CN109055395A

CN109055395A - A kind of photoperiod insensitiveness Hd1 allele and its molecular labeling and application

Info

Publication number: CN109055395A
Application number: CN201811041527.3A
Authority: CN
Inventors: 孙立亭; 林添资; 龚红兵; 景德道; 杨军; 余波; 钱华飞; 曾生元; 李闯; 姚维成; 杜灿灿; 胡庆峰; 巫章平; 周义文
Original assignee: Zhenjiang Institute of Agricultural Sciences Jiangsu Hilly Area
Current assignee: Zhenjiang Institute of Agricultural Sciences Jiangsu Hilly Area
Priority date: 2018-09-07
Filing date: 2018-09-07
Publication date: 2018-12-21
Anticipated expiration: 2038-09-07
Also published as: CN109055395B

Abstract

The present invention discloses a kind of photoperiod insensitiveness Hd1 allele and its molecular labeling and application.Photoperiod insensitiveness strain town round-grained rice 2400 (♀) is hybridized with photoperiod-sensitive kind Jiahe 218 (♂) and obtains F₂The genotype of single plant and corresponding F_2:3Genetic linkage analysis is carried out whether the photoperiod insensitiveness phenotype separation of family, by photoperiod at the heading stage insensitiveness assignment of genes gene mapping of town round-grained rice 2400 in the 6th article of chromosome, between label RM276 and I6-5, the detection of WGS- variant sites and gene sequencing analysis are carried out to town round-grained rice 2400 and Jiahe 218, it is found that there are the insertions of 123bp at first exon 517bp of Hd1 allele of town round-grained rice 2400；And molecular labeling H1 is developed whether to detect in town round-grained rice 2400 and its derivative strain containing photoperiod insensitiveness gene according to this difference, the photonasty of japonica rice variety can be improved, the Regional suitability and breeding efficiency of japonica rice variety are greatly improved.

Description

A kind of photoperiod insensitiveness Hd1 allele and its molecular labeling and application

Technical field

The invention belongs to molecular breeding field more particularly to a kind of photoperiod at heading stage insensitiveness Hd1 allele and draw The application of object.

Background technique

Heading stage is the important character of rice, determines area and the seasonal adaptation of rice varieties；It is short that rice belongs to high temperature Sunshine crop, kind heading stage determine by the response to temperature, photonasty and basic nutrition of kind, the photoperiod It (Photoperiod) is the essential condition adjusted growth period duration of rice.Japonica rice is the higher subspecies of rice evolution degree, relative to Xian Rice subspecies, the heading stage of most of japonica rice varieties show stronger photoperiod sensitivity, i.e., introduce a fine variety under different ecological condition, plant The nutrient growth and reproductive growth of strain show largely to change, and Other Main Agronomic Characters change, and such as plant height is broadcast and gone through together Phase, effective fringe, total grain panicle number etc., finally influence yield.Popularization of the kind in planted in different ecological areas, it is necessary to first Introduction And Trial, emphatically Investigate the regional adaptability of important agronomy and economic characters.Japonica rice is since photonasty is stronger, and the success rate of Introduction And Trial is lower, table Reveal relatively narrow adaptation planting area.

Paddy rice cross breeding breeding is through F₂After sufficient genetic recombination, it is necessary to the stabilization through the excessive generation is general to use 1 year Two seasons, Hainan are main south numerous added-generation bases.Japonica rice is due to there is stronger photonasty, and breeding material adds generation in Hainan, usually Show heading stage in advance, increment become smaller and total grain panicle number reduce, in the target single plant that Hainan is chosen, to adapt to targeted promotion The Thermo-Photo sensitive of the plantation of positive season in region, with certain blindness, breeding efficiency is not high；Meanwhile the kind of incubation is often only There is relatively narrow Production Zones Suitable, limits the utilization and extention in different ecological section.Therefore screening, initiative and breeding photoperiod at heading stage Insensitiveness germplasm or kind, to different ecological section introduce a fine variety application and eurytopicity breeding has great importance.

The site Hd1 is the response site at Photoperiod heading stage, and cDNA overall length 1557bp includes two exons, Coded product includes 395 amino acid, and containing Zinc finger domain, the CO family gene with arabidopsis with Zinc finger domain is same Source.Allele Hd1 from Kasalath is insensitive to the photoperiod, and the 2nd exon of Kasalath allele has one The missing of 2bp shows photoperiod insensitiveness so as to cause the forfeiture of Hd1 protein function.If according to this difference site development function mark Note, the segment amplified only have the difference of 2bp, the close inconvenient differentiation of band；If being developed according to the adjacent domain in this difference site The accuracy rate of linked marker, selection is not high.Therefore, it is necessary to find other excellent variations of Hd1 gene, and develop efficient function Large-scale application can be marked in molecular breeding.

Summary of the invention

The present invention be directed to the deficiency on breeding time breeding bottleneck and existing molecular labeling, to town rice series rice varieties into Go MNU (methyl-nitroso-urea) mutagenesis, the material after mutagenesis is investigated the neat fringe date and broadcast in Jiangsu and Hainan two places goes through the phase together, A photoperiod at heading stage insensitiveness strain town round-grained rice 2400 (deposit number is CCTCC NO:P201810) is screened, and rice product are provided It is 2400 photoperiod of town round-grained rice insensitiveness Hd1 allele and its molecule labelling method.

The purpose of the present invention can be achieved through the following technical solutions:

The present invention provides a kind of photoperiod insensitiveness Hd1 allele, encode the amino acid as shown in SEQ ID NO.2.

Specifically, the photoperiod insensitiveness Hd1 allelic nucleotide sequence is as shown in SEQ ID NO.1；Or and SEQ Sequence shown in ID NO.1 has 90% or more homology and encodes the protein of identical function.

The photoperiod insensitiveness Hd1 allele is isolated in rice strain town round-grained rice 2400.The rice strain town The complete name of round-grained rice 2400 is grass family Oryza japonica rice town round-grained rice 2400 (Oryza saliva L.Japonica 2400), classification Status is grass family Oryza, has been preserved in China typical culture collection center, and deposit number is CCTCC NO:P201810, is protected The hiding date is on January 17th, 2018, and preservation address is Wuhan, China Wuhan University, postcode 430072.

The present invention also provides a kind of photoperiod insensitiveness GAP-associated protein GAPs, and amino acid sequence is as shown in SEQ ID NO.2.

The present invention also provides the molecular labelings of the photoperiod insensitiveness Hd1 allele described in one kind, to include Hd1 base The segment for the 123bp sequence being inserted at first exon 517bp of cause.The molecular labeling is located at the inside of gene, Bu Huifa It is estranged high from, detection accuracy.By detecting whether to sentence containing 123bp sequence (the SEQ ID NO.5) segment being inserted into It is disconnected whether to contain the photoperiod insensitiveness Hd1 allele.

Invention further provides the Markers for Detection primers of the photoperiod insensitiveness Hd1 allele described in one kind, positive The base sequence of primer is as shown in SEQ ID NO.3, and the base sequence of reverse primer is as shown in SEQ ID NO.4.There are rice Amplified band when strain town round-grained rice 2400 photoperiod insensitiveness Hd1 allele is 456bp, and there are the Hd1 allele in Jiahe 218 When amplified band be 333bp.The DNA for being detected rice material is expanded with molecular labeling primer, if only with molecular labeling primer The segment of 456bp can be amplified, then there are 2400 photoperiod of town round-grained rice insensitiveness Hd1 allele for rice material, and are homozygote, Show as photoperiod insensitiveness；If the primer of 333bp can only be amplified, round-grained rice 2400 insensitiveness Hd1 allele in town is not present, and is Photoperiod-sensitive Hd1 gene pure, shows as photoperiod-sensitive；If the segment of 456bp and 333bp can be amplified simultaneously, Illustrate to be heterozygote, shows as photoperiod-sensitive.

The molecule labelling method of the screening rice strain town round-grained rice 2400 photoperiod insensitiveness Hd1 allele, comprising such as Lower step:

(1) informative population and family phenotypic evaluation: being female parent with town round-grained rice 2400, and photoperiod-sensitive kind Jiahe 218 is father This, hybridization F₁, construct the F comprising 247 parts of single plants₂Segregating population, each F₂Single plant selects method selfing to obtain accordingly by mixed F_2:3Family is divided to two parts, a F_2:3It is planted in Jiangsu, a F_2:3It is planted in Hainan, selection breeding time does not separate and broadcasts to be gone through together The corresponding F of the small family of phase luffing₂Single plant investigates the neat fringe date as extremists, and calculating broadcasts that go through the phase together (here from sowing The number of days of fringe), carry out phenotypic evaluation；

(2) extremists of photoperiod insensitiveness are chosen: according to F_2:3The phenotypic evaluation of family as a result, choose broadcasts that go through the phase together steady Fixed (luffing is small) and heading stage neat F_2:3F corresponding to family₂Single plant (target gene homozygous), with SDS method extract parent, The F of selection₂The DNA of single plant carries out two parents using simple sequence repeat marker SSR and insertion and deletion label InDel label Polymorphism screening, PCR amplification, amplified production carry out electrophoretic analysis on 8% polyacrylamide gel, there is polymorphic draw between parent Object is in F₂It is analyzed in single plant, obtains F₂Single plant genotype data；

(3) linkage analysis and genetic map is constructed: the list using MAPMARKER/EXP3.0 software to each molecular labeling Pnca gene type and corresponding family phenotype carry out linkage analysis；According to chain exchange rule, F is utilized₂The building of single plant genotype data The genetic map of rice；

(4) detection of WGS- variant sites and gene sequencing analysis: by the photoperiod insensitiveness gene of rice strain town round-grained rice 2400 It is positioned at the 6th article of chromosome, between molecular labeling RM276 and I6-5, WGS- variation is carried out to town round-grained rice 2400 and Jiahe 218 Site primer and gene sequencing analysis find that there are 123bp's at first exon 517bp of the Hd1 gene of town round-grained rice 2400 Insertion；

(5) H1, detection function labeling development and verifying: are marked according to the Hd1 difference site design function in town round-grained rice 2400 Whether mentioned significantly containing photoperiod insensitiveness gene to improve the photonasty of japonica rice variety in town round-grained rice 2400 and its derivative strain The Regional suitability and breeding efficiency of high japonica rice variety.

Invention further provides the molecular mark detection methods of the photoperiod insensitiveness Hd1 allele described in one kind, comprising:

(1) according to the nucleotide sequence design primer of the molecular labeling of the photoperiod insensitiveness Hd1 allele；

(2) PCR amplification is carried out as template using the genomic DNA for being detected rice；

(3) judge in pcr amplification product with the presence or absence of the molecular labeling.

Invention further provides the molecular labelings of the photoperiod insensitiveness Hd1 allele in assisted selection In application.The breeding is breeding photoperiod insensitiveness rice material or kind.The breeding can for biology can also be with For the method for molecular biology.

Invention further provides carrier, cell or hosts comprising the photoperiod insensitiveness Hd1 allele.

Invention further provides the photoperiod insensitiveness Hd1 allele or rice strain town round-grained rice 2400 in water Application in rice breeding.The breeding can be cultivation photoperiod insensitiveness or temperature insensitiveness rice varieties.

Invention further provides a kind of rice breeding methods, comprising: by the photoperiod insensitiveness Hd1 allele In Introduced into Rice material.Introduction method can be the means such as transgenosis of molecular biology, can also be using conventional biology Breeding method was waited such as using the rice material such as town round-grained rice 2400 of the Hd1 of insensitiveness containing photoperiod allele using hybridization, selfing Cheng Jinhang breeding.

The germplasm initiative of town round-grained rice 2400 is that positioning, clone and the breeding utilization of photoperiod insensitiveness gene provide the foundation material Material.2400 germplasm of rice strain town provided by the present invention round-grained rice and its photoperiod at heading stage insensitiveness allele site, molecule mark Note, has several advantages that in breeding utilization

(1) excellent variation germplasm can safe utilization.The Hd1 allele that the present invention is obtained by MNU chemical mutagenesis it is excellent Mutation is different, stablizes heredity, and belongs to the acquisition of the technologies such as non-transgenic, and improved variety (being) can be promoted and applied directly, saves and turns The security evaluation program of gene strain.

(2) H1 is the good functional label of polymorphism, and selection is accurate.First exon of the Hd1 gene of town round-grained rice 2400 There are the insertions of 123bp at 517bp, are 456bp there are the amplified band when Hd1 allele of town round-grained rice 2400, there are Jiahe Amplified band is 333bp when 218 Hd1 allele.

(3) gene function stablizes expression, and phenotype is obvious.The Hd1 allele that town round-grained rice 2400 carries, gene function stablize table Reach, phenotype is obvious: broadcasting together that go through the phase relatively stable in Jiangsu and Hainan two places, and broadcast go through year phase together between stablize, be conducive to Carry out breeding utilization and popularization and application in planted in different ecological areas.

(4) selection target is clear, and breeding results are high.The Derivative line that town round-grained rice 2400 is detected by functional label H1, can be pre- The photoperiod sensitivity for surveying rice plant, for rice varieties or the genotype detection of strain, to judge whether the product (being) have There is photoperiod insensitiveness gene, and then quickly breeding goes out photoperiod insensitiveness kind (being) and is applied to production.The application passes through detection light Period insensitiveness gene loci, substantially increases the Regional suitability of breeding japonica rice variety, to improve breeding results.

In addition, there are also unexpected the utility model has the advantages that

(1) 2400 heading stage of town round-grained rice also shows insensitiveness to temperature.Rice is high temperature short day crop, high temperature Accelerate bloom. In Transition In Jurong, Jiangsu positive season, sowing on May 15, in normal year in 2016, town round-grained rice 2400 was in August neat fringe on the 24th, and Jiahe 218 is in 9 Month neat fringe on the 4th；However, town round-grained rice 2400 was in August neat fringe on the 24th, and Jiahe 218 is in September neat fringe on the 12nd the low temperature time in 2015；Phase For Jiahe 218, the heading of town round-grained rice 2400 shows insensitiveness to temperature.Town round-grained rice 2400 and its Derivative line, due to warm Day neutral, difference Between year, different ecological section, heading stage stablize, be conducive to Variety distribution, variety popularization and kind Precise and quantitative cultivation management.

(2) be conducive to the selection of filial generation correlation of attributes minor gene.Due to breeding time light temperature insensitiveness, year at heading stage Between show consistent, the growth conditions being affected to quality responses --- temperature and illumination, also relatively reach unanimity between year, then it is right The minor gene of the relevant optical-and thermal-sensitive of quality important indicator carries out more stablize from generation to generation and selects, and genetic response is big, is conducive to quality The organic polymer of related major gene resistance and minor gene.

Detailed description of the invention

Fig. 1 is the electrophorogram of photoperiod insensitiveness gene genetic linkage analysis；Wherein, M:Marker；1: town round-grained rice 2400；2: Jiahe 218；3,6,9 represent F_2:3Family broadcasts together that going through the phase isolated F occurs₂Single plant；4,5,7,10,11,12,13,22,27 represent F₂ Single plant phenotype and town round-grained rice 2400 are consistent, and corresponding F_2:3Family is without separation；8,14-21,23-26,28-36 represent F₂Single plant table Type and Jiahe 218 are consistent, and corresponding F_2:3Family is without separation；

The Hd1 gene order in the town Tu2Wei round-grained rice 2400 and Jiahe 218 compares analysis result figure；

Fig. 3 is the electrophorogram of functional label H1 amplified production；Wherein, M:Marker；1: town round-grained rice 2400；2: Jiahe 218； Number in electrophoretogram is corresponding with Fig. 1, wherein 37 represent F₂Single plant phenotype and town round-grained rice 2400 are consistent, and corresponding F_2:3Family without Separation.

Specific embodiment

Combined with specific embodiments below, the present invention is furture elucidated, it should be understood that these embodiments are merely to illustrate the present invention Rather than limit the scope of the invention, after the present invention has been read, those skilled in the art are to various equivalences of the invention The modification of form falls within the application range as defined in the appended claims.

The acquisition of 1 photoperiod of embodiment insensitiveness rice mutant material

MNU (methyl nitroso has been carried out to town rice series rice varieties (town rice No. 18, town rice No. 19 and intermediate materials etc.) Urea) mutagenesis, method of mutagenesis is as follows:

(1) single plant that selection has been eared but only top is bloomed on a small quantity, preferably each single plant have 3-4 or so tassels；

(2) the clever flower shears that the plant chosen had opened flower was fallen in field in the first day afternoon, leaves and opens not yet Colored grain husk flower, it is listed to mark；

(3) the next morning allows single plant voluntarily to bloom, and is bloomed after finishing to afternoon, wipes out the grain husk flower not yet bloomed, only The grain husk flower for staying the same day to bloom, while trimming the tiller that do not ear；

(4) listed plant is dug out, guarantee root is complete as far as possible, and water planting saves in flowerpot, morning six By tassel bubble hour in the MNU solution of 1mM, not fracture point tassel, while guarantee dark treatment, try not to contact Illumination；

The tassel that processing is taken out after (5) 1 hours is rinsed with water the clean rear field for being selected in and setting that plants and carries out self-fertility；

(6) remaining MNU solution is added after a small amount of NaOH is stirred to be exposed to the sun after 12 hours under the sun and is outwelled.

Material after mutagenesis is investigated the neat fringe date and broadcast in Jiangsu and Hainan two places goes through the phase together, screens a heading stage light Period insensitiveness strain, is named as town round-grained rice 2400.

Town round-grained rice 2400 is 101 days in neat go through phase (number of days for sowing here fringe) of broadcasting of the positive season plantation in Jiangsu, in Hainan in Winter Plantation broadcast together go through the phase be 105 days, luffing 4 days；Photoperiod-sensitive kind Jiahe 218 is control, the positive season plantation in Jiangsu Broadcast together go through the phase be 112 days, Hainan in Winter plantation broadcast together go through the phase be 87 days, 25 days in advance ear；Hainan season of growth is opposite In Jiangsu, positive season is shorter sunshine time, and short-day promotes the effect of heading not significant town round-grained rice 2400.

The complete name of the rice strain town round-grained rice 2400 is 2400 (Oryza saliva of grass family Oryza japonica rice town round-grained rice L.Japonica 2400), classification position is grass family Oryza, has been preserved in China typical culture collection center, and preservation is compiled It number is CCTCC NO:P201810, the deposit date is on January 17th, 2018, preservation address was Wuhan, China Wuhan University, postcode 430072。

The acquisition of 2 photoperiod of embodiment insensitiveness Hd1 allele and the exploitation of molecular labeling

One, the F in 2400/ Jiahe 218 of town round-grained rice₂Informative population and phenotypic evaluation

It (1) is female parent with photoperiod insensitiveness strain town round-grained rice 2400, photoperiod-sensitive kind Jiahe 218 is male parent, is prepared miscellaneous Hand over kind of a F₁, construct the F in 2400/ Jiahe 218 of town round-grained rice₂Segregating population amounts to 247 parts；Each F₂Single plant selects method selfing to obtain by mixed Obtain the F in corresponding 2400/ Jiahe 218 of town round-grained rice_2:3Family.

(2) phenotypic evaluation

Group and family's based material to building plant in kind targeted promotion region and Hainan respectively, investigate Qi Sui Phase calculates phase of going through together of broadcasting (from the number of days for sowing here fringe), carries out phenotypic evaluation, specific as follows:

Rice material 1 year above-mentioned two seasons of plantation, the first season plant in Jiangsu Hilly Ground Zhenjiang Agriculture Science Research Institute It holds or participate in a prayer service at a temple proving ground, sowing on May 15, transplanting on June 15, single seedling plants, conventional fertilizer and water management；The second season plants in Hai Nanling Water test base, sowing on December 20, transplanting on January 20, local routine fertilizer and water management.Record F₂The neat fringe date of single plant, and Calculating, which is broadcast, goes through the phase together, F₂Divide single plant sowing, the F collected₂Seed is divided to two parts, a F_2:3Family is planted in Jiangsu, a F_2:3Family It is planted in Hainan, selection breeding time does not separate and broadcasts goes through the corresponding F of the small family of phase luffing together₂Homozygous single plant is fixed for gene Position.

Two, the F in 2400/ Jiahe 218 of town round-grained rice₂The genetic linkage analysis of group

Genetic linkage analysis is conventional method in that art process, primer involved in assignment of genes gene mapping process be SSR primer and Indel primer, wherein SSR primer has been announced on the net, and the SSR primer numbers used herein are as follows:

RM499

RM323

RM495

RM35

RM472

RM431

RM414

RM14

RM211

RM236

RM5356

RM3501

RM5390

RM322

RM5015

RM3366

RM3131

RM489

RM251

RM563

RM282

RM7117

RM8204

RM570

RM551

RM471

RM6172

RM142

RM1841

RM5473

RM470

RM567

RM6082

RM3969

RM164

RM4244

RM538

RM234

RM249

RM509

RM510

RM276

RM527

RM3827

RM3628

RM2008

RM162

RM275

RM295

RM481

RM125

RM501

RM478

RM429

RM172

RM420

RM6863

RM52

RM32

RM331

RM223

RM458

RM256

RM281

RM296

RM444

RM464

RM219

RM189

RM215

RM245

RM410

RM2504

RM216

RM239

RM333

RM496

RM590

RM591

RM147

RM286

RM332

RM4504

RM3625

RM7391

RM209

RM229

RM206

RM415

RM2935

RM519

RM2972

RM7120

RM7102

RM5479

RM6037

It is as follows for the Indel primer of the assignment of genes gene mapping herein:

(1) routine SDS method extracts parent, F₁And F₂The DNA of homozygous single plant.

(2) polymorphism sieve is carried out to two parents using simple sequence repeat marker SSR and insertion and deletion label Indel label Choosing, the system of PCR amplification are as follows: 1.5 μ l genomic DNAs, 0.5 μ l 2mM upstream primer, 0.5 μ l 2mM downstream primer, 1.2 μ l 10 × Taq Buffer, 0.3 μ l 1mM dNTP, 0.1 μ l 1000U Taq archaeal dna polymerase, ddH₂O complements to 10 μ l.PCR expands The program of increasing are as follows: 95 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, annealing temperature (55 DEG C) annealing 30s, 72 DEG C of extension 40s, operation 33 circulations；Last 72 DEG C of extensions 10min.Amplified production is detected with 8% polyacrylamide gel electrophoresis, with DNA Marker (I DNA ladder of 100bp-) is compareed as molecular weight, electrophoresis 1 hour under 240V constant pressure.Silver staining display DNA item Band, using there is the lamp box of fluorescent lamp to be observed.Record is as a result, there is polymorphic primer in F between parent₂Divided in homozygous single plant Analysis obtains F₂The genotype data of homozygous single plant；

(3) linkage analysis and genetic map is constructed: the list using MAPMARKER/EXP3.0 software to each molecular labeling Pnca gene type and corresponding family phenotype carry out linkage analysis；According to chain exchange rule, F is utilized₂The building of single plant genotype data The genetic map of rice.

Three, the detection of WGS- variant sites and gene sequencing analysis:

The DNA of town round-grained rice 2400 and Jiahe 218 is extracted, carries out the detection of full-length genome variant sites (by Beijing source Nuo Hezhi section The detection of skill limited liability company), find discrepant candidate gene in positioning section；Then expand discrepant candidate gene into Row sequencing, further verifies difference site.Discrepant candidate gene amplimer is as follows: (segmentation amplification)

Z504-1F:CCACAAGAGCCATGCGAGGTAG

Z504-1R:CATACTCATTTCATCTCATCACTGCTC

Z504-2F:GAGCCATGCGAGGTAGAGGAACAGG

Z504-2R:CATCTCATCACTGCTCTTTGCTTACTTC

Z504-1157-1F:CTACTGTCTAACAAAATGAAGGGATAAATAAAC

Z504-1367-1R:CAATTAGTGAGTTAGCATGGATCGTAT

Z504-1700-1R:GGCATATCTATCACCGTGCTGTCTG

Z504-877-1R:CACTCGCTCCCTTCCTTCTC

Four, result and analysis

Town round-grained rice 2400 broadcasts that go through the phase together be 101 days Jiangsu, broadcasts that go through the phase together be 105 days in Hainan, therefore Hainan is short Sunshine does not promote its Blooming, shows that round-grained rice 2400 strain in town shows photoperiod insensitiveness；And the phase is gone through together in broadcasting for Jiangsu in Jiahe 218 Be 112 days, Hainan broadcast together go through the phase be 87 days, show Jiahe 218 be photoperiod-sensitive kind.

The F that town round-grained rice 2400 and Jiahe 218 are prepared₁Group shows photoperiod-sensitive, illustrates 2400 photoperiod of town round-grained rice insensitiveness Gene shows as recessiveness.From above-mentioned F₂Chosen in group with consistent homozygous 10 plants of the single plant of 2400 phenotype of town round-grained rice, while choose with 10 plants of the consistent single plant of 218 phenotype of Jiahe, the polymorphism mark for carrying out 12 chromosomes of distribution carries out linkage analysis, in conjunction with phenotype Data and F₂The verifying of Day neutral and photaesthesia single plant is showed in group, finally contaminates the photoperiod insensitiveness assignment of genes gene mapping in the 6th article Colour solid, between molecular labeling RM276 and I6-5 (Fig. 1).

In conjunction with positioning result, the detection of full-length genome variant sites and gene sequencing point are carried out to town round-grained rice 2400 and Jiahe 218 Analysis finds there be 123bp (SEQ ID NO.5) at first exon 517bp of the Hd1 gene in town round-grained rice 2400

(GCAGCAACAACGGCATGTATTTTGGTGAAGTCGATGAGTACTTTGATCTTGTCAGGTACAATTCGTACTACGACAA CAACAATAACGACAACAGCAACAGCAACAGCAGCAACAACGACAACG insertion (Fig. 2)), 2400 photoperiod of town round-grained rice are blunt Feel the nucleotide sequence (SEQ ID NO.1) of Hd1 allele are as follows:

ATGAATTATAATTTTGGTGGCAACGTGTTCGACCAGGAGGTTGGAGTTGGAGGCGAAGGAGGAGGAGGA GGAGAGGGGAGCGGCTTCCCATGGGCGCGCCCGTGCGACGGCTGCCGCGCGGCGCCGAGCGTGGTGTACTGCCGCGC GGACGCGGCGTACCTGTGCGCGTCGTGCGACGCGCGGGTGCACGCGGCCAACCGCGTGGCGTCCCGCCACGAGCGCG TGCGGGTGTGCGAGGCCTGCGAGCAAGCCCCGGCCGCGCTCGCGTGCCGCGCCGACGCCGCCGCGCTGTGCGTGGCG TGCGACGTGCAGGTGCACTCCGCGAACCCGCTCGCCAGGCGCCACCAGCGCGTCCCCGTCGCGCCGCTCCCGGCCAT CACCATCCCGGCCACCTCCGTCCTCGCTGAGGCGGTGGTGGCCACCGCCACCGTCCTCGGCGGCAAGGACGAGGAGG TGGACTCTTGGATTATCCTCTCCAAAGATTCCAACAACAACAACAACAATAACAACAGCAACAGCAGCAACAACGGC ATGTATTTTGGTGAAGTCGATGAGTACTTTGATCTTGTCAGGTACAATTCGTACTACGACAACAACAATAACGACAA CAGCAACAGCAACAGCAGCAACAACGACAACGACAACGACAACGACAATAACAACAACAGCAACAGCAGCAACAACG GCATGTATTTTGGTGAAGTCGATGAGTACTTTGATCTTGTCGGGTACAATTCGTACTACGACAACCGCATCGAAAAC AACCAAGATCAGCAGTATGGGATGCATGAACAGCAAGAGCAGCAGCAGCAGCAGCAGGAGATGCAAAAGGAGTTTGC AGAGAAGGAAGGGAGCGAGTGTGTGGTACCTTCACAGATCACAATGCTGAGTGAGCAGCAGCATAGTGGTTATGGAG TTGTGGGAGCAGACCAGGCCGCCTCCATGACCGCCGGCGTCAGTGCTTACACAGATTCCATCAGCAACAGCGTGAGT TCATCTATTACTAGCTGCAACTATTTTTTTTTCAGAGAATGAACATCTATTACTGTTGTTAGTTAGTTGTTACTACA TGCCACGTTGTCAATGTTTTAGAGTTCATACTAGTACTTTTTAGTGGAAAAACATTCTCCAAACAAAAGCTACTGTC TAACAAAATGAAGGGATAAATAAACAGATCTCAACAAGAAAATAAAGATACTTTTCTACTTCCAAGCTGCGATCTTT AGGCTGATTAAATGGAACCGATAAAAAAAATACTTTAAAGAAAAGTACACAATTGATCTTTAGGCAGACCAGTTGAC TAGTTCCTGTATTTCTAAGCATATACCATCCATGCTAACTCACTAATTGAAAAGAAGTGAGTTTGTTAACCTTTTAT GTACACAGCAATCACCACACGAAAGACCTCATGAAAAGTAGGATAAGTGTAAGTGTAATTCATATTTTATCCCAGTG CATAAATTTAAAATATCTTACTTTTGCGACAGTAAAAAAGATATTGGAAGTTTTTCTTATGTATGTAAAATTAAATT AAGCCCATCTATATATCATTGCAGGGTCTCTGACACCTGCAATCTCCTTATGATTCGCATATTTCAGTGACCATTTG CCGATTCCATCTCAGATATCTCTCATCAATGGAGGCGGGTATAGTACCAGACAGCACGGTGATAGATATGCCAAATT CCAGCATCCTGACACCTGCTGGAGCAATCAATCTCTTCTCAGGTCCCTCGCTTCAGATGTCCCTTCACTTCAGCTCC ATGGACAGGGAGGCCAGGGTGCTCAGGTACAGGGAGAAGAAGAAGGCCAGGAAGTTTGAGAAGACAATACGTTATGA AACAAGAAAGGCGTATGCAGAGGCACGACCCCGGATCAAGGGCCGTTTCGCCAAGAGATCAGATGTGCAGATCGAAG TGGACCAGATGTTCTCCACTGCAGCTCTATCTGACAGTAGCTATGGTACTGTTCCATGGTTCTGATGGGACTCATGA GACGCTATCTTATAGGCATATATATGGGGACTTACTGAGTAGCAATAACATCGATCCAGTGGGAGTAGTTCTAGACA ATCTGTGTTATGAATAA

The amino acid sequence (SEQ ID NO.2) of 2400 photoperiod of town round-grained rice insensitiveness Hd1 are as follows:

MNYNFGGNVFDQEVGVGGEGGGGGEGSGCPWARPCDGCRAAPSVVYCRADAAYLCASCDARVHAANRVA SRHERVRVCEACEHAPAALACRADAAALCVACDVQVHSANPLARRHQRVPVAPLPAITIPATSVLAEAVVATATVLG GKDEEVDSWIILSKDSDNNNNNNNSNSSNNGMYFGEVDEYFDLVGYNSYYDNRIENNQDQQYGMHEQQEQQQQQQEM QKEFAEKEGSECVVPSQITMLSEQQHSGYGVVGADQAASMTAGVSAYTDSISNSISLINGGGYSTRQHGDRYAKFQH PDTCWSNQSLLRSLASDVPSLQLHGQGGQGAQVQGEEEGQEV

Molecular labeling H1 is designed according to this difference site (123bp insertion), using the labeled primer (H1 primer) to above-mentioned The Rice Population and family's based material of building are identified that the rice material of discovery performance photoperiod insensitiveness can amplify one The segment of 456bp, and the rice material for showing photoperiod-sensitive can amplify the segment of 333bp, or expand amplification simultaneously The segment of 456bp and 333bp, partial results are as shown in Figure 3 out.

The mutation for absolutely proving Hd1 gene above is the reason of leading to 2400 photoperiod of town round-grained rice insensitiveness, for mutated gene The insertion of the 123bp of (2400 photoperiod of town round-grained rice insensitiveness Hd1 allele), devises molecular labeling, with molecular labeling H1 primer The DNA of SEQ ID NO.1/SEQ ID NO.2 amplifying rice photoperiod insensitiveness kind or breeding material, if with molecular labeling H1 Primer is merely able to amplify the segment of 456bp, then rice varieties or breeding material town round-grained rice 2400 photoperiod insensitiveness Hd1 allele In the presence of, and be homozygote, show as photoperiod insensitiveness；If the primer of 333bp can only be amplified, 2400 insensitiveness Hd1 of town round-grained rice Allele is not present, and is photoperiod-sensitive Hd1 gene pure, shows as photoperiod-sensitive；If can expand while expand The segment of 456bp and 333bp out illustrates to show as photoperiod-sensitive for heterozygote.(Fig. 3).

H1 primer

Positive sequence SEQIDNO.3:CCTCTCCAAAGATTCC

Reverse sequence SEQIDNO.4:GCTCCCACAACTCCATA

Town round-grained rice 2400 and its spin-off are detected by the molecular labeling with above-mentioned photoperiod insensitiveness Hd1 allele site Whether contain photoperiod insensitiveness gene in system, to improve the photonasty of japonica rice variety, the region for greatly improving japonica rice variety is suitable Answering property and ripe phase breeding efficiency.

Sequence table

<110>Jiangsu Hilly Ground Zhenjiang Agriculture Science Research Institute

<120>a kind of photoperiod insensitiveness Hd1 allele and its molecular labeling and application

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2088

<212> DNA

<213>rice (Oryza sativa)

<400> 1

atgaattata attttggtgg caacgtgttc gaccaggagg ttggagttgg aggcgaagga 60

ggaggaggag gagaggggag cggcttccca tgggcgcgcc cgtgcgacgg ctgccgcgcg 120

gcgccgagcg tggtgtactg ccgcgcggac gcggcgtacc tgtgcgcgtc gtgcgacgcg 180

cgggtgcacg cggccaaccg cgtggcgtcc cgccacgagc gcgtgcgggt gtgcgaggcc 240

tgcgagcaag ccccggccgc gctcgcgtgc cgcgccgacg ccgccgcgct gtgcgtggcg 300

tgcgacgtgc aggtgcactc cgcgaacccg ctcgccaggc gccaccagcg cgtccccgtc 360

gcgccgctcc cggccatcac catcccggcc acctccgtcc tcgctgaggc ggtggtggcc 420

accgccaccg tcctcggcgg caaggacgag gaggtggact cttggattat cctctccaaa 480

gattccaaca acaacaacaa caataacaac agcaacagca gcaacaacgg catgtatttt 540

ggtgaagtcg atgagtactt tgatcttgtc aggtacaatt cgtactacga caacaacaat 600

aacgacaaca gcaacagcaa cagcagcaac aacgacaacg acaacgacaa cgacaataac 660

aacaacagca acagcagcaa caacggcatg tattttggtg aagtcgatga gtactttgat 720

cttgtcgggt acaattcgta ctacgacaac cgcatcgaaa acaaccaaga tcagcagtat 780

gggatgcatg aacagcaaga gcagcagcag cagcagcagg agatgcaaaa ggagtttgca 840

gagaaggaag ggagcgagtg tgtggtacct tcacagatca caatgctgag tgagcagcag 900

catagtggtt atggagttgt gggagcagac caggccgcct ccatgaccgc cggcgtcagt 960

gcttacacag attccatcag caacagcgtg agttcatcta ttactagctg caactatttt 1020

tttttcagag aatgaacatc tattactgtt gttagttagt tgttactaca tgccacgttg 1080

tcaatgtttt agagttcata ctagtacttt ttagtggaaa aacattctcc aaacaaaagc 1140

tactgtctaa caaaatgaag ggataaataa acagatctca acaagaaaat aaagatactt 1200

ttctacttcc aagctgcgat ctttaggctg attaaatgga accgataaaa aaaatacttt 1260

aaagaaaagt acacaattga tctttaggca gaccagttga ctagttcctg tatttctaag 1320

catataccat ccatgctaac tcactaattg aaaagaagtg agtttgttaa ccttttatgt 1380

acacagcaat caccacacga aagacctcat gaaaagtagg ataagtgtaa gtgtaattca 1440

tattttatcc cagtgcataa atttaaaata tcttactttt gcgacagtaa aaaagatatt 1500

ggaagttttt cttatgtatg taaaattaaa ttaagcccat ctatatatca ttgcagggtc 1560

tctgacacct gcaatctcct tatgattcgc atatttcagt gaccatttgc cgattccatc 1620

tcagatatct ctcatcaatg gaggcgggta tagtaccaga cagcacggtg atagatatgc 1680

caaattccag catcctgaca cctgctggag caatcaatct cttctcaggt ccctcgcttc 1740

agatgtccct tcacttcagc tccatggaca gggaggccag ggtgctcagg tacagggaga 1800

agaagaaggc caggaagttt gagaagacaa tacgttatga aacaagaaag gcgtatgcag 1860

aggcacgacc ccggatcaag ggccgtttcg ccaagagatc agatgtgcag atcgaagtgg 1920

accagatgtt ctccactgca gctctatctg acagtagcta tggtactgtt ccatggttct 1980

gatgggactc atgagacgct atcttatagg catatatatg gggacttact gagtagcaat 2040

aacatcgatc cagtgggagt agttctagac aatctgtgtt atgaataa 2088

<210> 2

<211> 342

<212> PRT

<213>rice (Oryza sativa)

<400> 2

Met Asn Tyr Asn Phe Gly Gly Asn Val Phe Asp Gln Glu Val Gly Val

1 5 10 15

Gly Gly Glu Gly Gly Gly Gly Gly Glu Gly Ser Gly Cys Pro Trp Ala

20 25 30

Arg Pro Cys Asp Gly Cys Arg Ala Ala Pro Ser Val Val Tyr Cys Arg

35 40 45

Ala Asp Ala Ala Tyr Leu Cys Ala Ser Cys Asp Ala Arg Val His Ala

50 55 60

Ala Asn Arg Val Ala Ser Arg His Glu Arg Val Arg Val Cys Glu Ala

65 70 75 80

Cys Glu His Ala Pro Ala Ala Leu Ala Cys Arg Ala Asp Ala Ala Ala

85 90 95

Leu Cys Val Ala Cys Asp Val Gln Val His Ser Ala Asn Pro Leu Ala

100 105 110

Arg Arg His Gln Arg Val Pro Val Ala Pro Leu Pro Ala Ile Thr Ile

115 120 125

Pro Ala Thr Ser Val Leu Ala Glu Ala Val Val Ala Thr Ala Thr Val

130 135 140

Leu Gly Gly Lys Asp Glu Glu Val Asp Ser Trp Ile Ile Leu Ser Lys

145 150 155 160

Asp Ser Asp Asn Asn Asn Asn Asn Asn Asn Ser Asn Ser Ser Asn Asn

165 170 175

Gly Met Tyr Phe Gly Glu Val Asp Glu Tyr Phe Asp Leu Val Gly Tyr

180 185 190

Asn Ser Tyr Tyr Asp Asn Arg Ile Glu Asn Asn Gln Asp Gln Gln Tyr

195 200 205

Gly Met His Glu Gln Gln Glu Gln Gln Gln Gln Gln Gln Glu Met Gln

210 215 220

Lys Glu Phe Ala Glu Lys Glu Gly Ser Glu Cys Val Val Pro Ser Gln

225 230 235 240

Ile Thr Met Leu Ser Glu Gln Gln His Ser Gly Tyr Gly Val Val Gly

245 250 255

Ala Asp Gln Ala Ala Ser Met Thr Ala Gly Val Ser Ala Tyr Thr Asp

260 265 270

Ser Ile Ser Asn Ser Ile Ser Leu Ile Asn Gly Gly Gly Tyr Ser Thr

275 280 285

Arg Gln His Gly Asp Arg Tyr Ala Lys Phe Gln His Pro Asp Thr Cys

290 295 300

Trp Ser Asn Gln Ser Leu Leu Arg Ser Leu Ala Ser Asp Val Pro Ser

305 310 315 320

Leu Gln Leu His Gly Gln Gly Gly Gln Gly Ala Gln Val Gln Gly Glu

325 330 335

Glu Glu Gly Gln Glu Val

340

<210> 3

<211> 16

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

cctctccaaa gattcc 16

<210> 4

<211> 17

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gctcccacaa ctccata 17

<210> 5

<211> 123

<212> DNA

<213>rice (Oryza sativa)

<400> 5

gcagcaacaa cggcatgtat tttggtgaag tcgatgagta ctttgatctt gtcaggtaca 60

attcgtacta cgacaacaac aataacgaca acagcaacag caacagcagc aacaacgaca 120

acg 123

Claims

1. a kind of photoperiod insensitiveness Hd1 allele, which is characterized in that coding amino acid as shown in SEQ ID NO.2.

2. photoperiod insensitiveness Hd1 allele according to claim 1, which is characterized in that nucleotide sequence such as SEQ ID Shown in NO.1；Or there is 90% or more homology with sequence shown in SEQ ID NO.1 and encode the protein of identical function.

3. a kind of photoperiod insensitiveness GAP-associated protein GAP, which is characterized in that amino acid sequence is as shown in SEQ ID NO.2.

4. the molecular labeling of photoperiod insensitiveness Hd1 allele according to claim 1, which is characterized in that its be comprising The segment for the 123bp sequence being inserted at first exon 517bp of Hd1 gene.

5. the Markers for Detection primer of photoperiod insensitiveness Hd1 allele according to claim 4, which is characterized in that The base sequence of forward primer is as shown in SEQ ID NO.3, and the base sequence of reverse primer is as shown in SEQ ID NO.4.

6. the molecular mark detection method of photoperiod insensitiveness Hd1 allele according to claim 4, which is characterized in that Include:

(1) the nucleotide sequence design of the molecular labeling of photoperiod insensitiveness Hd1 allele according to claim 4 is drawn Object；

7. molecular labeling the answering in assisted selection of photoperiod insensitiveness Hd1 allele according to claim 4 With.

8. including carrier, cell or the host of photoperiod insensitiveness Hd1 allele as claimed in claim 1 or 2.

9. photoperiod insensitiveness Hd1 allele according to claim 1 or 2 or rice strain town round-grained rice 2400 are educated in rice Application in kind.

10. a kind of rice breeding method characterized by comprising by photoperiod insensitiveness Hd1 equipotential of any of claims 1 or 2 In channel genes rice material.