CN100393740C - High yield rice cultivation method and specific molecular mark - Google Patents
High yield rice cultivation method and specific molecular mark Download PDFInfo
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- CN100393740C CN100393740C CNB2005101050051A CN200510105005A CN100393740C CN 100393740 C CN100393740 C CN 100393740C CN B2005101050051 A CNB2005101050051 A CN B2005101050051A CN 200510105005 A CN200510105005 A CN 200510105005A CN 100393740 C CN100393740 C CN 100393740C
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Abstract
The present invention discloses a method for culturing high-yield rice and a special molecular marker thereof. The special molecular marker is obtained by carrying out PCR amplification to one of the following primers: a primer pair comprising SEQ ID No: 1 and SEQ ID No: 2 in the sequence table, and a primer pair comprising SEQ ID No: 3 and SEQ ID No: 4 in the sequence table. The method for culturing new high-yield rice varieties has the advantages of simple operation, low cost and short period and is suitable for being popularized and used. Because the method is free from the influence of other gene effects and environmental factors, the method can carry out selection in early generations, shorten seed breeding time and enhance seed breeding efficiency. The method has important significance for the variety improvement of rice.
Description
Technical field
The present invention relates to a kind of method and specific molecular mark of cultivating high-yield rice.
Background technology
Paddy rice is an important crops, and over nearly 20 years, its potentiality aspect output do not obtain big breakthrough.Rangel et al (Rangel PH, Guimaraes EP, Neves PCF.Base genetics DNA cultivatedde arroz (oryza sativa L.) irrigado do Brasil.Pesq Agropec Bras, 1996,31:349-357) and Tanksley (Tanksleys SD, McCouch SR.Seed banks and molecularmaps:Unlocking genetic potential from the wild.Science, 1997,277:1063-1066) point out the breeding resource narrow be the basic reason that causes this situation.Therefore current crop breeding demands excavating a collection of new gene urgently, to widen the basis of crop genetic.And excavate new gene, utilize molecular marking technique that rice yield traits is selected just present research focus and difficult point both at home and abroad.Along with the saturated molecule linkage maps of paddy rice, utilize molecule marker to locate the quantitative trait locus of a large amount of control rice yield traits (QTL), yet the report that utilizes molecular marking technique that its yield traits is improved is very few.
Common wild-rice is ancestors' kind of cultivated rice, and its genetic diversity is greater than cultivated rice, and wild-rice is in being evolved into the cultivated rice process, and through natural selection and artificial selection, gene diversity reduces, and the allelotrope number reduces.According to the study, the allelotrope number of cultivated rice is about 60% (Sun CQ of wild-rice, Wang XK, Li ZC, Yoshimura A.Comparison of the genetic diversity of common wild rice (Oryza rufipogon Griff.) and cultivated rice (O.sativa L.) using RFLP markers.Theor Appl Genet, 2001,102:157-162).From wild-rice, excavate and utilize the excellent gene of in cultivated rice, having lost or having weakened, and they are applied to have important theoretical meaning and more practical value in the rice breeding production.The high resistance to hoja blanca gene Xa21 that has cloned is found in long flower pesticide wild-rice (O.longistaminata).(Zhang Qi, ZhaoBing Yu, Zhao Kaijun such as Zhang Qi, Wang Chunlian, Yang Wencai, Lin Shicheng, fault is more living, Zhou Yongli, Li Daoyuan, Chen Chengbin, Zhu Lihuang. the evaluation of the new gene Xa-23 of the anti-bacterial blight of rice of common wild-rice (t) and molecule marker location. Acta Agronomica Sinica, 2000,26 (5): 536-542) from the common wild-rice of Guangxi, find a high anti-gene Xa23 of wide spectrum in the time of infertility.(Xiao J such as Xiao, Grandillo S, Ahn S N et al.Gene from wild rice improveyield.Nature, 1996,384:223-224) from the Malaysian common wild-rice of low yield, find and can increase production two QTL of 17% and 18% respectively.(Li DJ such as Li, Sun CQ, Fu YC, Chen L, Zhu ZF, Li C, Cai HW, Wang XK.Identification and mapping of genes for improvingyield from Chinese common wild rice (O.rufipogon Griff.) using advancedbackcross QTL analysis.Chinese Science Bulletin, 2002,18:1533-1537) from China's Jiangxi Dongxiang Wild Rice, find and located 2 high yield QTL.But the locus (QTL) for rice yield traits only rests on the localized stage at present.
Utilize molecule marker that the technology of the molecular marker assisted selection of rice bacterial blight resistance gene, rice blast gene is all had report both at home and abroad, but up to now, yet there are no report about the molecule marker selection technology of rice yield traits QTLs.
Wild-rice is the low yield plant, but have the high yield gene, how to excavate the high yield gene in the wild-rice, and to set up wild-rice high yield gene molecule marker selection technology be a global difficult problem.China's wild-rice aboundresources; from wild-rice, excavate, locate the high yield gene; seek and the closely linked molecule marker of high yield gene; set up the high yield gene molecule marker selection technology and the protection that patents; not only provide new gene and new technology for cultivating the super high-yielding new variety; and to strengthening the protection of China's wild-rice genetic resources, it is significant that resources advantage is become economic advantages.
Summary of the invention
The purpose of this invention is to provide a kind of method and specific molecular mark of cultivating high-yield rice.
The specific molecular mark of cultivation high-yield rice provided by the present invention, be by following primer to one of obtain through pcr amplification:
1) by SEQ ID № in the sequence table: 1 and SEQ ID №: 2 primers of forming are right, called after lrk1;
2) by SEQ ID № in the sequence table: 3 and SEQ ID №: 4 primers of forming are right, called after nPM4.
In the molecule marker RM279 and the 209kb scope between the Indel12 of described specific molecular mark on Jiangxi Dongxiang Wild Rice the 2nd the short arm of a chromosome, and in this scope, navigate to a QTL that can improve rice yield, called after qGY2-1 experimental results show that the introgression line that has this QTL also can increase production 5-12% than super high-yielding kind osmanthus towards No. 2.
Second purpose of the present invention provides a kind of method of cultivating high-yield rice.
The method of cultivation high-yield rice provided by the present invention, be to make male parent, need the kind of improvement to make hybridization of female parent with the kind of QTL with the described molecule marker of claim 1, cross-fertilize seed is backcrossed with need improving the breed, from the kind of backcrossing that obtains, filter out the individual plant that contains described QTL, improve the breed with need again and backcross at least 2 times, last selfing obtains the rice varieties of high yield; Wherein, from the kind of backcrossing, filter out the method for the individual plant that contains described QTL, be to be template with oryza sativa genomic dna to be measured, use by SEQ ID № in the sequence table: 1 and SEQ ID №: 2 primers of forming are right, carry out pcr amplification, detect the band whether the 812bp size is arranged in the amplified production, be template with oryza sativa genomic dna to be measured simultaneously, use by SEQ ID № in the sequence table: 3 and SEQ ID №: 4 primers of forming are right, carry out pcr amplification, detect the band whether the 1178bp size is arranged in the amplified production.
When lrk1 and nPM4 being screened,, contain the gene that can improve rice yield in the paddy rice to be measured as the band of 812bp and 1178bp size is arranged respectively in the amplified production with primer.
The reaction system of pcr amplification can be: oryza sativa genomic dna template 20ng, Taq enzyme 0.5U, 2.0 μ l, 10 * PCR damping fluid (100mM TrisCl pH9.0,500mM KCl, 15mM Mg
2+, 1%Triton X-100), 100 μ MdNTPs, forward primer 0.2 μ M, reverse primer 0.2 μ M is with DEPC water postreaction system to 20 μ l.
The PCR reaction conditions can be: 94 ℃ of 30sec of elder generation, 60 ℃ of 30sec, 72 ℃ of 1min, totally 35 circulations; 72 ℃ of 10min again.
Can detect the band whether 812bp and 1178bp size are arranged in the amplified production by 1% agarose gel electrophoresis.
The molecule marker that the present invention develops in the Dongxiang Wild Rice QTL Fine Mapping of Jiangxi can be used for differentiating whether have the gene that can improve rice yield in common wild-rice, the cultivated rice, realizes the selection to the high-yield rice plant.Cultivate new high-yielding rice varieties with method of the present invention and have simple to operately, with low cost, the advantage that the cycle is short is suitable for applying.And, can shortening the breeding cycle, improve breeding efficiency in generation selection early because of not being subjected to other genetic effect and Effect of Environmental.The present invention is significant in the breed improvement of paddy rice.
The present invention will be further described below in conjunction with drawings and the specific embodiments.
Description of drawings
Fig. 1 is template for Dongxiang Wild Rice and osmanthus towards No. 2 genomic dna, respectively at the banding pattern of primer to the pcr amplification product under the guiding of lrk1 and nPM4 (molecule marker)
Fig. 2 be primer under the nPM4 guiding to the qualification result of some kinds
Fig. 3 be primer under the lrk1 guiding to the qualification result of some kinds
The phenotype of the individual plant of Fig. 4 is super high-yielding kind " osmanthus is towards No. 2 " and transformation QTL qGY2-1
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
Embodiment 1, discovery and the location that can improve rice yield QTL qGY2-1
At first Dongxiang, Jiangxi common wild-rice and super high-yielding kind " osmanthus is towards No. 2 " are hybridized and backcross, made up respectively with osmanthus towards being for No. 2 that the height of genetic background is for backcross population and wild-rice gene introgression line, utilize the SSR mark to adopt Mapmanage QTX software near the 2nd karyomit(e) RM233A of Jiangxi common wild-rice again, find 1 high yield QTL (Li DJ that can make osmanthus towards No. 2 output raisings, Sun CQ, Fu YC, Chen L, Zhu ZF, Li C, Cai HW, Wang XK.Identification and mapping of genes for improving yieldfrom Chinese common wild rice (O.rufipogon Griff.) using advanced backcrossQTL analysis.Chinese Science Bulletin, 2002,18:1533-1537).On this basis, selection contains the high yield introgression line of this QTL section, backcross towards No. 2 with osmanthus, structure is at the near isogenic line of this QTL section, utilization iterates localization method, this QTL is carried out Fine Mapping, be located in the molecule marker RM279 and the 209kb scope between the Indel12 on Jiangxi Dongxiang Wild Rice the 2nd the short arm of a chromosome, with this QTL called after qGY2-1, field cultivation experimental results show that the plant of carrying this high yield gene is 5-30% than the plant amount of increase in production of this gene not, and the introgression line that has this QTL also can increase production 5-12% than super high-yielding kind osmanthus towards No. 2.The inquiry of rice genome sequence is carried out in the interval of this 209kb, the result shows that this interval contains 8 class plant sulfo-kinetin receptor proteins (LRR-Kinase) gene, this genoid has been proved to be in Radix Dauci Sativae has function (the Yoshikatsu M.Mari O.Akiko M.Youji S.An LRR receptor kinase involved inperception of peptide plant hormone.Science that promotes hyperplasia, 2002,296:1470-1472).
Embodiment 2, the screening and the application that can improve rice yield QTL qGY2-1
1, design of primers and banding pattern analysis
2) and nPM4 (SEQ ID № in the sequence table: 3 and SEQ ID №: 4) the Japanese fine genome sequence of the QTL qGY2-1 region that obtains according to embodiment 1 utilizes DNAsist software design PCR primer to lrk1 (SEQ ID № in the sequence table: 1 and SEQ ID №:.
Concrete grammar is: is template with Dongxiang Wild Rice and osmanthus towards No. 2 genomic dna, respectively under the guiding of primer to lrk1 and nPM4, carry out pcr amplification, the PCR reaction system is: oryza sativa genomic dna template 20ng, Taq enzyme 0.5U, 2.0 μ l 10 * PCR damping fluid (100mM TrisCl pH9.0,500mM KCl, 15mM Mg
2+, 1%Triton X-100), 100 μ MdNTPs, forward primer 0.2 μ M, reverse primer 0.2 μ M is with DEPC water postreaction system to 20 μ l.The PCR reaction conditions is: 94 ℃ of 30sec of elder generation, 60 ℃ of 30sec, 72 ℃ of 1min, totally 35 circulations; 72 ℃ of 10min again.Reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis to be detected, detected result as shown in Figure 1, lrk1 can amplify the fragment of 812bp in Dongxiang Wild Rice, and this fragment osmanthus towards No. 2 in the disappearance, nPM4 can amplify the fragment of 1178bp in Dongxiang Wild Rice, the fragment that amplifies in towards No. 2 in osmanthus has only 609bp.In conjunction with genotype and phenotype analytical, confirm this two mark and this QTL qGY2-1 be divided into from.Therefore can be according to whether there being the band of 812bp and 1178bp size that rice varieties is screened in the product that carries out pcr amplification with aforesaid method simultaneously.
2, can improve the screening of rice yield QTL qGY2-1 and the cultivation of high-yield rice
Utilize specific molecular mark of the present invention that collected rice varieties is carried out QTL (qGY2-1) screening, the kind that contains qGY2-1 that screens is made donor parents, backcross after the target variety hybridization of needs improvement with not containing this QTL, utilize specific molecular mark of the present invention to carry out assisted Selection, this QTL transformation is gone to the target variety that needs improvement.
Concrete steps are: at first utilize specific molecular mark of the present invention, method with step 1, rice varieties to the kind of the good plant type of need improvement and the various different sourcess collected is identified, contain in the collected kind of result and need the kind of improvement not contain this QTL, show and to carry out the transformation improvement.Next, the kind that contains QTL (qGY2-1) that filters out with need improve the breed by breeding time length plant successively, mix up the florescence.At full heading time, make male parent, need the kind of improvement to make hybridization of female parent with the kind that contains QTL (qGY2-1), the cross-fertilize seed that is obtained is improved the breed with need and backcrosses, and is called BC
1F
1, utilize molecule marker of the present invention at BC
1F
1Filter out the individual plant that contains this QTL in the colony and improve the breed with need again and backcross, so circulation several times, last selfing once, it is higher and other economical character and the strain system that need improve the breed similar can be promoted after this strain system stablizes to select output.
Screening and the utilization that utilizes specific examples to demonstrate to improve rice yield QTL qGY2-1 with regard to aforesaid method below
The collection kind is as follows: K17A, K17B, golden 23A, golden 23B, special blue or green, bright extensive 63, the Lu is extensive 17, Dongxiang Wild Rice, Wang Nan 25, and osmanthus is towards No. 2, E32,8015s, middle work 8923, middle work 123, middle work 9017, H2W44, Y22s, 377-60, C418, the safe A in Guangdong, win safe No. 1, Japan fine, examine rich No. 11, early clear 005, town 88, middle round-grained rice 83-D.
Extract the genomic dna of above-mentioned different rice varieties respectively,, under the guiding of primer, carry out pcr amplification respectively lrk1 and nPM4 with the method for step 1.Reaction finishes, and the PCR product is carried out 1% agarose gel electrophoresis detect, and (swimming lane A is K17A to detected result among Fig. 2 as shown in Figures 2 and 3, swimming lane B is K17B, and swimming lane C is golden 23A, and swimming lane D is golden 23B, swimming lane E is special blue or green, and swimming lane F is bright extensive 63, and swimming lane G is Lu extensive 17, swimming lane H is a Dongxiang Wild Rice, and swimming lane I is Wang Nan 25, and swimming lane J is that osmanthus is towards No. 2, swimming lane K is E32, swimming lane L is 8015s, and swimming lane M is middle work 8923, and swimming lane N is middle work 123, swimming lane O is middle work 9017, swimming lane P is H2W44, and swimming lane Q is Y22s, and swimming lane R is 377-60, swimming lane S is C418, swimming lane T is the safe A in Guangdong, and swimming lane U is for winning safe No. 1, and swimming lane V is that Japan is fine; Swimming lane A is for examining rich No. 11 among Fig. 3, swimming lane B is morning clear 005, swimming lane C is town 88, swimming lane D is middle round-grained rice 83-D, swimming lane E-V is identical with kind among Fig. 2), recover special blue or green, bright extensive 63, Lu extensive 17 of system and excellent sterile and be the safe A in 8015s, Y22s and Guangdong under the guiding of primer to lrk1 and nPM4, can amplify the fragment of 812bp and 1178bp respectively, have the genotype identical, show and contain the QTL qGY2-1 that can improve rice yield in these rice varieties with Dongxiang Wild Rice.
Oneself knows contains in the common wild-rice of Dongxiang and does not contain QTL qGY2-1 in the rice variety " osmanthus is towards No. 2 ", Dongxiang common wild-rice and " osmanthus is towards No. 2 " are hybridized and backcross, utilizing molecule marker of the present invention to carry out assisted Selection, is the BC of donor at the Dongxiang common wild-rice
4F
5Choose one in the colony and contain QTL qGY2-1 and other background is the strain of " osmanthus is towards No. 2 " system, this strain system is similar to the economical character of " osmanthus is towards No. 2 ", but growing way is more prosperous, and output is higher, and its phenotype as shown in Figure 4.
Sequence table
<160>4
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
tcgaggttgg?agatgctggt?t 21
<210>2
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
aaatgatctg?aattggtgtt?cgg 23
<210>3
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
atttcatcgt?tggcttatca?gtc 23
<210>4
<211>22
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
aatcgttcta?tgcgcaaagg?tt 22
Claims (5)
1. specific molecular mark of cultivating high-yield rice, be by following primer to one of obtain through pcr amplification:
1) by SEQ ID № in the sequence table: 1 and SEQ ID №: 2 primers of forming are right;
2) by SEQ ID № in the sequence table: 3 and SEQ ID №: 4 primers of forming are right.
2. method of cultivating high-yield rice, be to make male parent, need the kind of improvement to make hybridization of female parent with the kind of QTL with the described molecule marker of claim 1, cross-fertilize seed is backcrossed with need improving the breed, from the kind of backcrossing that obtains, filter out the individual plant that contains described QTL, improve the breed with need again and backcross at least 2 times, last selfing obtains the rice varieties of high yield; Wherein, from the kind of backcrossing, filter out the method for the individual plant that contains described QTL, be to be template with oryza sativa genomic dna to be measured, use by SEQ ID № in the sequence table: 1 and SEQ ID №: 2 primers of forming are right, carry out pcr amplification, and the band of 812bp size is arranged in the amplified production, be template with oryza sativa genomic dna to be measured simultaneously, use by SEQ ID № in the sequence table: 3 and SEQ ID №: 4 primers of forming are right, carry out pcr amplification, and the individual plant of the band of 1178bp size is arranged in the amplified production.
3. method according to claim 2, it is characterized in that: the reaction system of described pcr amplification is: oryza sativa genomic dna template 20ng, Taq enzyme 0.5U, 2.0 μ l 10 * PCR damping fluid, 100 μ M dNTPs, forward primer 0.2 μ M, reverse primer 0.2 μ M is with DEPC water postreaction system to 20 μ l.
4. according to claim 2 or 3 described methods, it is characterized in that: described PCR reaction conditions is: 94 ℃ of 30sec of elder generation, 60 ℃ of 30sec, 72 ℃ of 1min, totally 35 circulations; 72 ℃ of 10min again.
5. according to claim 2 or 3 described methods, it is characterized in that: the method for described detection amplified production is 1% agarose gel electrophoresis.
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| CN106893728B (en) * | 2015-12-18 | 2020-12-18 | 中国种子集团有限公司 | Recombinant nucleic acid fragment RecCR033208 and detection method thereof |
| CN105684889A (en) * | 2016-03-14 | 2016-06-22 | 四川省农业科学院生物技术核技术研究所 | Orange-red hull rice breeding material obtained through rapid transformation with molecular markers and transformation method of breeding material |
| CN105671057A (en) * | 2016-03-18 | 2016-06-15 | 浙江大学 | Application of gene OsTPPC in regulating total phosphorus and phytic acid levels in rice kernels |
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| CN1475106A (en) * | 2002-08-14 | 2004-02-18 | 华中农业大学 | Method of quickly improving paddy rice quality |
| CN1566360A (en) * | 2003-07-02 | 2005-01-19 | 中国科学院上海生命科学研究院 | Species molecular fingerprint technique based on rice super gene family |
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| CN1475106A (en) * | 2002-08-14 | 2004-02-18 | 华中农业大学 | Method of quickly improving paddy rice quality |
| CN1566360A (en) * | 2003-07-02 | 2005-01-19 | 中国科学院上海生命科学研究院 | Species molecular fingerprint technique based on rice super gene family |
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