CN103981181B - SSR marker chain with aphelenchoides besseyi Resistance QTL on No. 3 chromosomes and application thereof - Google Patents
SSR marker chain with aphelenchoides besseyi Resistance QTL on No. 3 chromosomes and application thereof Download PDFInfo
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- CN103981181B CN103981181B CN201410239721.8A CN201410239721A CN103981181B CN 103981181 B CN103981181 B CN 103981181B CN 201410239721 A CN201410239721 A CN 201410239721A CN 103981181 B CN103981181 B CN 103981181B
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- aphelenchoides besseyi
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Abstract
The invention discloses a kind of molecular labeling chain with aphelenchoides besseyi Resistance QTL.The essence of the present invention is according to chain law of segregation, with aphelenchoides besseyi disease-resistant variety Tetep and No. 5 filial generation F of susceptible variety Huaihe River rice2Colony is mapping population, by the linkage analysis between aphelenchoides besseyi Resistance Identification result and SSR marker data, it is thus achieved that SSR marker RM5626 chain with aphelenchoides besseyi Resistance QTL on No. 3 chromosomes and RM7097.By detection Tetep and derived varieties (being) thereof and the SSR marker banding pattern of the aphelenchoides besseyi susceptible variety single rice plant of cross combination offspring, may determine that whether this plant has aphelenchoides besseyi resistance, it is applied in the assisted selection of Tetep and derived varieties (being)/susceptible variety aphelenchoides besseyi disease-resistant variety thereof, aphelenchoides besseyi Resistance Identification stability and poor repeatability and the shortcoming such as waste time and energy can be overcome in conventional breeding, its result can simplify system of selection and improve breeding efficiency, and then accelerate the seed selection process of water resistant Aphelenchoides oryzae Yokoo paddy rice disease kind.
Description
Technical field:
The present invention relates on No. 3 chromosomes a kind of molecular labeling RM5626 chain with aphelenchoides besseyi Resistance QTL and
RM7097, can be applicable to molecular mark, belongs to paddy disease-resistant breeding and biology field.
Background technology:
Aphelenchoides besseyi is a kind of ectoparasite pathogen of paddy rice, and its classical symptom is that blade tip is twisted into dry point, should
Disease once generation can make grain yield suffer heavy losses (Journal ofthe faculty of Agriculture, Kyushu,
University, 1950,9 (3): 309-333；Nematologica, 1975,21 (3): 351-357；Journal
OfAgricultural Technology, 2011,337-344).Aphelenchoides besseyi traditional control method cost is big, also can make
Become serious environmental problem (Joumal of Agricultural Science and Technology, 2011,14 (1):
195-203).Utilize rice varieties self resistance be control that aphelenchoides besseyi is most economical, environmental protection, effective strategy (Annual
Review ofphytopathology, 2001,39 (1): 285-312；Plant resistance to parasitic
Nematodes, 2002,141-151).
Breeding man once screened some aphelenchoides besseyi resistant varieties, such as cvs Arkansas Fortuna, Nira43,
Asa-Hi, Binam, Domsiah etc. (Phytopathology, 1949,39；Bulletin of the Kyushu
Agricultural Experiment Station, 1953,1 (3): 339-349；Journal of Agricultural
Science and Technology, 2011,14 (1): 195-203).But some aphelenchoides besseyi resistant variety or only exist
A special regional representation resistance, or only aphelenchoides besseyi is showed resistance, extremely sensitive or the most relatively low to other pathogens
Yield (Plant parasitic nematodes in subtropical and tropical agriculture,
2005,87-130).It addition, the hereditary variation of aphelenchoides besseyi population can reduce effective resistance of resistant variety, even cause
The disappearance of varietal resistance.At present, rice varieties is the most unclear to hereditary basis and the molecular mechanism thereof of aphelenchoides besseyi resistance.
Illustrate paddy rice the genetics of resistance of nematode aphelenchoide basis and molecular mechanism thereof are contributed to deeper into understand the mutual of nematode and paddy rice
Make mechanism, lay the foundation (The Plant Journal2004,38 (2): 285-297) for nematode resistance breeding.It addition, for posting
Being identified and isolated from and study the mutual of nematode and plant further on molecule and biochemistry level of main Plant nematode resistant gene
Make mechanism and provide the new visual field (The Plant Journal, 2002,31 (2): 127-136).So far, beet, tomato,
Potato, Soybean and Other Crops navigate to many nematode resistance sites such as H1, GroV1, Cre, Mi3 (Genome, 1993,36
(1): 152-156；Molecular Breeding, 1996,2 (1): 51-60；Theoretical and Applied
Genetics, 1994,89 (7-8): 927-930；Theoretical and Applied Genetics, 1995,91 (3):
457-464).And, it is cloned into several nematode resistance genes such as Hslpro-1, Mi-1, Hero A and Gpa2 and these have been resisted
Property gene carried out heredity and functional analysis (Science1997,275 (5301): 832-834；Nature
Biotechnology, 1998,16 (13): 1365-1369；The Plant Journal, 2002,31 (2): 127-136；The
Plant Journal, 2000,23 (5): 567-576), this enhances us greatly to these host crop nematode resistances
The understanding of molecular mechanism.But, have no at present about the rice varieties quantitative trait locus to aphelenchoides besseyi resistance
(Quantitative Trait Locus, QTL) or the report of gene, only resist in kind the Resistence research of aphelenchoides besseyi
Property identification level on, this significantly limit the effective utilization in resistance breeding of the aphelenchoides besseyi Resistance resource.
Molecular mark technology effectively solves this problem, by building the genetic linkage map in important anti-source
Spectrum and qtl analysis, can effectively find the molecular labeling chain with aphelenchoides besseyi Resistance QTL, and using these to mark can
To tie up to the offspring in this anti-source and spin-off thereof carry out screening the most from generation to generation seedling stage, eliminate disease plant, not only saved cost,
Also improve breeding efficiency.
Summary of the invention:
The present invention is directed to the studies above background, with the aphelenchoides besseyi disease-resistant variety Tetep screened and susceptible main cultivation
Kind Huaihe River rice No. 5 is material, 842 to (http://www.gramene.org) microsatellite marker in screen 160 polymorphic
Property SSR marker, therefrom select 127 SSR marker to Huaihe River rice No. 5/Tetep F2Colony is analyzed, and builds rice genetic linkage
Collection of illustrative plates, by itself and F2∶3Hundred nematode number (The number of after family aphelenchoides besseyi artificial infection idenfication
A.besseyi in100grains, NA) between carry out linkage analysis, it is thus achieved that with aphelenchoides besseyi resistance on No. 3 chromosomes
SSR marker RM5626 that QTL is chain and RM7097, the molecule assisted Selection that can be applicable to aphelenchoides besseyi disease-resistant variety is educated
Kind.
On No. 3 chromosomes provided by the present invention SSR marker RM5626 chain with aphelenchoides besseyi Resistance QTL and
RM7097, is prepared by the following:
1) susceptible variety Huaihe River rice No. 5 (♀) obtains hybrid F with disease-resistant variety Tetep (♂) hybridization1, F1Selfing obtains F2Colony,
Individual plant results F2∶3Family seed is used for aphelenchoides besseyi Resistance Identification；
2) CTAB method extracts parent, F1And F2The DNA of 138 individual plants of colony；
3) utilize the 127 pairs of SSR primers selected to parent, F1And F2Colony carries out PCR amplification, and amplified production is by poly-third
Acrylamide gel electrophoresis analysis, it is thus achieved that molecular marker data, builds genetic linkage map；
4) parent Huaihe River rice No. 5, Tetep and filial generation F thereof1And F2∶3Family carries out aphelenchoides besseyi artificial infection mirror
Fixed, after seed maturity, single fringe sowing, adds up NA.
5) calculate linkage inheritance distance between mark according to marker bands MAPMAKER/EXP3.0 software, and use
Mapdraw draws according to each mark position on chromosome, builds rice genetic linkage map；
6) use based on composite interval mapping method software Windows QTL Cartographer V2.5 detection LIPIDS OF DRY RICE EMBRYO point
The Resistance QTL of nematode.The threshold value of LOD value is set to 2.5, by the number of P=0.05 probable value detection Resistance QTL and at chromosome
On position.
The application bag of molecular labeling RM5626 and RM7097 chain with aphelenchoides besseyi Resistance QTL on No. 3 chromosomes
Include: with the single rice plant of Tetep and derived varieties (being) thereof with aphelenchoides besseyi susceptible variety cross combination offspring be
Object, by detecting the banding pattern data of RM5626 and RM7097 mark on its No. 3 chromosomes, can predict that this plant is to LIPIDS OF DRY RICE EMBRYO
The resistance of point nematode.
The present invention can overcome aphelenchoides besseyi Resistance Identification stability in conventional breeding, repeatability is poor and wastes time and energy
Etc. shortcoming, its result can simplify system of selection and improve breeding efficiency, and then accelerates the breeding process of disease-resistant variety.
Accompanying drawing illustrates:
Fig. 1 utilizes Huaihe River rice No. 5/Tetep F2The Genetic Linkage Map spectrum of informative population
Aphelenchoides besseyi Resistance QTL genetic linkage group's positioning analysis schematic diagram on No. 3 chromosomes of Fig. 2
Note: ■ Resistance QTL linkage group section；Left side is genetic distance (cM) between mark；Right side is mark title
Detailed description of the invention:
In example below, method therefor is conventional method if no special instructions.
Embodiment 1, the acquisition of the molecular labeling chain with aphelenchoides besseyi Resistance QTL
1, vegetable material
2008 field planting Huaihe River, academy of agricultural sciences of Jiangsu Province rice No. 5 and Tetep, and hybridize acquisition hybrid F1, next year F1Selfing
Obtain F2Seed, 2009 at Hainan breeding F2Colony, as mapping population.F2Individual plant is numbered, and takes parent, hybrid F tillering stage1And F2
The partial blade of each individual plant of colony ,-70 DEG C of Refrigerator stores are for ssr analysis, F2Colony's individual plant sowing, in case phenotypic evaluation.
2, the cultivation of nematode with separate
The pathogen of Botrytis cinerea (Botrytis cinerea) bacterium block is seeded in Potato Dextrose Agar (PDA) cultivate
25 DEG C of cultivations on base, after the pathogen of Botrytis cinerea covers with culture medium, with 3% hydrogen peroxide to aphelenchoides besseyi surface sterilization 10min,
After sterilizing milli-Q water 3 times, about 400 aphelenchoides besseyis are inoculated on the pathogen of Botrytis cinerea covering with culture medium, in 25
DEG C cultivate about 20d (Journal of nematology, 2009,41 (1): 17).It is separately cultured by Baermann funnel technique
Aphelenchoides besseyi, and with 3% hydrogen peroxide surface sterilization (Rice Science, 2009,16 (4): 301-306), sterilized water
Nematode is collected, as inoculation material after flushing.
3, CTAB method extracts DNA
1) weigh 500mg rice leaf to be placed in 2.0mL Eppendorf pipe, centrifuge tube is placed in liquid nitrogen cooling, fill
Take out rapidly after full liquid nitrogen, be ground into powder with grinding rod；
2) water-bath 1-1.5h, 15min shake 1 time under the conditions of 65 DEG C；
3), under the conditions of 4 DEG C 13,000rpm is centrifuged 10min, takes upper strata aqueous phase；
4) 900 μ L chloroforms are added: isoamyl alcohol (24: 1), fully mixing concussion is become white to solution colour from green
Look；
5), under the conditions of 4 DEG C 13,000rpm is centrifuged 10min, takes upper strata aqueous phase；
6) add equal-volume isopropanol, stand 20-30min under the conditions of-20 DEG C, be settled out cotton-shaped DNA；
7), under the conditions of 4 DEG C 13,000rpm is centrifuged 10min, abandons supernatant, adds the washing of 1mL70% ethanol, under the conditions of 4 DEG C 13,
000rpm is centrifuged 5min, abandons supernatant, is placed on super-clean bench and dries；
8) adding 30 μ L deionized water dissolving DNA, 4 DEG C save backup.
OD value and concentration is measured, by each sample DNA with EppendorfBioPhotometer Plus nucleic acid-protein analyzer
It is diluted to 20ng/ μ L standby.
4, SSR marker analysis
(1) PCR amplification
Use the PCR reaction system of 10 μ L: DNA (10ng/ μ L) 1.3 μ L, Primer (4pmol/ μ L) 1.5 μ L, 10 × PCR
Buffer (w/Mg) 1.5 μ L, dNTP (2.5mM) 0.2 μ L, Taq (5U/ μ L) 0.1 μ L, ddH2O5.4μL.PCR response procedures: 95
DEG C sex change 5min；95 DEG C of sex change 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 1min, carry out 35 circulations；72 DEG C extend 10min.Produce
Thing adds loading buffer stopped reaction, stand-by.
(2) polyacrylamide gel electrophoresis
Using 8% polyacrylamide gel to detect above-mentioned PCR product, electrophoresis apparatus used is that double plate holder core is the most electric
Swimming groove DYCZ-30 type (Liuyi Instruments Plant, Beijing).Concrete operation step is as follows:
1) with liquid detergent glass plate scrub repeatedly, with distilled water flushing, oven for drying, can before assembling glass plate
Use alcohol wipe；
2) putting in rubber seal strip by two pieces of glass plates, Ago-Gel seals bottom and installs to electrophoresis tank；
3) each liquid storage preparation acrylamide gel is added by table 1 order, with pipettor by equal for the glue prepared after fully shaking up
Between even injection two glass plate, it is inserted into comb, notes preventing from producing bottom comb bubble.Stand about 1h and treat that it solidifies；
The volume of polyacrylamide gel agents useful for same prepared by table 1
4) in electrophoresis tank, add the electrophoretic buffer of 1 × TAE, extract comb, PCR primer is joined in loading wells；
5) 200V constant voltage electrophoresis 1-2h.
(3) dyeing (argentation) of polyacrylamide gel
1) with spirituosity 10%, the solution of glacial acetic acid 0.5% fixes 2 times, 6min/ time；
2) abandon fixed solution, add 0.2%AgNO3Solution infiltration 10-12min；
3) ddH is used after infiltration2O cleans；
4) carry out putting generation about 30s with 0.002% hypo solution；
5) Dai Houyong is put containing 1.5%NaOH, the solution colour developing of 0.4% formaldehyde；
6), after band is clear, distilled water flushing, color development stopping is reacted；
7) with the encapsulated preservation of preservative film, and on white light, experimental result is recorded.
5, aphelenchoides besseyi evaluation of resistance
F2∶3Family aphelenchoides besseyi evaluation of resistance was carried out in experimental plot, Jiangsu Province Agriculture Science Institute summer in 2012.
F2∶3The dry seed of family and parent Huaihe River rice No. 5 and Tetep soaks 15min, to guarantee nothing in dry seed in 55 DEG C of warm water
Survival aphelenchoides besseyi (Joumal of Agricultural Science and Technology, 2011,14 (1):
195-203).By the seed-soaking processed, vernalization and be seeded in seedlings nursing plate, by shoot transplanting equipment to land for growing field crops after about 20d.Wait to plant
Strain length is to the nematode of cultivation was configured to during the jointing stage nematode suspension of 10000/mL, with water suction absorbent cotton parcel Rice Leaf
Sheath portion, then draws the nematode hanging drop of 40 μ L on leaf sheath top with liquid-transfering gun, wraps up rice leaf sheath portion with absorbent cotton equally,
Draw 40 μ L sterilized waters with liquid-transfering gun and drop in leaf sheath top as comparison (Plant resistance to parasitic
Nematodes, 2002,141-151；Journal of Integrative Agriculture, 2013, Doi:10.1016/
S2095-3119 (13) 60608-5), single fringe sowing after seed maturity, the single fringe setting percentage of statistics and single fringe seed weight.Strip off water
Rice grain husk shell, is immersed in grain husk shell and the grain of rice in the mesh screen filling in hyponome, soaks 24h, treats that nematode dissociates to funnel
After bottom footpath, take bottom footpath that the water of about 15mL is in the culture dish of a diameter of 6cm being divided into 12 regions, with counter at body
Microscopy list fringe paddy nematode amount under formula microscope, adds up NA.Use SPSS20.0 software that phenotypic data is analyzed.
6, the structure of genetic map and qtl analysis
Synthesize 842 to SSR primer according to microsatellite database (http://www.gramene.org), carry out between parent
Polymorphic detection, has polymorphic 127 SSR marker and analyzes F between selected parent2Colony.
MAPMAKER/EXP3.0 software is utilized to carry out linkage analysis.Wherein identical with No. 5 banding patterns of Huaihe River rice it is entered as " A ",
Identical with Tetep banding pattern is entered as " B ", and the banding pattern of heterozygous is entered as " H ", and special or disappearance banding pattern is entered as "-".According to
Linkage relationship between each mark, with " group " order when LOD value is more than 2.5, is grouped mark.Use Kosambi again
Between function will mark in each group, recombination fraction is converted into genetic distance (Centimorgan, cM).Little linkage group is directly used
" compare " orders, and selects the structure locking frame figure that puts in order with maximum likelihood value；For bigger linkage group, then
First by the method for 2 tests, calculate the LOD value between marking the most two-by-two, use " compare " order to obtain its suggestion most preferably
Put in order, build basic framework figure, then " try " order, find out the most suitable position of each mark (maximum likelihood value method).With
Time with " ripple " verify.Finally obtain genetic linkage maps with " map " order.Construct the water comprising 12 genetic linkage groups
Rice collection of illustrative plates, covers rice genome 2179.6cM, and between 127 SSR marker, average headway is 17.16cM (Fig. 1).
Use based on composite interval mapping method (Windows QTL Cartographer V2.5) to build genetic map
And F2∶3Phenotypic data is analyzed, and the threshold value of LOD value is set to 2.5, by P=0.05 probable value detection Resistance QTL number and
Position on chromosome.No. 3 chromosomes detect 1 the water resistant Aphelenchoides oryzae Yokoo QTL, named qNA3 relevant to NA
(the principle Theor Appl Genet of name reference McCouch, 1988,76 (6): 815-829), is positioned at the 3rd chromosome
Between RM5626-RM7097, LOD value is 3.04, and contribution rate is 16.91% (Fig. 2).
Embodiment 2, molecular labeling chain with aphelenchoides besseyi Resistance QTL on No. 3 chromosomes with Huaihe River rice No. 5 with
Tetep is the application in the cross combination offspring of parent
The SSR marker chain with aphelenchoides besseyi Resistance QTL obtained on No. 3 chromosomes, to Huaihe River rice No. 5 and Tetep
The F of hybridization2∶3The part individual plant of family has carried out aphelenchoides besseyi resistance prediction, extracts each individual plant DNA respectively, then uses SSR
The primer of mark RM5626 and RM7097 carries out PCR Amplification Analysis, determines whether there is corresponding mark by band analysis,
There is mark illustrates that this strain aphelenchoides besseyi reaches anti-level, does not exists, is susceptible.Subsequently, LIPIDS OF DRY RICE EMBRYO point is utilized
Nematode artificial infection idenfication method measure the tested strain actual resistance to aphelenchoides besseyi, and with disease-resistant variety, susceptible product
Planting and compare, result shows match with actually detected result (table 2) that predict the outcome.
Huaihe River rice No. 5/Tetep filial generation F predicted by table 22∶3The resistance of family aphelenchoides besseyi
Above-mentioned enforcement limits the present invention the most in any form.
Claims (1)
1. molecular labeling RM5626 and RM7097 is at qualification rice varieties to the application in aphelenchoides besseyi resistance, and its feature exists
In with the single rice plant of Tetep and the Huaihe River rice No. 5 cross combination offspring as parent as object, the banding pattern marked by detection
Data, it was predicted that this plant resistance to aphelenchoides besseyi.
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| CN103555718A (en) * | 2013-11-18 | 2014-02-05 | 江苏省农业科学院 | SSR (simple sequence repeat) markers on No.3 chromosome, closely linked to RBSDV (rice black-streaked dwarf virus) resistant QTL (quantitative trait locus) and application thereof |
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| CN103555718A (en) * | 2013-11-18 | 2014-02-05 | 江苏省农业科学院 | SSR (simple sequence repeat) markers on No.3 chromosome, closely linked to RBSDV (rice black-streaked dwarf virus) resistant QTL (quantitative trait locus) and application thereof |
Non-Patent Citations (4)
| Title |
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| Genetic dissection of biomass production, harvest index and panicle characteristics in indica-indica crosses of Iranian rice (Oryza sativa L.) cultivars;Hossein等;《Australian Journal of Crop Science》;20091231;第3卷(第3期);全文 * |
| Mapping QTLs for nitrogen-deficiency tolerance at seedling stage in rice(Oryza sativa L.);FENG等;《Plant Breeding》;20100210;第129卷(第6期);全文 * |
| Reduction of N-Oxides and Sulfoxide by the Same Terminal Reductase in Proteus mirabilis;Adeline等;《CURRENT MICROBIOLOGY》;19911231;第23卷;全文 * |
| Toward the mapping of Agronomic characters on a rice genetic map:quantitative trait loci analysis under saline condition;Gonbad等;《Biotechnology》;20091231;第8卷(第1期);全文 * |
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