CN102676685B - Primers and method for improving resistance of susceptible rice varieties to black streaked dwarf viral disease - Google Patents
Primers and method for improving resistance of susceptible rice varieties to black streaked dwarf viral disease Download PDFInfo
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Abstract
The invention provides close linkage simple sequence repeat (SSR) molecular markers of a main effect quantitative trait locus (QTL), namely qRBSDV-6 for resistance to rice black streaked dwarf viral disease, and a method for improving the resistance of susceptible rice varieties through marker assistant selection. For derived line segregating population obtained by hybridizing and back-crossing Minghui 63 serving as a resistance source and a susceptible receptor parent, genotypes of the close linkage markers RM7158 and RM587 on two sides of the main effect quantitative trait locus (QTL), namely qRBSDV-6 for resistance to black streaked dwarf viral disease on the short arm of the sixth chromosome of Minghui 63 are detected, the close linkage markers can be successfully imported into the genetic background of the susceptible rice varieties, the resistance of the susceptible rice varieties to the black streaked dwarf viral disease is effectively improved and the selection efficiency reaches 92.31 percent. The method is easy to implement, quick and efficient, and is an effective method for improving the resistance to the rice black streaked dwarf viral disease.
Description
Technical field
The present invention is directed to black streaked dwarf virus of rice main effect Resistance QTL---
qRBSDV-6, provide a kind of and utilized its compact linkage molecule mark to carry out assisted Selection and improve the method for rice varieties black streak dwarf resistance, belong to molecular genetics field.
Background technology
Black streaked dwarf virus of rice be a kind of mainly by small brown rice planthopper (
laodelphax striatellusfallen) serve as medium and the virus disease propagated, the general performance of disease plant is seriously stunt, leaf look dark green, short-term white stripes appears in blade back vein, can not ear or wrap neck fringe, fringe particle shape diminishes even deformity and the symptoms such as remarkably influenced economic yield.Once just cannot prevent and treat because paddy rice infects this virus, thereby quilt analogy be paddy rice " cancer ".This disease occurs at first Chinese the sixties in last century, in recent years, along with the variation of cropping system and the lasting appearance in time warm winter, black streak dwarf in Zhejiang, the ground such as Jiangsu, Shanghai is again rampant popular, partial area seriously occur, and that most kinds of producing application all show this disease is susceptible, to Rice Production, cause significant damage.
At present, although do not have to find black streak dwarf to show immune rice germplasm, the report of relevant its genetics of resistance, breeding research is also less, has been reported and shows that Different Types of Rice kind has notable difference to the resistance of black streak dwarf.This research group utilizes the recombinant inbred lines of " precious Shan 97/ bright extensive 63 ", by the natural inoculated identification of 2 regions of disease, the Resistance QTL of 6 black streaked dwarf virus of rices on the 6th, 7,9 and 11 karyomit(e)s, detected respectively, all come from parent bright extensive 63, especially 2 closely linked QTL(that on the 6th karyomit(e), R2869-Waxy locates in interval are because be Primary Location, actually a QTL that effect is larger, Hai Shi two closely linked QTL can't determine, by its preliminary designation, be
qRBSDV-6), there is the features such as LOD value is large, effect is remarkable, have important breeding be worth (Pan Cunhong, Li Aihong etc., black streaked dwarf virus of rice Resistance QTL is analyzed, Acta Agronomica Sinica, 2009,35(12): 1-5).
In production, at present the control of black streaked dwarf virus of rice is mainly improved in conjunction with cropping system adjustment, cultivation technique and optimum period prevention and elimination of disease and pests etc., but be often difficult to produce a desired effect, and environment is caused to larger pollution.Cultivating disease-resistant variety is most economical, the effective means of all kinds of diseases of control, has most important theories and practice significance.Main effect Resistance QTL for location, by the method for molecular marker assisted selection, is imported in the genetic background of susceptible variety, can effectively improve its resistance.
Summary of the invention
The object of the invention is for the black streak dwarf main effect Resistance QTL on Short arm of chromosome 6---
qRBSDV-6, the bilateral compact linkage molecule mark in its PCR-based technical foundation is provided, by molecular marker assisted selection, imported in the genetic background of susceptible variety and improved the method for its black streak dwarf resistance.
qRBSDV-6primary Location is between mark R2869 and Waxy, because R2869 and Waxy are RFLP marks, if directly apply to marker assisted selection, exist workload large, operate the shortcomings such as loaded down with trivial details, thereby we are according to above-mentioned physical location (the RGP website being marked in the fine genome sequence of paddy rice Japan, http://rgp.dna.afrc.go.jp/)), in conjunction with Gramene website (http://www.gramene.org) the upper paddy rice SSR mark of announcing, in the outside of mark R2869 and Waxy, screened 2 at Resistance QTL donor parents bright extensive 63 and part Susceptible parent (as Huaihe River rice No. 5, No. 1, Wulin tomb round-grained rice, raise round-grained rice 9538 etc.) between there is SSR mark RM7158 and the RM587 of good polymorphism, for target Resistance QTL (
qRBSDV-6) molecular marker assisted selection.
The invention discloses paddy rice SSR mark RM7158 and the RM587 application in rice varieties black streak dwarf resistance molecular marker assisted selection.
Molecule marker RM7158 provided by the invention and RM587, obtained through pcr amplification by following primer pair respectively:
1) labeled primer RM7158,
Forward primer sequence, 5 '-CGTCCATGGACTTGTTAGC-3 ' (SEQ ID NO.1)
Reverse primer sequence, 5 '-ACGGATACGCCATCCACAT-3 ' (SEQ ID NO.2);
2) labeled primer RM587,
Forward primer sequence, 5 '-ACGCGAACAAATTAACAGCC-3 ' (SEQ ID NO.3)
Reverse primer sequence, 5 '-CTTTGCTACCAGTAGATCCAGC-3 ' (SEQ ID NO.4).
In above-mentioned mark, the amplified production of RM7158 in Resistance QTL donor parents bright extensive 63 is 128 bp, and amplified production in Susceptible parent is 144 bp; The amplified production of RM587 in Resistance QTL donor parents bright extensive 63 is 222 bp, and amplified production in Susceptible parent is 210bp.
The present invention also provides the method for molecular marker assisted selection improvement rice varieties black streak dwarf, to doing anti-source with bright extensive 63, by the derivative segregating population plant of method acquisitions such as hybridizing or backcross, the genomic dna of paddy rice to be detected of take is template, 2 pairs of primers by mark RM7158 and RM587 carry out pcr amplification, as there being respectively the band of 128 bp and 222 bp sizes in the amplified production of paddy rice to be detected, this rice plant carry target Resistance QTL "
qRBSDV-6".
The extraction of DNA adopts conventional CTAB method (Murray & Thompson, 1980 Rapid isolation of high-molecular-weight plant DNA.
nucleic Acids Res8:4321-4325).
PCR reaction is in EPPENDORF grads PCR instrument (model: carry out Mastercycler Pro), amplified production is electrophoretic separation in 3% sepharose, bromination second is formed sediment after (EB) dyeing, BIO-RAD gel imaging instrument (model: Gel Doc XR) under ultraviolet lamp, scanning is taken pictures and analyzes.
Described pcr amplification system is: 0.2 μ mol L
-1each 1.5 μ L of forward and reverse primer, 200 μ mol L
-1dNTPs 2 μ L, 10 * PCR buffer, 2 μ L, the DNA profiling 2 μ L of 50 ~ 100 ng, 1 U μ L
-1's
tagenzyme 0.3 μ L, ddH
2o 10.7 μ L.
Described PCR reaction conditions is: 94 ° of C denaturation 5 min, and 94 ° of C sex change 1 min, 55 ° of C renaturation 1 min, 72 ° of C extend 1 min, 35 circulations, last 72 ° of C extend 5 min.
Beneficial effect of the present invention:
1) by the present invention first with SSR mark define black streak dwarf main effect Resistance QTL in rice varieties bright extensive 63 "
qRBSDV-6";
The main effect Resistance QTL that 2) molecule marker of the present invention limits "
qRBSDV-6" locality specific, it is convenient to identify.By detecting and the closely linked molecule marker of this Resistance QTL, can predict that rice plant is whether containing target Resistance QTL again, and then screening black streak dwarf resistance individual plant or strain are for rice breeding.Its detection method is convenient and swift, not affected by environment, accelerates target variety and cultivates process.
Accompanying drawing explanation
Fig. 1 is molecule marker RM7158 provided by the invention, RM587 to bright extensive 63 making donor, Huaihe River rice is for No. 5 the BC of recurrent parent
3f
3the detected result of generation strain.In figure, P1: bright extensive 63; P2: Huaihe River rice No. 5; 1-18:BC
3f
3generation different genotype strain; R1: disease-resistant gene type isozygotys; S3: the susceptible genotype of isozygotying.
Embodiment
Following instance further illustrates content of the present invention by reference to the accompanying drawings, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to the scope of the invention.
In the embodiment of the present invention, rice varieties used all has present kind, and wherein No. 5 Jiangsu Xuhuai Zone Huaiyin Agricultural Sciences Institute of You Gai breed breeding unit of Huaihe River rice provide; The Sanming City, Fujian Province institute of agricultural sciences of bright Hui63You Gai breed breeding unit provides; No. 1 Yangzhou University of You Gai breed breeding unit of Wulin tomb round-grained rice provides.
Embodiment 1:
1) under No. 5 genetic backgrounds of Huaihe River rice, carry "
qRBSDV-6" near isogenic line build
Take highly susceptible " Huaihe River rice No. 5 " as maternal, obtain F with donor parents " bright extensive 63 " hybridization
1, then take " Huaihe River rice No. 5 " carry out continuous backcross as recurrent parent, from BC
1f
1segregating generation starts, for different paddy rice individual plants, utilize target Resistance QTL provided by the invention "
qRBSDV-6" SSR mark RM7158 and the RM587 of both sides carry out genotype detection.The extraction of DNA adopts conventional CTAB method.
Pcr amplification system is: 0.2 μ mol L
-1each 1.5 μ L of forward and reverse primer, 200 μ mol L
-1dNTPs 2 μ L, 10 * PCR buffer, 2 μ L, the DNA profiling 2 μ L of 50 ~ 100 ng, 1 U μ L
-1's
tagenzyme 0.3 μ L, ddH
2o 10.7 μ L.
PCR reaction conditions is: 94 ° of C denaturation 5 min, and 94 ° of C sex change 1 min, 55 ° of C renaturation 1 min, 72 ° of C extend 1 min, 35 circulations, last 72 ° of C extend 5 min.
Select two marks to be the approaching individual plant of heterozygosis banding pattern and economical character and recurrent parent and backcross, through continuous backcrossing for 3 times, obtain BC
3f
1after offspring, (Yangzhou, 1 year two Ji Jiadai in Hainan), carry out selfing, BC
3f
2segregating generation selects bilateral mark to be isozygoty resistant gene type and good 18 of the individual plants (Fig. 1) of economical character, BC
3f
3plant into from generation to generation strain, through economical character, differentiate and evaluate, finally determine that 13 are used for follow-up black streak dwarf Resistance Identification with the most approaching strain of recurrent parent.
2) black streak dwarf Resistance Identification and the evaluation of the near isogenic line obtaining
Positive season in 2011, in Yangzhou bayhead town, going to river regional institute of agricultural sciences test base to above-mentioned 13 near isogenic lines and contrast divides two dates of seeding to carry out spontaneous induction evaluation, with cave sickness rate evaluate its resistance level (its method referring to: Li Aihong, wear positive element etc., the initial analysis of different genotype rice germplasm to black streak dwarf resistance, Yangzhou University's journal (agricultural and life science version), 2008,29(3): 18-22).Result shows, two susceptible contrast Huaihe River rice No. 5, Wulin tomb round-grained rice No. 1 sickness rate in the natural qualification test of 2 dates of seeding, all over 50%, shows that incidence is abundant.Its resistance of near isogenic line that variance analysis demonstration is cultivated has been compared remarkable improvement (table 1) with recurrent parent " Huaihe River rice No. 5 ", the near isogenic line of sowing on May 5, except MTJ-12, all the other its black streak dwarf sickness rate of 12 strains generally between 20-30%, the sickness rate of " Huaihe River rice No. 5 " 56% left and right 25% left and right that declines; The near isogenic line of sowing on May 12, equally except MTJ-12, all the other strain sickness rate are generally at 25-40%, sickness rate is generally than the raising approximately 10% of sowing on May 5, equally, the sickness rate of susceptible contrast " Huaihe River rice No. 5 " also obviously rises, and reaches 65.85%, the sickness rate of great majority improvement near isogenic lines still, compared with recurrent parent 25% left and right that declines, has fully proved the validity that target QTL selects in the practices of breeding.
The method that adopts the inoculation of artificial capture method striatellus imago is identified in artificial inoculation, and strain to be identified was sowed between 5 to 15 May, and suitably thinning after emerging, remains on the population size about 120 strains, with 60 object nylon mesh sealing covers.2 leaf phases started from the striatellus imago of rice seedling bed collection around, it is carried out to small brown rice planthopper and take the rear (Lv Yongping of malicious rate mensuration, Lei Juanli etc., the RT-PCR of black streaked dwarf virus of rice detects, Zhejiang Agriculture journal, 2002,14(2): 117-119), by the worm amount inoculation of 5 small brown rice planthoppers of every seedling, manually stir every day, after 5 days, spray sterilant, be transplanted in the fly net of field.Test-results and twice spontaneous induction qualification result is basically identical by stages, relation conefficient reaches 0.88 and 0.87, has verified the reliability of natural qualification result.
Because the distance between molecule marker RM7158, RM587 is larger, with reference to the fine genome sequence of Japan, the physical distance between two marks reaches 2000Kb left and right, thereby exchange restructuring easily occurs therebetween.Although strain MTJ-12 Marker selection is positive, both sides mark is disease-resistant gene type, shows susceptible phenotype, may exchange occur with chromosome segment between these strain two marks and recombinate relevant.
In this example, the identical rate of Marker selection and resistant phenotype reaches 92.31%, has fully proved the validity of present method.
the black streak dwarf resistance performance of the different near isogenic lines of table 1
In same row, different lowercases are illustrated in the significance of difference of 0.05 level.
SEQUENCE LISTING
<110> Inst. of Agricultural Science, Lixiahe Prefecture, Jiangsu Prov.
Mono-kind of <120> is for improveing primer and the method for susceptible rice varieties black streak dwarf resistance
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> artificial sequence
<400> 1
cgtccatgga cttgttagc 19
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<400> 2
acggatacgc catccacat 19
<210> 3
<211> 20
<212> DNA
<213> artificial sequence
<400> 3
acgcgaacaa attaacagcc 20
<210> 4
<211> 22
<212> DNA
<213> artificial sequence
<400> 4
ctttgctacc agtagatcca gc 22
Claims (3)
1. paddy rice SSR mark RM7158 and the RM587 application in rice varieties black streak dwarf resistance molecular marker assisted selection; The primer sequence that it is characterized in that described RM7158 is:
Forward primer, 5 '-CGTCCATGGACTTGTTAGC-3 '
Reverse primer, 5 '-ACGGATACGCCATCCACAT-3 ';
The primer sequence of described RM587 is:
Forward primer, 5 '-ACGCGAACAAATTAACAGCC-3 '
Reverse primer, 5 '-CTTTGCTACCAGTAGATCCAGC-3 '.
2. for the primer pair of black streaked dwarf virus of rice main effect Resistance QTL molecular marker assisted selection, it is characterized in that,
A pair of is the primer of molecule marker RM7158, and sequence is:
Forward primer, 5 '-CGTCCATGGACTTGTTAGC-3 '
Reverse primer, 5 '-ACGGATACGCCATCCACAT-3 ';
Another is to being the primer of molecule marker RM587, and sequence is:
Forward primer, 5 '-ACGCGAACAAATTAACAGCC-3 '
Reverse primer, 5 '-CTTTGCTACCAGTAGATCCAGC-3 '.
3. a molecular marker assisted selection has the method for the improvement rice varieties of black streak dwarf resistance, it is characterized in that, to doing anti-source with bright extensive 63, by the derivative segregating population plant hybridizing or the method that backcrosses obtains, the genomic dna of paddy rice to be detected of take is template, 2 pairs of primers by mark RM7158 claimed in claim 2 and RM587 carry out pcr amplification, as there being respectively the band of 128 bp and 222 bp sizes in the amplified production of paddy rice to be detected, this rice plant carries target Resistance QTL
qRBSDV-6.
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| CN104450694B (en) * | 2014-11-24 | 2017-08-11 | 广西壮族自治区农业科学院水稻研究所 | Anti- southern rice black-streaked dwarf disease site qSRBSDV6 and its molecule labelling method |
| CN106834281A (en) * | 2015-12-04 | 2017-06-13 | 无锡南理工科技发展有限公司 | SSR marker with black streaked dwarf virus of rice Resistance QTL close linkage and its application on No. 4 chromosomes |
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| CN101294161A (en) * | 2008-04-22 | 2008-10-29 | 中国水稻研究所 | Numerator mark concatenated with whitebacked planthopper resistance genes of rice, and developing method thereof |
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| #8722 * |
| 1264页. * |
| 289页. * |
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