain of plant pair
Sick plant number meets 3:1 segregation ratio, shows that G252 is controlled the resistance of bacterial leaf-blight by dominant gene.Applicant seminar is logical
It crosses the assignment of genes gene mapping and obtains 1 wide spectrum highly resistance gene all resistant to above-mentioned 11 bacterial strains, be temporarily named as Xa45 (t).
Since Xa45 (t) is a wide spectrum high resistance to hoja blanca dominant gene, which has in cultivation resistance new varieties
Wide application prospect.Therefore, it is necessary to find the close linkage label of Xa45 (t), it is anchored on chromosome, is conducive to
It by the function of close linkage marker research Xa45 (t) on chromosome, while being to clone and cultivate new resist using Xa45 (t)
Sick rice varieties are of great significance.
Summary of the invention
For the studies above background, the object of the present invention is to provide with rice wide spectrum high resistance to hoja blanca gene Xa45 (t)
Gene Xa45 (t) can be accurately anchored on chromosome by the molecular labeling Hxjy-14 of close linkage.Pass through detection and Xa45
(t) the molecular labeling Hxjy-14 of close linkage, can detecte the bacterial leaf spot of paddy gene Xa45 (t) Monogenic lines progeny material
Sick resistance, it is not only easy to operate, and accuracy rate is up to 99.45% or more, and then can accelerate the breeding of bacterial blight-resisting rice varieties
Progress.
To achieve the above object, provided by the invention a kind of close with rice wide spectrum high resistance to hoja blanca gene Xa45 (t)
Linkage molecule label Hxjy-14 is made of Hxjy-14-F primer and Hxjy-14-R primer, and target fragment length 170bp is described
The nucleotide sequence of Hxjy-14-F primer is as shown in SEQ ID NO:1, the nucleotide sequence of Hxjy-14-R primer such as SEQ ID
Shown in NO:2.
A kind of and rice wide spectrum high resistance to hoja blanca gene Xa45 (t) compact linkage molecule provided by the invention marks
Physical distance of the Hxjy-14 on rice chromosome is 27.983Mb, and gene Xa45 (t) can be anchored on to No. 11 chromosomes of rice
On long-armed end, it can also be used to single plant of the screening containing target gene.
It is marked the present invention also provides above-mentioned with rice wide spectrum high resistance to hoja blanca gene Xa45 (t) compact linkage molecule
Application of the Hxjy-14 in rice bacterial blight resistance molecular marker assisted selection breeding.
In above-mentioned application, compact linkage molecule label Hxjy-14 can amplify the amplified fragments of 170bp, then target
Rice material contains the site Bacterial blight resistance gene Xa45 (t).
The amplification carries out PCR amplification using 15 μ L PCR systems；15 μ L PCR reaction systems are as follows: 5U/ μ L Taq enzyme
0.1 μ L contains Mg2+10 × PCR Buffer, 1.5 μ L, 2.5mmol/L dNTP, 1.2 μ L, the 10 μm of upstream and downstream ol/L primers are each
0.4 μ L, 20ng/ μ L template DNA 2 μ L, ddH2O 9.4μL；PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation
30sec, 55 DEG C of renaturation 30sec, 72 DEG C of extension 1min, totally 35 recycle；72 DEG C of extension 10min；5 μ L pcr amplification products are taken to exist
5V/cm constant pressure electrophoresis on 4% Ago-Gel, is then imaged and is saved by gel imaging system.
To prevent the 15 μ L PCR reaction system liquid evaporations, the 15 μ L PCR reaction systems described in configuration well are most
1 drop paraffin oil is added afterwards.
It is marked the present invention also provides described with rice wide spectrum high resistance to hoja blanca gene Xa45 (t) compact linkage molecule
Application of the Hxjy-14 in molecular marker assisted selection.
Compared with prior art, the beneficial effects of the present invention are:
(1) present invention excavates out the high bacterial leaf spot resistant of wide spectrum using Yuanjiang River common wild-rice and molecule labelling method for the first time
Ospc gene Xa45 (t).
(2) the molecular labeling Hxjy-14 developing with gene Xa45 (t) close linkage of the invention is real in solid heredity
It tests and is verified in result, and mark specificity high, difference is obvious between parents, strong operability, the target fragment list that amplifies
One, it is easy detection, as long as passing through simple PCR amplification and agarose gel electrophoresis detection technique, so that it may be accurately positioned
To target gene, without polyacrylamide gel electrophoresis detection cumbersome, that toxicity is big, time-consuming, detect it is environmentally friendly, convenient, fast,
Efficiently.
(3) for the present invention by No. 11 chromosome long arm ends of gene Xa45 (t) finely positioning rice, orientation range is fine,
Gene Xa45 (t) has been limited in a genome range as small as possible, the efficiency of candidate gene screening has been improved, after being
Continuous clone using gene Xa45 (t) and studies its function and has established good theoretical basis.
(4) assist-breeding is with clearly defined objective, effectively increases the assist-breeding efficiency of conventional breeding.Conventional breeding often through
Phenotypic Selection single plant is used for subsequent hybridization or backcross transformation.But the bacterial leaf spot resistance evaluation program of rice is complicated, and the period is long,
It is influenced simultaneously vulnerable to environmental factor and human factor.Such as, bacterial blight of rice identification will generally grow to boot stage progress to plant
Bacterium is connect, so the cultivation cycle of plant is long before Resistance Identification, and bothersome laborious；Furthermore bacterial strain, artificial infection, field are cultivated
Between investigate etc. all need to spend a large amount of manpower and material resources, also need to avoid rainy weather.Therefore resistance breeding is carried out using conventional means
Difficulty is larger, low efficiency, at high cost.However molecular labeling Hxjy-14 detects the bacterial leaf-blight in target plant through the invention
Resistant gene site, only in seedling stage can Rapid identification go out the single plant of highly resistance homozygous genotype, eliminate susceptible single plant or miscellaneous in time
Genotype single plant is closed, not only saves production cost, but also greatly improve the efficiency of selection of resistant material, greatly shortening rice product
The breeding cycle of kind.
It is the nucleotide sequence of Hxjy-14-F primer shown in SEQ ID NO:1 in sequence table.
It is the nucleotide sequence of Hxjy-14-R primer shown in SEQ ID NO:2 in sequence table.
It is the nucleotide sequence of R13I14-F primer shown in SEQ ID NO:3 in sequence table.
It is the nucleotide sequence of R13I14-R primer shown in SEQ ID NO:4 in sequence table.
It is the nucleotide sequence of RM224-F primer shown in SEQ ID NO:5 in sequence table.
It is the nucleotide sequence of RM224-R primer shown in SEQ ID NO:6 in sequence table.
It is the nucleotide sequence of RM27322-F primer shown in SEQ ID NO:7 in sequence table.
It is the nucleotide sequence of RM27322-R primer shown in SEQ ID NO:8 in sequence table.
Detailed description of the invention
Fig. 1: the F of 21 parts of G252/02428 is identified using compact linkage molecule label Hxjy-142The susceptible offspring Xa45 of group
(t) Gene Isolation situation electrophoretogram.M is represented Standard molecular weight markers (DL1000 Marker), G252 be disease-resistant parent, 02428
For Susceptible parent, F1Hybridize the generated first generation, the digital generation of number 1-21 for disease-resistant parent G252 and Susceptible parent 02428
21 F that table is randomly selected from G252/02428 group2Susceptible single plant.The corresponding sick grade of each single plant is labeled in each single plant number
Top, S indicate susceptible (scab length is greater than 6cm) that R indicates disease-resistant (scab length is less than or equal to 6cm).
Fig. 2: the F of 21 parts of G252/02428 is identified using compact linkage molecule label Hxjy-142The disease-resistant offspring Xa45 of group
(t) Gene Isolation situation electrophoretogram.M is represented Standard molecular weight markers (DL1000 Marker), G252 be disease-resistant parent, 02428
For Susceptible parent, F1Hybridize the generated first generation, the digital generation of number 1-21 for disease-resistant parent G252 and Susceptible parent 02428
21 F that table is randomly selected from G252/02428 group2Disease-resistant single plant.The corresponding sick grade of each single plant is labeled in each single plant number
Top, R indicate disease-resistant (scab length is less than or equal to 6cm).
Specific embodiment
Embodiment of the present invention is described below in conjunction with embodiment, without specified otherwise is conventional side in embodiment
Method.Agents useful for same or instrument are commercially available.
The compact linkage molecule of the rice wide spectrum high resistance to hoja blanca gene Xa45 (t) of the present invention of embodiment 1 marks Hxjy-14
Acquisition, include the following steps:
(1) F2Informative population and phenotypic evaluation
(1) bacterial leaf spot resistance identification is carried out from the introgressive line of Yuanjiang River common wild-rice (picking up from Yuanjiang county),
The introgressive line of the Yuanjiang River common wild-rice be Yuanjiang River common wild-rice is hybridized with collaboration 35, and with collaboration 35 be circulation parent
This, is bred as taking collaboration 35 as genetic background and the introgression line BC for carrying Bacterial blight resistance gene2F16Material.
With following 11 collected from Philippine, Japan, China the strong pathogenic bacteria of bacterial leaf-blight to introgression line BC2F16Material connects
Kind carries out bacterial leaf spot resistance identification, in introgression line BC2F16Material plant boot stage carry out artificial infection, identify each introgression line and
The bacterial leaf spot resistance of parents.It was found that strain G252 (i.e. Yuanjiang River common wild-rice introgression line G252) and donor parents Yuanjiang River are common
Wild rice highly resistance 11 bacterial strains, receptor parent collaboration 35 is high to feel 11 kinds of bacterial strains, show G252 to bacterial leaf-blight have with
It is commonly wild to illustrate that the bacterial leaf spot resistance gene of G252 carrying may be from Yuanjiang River for the identical Resistant reaction of Yuanjiang River common wild-rice
Raw rice filters out and all has wide spectrum, resistance anti-source G252 to 11 strong pathogenic bacteria of bacterial leaf-blight.
For G252 as disease-resistant parent, japonica rice 02428 is used as Susceptible parent, when G252 and 02428 are bloomed, by G252 with
02428 carries out conventional hybridization, and bagging processing and single plant are collected, and obtains F1For seed, F1After germination seedling, DNA is extracted respectively
(using CTAB method) detects F using SSR marker1The authenticity of plant is by true F1Plant selfing, bagging processing and single plant are collected,
Obtain F2For seed, F2For constituting F after germination seedling2Segregating population, by F2Segregating population is as assignment of genes gene mapping group material
In Yuanjiang county base, single plant is planted for material, plantation, and every row plants 10 plants, and seeding row spacing is 12cm × 24cm, conventional liquid manure
Management around sets protection row.
The strong pathogenic bacteria of 11 bacterial leaf-blights be PXO99, T7147, YN18, YN1, GD414, HEN11, ScYc-b,
YN7, FuJ, YN24 and Y8.
Above-mentioned each bacterial strain and associated materials are disclosed in following non-patent literature, and applicant has preservation, from present patent application day
Rising in 20 years can provide.
PXO99, Philippine's reference culture 6 acquire from Philippine, are published in document " such as Zheng Wei China, Japan and luxuriant and rich with fragrance rule
Guest's Genetic Diversity of Xanthomonas oryzae pv. oryzae microorganism journal, 2008,35 (4): 519~523 ".
T7147, Nippon Standard bacterial strain 2, acquisition is published in document " such as Zheng Wei China, Japan and Philippine from Japan
Genetic Diversity of Xanthomonas oryzae pv. oryzae microorganism journal, 2008,35 (4): 519~523 "
YN18 (Chinese Industrial Standards (CIS) bacterium strain No.1), YN1 (Chinese Industrial Standards (CIS) bacterial strain 2), GD414 (Chinese Industrial Standards (CIS) bacterial strain 3),
HEN11 (Chinese Industrial Standards (CIS) bacterial strain 4), ScYc-b (Chinese Industrial Standards (CIS) bacterial strain 5), YN7 (Chinese Industrial Standards (CIS) bacterial strain 6), FuJ (China
Reference culture 8) and YN24 (Chinese Industrial Standards (CIS) bacterial strain 9) acquisition from Northeast China rice region, be published in the document " northeast the such as Wu Xian
Rice leaf spot bacteria strain analysis of genetic diversity and kind are to bacterial leaf-blight evaluation of resistance Jilin Auto Industry, and 2015,
37 (3): 290~295 ".
Y8, the strong pathological form biological strain in Yunnan, acquisition is from Chinese yunnan, and being published in document, " wealth has equal common wild-rice
Cultivated rice filial generation bacterial leaf spot resistance evaluates Agriculture in Jiangxi journal, 2010,22 (8): 81~84 "
Collaboration 35 (commercially available), cultivated rice, the high yield and high quality japonica rice variety of China's breeding, being published in document, " wealth has equal general
Common wild rice cultivated rice filial generation bacterial leaf spot resistance evaluates Agriculture in Jiangxi journal, 2010,22 (8): 81~84 ".
Japonica rice 02428 is the wide affine japonica rice (cultivated rice) of short stem of China's breeding, is published in document and " thanks to valiant Deng japonica rice
02428 mutant recombinant inbred lines brown rice functional component content and its agriculture journal in the southwest correlation analysis with economical character,
5 phases of volume 24 in 2011: 1620~1624 ".
(2) bacterium processing is connect to parent, F in plant boot stage with the strong pathogenic bacteria of above-mentioned 11 bacterial leaf-blights2Family is resisted
Characteristic of disease identification.Before inoculation, by strain inoculated on NA culture medium (NA culture medium prescription: beef extract 3g, yeast extract 1g,
Peptone 5g, sucrose 10g, agar 17g add water to be settled to 1000ml, adjustPH to 6.8~7.0.), it is placed in 28 DEG C of ± 2 DEG C of cultures
48h~72h.Bacterial strain is eluted with sterile distilled water when inoculation, suspends uniformly, is configured to 3 × 108cfu/mL(OD600=0.5).In
Plant boot stage (after rice transplanting about 40 days) dips the germ suspension prepared after operating scissors sterilizing, chooses sword-like leave, and scissors is flat
It sets, point of a knife is upward, cuts off 1~3cm of blade tip, 1 plant of plant of each strain inoculated, and 1 bacterial strain of every inoculation changes a scissors, carries out mark
Note is observed under natural conditions after inoculation and is recorded.
Inoculation 15 days or so when the progression of the disease for material of participating in the experiment tends towards stability, often select good strains in the field for seed take scab longest 3 it is lossless
Evil leaf (no insect pest, in addition to bacterial leaf-blight without other diseases and mechanical damage) measures scab length.Demarcate using 6cm as anti-sense,
It is disease-resistant that i.e. scab length, which is less than or equal to 6cm, is susceptible greater than 6cm.
(2) resistance pattern is analyzed
F2The Resistance Identification of plant can determine whether the gene type that disease-resistant parent is contained, it may be assumed that if F2The theoretical anti-sense segregation ratio of plant
For 3:1, then controlled for 1 dominant gene；1:3 is then 1 recessive gene control；15:1 is then 2 dominant gene controls；1:15
It is then 2 recessive gene controls, and so on, it will be appreciated that the disease-resistant characteristic of disease-resistant parent is how much to show recessive gene control.
Theoretical anti-sense segregation ratio can be analyzed by Chi-square Test, it may be assumed that between the actual observed value and theoretical implications value of statistical sample
Departure degree by [X2=Σ (actual observation value-theoretical implications value) 2/ theoretical implications value] formula calculate chi-square value, by card side
Value by CHIDIST function calculate P value, if P value be greater than 0.05, difference is not significant, receive it is assumed that if P value less than 0.05, difference
Significantly, do not receive hypothesis.
(3) extracting genome DNA and PCR reaction
(1) parent and F are extracted respectively with CTAB method2The DNA of each single-strain blade of group, DNA use 0.8% agarose after purification
Gel detection, to determine its quality.DNA concentration is adjusted to 20ng/ μ L using ultraviolet specrophotometer, is stored in -20 DEG C respectively
Refrigerator is spare.
(2) PCR amplification and electrophoresis
PCR reaction carries out in PCR amplification instrument.PCR reaction system (15 μ L) are as follows: 0.1 μ L of 5U/ μ L Taq enzyme contains Mg2+'s
10 × PCR Buffer, 1.5 μ L, 2.5mmol/L dNTP 1.2 μ L, each 0.4 μ L, 20ng/ μ L of the 10 μm of upstream and downstream ol/L primers
Template DNA 2 μ L, ddH29.4 μ L of O, finally plus 1 drop paraffin oil prevents from evaporating；PCR reaction condition are as follows: 94 DEG C of initial denaturation 5min；
94 DEG C of denaturation 30sec, 55 DEG C of renaturation 30sec, 72 DEG C of extension 1min, totally 35 recycle；72 DEG C of extension 10min；5 μ L PCR are taken to expand
Increase production object 5V/cm constant pressure electrophoresis on 4% Ago-Gel, is then imaged and saves by gel imaging system.
The upstream primer refers to the primer positioned at 5 ' ends of entire amplicon (being exactly the aim sequence being amplified).Downstream
Primer refers to the primer positioned at 3 ' ends of entire amplicon (being exactly the aim sequence being amplified).
(4) assignment of genes gene mapping
(1) polymorphism mark screening
The SSR molecular marker announced according to the website Gramene (http://www.gramene.org/) is according to relatively uniform
Physical distance select 309 labels, to parents (G252 and 02428) by 1 step (3) PCR reaction system of above-described embodiment and
PCR reaction condition carries out PCR amplification and the detection of 4% agarose gel electrophoresis, and it is good to be screened out from it polymorphism, and banding pattern is easy to determine
SSR marker.
(2) gene just positions
Assignment of genes gene mapping population material (the F described in 1 step (1) of above-described embodiment2Segregating population) in choose it is extreme anti-
10 plants of 10 plants of sick plant and extreme disease plant, extract nuclear DNA, disease-resistant plant and disease plant using CTAB method respectively
Mixed in equal amounts respectively respectively constitutes anti-, the sense pond DNA, the polymorphism SSR marker screened with 1 step (4) of above-described embodiment (1)
Molecular Detection carried out respectively to anti-, the sense pond DNA of target group, G252 and 02428 liang of parent is as control, preliminary screening and anti-
Then the label of bacterial leaf-blight gene linkage is verified linked marker with the susceptible single plant in part.When in two parents and anti-, sense
Polymorphism is detected between the pond DNA, illustration purpose gene is located between this two label.This two label is susceptible to target group
Single plant carries out PCR amplification, analyzes result according to PCR and (carries out by 1 step (3) extracting genome DNA of embodiment and PCR reaction
PCR), the banding pattern of disease-resistant parent is denoted as " A ", the banding pattern of Susceptible parent is denoted as " B ", and heterozygosis banding pattern is denoted as " H ", by each SSR
Mark PCR banding pattern combination parents, the F of amplification2The phenotype of susceptible single plant, detection two mark whether it is chain with target gene, thus
On chromosome by gene Primary Location.It is described extremely disease-resistant to refer in all F2In family, scab is most short, disease-resistant strongest
Plant；It is described extremely susceptible to refer in all F2In family, scab longest, disease-resistant most weak plant.
(3) gene finely positioning
According to first positioning result, referring to OryzasativaLcv.Nipponbare genome, by the rice Japan between the chain label of target gene
Fine genome sequence downloading, uploads these sequences in ncbi database (https: //www.ncbi.nlm.nih.gov/), searches
Rope simple repeated sequence designs SSR marker using Primer premier 5.0.In ncbi database, pass through Blast journey
The sequence of downloading is compared sequence with 9311 genome sequence of rice, searches insertion and deletion region, design InDel label.It will
Designed SSR marker and InDel label carry out polymorphism screening, and the polymorphism mark screened is each susceptible in target group
Linkage analysis is carried out between single plant, finally on chromosome by target gene finely positioning.
(5) result and analysis
(1) Yuanjiang River common wild-rice introgression line G252 resistant analysis
By F2Plant is divided into 11 microcommunities (G1~G11), and each microcommunity is inoculated with institute in 1 step (1) of above-described embodiment
1 bacterial strain in 11 bacterial strains stated, while G252 is used as negative control as positive control and japonica rice 02428, as G252 and
When 02428 scab of japonica rice is stablized, investigation statistics F2Incidence, the results show that each microcommunity is poor to the anti-sense of corresponding bacterial strain
It is different all more apparent.According to dientification of bacteria situation is connect, each microcommunity disease-resistant plant and disease plant number, preliminary analysis hair are counted
Now each microcommunity is in the separation of 3 anti-: 1 sense to the theoretical implications value of corresponding bacterial strain, thus it is speculated that it is aobvious that G252 may carry 1 wide spectrum
Property Bacterial blight resistance gene or multiple resistant gene interactions promote G252 to bacterial leaf-blight have resistance of wide spectrum.
(2) gene Xa45 (t) is just positioned
From each microcommunity, 10 plants of 10 plants of extreme disease-resistant plant and extreme disease plant are chosen, is distinguished using CTAB method
DNA is extracted, anti-sense plant distinguishes mixed in equal amounts, anti-, the sense pond DNA is respectively constituted, with the 86 pairs of SSR polymorphism marks pair screened
The anti-sense pond of each microcommunity carries out Molecular Detection, and 02428 liang of parent of G252 and japonica rice is as control, as a result No. 11 chromosome
SSR marker can detect polymorphism between two parents and the anti-sense pond of each microcommunity, and illustration purpose gene is likely located at
On the chromosome.Choose the F of each microcommunity2Susceptible single plant carries out linkage analysis, finds the target gene of each microcommunity
Between RM224 and RM27322, show the consistent resistance of hereditary effect that G252 carries 1 to each bacterial strain of participating in the experiment
The gene is temporarily named as Xa45 (t) by gene, is further demonstrated that gene Xa45 (t) wide spectrum high resistance to hoja blanca, is also shown structure
11 microcommunities built can merge into 11 microcommunities 1 big respectively to the Resistant reaction indistinction of each bacterial strain of participating in the experiment
Group, follow-up test are all studied with big group.
Linked marker RM224 is made of RM224-F primer and RM224-R primer, the nucleotides sequence of the RM224-F primer
Column are as shown in SEQ ID NO:5, and the nucleotide sequence of RM224-R primer is as shown in SEQ ID NO:6.
Linked marker RM27322 is made of RM27322-F primer and RM27322-R primer, the RM27322-F primer
Nucleotide sequence is as shown in SEQ ID NO:7, and the nucleotide sequence of RM27322-R primer is as shown in SEQ ID NO:8.
(3) gene Xa45 (t) finely positioning
In order to further position Xa45 (t) gene, new 131 couples of SSR and 136 couple of InDel label is developed, is screened out from it
The polymorphism mark of acquisition is carried out high-precision by 5 pairs of SSR and 9 pair of InDel polymorphism marks between each susceptible single plant of target group
Linkage analysis, finally gene Xa45 (t) finely positioning in rice No. 11 chromosome long arm end R13I14 and Hxjy-14 two
Between label within the scope of the physical distance of 26kb, and find the gene that InDel label Hxjy-14 is expanded between each susceptible single plant
Type is all consistent with 02428 genotype of Susceptible parent, shows Hxjy-14 and gene Xa45 (t) close linkage (being shown in Table 1).
The linked marker of 1 rice wide spectrum high resistance to hoja blanca gene Xa45 (t) of table
a: for the physical distance on OryzasativaLcv.Nipponbare genome.
Embodiment 2
The verifying of molecular labeling Hxjy-14
(1) material and method
(1) material
Negative kind: sense bacterial leaf-blight kind 02428 (as negative control), OryzasativaLcv.Nipponbare and G252/02428 filial generation
296 parts of not disease-resistant material, totally 299 parts.
Positive kind: 63 parts of G252 (as positive control) and the disease-resistant material of G252/02428 filial generation, totally 64 parts.
Compact linkage molecule label: Hxjy-14
OryzasativaLcv.Nipponbare, the round-grained rice type conventional rice of Japanese breeding, being published in document, " the .CBF regulator such as Pan Xiaowu is in rice varieties
Difference regulatory mechanism rice in China science during OryzasativaLcv.Nipponbare and 93-11 domestication by low temperature, 2012,26 (5): 521~528 ".
(2) method
Each oryza sativa genomic dna extracts and carries out PCR to each genomic DNA using compact linkage molecule label Hxjy-14
The method of amplification is the same as embodiment 1.
(2) result
PCR amplification is carried out to the equal 363 parts of feminine genders of rice material 02428 and positive DNA respectively.The result shows that: Hxjy-14
Label can amplify corresponding target fragment (target fragment length 170bp) in positive sample, there is 2 parts in negative sample
Corresponding target fragment is amplified, does not amplify the identical segment of size, verification and measurement ratio 99.45% in remaining negative sample
(361/363).As a result illustrate, compact linkage molecule label Hxjy-14 provided by the invention and its detection method can be sieved accurately
The material containing Xa45 (t) gene is selected, whether resistance of the rice plant to bacterial leaf-blight can be effectively predicted, can be greatly speeded up anti-
The rice material of bacterial leaf-blight screens progress.
Sequence table
<110>KUNMING INST OF BOTANY CAS
<120>with the molecular labeling Hxjy-14 of rice wide spectrum high resistance to hoja blanca gene Xa45 (t) close linkage
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
ggagacatcc tcaactcttg 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gattagttgg ggaatgacgg 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
agctgagagc cagagatcag 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gtccatcaaa caccaagatc 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
atcgatcgat cttcacgagg 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
tgctataaaa ggcattcggg 20
<210> 7
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
agagcccatg tagctacgcc ttcg 24
<210> 8
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
aatcatgccg gctgaaattg tacc 24
Claims (7)
1. a kind of mark Hxjy-14 with rice wide spectrum high resistance to hoja blanca gene Xa45 (t) compact linkage molecule, feature exists
In the compact linkage molecule label Hxjy-14 is made of Hxjy-14-F primer and Hxjy-14-R primer, target fragment length
170bp, the nucleotide sequence of the Hxjy-14-F primer is as shown in SEQ ID NO:1, the nucleotides sequence of Hxjy-14-R primer
Column are as shown in SEQ ID NO:2.
2. compact linkage molecule according to claim 1 marks Hxjy-14, which is characterized in that the compact linkage molecule
Marking physical distance of the Hxjy-14 on rice chromosome is 27.983Mb.
3. compact linkage molecule label Hxjy-14 of any of claims 1 or 2 assists selecting in rice bacterial blight resistance molecular labeling
Select the application in breeding.
4. application according to claim 3, which is characterized in that compact linkage molecule label Hxjy-14 can be amplified
The amplified fragments of 170bp, then target rice material contains the site Bacterial blight resistance gene Xa45 (t).
5. application according to claim 4, which is characterized in that the amplification carries out PCR expansion using 15 μ L PCR systems
Increase；15 μ L PCR reaction systems are as follows: 0.1 μ L of 5U/ μ L Taq enzyme contains Mg2+10 × PCR Buffer 1.5 μ L, 2.5mmol/L
1.2 μ L of dNTP, the 10 μm of upstream and downstream ol/L primers each 0.4 μ L, 20ng/ μ L template DNA 2 μ L, ddH2O 9.4μL；PCR reaction
Condition are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30sec, 55 DEG C of renaturation 30sec, 72 DEG C of extension 1min, totally 35 recycle；72
DEG C extend 10min；5 μ L pcr amplification products 5V/cm constant pressure electrophoresis on 4% Ago-Gel is taken, gel imaging is then passed through
System imaging simultaneously saves.
6. application according to claim 5, which is characterized in that in 15 μ L PCR reaction systems plus 1 drips paraffin oil.
7. application of the compact linkage molecule label Hxjy-14 of any of claims 1 or 2 in molecular marker assisted selection.
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| CN114438100A (en) * | 2022-03-01 | 2022-05-06 | 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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