CN106191046A - A kind of construction method of wild rice CSSL population - Google Patents
A kind of construction method of wild rice CSSL population Download PDFInfo
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Abstract
The invention discloses the construction method of a kind of wild rice CSSL population (CSSL).The present invention constructs the substitution line colony that can cover whole chromogene group, and the genetic background of substitution line is the most clear, for some substitution line, its genetic background is roughly the same with receptor parent, only there are differences with donor parents at certain known mark section, and for a complete set of substitution line, the chromosomal fragment of all wild rices has corresponding labelling section, and avoids repetition and disappearance.The wild rice CSSL population that the present invention builds the most only is set up a platform comprehensively carrying out wild rice genomics research and is provided basis, also provides the most favourable help for Rice Genomics research, Innovation Germplasm resource.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the construction method of a kind of wild rice CSSL population.
Background technology
Common wild-rice (Oryza.rufipogon Griff.) is ancestors' kind of Asian Cultivated Rice (O.sativa L.),
It is the important germ plasm resource of rice varieties improvement.Have been found that wild rice has Resistant, degeneration-resistant, high yield, high-quality, phosphorus efficiency profit
With valuable genes such as, sterile and recoveries.Therefore, from wild rice, excavate the excellent base lost in cultivated rice or weakened
Cause, finds and utilizes new beneficial gene resource to become the new breakthrough mouth of rice varieties improvement.
Many important economical characters such as crop yield, quality, resistance, belong to complicated quantitative inheritance character more.By
Complicated in its genetic background, utilize molecular marker analysis by genetical population, the degree of accuracy of QTL location is the highest, it is also difficult to QTL
Carry out Effect Estimation and reliable marker assisted selection accurately, more can not realize the separating clone of QTL.Chromosome segment is replaced
System (CSSL, chromosome segment substitution line), is with same parent as genetic background, has replaced confession
The colony that the serial strain of body parent one or minority chromosome segment is formed, utilizes this colony without considering other genomes
The hereditary effect in region, can be greatly improved the accuracy to complicated agronomic trait gene location.Further, it is also possible to it is fixed in primary
On the basis of Wei, by utilizing the CSSL population containing target QTL and background parent to set up F2Secondary segregating population and
Its highdensity genetic map, can be resolved into QTL site single Mendelian factor, and then be separated by map-based cloning
Target gene.
Beneficial gene in wild rice germplasm resources to be utilized, first has to that it is carried out genetic analysis and gene mapping is ground
Study carefully, provide hereditary information for exogenous gene transfer and utilization, thus select and gene gram for carrying out marker assisted selection further
Grand lay the first stone.But wild rice comprehensive agronomy character is poor, the unfavorable gene frequency of occurrences is high and the most chain with beneficial gene, and these are all
Limit being directly accurately positioned and utilizing wild rice excellent genes, by means of the cultivated rice platform of known group sequence,
Set up the CSSL population that a set of many generations with wild rice as donor parents backcross, make the genetic background of cultivated rice only take
Band portion wild rice chromosome segment or gene, be a kind of quick side accurately excavating, position, clone wild rice excellent genes
Method.
The existing wild rice substitution line having been built up and introgressive line have a lot, and main method is to utilize continuous backcross
The method combined with molecule assisted Selection.As: Tian utilizes 126 SSR molecular marker to construct 156 introgression lines (IL),
Tan utilize 179 SSR molecular marker construct 120 IL, Hirabayashi et al. utilize japonica rice Japan fine 3 sets that construct ooze
Entering and be, donor parents is 2 wild seed rice.But it is excessive that the shortcoming of introgression line is introduced into fragment, colony's chromogene group is covered
Lid not exclusively, to next step QTL to be finely positioned gene cloning efficiency the highest.Furuta et al. utilizes 149 SNP molecules
Labelling constructs 33 wild rice chromosomes partially solely substitution line, but this small group is insufficient to the genomics to wild rice and carries out
Comprehensively research and analyse.
Summary of the invention
It is an object of the present invention to provide a kind of primer set for building wild rice CSSL population.
What the present invention provided is used for building the primer set of wild rice CSSL population by primer sets 1 and primer sets
2 compositions；
Described primer sets 1 by 181 primers to forming；
Described primer sets 2 by 100 primers to forming；
1-primer is formed 181 by described 181 primers to by primer, and primer is to the 1-primer nucleotide sequence to 181
As shown in table 1；
Described 100 primers to by described primer to 1, described primer to 3, described primer to 5, described primer to 7, described
Primer to 9, described primer to 11, described primer to 13, described primer to 15, described primer to 17, described primer to 19, described
Primer to 21, described primer to 23, described primer to 25, described primer to 27, described primer to 29, described primer to 31, institute
State primer to 33, described primer to 35, described primer to 37, described primer to 39, described primer to 41, described primer to 43,
Described primer to 45, described primer to 47, described primer to 49, described primer to 51, described primer to 53, described primer pair
55, described primer to 57, described primer to 59, described primer to 60, described primer to 61, described primer to 62, described primer
To 64, described primer to 66, described primer to 68, described primer to 70, described primer to 71, described primer to 72, described in draw
Thing to 74, described primer to 76, described primer to 78, described primer to 79, described primer to 81, described primer to 83, described
Primer to 85, described primer to 87, described primer to 89, described primer to 91, described primer to 93, described primer to 96, institute
State primer to 98, described primer to 99, described primer to 101, described primer to 105, described primer to 106, described primer pair
108, described primer to 112, described primer to 114, described primer to 116, described primer to 118, described primer to 120, institute
State primer to 122, described primer to 124, described primer to 126, described primer to 128, described primer to 130, described primer
To 132, described primer to 134, described primer to 136, described primer to 137, described primer to 138, described primer to 139,
Described primer to 141, described primer to 142, described primer to 144, described primer to 145, described primer to 146, described in draw
Thing to 148, described primer to 150, described primer to 152, described primer to 153, described primer to 154, described primer pair
156, described primer to 157, described primer to 159, described primer to 160, described primer to 162, described primer to 164, institute
State primer to 165, described primer to 166, described primer to 168, described primer to 170, described primer to 172, described primer
To 173, described primer to 174, described primer to 175, described primer to 176, described primer to 178 and described primer to 180
Composition.
It is a further object to provide the complete PCR reagent for building wild rice CSSL population or
Test kit.
What the present invention provided is used for building the complete PCR reagent of wild rice CSSL population by PCR reagent 1 He
PCR reagent 2 forms；
Described PCR reagent 1 includes described 181 primers pair；
Described PCR reagent 2 includes described 100 primers pair.
It is a still further object of the present invention to provide the new use of above-mentioned primer set or above-mentioned complete PCR reagent or test kit
On the way.
The invention provides above-mentioned primer set or above-mentioned complete PCR reagent or test kit and build wild rice chromosome sheet
Application in section substitution line.
It is a still further object of the present invention to provide the construction method of a kind of wild rice CSSL population.
The construction method of the wild rice CSSL population that the present invention provides comprises the steps:
(1) with cultivated rice as nonrecurrent parent, with wild rice as recurrent parent, backcross three times, obtain BC3F1For colony；
(2) by above-mentioned primer sets 2 to described BC3F1Screen for colony, obtain each primer pair in described primer sets 2
Representative strains, the representative strains that selection is obtained composition colony be denoted as BC3F1-A is for colony；
The representative strains of described each primer pair is the individual plant meeting following condition: this primer is to the mark on corresponding genome
Note fragment is consistent with in described wild rice in this representative strains；
(3) by described BC3F1-A carries out with described cultivated rice the 4th time backcrossing for the individual plant of colony respectively, obtains BC4F1Generation
Colony；
(4) with each primer in above-mentioned primer sets 1 to described BC4F1Screen for colony, select to meet following bar
The individual plant of part is as target individual plant: this individual plant only has 1 to or 2 to or the 3 pairs of primers to corresponding labeled fragment with in described open country
In raw rice consistent, and in primer sets 1, remaining primer is consistent with in described cultivated rice to labeled fragment corresponding in this individual plant；
The colony being made up of target individual plant is denoted as BC4F1-A for colony, i.e. for the purpose of member in wild rice CSSL population.
In said method, described step also comprises the steps () in (4): by described BC4F1For non-targeted list in colony
The residue individual plant of strain is denoted as BC4F1-B is for colony；By described BC4F1-B, for the step of individual plant repetition (3)-(4) of colony, obtains
Target individual plant, the colony being made up of target individual plant is as the member in purpose wild rice CSSL population；
Described BC4F1-A also includes the step of selfing for the individual plant of colony；The number of times of described selfing is 3-6 time, described selfing
Every generation in screening containing the corresponding primer individual plant to corresponding labeled fragment.
In said method,
The method of described step (2) comprises the steps:
A1) described BC is arbitrarily chosen3F1For three individual plants in colony, it is denoted as individual plant X, individual plant Y and individual plant Z respectively；Arbitrarily
Choose a certain primer pair in described primer sets 2, be denoted as primer to M；
A2) use described primer that described individual plant X, described individual plant Y and described individual plant Z are identified by M respectively, until
To the primer representative strains to M；
A3) the like, until obtaining the representative strains of each primer pair in described primer sets 2, and draw described in all
The representative strains of each primer pair in thing group 2 is denoted as BC3F1-A is for colony；
The method of described step (4) comprises the steps:
B1) use each primer in described primer sets 1 to identify to the individual plant in described colony O, select to meet
The individual plant of following condition is as the primer target individual plant to M: in this individual plant except described primer to labeled fragment corresponding for M with
In described wild rice consistent beyond, other primers of primer sets 1 be centering to many containing 2 pairs of primers to corresponding labeled fragment with in institute
State in wild rice consistent, and in primer sets 1, remaining primer is to labeled fragment corresponding in this individual plant and in described cultivated rice
Unanimously；
Described colony O is that the representative strains of M is backcrossed by described primer with described cultivated rice, the progeny population obtained；
B2) the like, to described BC4F1Identify for other individual plants in colony, until obtaining described primer sets 2
In the target individual plant of each primer pair, and the target individual plant of each primer pair in all described primer sets 2 is denoted as
BC4F1-A is for individual plant.
In said method, described b1) in, described other primers of primer sets 1 are centering to many marks containing 2 pairs of primers to correspondence
Note fragment consistent with described wild rice be following N1) N2) or N3):
N1) other primer centerings of primer sets 1 do not have primer consistent with in described wild rice to corresponding labeled fragment；
N2) other primer centerings of primer sets 1 contain only 1 pair of primer to corresponding labeled fragment and in described wild rice one
Cause；
N3) other primer centerings of primer sets 1 contain only 2 pairs of primers to corresponding labeled fragment and in described wild rice one
Cause.
In said method,
Described cultivated rice is cultivated rice 9311；
Described wild rice is common wild-rice.
It is a still further object of the present invention to provide the wild rice CSSL population that said method prepares.
Above-mentioned wild rice CSSL population is falling within the protection of the present invention as the application in NIL
Scope.
Final object of the present invention is to provide above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box or above-mentioned
Method or the new application of above-mentioned wild rice CSSL population.
The invention provides above-mentioned primer set or above-mentioned PCR reagent or mentioned reagent box or said method or above-mentioned wild
Rice CSSL population is at following c1) or c2) in application:
C1) quantitative trait locus location；
C2) gene clone.
The advantage of the construction method of the present invention is as follows:
1, the present invention is on the basis of CSSL population construction method used by forefathers, is improved, at BC3F1Generation
Just proceeding by molecular marker assisted selection (MAS, marker-assisted selection), early stage expands heredity with hybridization
Based on colony.BC3F1In generation, selects abundant individual plant so that colony be enough to cover the chromogene group that wild rice is whole.
BC4F1In generation, detects the genetic background of all individual plants, and this step workload is huge, but, based on this, the route of structure is
Very clear, hereafter no matter field backcrosses or selfing, and workload has narrowed down to minimum.
2, the CSSL population colony that the present invention builds, covers donor parents wild rice the most to greatest extent
Chromogene group, up to 100%, though in building process occur molecular marker lose produce room (GAP), also can review
Previous generation material, selects individual plant continue selfing or backcross, makes up GAP.
3, the CSSL population colony that the present invention builds, the displacement fragment contained by each strain is averagely less than 3
Individual, each displacement fragment length is less than 5cM.Most only one of which displacement fragments, i.e. single slice substitution line, can wait base as near
Because of be (NIL, near isogenic line) utilize, substantially increase next step carry out quantitative trait locus (QTL) location and
The efficiency of gene clone.
4, current, that the present invention builds wild rice CSSL population, no matter group size (about 200), dye
(contained by the most each strain, displacement fragment is many for clip size contained by colour solid genome coverage rate, single strain, heredity noise
Few), the better than substitution line colony constructed by forefathers.Can efficiently utilize and carry out wild rice QTL location and favorable genes clone.
The present invention constructs the substitution line colony that can cover whole chromogene group, and the genetic background of substitution line is non-
Often clear, for some substitution line, its genetic background is roughly the same with receptor parent, only at certain known mark section and
Donor parents there are differences, and for a complete set of substitution line, the chromosome segment of all wild rices has corresponding labelling
Section, and avoid repetition and disappearance.The wild rice CSSL population that the present invention builds the most only sets up one entirely
Face carries out the platform of wild rice genomics research provides basis, also for Rice Genomics research, Innovating Rice germ plasm resource
Provide the most favourable help.
Accompanying drawing explanation
Fig. 1 is the structure flow chart of wild rice CSSL population.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Quantitative test in following embodiment, is respectively provided with three times and repeats experiment, results averaged.
The rice restorer kind that female parent material cultivated rice 9311 is large-scale promotion in following embodiment, is order-checking product
Kind, its whole genome sequence can be downloaded from website and obtain: http://www.ricedata.cn/ (country's Oryza sativa L. number
According to center, storehouse), can be provided free by country rice germplasm storehouse, as business breeding purposes, need to national genebank (garden) with
And germ plasm resource collector signs sharing mutual interests agreement.
Male parent material common wild-rice in following embodiment can be free by wild rice germplasm resources national genebank (garden)
There is provided, as business breeding purposes, sharing mutual interests agreement need to be signed with national genebank (garden) and germ plasm resource collector.
Embodiment 1, the qualification of polymorphism SSR molecular marker and screening
1, select to be distributed in 780 pairs of SSR primers of 12 chromosomes, 175 pairs of InDel primers, be used for screening structure displacement
Primer used by system, all of primer genetic distance (unit: cM cM) comes from website http: //
www.gramene.org。
2, list of references " Eshed Y, Zamir D:A genomic library of Lycopersicon
pennellii in L.esculentum:a tool for fine mapping of genes.Euphytica,1994,79
(3) in: 175-179. ", 369 pairs of SSR primers detect there is polymorphism, finally between parents' (wild rice and cultivated rice 9311)
Choose and be uniformly distributed in 119 pairs of SSR primers of 12 chromosomes and 62 pairs of InDel primer totally 181 pairs of primers for building displacement
System.The information of 181 pairs of primers and sequence are as shown in table 1, the genetic distance average out to 5.7cM between every two primers.
1,119 pairs of SSR primers of table and 62 pairs of InDel primer information
Embodiment 2, the construction method of wild rice CSSL population
The construction method of the wild rice CSSL population of the present invention is to utilize continuous backcross to combine MAS, from BC3 generation
Proceeding by Markers for Detection, carry out Selective backcross according to the distribution of heterologous DNA fragments, in continuous tracing detection paramount generation, returns
Hand over offspring, set up the CSSL population of wild rice full-length genome series.The wild rice chromosome segment displacement of the present invention
System builds flow chart as it is shown in figure 1, the construction method of wild rice CSSL population of the present invention is specific as follows:
1, with cultivated rice 9311 for maternal, common wild-rice is male parent, hybridizes, obtains F1For seed.
2, by F1After planting for seed, and the field test hybrid true and false (due to cultivated rice as female parent, wild rice and cultivation
The hybrid field phenotype of rice differs markedly from cultivated rice, self progeny and filial generation and is easily discriminated in field).Choose wherein
One strain hybridization F1For plant, it is backcrossed with cultivated rice 9311, obtains BC1F1For seed.
3, by BC1F1Plant for seed simple grain, obtain about 200 BC1F1For individual plant, every strain respectively with cultivated rice 9311
Backcrossing, every plant averagely gathers in the crops 10 BC2F1For seed, results selfed seed preserves simultaneously；By BC2F1Plant for seed simple grain,
Obtain about 2000 BC2F1For individual plant；Each plant selects 1-2 BC2F1Individual plant backcrosses with cultivated rice 9311 the most respectively,
To BC3F1For seed；By BC3F1Plant for seed simple grain, obtain 1000 BC3F1For individual plant.
4, from BC3F1In generation, starts, and selects the individual plant of certain molecular marker wild rice genotype, selfing or backcross, lower Dai Zhi
Strain is still screened with this molecular marker, i.e. chooses the individual plant of this molecular marker wild rice genotype, selfing or backcross, directly
Stable to strain.Specifically comprise the following steps that
(1) the method detection BC that single labelling is followed the trail of3F1For plant genotype
The method using single labelling to follow the trail of, randomly selects 100 primers being uniformly distributed on 12 chromosomes to (by drawing
Thing to 1, primer to 3, primer to 5, primer to 7, primer to 9, primer to 11, primer to 13, primer to 15, primer to 17, draw
Thing to 19, primer to 21, primer to 23, primer to 25, primer to 27, primer to 29, primer to 31, primer to 33, primer pair
35, primer to 37, primer to 39, primer to 41, primer to 43, primer to 45, primer to 47, primer to 49, primer to 51, draw
Thing to 53, primer to 55, primer to 57, primer to 59, primer to 60, primer to 61, primer to 62, primer to 64, primer pair
66, primer to 68, primer to 70, primer to 71, primer to 72, primer to 74, primer to 76, primer to 78, primer to 79, draw
Thing to 81, primer to 83, primer to 85, primer to 87, primer to 89, primer to 91, primer to 93, primer to 96, primer pair
98, primer to 99, primer to 101, primer to 105, primer to 106, primer to 108, primer to 112, primer to 114, primer
To 116, primer to 118, primer to 120, primer to 122, primer to 124, primer to 126, primer to 128, primer to 130,
Primer to 132, primer to 134, primer to 136, primer to 137, primer to 138, primer to 139, primer to 141, primer pair
142, primer to 144, primer to 145, primer to 146, primer to 148, primer to 150, primer to 152, primer to 153, draw
Thing to 154, primer to 156, primer to 157, primer to 159, primer to 160, primer to 162, primer to 164, primer pair
165, primer to 166, primer to 168, primer to 170, primer to 172, primer to 173, primer to 174, primer to 175, draw
Thing to 176, primer to 178 and primer to 180 compositions) carry out molecular marker assisted selection (MAS), come what detecting step 3 obtained
1000 BC3F1For the genotype of individual plant, choose the BC of the wild-type fragment corresponding containing every a pair SSR primer respectively3F1Dai Dan
Strain.
Illustrate with a pair SSR primer of certain in 100 pairs of SSR primers: by certain a pair SSR primer named SSR primer
A, uses SSR primer A respectively to any 3 BC3F1Genome for individual plant (being respectively designated as plant 1, plant 2 and plant 3)
DNA carries out PCR amplification, detects pcr amplification product respectively, if SSR primer A to the amplification of plant 2 with SSR primer A to open country
The amplification of raw rice is identical, chooses plant 2, and plant 2 is the individual plant of the wild rice fragment corresponding containing SSR primer A, gives up
Plant 1 and plant 3.
In this way, obtain the individual plant of the wild rice fragment that every a pair SSR primer is corresponding in remaining 100 pairs of SSR primer,
Final from 1000 BC3F1Have chosen 236 BC altogether for individual plant3F1For individual plant.By 236 BC3F1For individual plant respectively with cultivated rice
9311 backcross, and obtain BC4F1For seed, simple grain is planted, is obtained 376 BC4F1For individual plant.
(2) the method detection BC that single labelling is followed the trail of4F1For plant population genotype (genetic background)
Use whole 181 pairs of SSR primers to carry out molecular marker assisted selection (MAS), detect 376 BC4F1For individual plant base
Because of type, and then determine the genetic background of all substitution line individual plants.Later generation individual plant, utilizes the genetic background information obtained,
In addition to detecting the labeled fragment except following the trail of, whether other wild rice fragment displaces, and i.e. selects the individual plant that heredity noise is few, continues
Continue and backcross or selfing.
Illustrate with the individual plant of the wild rice fragment corresponding containing SSR primer A: be respectively adopted 181 pairs of SSR primers to containing
The individual plant having wild rice fragment corresponding for SSR primer A carries out PCR amplification, detects pcr amplification product,
If this individual plant is in addition to the wild rice fragment corresponding containing SSR primer A, possibly together with the open country that other SSR primers are corresponding
The number of raw rice fragment is not more than 2, then by this individual plant selfing, and selfing always to BC4F4Or BC4F5, until stable, in many generations, are certainly
Combining MAS in friendship, in the plant of future generation of selfing, still screening is containing wild rice fragment corresponding for SSR primer A and variable rate technology
Stable plant；
If this individual plant is in addition to the wild rice fragment corresponding containing SSR primer A, containing corresponding wild of other SSR primers
The number more than two of rice fragment, then backcross this individual plant with cultivated rice 9311, is returned to BC always5F1Or BC6F1Or BC7F1, directly
The number containing wild rice fragment corresponding to other SSR primers to this individual plant is less equal than 2, according still further to said method many generations
Selfing combines MAS, and in selfing and the plant of future generation that backcrosses, still screening is containing wild rice fragment corresponding for SSR primer A and field
Between show stable plant.
Per generation is often plantation 20-30 strain, selects 20 individual plants to take blade and extracts DNA, and fixed member labelling is followed the trail of.Until being respectively
Material variable rate technology is stable, 200 strains of final acquisition, and whole colony covers whole wild rice chromogene groups.Pass through
Showing with rice sequences comparison and Function detection result, the CSSL population that the present invention obtains can not only be carried out comprehensively
Wild rice genomics research, but also a series of Oryza sativa L. Innovation Germplasms with wild rice excellent genes may be obtained.
Claims (10)
1., for building a primer set for wild rice CSSL population, it is made up of primer sets 1 and primer sets 2；
Described primer sets 1 by 181 primers to forming；
Described primer sets 2 by 100 primers to forming；
1-primer is formed 181 by described 181 primers to by primer；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 1 and sequence 21；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 3 and sequence 42；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 5 and sequence 63；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 7 and sequence 84；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 9 and sequence 10 5；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 11 and sequence 12 6；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 13 and sequence 14 7；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 15 and sequence 16 8；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 17 and sequence 18 9；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 19 and sequence 20 10；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 21 and sequence 22 11；
Described primer is made up of the single strand dna shown in sequence 23 and the single strand dna shown in sequence 24 12；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 25 and sequence 26 13；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 27 and sequence 28 14；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 29 and sequence 30 15；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 31 and sequence 32 16；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 33 and sequence 34 17；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 35 and sequence 36 18；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 37 and sequence 38 19；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 39 and sequence 40 20；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 41 and sequence 42 21；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 43 and sequence 44 22；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 45 and sequence 46 23；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 47 and sequence 48 24；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 49 and sequence 50 25；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 51 and sequence 52 26；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 53 and sequence 54 27；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 55 and sequence 56 28；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 57 and sequence 58 29；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 59 and sequence 60 30；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 61 and sequence 62 31；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 63 and sequence 64 32；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 65 and sequence 66 33；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 67 and sequence 68 34；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 69 and sequence 70 35；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 71 and sequence 72 36；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 73 and sequence 74 37；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 75 and sequence 76 38；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 77 and sequence 78 39；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 79 and sequence 80 40；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 81 and sequence 82 41；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 83 and sequence 84 42；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 85 and sequence 86 43；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 87 and sequence 88 44；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 89 and sequence 90 45；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 91 and sequence 92 46；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 93 and sequence 94 47；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 95 and sequence 96 48；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 97 and sequence 98 49；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 99 and sequence 100 50；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 101 and sequence 102 51；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 103 and sequence 104 52；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 105 and sequence 106 53；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 107 and sequence 108 54；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 109 and sequence 110 55；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 111 and sequence 112 56；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 113 and sequence 114 57；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 115 and sequence 116 58；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 117 and sequence 118 59；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 119 and sequence 120 60；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 121 and sequence 122 61；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 123 and sequence 124 62；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 125 and sequence 126 63；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 127 and sequence 128 64；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 129 and sequence 130 65；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 131 and sequence 132 66；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 133 and sequence 134 67；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 135 and sequence 136 68；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 137 and sequence 138 69；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 139 and sequence 140 70；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 141 and sequence 142 71；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 143 and sequence 144 72；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 145 and sequence 146 73；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 147 and sequence 148 74；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 149 and sequence 150 75；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 151 and sequence 152 76；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 153 and sequence 154 77；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 155 and sequence 156 78；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 157 and sequence 158 79；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 159 and sequence 160 80；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 161 and sequence 162 81；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 163 and sequence 164 82；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 165 and sequence 166 83；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 167 and sequence 168 84；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 169 and sequence 170 85；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 171 and sequence 172 86；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 173 and sequence 174 87；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 175 and sequence 176 88；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 177 and sequence 178 89；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 179 and sequence 180 90；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 181 and sequence 182 91；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 183 and sequence 184 92；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 185 and sequence 186 93；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 187 and sequence 188 94；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 189 and sequence 190 95；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 191 and sequence 192 96；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 193 and sequence 194 97；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 195 and sequence 196 98；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 197 and sequence 198 99；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 199 and sequence 200 100；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 201 and sequence 202 101；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 203 and sequence 204 102；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 205 and sequence 206 103；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 207 and sequence 208 104；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 209 and sequence 210 105；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 211 and sequence 212 106；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 213 and sequence 214 107；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 215 and sequence 216 108；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 217 and sequence 218 109；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 219 and sequence 220 110；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 221 and sequence 222 111；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 223 and sequence 224 112；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 225 and sequence 226 113；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 227 and sequence 228 114；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 229 and sequence 230 115；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 231 and sequence 232 116；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 233 and sequence 234 117；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 235 and sequence 236 118；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 237 and sequence 238 119；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 239 and sequence 24 0 120；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 24 1 and sequence 24 2 121；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 24 3 and sequence 24 4 122；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 24 5 and sequence 24 6 123；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 24 7 and sequence 24 8 124；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 24 9 and sequence 250 125；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 251 and sequence 252 126；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 253 and sequence 254 127；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 255 and sequence 256 128；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 257 and sequence 258 129；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 259 and sequence 260 130；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 261 and sequence 262 131；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 263 and sequence 264 132；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 265 and sequence 266 133；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 267 and sequence 268 134；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 269 and sequence 270 135；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 271 and sequence 272 136；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 273 and sequence 274 137；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 275 and sequence 276 138；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 277 and sequence 278 139；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 279 and sequence 280 140；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 281 and sequence 282 141；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 283 and sequence 284 142；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 285 and sequence 286 143；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 287 and sequence 288 144；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 289 and sequence 290 145；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 291 and sequence 292 146；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 293 and sequence 294 147；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 295 and sequence 296 148；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 297 and sequence 298 149；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 299 and sequence 300 150；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 301 and sequence 302 151；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 303 and sequence 304 152；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 305 and sequence 306 153；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 307 and sequence 308 154；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 309 and sequence 310 155；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 311 and sequence 312 156；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 313 and sequence 314 157；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 315 and sequence 316 158；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 317 and sequence 318 159；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 319 and sequence 320 160；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 321 and sequence 322 161；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 323 and sequence 324 162；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 325 and sequence 326 163；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 327 and sequence 328 164；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 329 and sequence 330 165；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 331 and sequence 332 166；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 333 and sequence 334 167；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 335 and sequence 336 168；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 337 and sequence 338 169；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 339 and sequence 340 170；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 341 and sequence 342 171；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 343 and sequence 344 172；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 345 and sequence 346 173；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 347 and sequence 348 174；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 349 and sequence 350 175；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 351 and sequence 352 176；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 353 and sequence 354 177；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 355 and sequence 356 178；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 357 and sequence 358 179；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 359 and sequence 360 180；
Described primer is made up of the single strand dna shown in the single strand dna shown in sequence 361 and sequence 362 181；
Described 100 primers to by described primer to 1, described primer to 3, described primer to 5, described primer to 7, described primer
To 9, described primer to 11, described primer to 13, described primer to 15, described primer to 17, described primer to 19, described primer
To 21, described primer to 23, described primer to 25, described primer to 27, described primer to 29, described primer to 31, described in draw
Thing to 33, described primer to 35, described primer to 37, described primer to 39, described primer to 41, described primer to 43, described
Primer to 45, described primer to 47, described primer to 49, described primer to 51, described primer to 53, described primer to 55, institute
State primer to 57, described primer to 59, described primer to 60, described primer to 61, described primer to 62, described primer to 64,
Described primer to 66, described primer to 68, described primer to 70, described primer to 71, described primer to 72, described primer pair
74, described primer to 76, described primer to 78, described primer to 79, described primer to 81, described primer to 83, described primer
To 85, described primer to 87, described primer to 89, described primer to 91, described primer to 93, described primer to 96, described in draw
Thing to 98, described primer to 99, described primer to 101, described primer to 105, described primer to 106, described primer to 108,
Described primer to 112, described primer to 114, described primer to 116, described primer to 118, described primer to 120, described in draw
Thing to 122, described primer to 124, described primer to 126, described primer to 128, described primer to 130, described primer pair
132, described primer to 134, described primer to 136, described primer to 137, described primer to 138, described primer to 139, institute
State primer to 141, described primer to 142, described primer to 144, described primer to 145, described primer to 146, described primer
To 148, described primer to 150, described primer to 152, described primer to 153, described primer to 154, described primer to 156,
Described primer to 157, described primer to 159, described primer to 160, described primer to 162, described primer to 164, described in draw
Thing to 165, described primer to 166, described primer to 168, described primer to 170, described primer to 172, described primer pair
173, described primer to 174, described primer to 175, described primer to 176, described primer to 178 and described primer to 180 groups
Become.
2. for building complete PCR reagent or the test kit of wild rice CSSL population,
Described complete PCR reagent is made up of PCR reagent 1 and PCR reagent 2；
Described PCR reagent 1 includes 181 described in claim 1 primer pair；
Described PCR reagent 2 includes 100 described in claim 1 primer pair.
3. the primer set described in claim 1 or the complete PCR reagent described in claim 2 or test kit are building wild rice
Application in CSSL population.
4. a construction method for wild rice CSSL population, comprises the steps:
(1) with cultivated rice as nonrecurrent parent, with wild rice as recurrent parent, backcross three times, obtain BC3F1For colony；
(2) by primer sets 2 described in claim 1 to described BC3F1Screen for colony, obtain in described primer sets 2 each
The representative strains of primer pair, the colony of representative strains selection obtained composition is denoted as BC3F1-A is for colony；
The representative strains of described each primer pair is the individual plant meeting following condition: this primer is to the marker slip on corresponding genome
Section is consistent with in described wild rice in this representative strains；
(3) by described BC3F1-A carries out with described cultivated rice the 4th time backcrossing for the individual plant of colony respectively, obtains BC4F1For colony；
(4) with each primer in primer sets 1 described in claim 1 to described BC4F1Screen for colony, select full
The individual plant of the following condition of foot is as target individual plant: this individual plant only has 1 to or 2 to or the 3 pairs of primers to corresponding labeled fragment with
In described wild rice consistent, and in primer sets 1 remaining primer to labeled fragment corresponding in this individual plant with in described cultivation
In rice unanimously；The colony being made up of target individual plant is denoted as BC4F1-A for colony, i.e. for the purpose of wild rice CSSL population
In member.
Method the most according to claim 4, it is characterised in that: described step also comprises the steps () in (4): by institute
State BC4F1It is denoted as BC for the residue individual plant of non-targeted individual plant in colony4F1-B is for colony；By described BC4F1-B is for the individual plant of colony
Repeating the step of (3)-(4), obtain target individual plant, the colony being made up of target individual plant puts as purpose wild rice chromosome segment
Change the member in being；
Described BC4F1-A also includes the step of selfing for the individual plant of colony；The number of times of described selfing is 3-6 time.
6. according to the method described in claim 4 or 5, it is characterised in that:
The method of described step (2) comprises the steps:
A1) described BC is arbitrarily chosen3F1For three individual plants in colony, it is denoted as individual plant X, individual plant Y and individual plant Z respectively；Arbitrarily choose
A certain primer pair in described primer sets 2, is denoted as primer to M；
A2) use described primer that described individual plant X, described individual plant Y and described individual plant Z are identified, until being drawn by M respectively
The thing representative strains to M；
A3) the like, until obtaining the representative strains of each primer pair in described primer sets 2, and by all described primer sets 2
In the representative strains of each primer pair be denoted as BC3F1-A is for colony；
The method of described step (4) comprises the steps:
B1) use each primer in described primer sets 1 to identify to the individual plant in described colony O, select satisfied as follows
The individual plant of condition is as the primer target individual plant to M: in this individual plant except described primer to labeled fragment corresponding for M with described
In wild rice consistent beyond, other primers of primer sets 1 be centering to many containing 2 pairs of primers to corresponding labeled fragment with in described open country
In raw rice consistent, and in primer sets 1, remaining primer is consistent with in described cultivated rice to labeled fragment corresponding in this individual plant；
Described colony O is that the representative strains of M is backcrossed by described primer with described cultivated rice, the progeny population obtained；
B2) the like, to described BC4F1Identify for other individual plants in colony, until obtaining in described primer sets 2 every
The target individual plant of one primer pair, and the target individual plant of each primer pair in all described primer sets 2 is denoted as BC4F1-A
For individual plant.
7. according to described method arbitrary in claim 4-6, it is characterised in that:
Described cultivated rice is cultivated rice 9311；
Described wild rice is common wild-rice.
8. the wild rice CSSL population prepared according to described method arbitrary in the claims 4-7.
9. the wild rice CSSL population described in claim 8 is as the application in NIL.
10. the primer set described in claim 1 or the PCR reagent described in claim 2 or the test kit described in claim 2
Or the method described in arbitrary in claim 4-7 or the wild rice CSSL population described in claim 8 are at following c1)
Or c2) in application:
C1) quantitative trait locus location；
C2) gene clone.
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| CN201610719097.0A CN106191046B (en) | 2016-08-24 | 2016-08-24 | A kind of construction method of wild rice CSSL population |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107372095A (en) * | 2017-08-10 | 2017-11-24 | 福建省农业科学院水稻研究所 | A kind of method based on CSSL population positioning crop dominant gene |
| US20210363600A1 (en) * | 2020-05-19 | 2021-11-25 | Institute of Food Crops, Hubei Academy of Agricultural Sciences | Primer groups for detecting hybrid rice backbone parent and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773883A (en) * | 2014-02-12 | 2014-05-07 | 中国农业科学院作物科学研究所 | Method and special primer for assisting-culturing excellent-quality and high-yield wheat variety by using molecular marker |
-
2016
- 2016-08-24 CN CN201610719097.0A patent/CN106191046B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103773883A (en) * | 2014-02-12 | 2014-05-07 | 中国农业科学院作物科学研究所 | Method and special primer for assisting-culturing excellent-quality and high-yield wheat variety by using molecular marker |
Non-Patent Citations (2)
| Title |
|---|
| WEIHUA QIAO ET AL: "Development and characterization of chromosome segment substitution lines derived from Oryza rufipogon in the genetic background of O. sativa spp. indica cultivar 9311", 《BMC GENOMICS》 * |
| YUVAL ESHED & DANI ZAMIR: "A genomic library of Lycopersicon pennellii in L. esculentum: A tool for fine mapping of genes", 《EUPHYTICA》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107372095A (en) * | 2017-08-10 | 2017-11-24 | 福建省农业科学院水稻研究所 | A kind of method based on CSSL population positioning crop dominant gene |
| CN107372095B (en) * | 2017-08-10 | 2019-04-16 | 福建省农业科学院水稻研究所 | A method of crop dominant gene is positioned based on CSSL population |
| US20210363600A1 (en) * | 2020-05-19 | 2021-11-25 | Institute of Food Crops, Hubei Academy of Agricultural Sciences | Primer groups for detecting hybrid rice backbone parent and application thereof |
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| Publication number | Publication date |
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| CN106191046B (en) | 2019-05-28 |
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