CN108300801A - The molecular labeling and application that a kind of and rape grain weight and Pod length are closely related - Google Patents
The molecular labeling and application that a kind of and rape grain weight and Pod length are closely related Download PDFInfo
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Abstract
The present invention relates to rapeseed breeding and molecular biology fields, and in particular to the molecular labeling and application that a kind of and rape grain weight and Pod length are closely related.A kind of molecular labeling being closely related with rape grain weight and Pod length, which is characterized in that the molecular labeling is to the molecular labeling of gene BnaA.ARF18a, and nucleotide sequence is as shown in SEQ ID No.1.The molecular labeling solves the weight of grain present in conventional breeding methods and Pod length can only be identified in the later stage, so that the problem that breeding cycle is long, easily affected by environment, efficiency of selection is low, improve the efficiency of rapeseed breeding, the Brassica Napus Seedling phase just can be fast and reliable identification different cultivars rape single plant grain weight and Pod length, rapeseed breeding efficiency is improved, the rapeseed breeding time limit is greatly shortened.
Description
Technical field
The present invention relates to rapeseed breeding and molecular biology fields, and in particular to a kind of tight with rape grain weight and Pod length
Close relevant molecular labeling and application.
Background technology
Cabbage type rape is the important oil crops in China, not only provides edible oil for the mankind, but also can provide richness
Feed containing protein, therefore it is always breeders' primary goal to improve yield of rape.Seed size (weight) and silique are long
Degree is the important component that yield of rape is constituted, and is found that hundreds of weights and Pod length in rape etc. in the latest 20 years
QTL, but these QTL heredity and molecular regulation network it is not clear.
Seed size/weight is considered as one of most important character, product of the seed as higher plant zoogamy,
It is not only the main form of breeding plant and the main source of grain for the survival of mankind.Rape seed is not only
The most important harvest substance of the storage organ of oil and protein, even more rape, research seed size/weight have extremely important
Meaning.(1) the single plant total output of rape mainly weighs three principal elements by single plant silique number, Seed number per pod and grain and constitutes,
Wherein the genetic force highest of grain weight, minimum affected by environment are an important economical traits of rape and yield of rape is in positive
Close (Clarke and Simpson, 1978；Butruille et al., 1999；Shi et al., 2009).(2) seed weight
Have an effect on the characters such as seed oil content and protein content (Morgan et al., 1998；Adamskia et al., 2009；
Lionneton et al., 2004).(3) its germinating power of the big seed of grain is better than small-sized seed, and grown up to by large seed
Its environmental suitability of plant is higher than the plant grown up to by small-sized seed.Therefore, the hereditary basis for illustrating rape Grain filling has
Conducive to the output increased and quality-improving of rape.
Seed size/weight is important one of the yield traits of plant.In angiosperm, ripe seed include embryo,
Endosperm and kind skin, respectively from fertilized eggs, fertilization polar core and integument development.The formation of grain crop weight is cell division to increase
Cell number, cell extend with increase volume and the constantly substantial process of accumulation of photosynthate, content (Egli et al.,
1989；Yu Zhenwen, 1995；Guo Wenshan, 1997；Li Baihang, 1998；Li Na, 2015).In many plants, cotyledon or endosperm
Dry-matter accumulation and cell number and seed size correlation (Yu Zhenwen, 1995；Guo Wenshan, 1997；Li Baihang,
1998；Brocklehurst et al.,1978；Herzog et al.,1982；Guldan et al.,1985；Reddy et
al.,1983；Zhang Zujian, 1998；Wei Lingji, 2004；Li Na, 2015).So far, some signal pathways have been reported logical
Crossing influences the growth of endosperm or parent to control seed size/weight, including：IKU approach, Ubiquitin-Proteasome Pathway, G- eggs
Different signal pathway (the Li such as white signal approach, MAPK signal transduction, plant hormone and transcription factor
et al.,2016)。
In recent years, with the development of molecular marking technique, the QTL positioning of rape mass of 1000 kernel makes great progress.
Quijada etc. (2006) has identified 3 QTLs (N7, N17 and N19) about grain weight in the experiment of 2 years 2: 4 groups,
But neither one QTL can repeat to detect in multiple groups.Udall etc. (2006) is in Hua-DH, SYN-DH and one
6,4 and 5 QTLs are identified in test cross group respectively, and it was found that be located at N14 linkage groups QTL can in different groups and
Stablize heredity in environment.Shi etc. (2009) is reflected using a TNDH group and a RC-F2 group in 10 natural environments altogether
The fixed QTLs to 159 mass of 1000 kernel, but only 4 main effect QTL s and only can be in 10 rings there are one QTL-qSW.A7-2
It can be detected in border.Fan etc. (Fan et al.2010) constructs a DH group and a F2 group for mass of 1000 kernel
QTL is positioned.In the QTLs of 9 mass of 1000 kernel, the QTL-TSWA07a and TSWA07b of two main effects can be repeated in different year
It detects, explains the phenotypic variation of 27.6-37.9%, and the two main effect QTLs can be simultaneously in DH groups and F2 groups
In detect, it was demonstrated that important function of the QTL to mass of 1000 kernel.Yang etc. (2012) is selfed using the recombination containing 186 strains
It is the QTLs that group has navigated to 9 grains weight, wherein be located at the phenotype that a main effect QTL-cqSWA9 of A09 chromosomes is explained
Variation is 28.2%, and the main effect QTL-cqSLA9 of the QTL and control Pod length is located at same section, implys that the section
The gene of control mass of 1000 kernel and Pod length may be contained simultaneously, or have that gene can influence mass of 1000 kernel simultaneously and silique is long
Degree.
Although the QTL of a collection of influence grain weight and Pod length has been identified in current research, about rape
The Functional marker of grain weight and Pod length is less, and does not find to distinguish the label of rape seed height grain weight.
Invention content
The present invention provides a kind of molecular labeling being closely related with rape grain weight and Pod length in order to solve the above problem, should
Molecular labeling solves the weight of grain present in conventional breeding methods and Pod length can only be identified in the later stage so that breeding cycle length,
Problem easily affected by environment, efficiency of selection is low, improves the efficiency of rapeseed breeding.
A kind of molecular labeling being closely related with rape grain weight and Pod length, which is characterized in that the molecular labeling pair
The molecular labeling of gene BnaA.ARF18a, the nucleotide sequence are nucleotide sequence or the sequence shown in SEQ ID No.1
Row are through replacement, missing or increase one or more nucleotide, and coding is same or similar, and amino acid sequence with the same function
The nucleotide sequence of row.
Further, said gene BnaA.ARF18a contains 9 SNP, and initiation codon ATG is defined as+1, wherein 6
SNP be located at+186 ,+193 ,+262 ,+1045 ,+1303 ,+1345,3 SNP of exon region be located at include subregion+
59 ,+931 ,+1429,9 SNP constitute Hap A, Hap B and tri- kinds of haplotypes of Hap C, Hap A types in different single plant DNA
Nucleotide sequence as shown in SEQ ID No.1, the nucleotide sequence of Hap Type Bs as shown in SEQ ID No.2, Hap c-types
Nucleotide sequence is as shown in SEQ ID No.3.
In above three haplotype, its mass of 1000 kernel (3.02 ± 0.29g) of the material containing Hap A and Pod length (5.09
± 0.35cm) all it is minimum, maximum is material (4.05 ± 0.46g containing Hap C；7.37 ± 1.81cm), contain Hap
The material of B is located at by-level (3.44 ± 0.42g；5.38±0.36cm).
Further, the molecular labeling is the primer pair according to SNPs designs different between three kinds of haplotypes.
Further, above-mentioned primer pair is BnaA.ARF18a-186 or/and BnaA.ARF18a-262, primer pair
The nucleotide sequence of the forward primer of BnaA.ARF18a-186 is as shown in SEQ ID No.4, the nucleosides of reverse primer
Acid sequence is as shown in SEQ ID No.5；The nucleotide sequence of the forward primer of BnaA.ARF18a-262 such as SEQ ID No.6 institutes
Show, the nucleotide sequence of reverse primer is as shown in SEQ ID No.7.
Application of the above-mentioned molecular labeling in rapeseed breeding is also protection scope of the present invention, and the rapeseed breeding includes point
The breeding methods such as sub- marker assisted selection breeding, gene pyramiding breeding and transgenic breeding.
The present invention also provides the methods that above-mentioned molecular labeling is applied in rapeseed breeding, include the following steps：
(1) PCR amplification system is established：Using any right of primer pair BnaA.ARF18a-186 and BnaA.ARF18a-262
Or two pairs, using cabbage type rape single plant DNA as template, carry out PCR amplification.
(2) digestion is carried out to the product of above-mentioned PCR amplification：
It is expanded according to primer pair BnaA.ARF18a-186, amplified production is subjected to EcoRV digestions, electrophoretic separation cannot
Observe the band of 211bp length, then the single plant is high grain weight and siliqua single plant, observes the band of 211bp length, then should
Single plant is not high grain weight and siliqua single plant.
It is expanded according to primer pair BnaA.ARF18a-262, amplified production is subjected to BsmAI digestions, electricity separation, observation
To the band of 125bp length, then the single plant is low grain weight and silicle single plant, if not it is observed that the band of 125bp length,
The single plant be not low grain weight and silicle single plant.
PCR amplification is carried out respectively according to primer pair BnaA.ARF18a-186 and BnaA.ARF18a-262, using primer
EcoRV digestions are used to the product of BnaA.ARF18a-186 amplifications, using the product of primer pair BnaA.ARF18a-262 amplifications
It using BsmAI digestions, is separated by electrophoresis respectively, not it is observed that the band of 211bp length, the list in the product of EcoRV digestions
Strain is high grain weight and siliqua single plant；The band of 125bp length is observed in the product of BsmAI digestions, then the single plant is low
Grain weight and silicle single plant；211bp bands are observed in the product of EcoRV digestions, and cannot in the product of BsmAI digestions
Observe the band of 125bp, then the single plant is the single plant of medium grain weight and moderate-length silique.
Breeder can predict, screen or identify that the grain of single plant is heavy and silique is long and chooses in above-mentioned primer pair as needed
The DNA of one or more pairs of single plants is appointed to carry out amplification and digestion, the grain weight and silique to obtain single plant are long.
The beneficial effects of the present invention are：
The molecular labeling of the present invention, the Brassica Napus Seedling phase just can be fast and reliable identification different cultivars rape single plant
Grain weight and Pod length, improve rapeseed breeding efficiency, greatly shorten the rapeseed breeding time limit；It is reflected using molecular labeling
The grain weight and Pod length of group, can be respectively increased 17% and 34% by fixed and selection, and the workload of later stage Test Identification is reduced
33% or more.
Description of the drawings
Fig. 1 be cabbage type rape grain weight (on) and Pod length (under) result of association analysis, wherein abscissa is to dye
Body physical distance, ordinate are the-log of each SNP site10(P) numerical value, the representative of horizontal dotted line reach significant-log10
(P) numerical value, mass of 1000 kernel 6.02, Pod length 6.3；
Fig. 2 is the haplotyping result schematic diagram of BnaA.ARF18a in natural population, wherein (a) BnaA.ARF18a bases
Because of three kinds of different haplotype schematic diagrames that structure and the sites SNPs are constituted, the box of grey and the horizontal line of black respectively represent outside
Aobvious son and introne.(b) phenotypic difference of the mass of 1000 kernel and Pod length between three kinds of different monomers types, asterisk represent Hap B and
The significance of difference between Hap C and Hap A, P≤0.001 * P≤0.05, * * *；
Fig. 3 is the exploitation that two couples of dCAPS are marked in BnaA.ARF18a, and dCAPS flags sequence are shown in box, include
The position of digital representation gene order in number, blue letters represent the SNP between different monomers type, and red letters representative is designing
The base changed for introducing restriction enzyme site in dCAPS labeling process.
Fig. 4 marks the detection case schematic diagram in different grains weight and Pod length material for dCAPS, wherein 1-5 is certainly
Hap A materials in right group, 6-10 are the Hap B materials in natural population, and 11-15 is the Hap C-materials in natural population.
Specific implementation mode
Be a kind of specific implementation mode of the method for the present invention below, but be not the restriction to the method for the present invention, it is any not
Surpass the transformation from substantive content of the present invention, should belong to the scope of protection of the present invention.
Embodiment 1:
A kind of molecular labeling BnaA.ARF18a-186 that weight and Pod length are closely related with rape grain and
BnaA.ARF18a-262 is prepared by the following：
The present embodiment is made a variation by 157 parts of genetic origin differences, grain weight and Pod length for larger rape variety, structure
The method for obtaining the molecular labeling being closely related with rape grain weight and Pod length is described in detail, specifically in one natural population
It is as follows：
(1) informative population：
It is made a variation larger (1.75-5.71g/4.02-10.62cm) with 157 parts of genetic origin differences, grain weight and Pod length
The natural population of rape variety composition is research material (being collected in world wide).
(2) measurement of grain weight and Pod length：
(1) vegetable material is in field planting, 3 row of each strain, often row 8-10 plants, RANDOMIZED BLOCK DESIGN；The Yangtze river basin one
As September part be seeded in seedbed, late October transplants to crop field；Conventional method carries out field management, until seed maturity (next year
At the beginning of 5 months)；
(2) at the maturity period, each strain randomly selects 5 single plants, and 20 are respectively taken upwards in turn from the bottom of main inflorescence
Difference unobvious silique, strain each in this way share 100 siliques.
Measure length：The silique of rape is not complete straight line, most of to have buckling phenomenon.A root long cotton thread is taken,
With lowermost end (being free of carpopodium) alignment cotton thread starting point of first silique, the trend for cotton thread being bonded silique extends forward, until
The front end of silique fruit beak is cotton thread terminal, i.e., we only take the length of silique fruit body, write down the value of cotton thread origin-to-destination, this
A value is the length value of single silique, and it is the Pod length (cm) of each kind to take the average value of 100 siliques.
Measure mass of 1000 kernel：The seed that each strain harvests is put in baking oven drying, 1000 seeds is therefrom counted at random and claims
Weight, is repeated 3 times, and it is the grain weight (g) of each kind to take the average value of 3 measurements.
(3) DNA is extracted
For all plant research materials, young leaflet tablet is acquired in seedling stage, using " CTAB methods extract plant genome DNA "
Experimental procedure extracts genomic DNA.
(1) appropriate young leaflet tablet is acquired, with liquid N2It smashes, 0.4g is fitted into 2mL centrifuge tubes (- 20 DEG C of precoolings).
(2) preheating 2%CTAB (sterilizing) adds in 700 μ L to the centrifuge tube equipped with blade powder, to 70 DEG C (for anti-block
Change, the beta -mercaptoethanol of 14 μ L can also be added) mixing (preventing freeze thawing).
(3) 65 DEG C of water-bath 45min are immediately placed on, per 5min, are turned upside down 1 time；
(4) isometric (600 μ L) chloroform/isoamyl alcohol (24 is added:1) it, turns upside down for several times, until lower layer's liquid phase is dark green
Until.12000g is centrifuged 5 minutes.
(5) supernatant is taken, it is primary (can save) to repeat 4 steps.
(6) it takes 450 μ L supernatants in a new 2mL centrifuge tubes, is added 95% ethyl alcohol of 1mL and 20 μ L 10M NH4AC, mixing,
It is placed at room temperature for 10min.
(7) 12000g centrifuges 10min, removes supernatant, embathes precipitation with 75% absolute ethyl alcohol, spontaneously dries about 30min.
(8) 50 μ L 0.5TE or sterile water (containing 20 μ g/RNase) is added, is placed in 4 DEG C overnight, after DNA dissolvings, detection
DNA concentration and quality.
(4) genome resurveys sequence
Hundred Mai Ke companies of Beijing are sent to be sequenced DNA sample.The standard that experiment flow is provided according to Illumina companies
Protocol is executed, including the flows such as sample quality detection, library construction, Library Quality detection and library sequencing.Sample gene
After group DNA detections are qualified, then DNA fragmentation is carried out segment by the method (ultrasonic wave) interrupted with machinery to the DNA of fragmentation
Purifying, end are repaired, 3 ' ends add A, connection sequence measuring joints, then carry out clip size selection with agarose gel electrophoresis, carry out PCR
Amplification forms sequencing library, the advanced style of writing library quality inspection in the library built up, the library Illumina HiSeqTM of quality inspection qualification
4000 are sequenced, and sequencing length is PE125, and sequencing depth is 5.
The initial data that sequencing obtains is filtered, Clean Reads are obtained.With BWA (http://bio-
Bwa.sourceforge.net/) software is by clean reads and rape reference gene group Brassica_
napus.annotation_v5(http://www.genoscope.cns.fr/brassicanapus/data/) it is compared；
SNP and Indel detections are carried out with GATK softwares, detailed process refers to the BestPractice of GATK official websites：
https://www.broadinstitute.org/gatk/guide/best-practices.php.Delete missing
Rate>0.6 site, site of the miss rate less than 0.6 use software beagle v4.1 (https://
Faculty.washington.edu/browning/beagle/beagle.html#downl oad) it is filled up.Finally, it goes
Except heterozygosis rate is more than 0.25 and minimum site of the gene frequency (minor allele frequency, MAF) less than 0.05,
Remaining site is used for whole-genome association.
(5) association analysis
GWAS analyses (Team et al., 2014) are carried out using the mrMLM v1.3 software packages of R language, using the mixed of Q+K
Linear model is associated character and label the detection in site, and rMLM threshold values are set as P ＜-log10 (0.05/me) (me
For the number of significant notation number, specific number is with reference to software analysis method), the threshold value of mrMLM is set as LOD>3.The knot of rMLM
Fruit passes through R software package qqman (https://cran.r-project.org/web/packages/qqman/) it carries out
Manhattan schemes and Q-Q figures are shown.One and grain weight and Pod length are detected in the 27.78Mb-28.61Mb of A09 chromosomes
Significantly correlated section (figure one).
(6) candidate gene is predicted
It is the significant positions SNP is each downwards upwards to extend 100kb or be in same haplotype area with notable SNP with selection
(r2＞ 0.5) section, we by such section definition be candidate association section.The pre- astronomical observation of the following conditions is referred in this section
Select gene：1) with reference to the result for having reported QTL positioning；2) SNP directly falls in gene internal and causes the change of amino acid；3) exist
Gene on cabbage type rape or arabidopsis reference gene group with the relevant known function of character.One is screened according to above method
It is a it has been reported that with rape grain weight and the relevant gene BnaA.ARF18a of Pod length (Liu et al., 2015).
(7) functional label conversion and detection
Contain 9 SNPs in total in BnaA.ARF18.a gene internals, 3 are located at introne 6 and are located at exon and only
There are 2 SNPs to cause the change of amino acid；9 SNPs may be constructed 3 kinds of different types of haplotypes in different storerooms
(Haplotype) Hap A, Hap B and Hap C (figure two).In three haplotypes, its mass of 1000 kernel (3.02 of the material containing Hap A
± 0.29g) and Pod length (5.09 ± 0.35cm) be all it is minimum, it is maximum be containing Hap C material (4.05 ±
0.46g；7.37 ± 1.81cm), the material containing Hap B is located at by-level (3.44 ± 0.42g；5.38±0.36cm).Root
According to two pairs of dCAPS labels of SNP information designs different between three kinds of haplotypes, (design of primers is the principle marked according to dCAPs
By introducing base mismatch in amplimer on the basis of CAPS is marked, then new limitation can be introduced in conjunction with SNP site
Property restriction endonuclease action site, generate and CAPS marking classes as polymorphism.Here, we utilize online website dCAPS Finder
2.0(http://helix.wustl.edu/dcaps/dcaps.html) Computer Aided Design)：The forward direction of BnaA.ARF18a-186 is drawn
Object sequence is 5 '-ATGGCGAATGTAGATGGAGAT-3 ', reverse primer sequences 5 '-
CTCTGTGTACAGCTGATCTTGATAT-3’；The forward primer sequence of BnaA.ARF18a-262 is 5 '-
GTTGTTTGTTTGATTTTCAGGTT-3 ', reverse primer sequences 5 '-GACCCTGAGGGAAGTAGAAAAGTCT-3 ' (figure
Three).
According to natural population's haplotyping as a result, each haplotype has chosen 5 different strain (1-5 respectively：
Hap A, 6-10：Hap B, 11-15：Hap C) using two pairs of dCAPS label progress PCR amplifications, PCR product is used corresponding respectively
Digestion with restriction enzyme processing.The result of 3% agarose gel electrophoresis is shown：As being imagined experiment, EcoRV
The BnaA.ARF18a-186PCR products of 11-15 materials can be cut off and the PCR product of 1-10 materials cannot but be cut；
The BnaA.ARF18a-262PCR products of 6-15 materials can be cut off and cannot but be cut the PCR product of 1-5 materials by BsmAI
It opens (figure four).The selection marked by two couples of dCAPS, people can select the optimal haplotype of mass of 1000 kernel and Pod length.
Embodiment two：
Application of the molecular labeling that a kind of and rape grain weight and Pod length are closely related in yield of Brassica napus L breeding, step
Suddenly it is：
With (the high grain long angle again European winter variety Express (low grain weight silicle) and China semi-winterness kind House7
Fruit) it is parent, structure DH segregating populations are research material, utilize two kinds of BnaA.ARF18a-186 and BnaA.ARF18a-262
DCAPS labels further expand detection range, and detection method is as follows：
(1) DH groups and parent DNA are extracted, method is the same as embodiment two.
(2) the haplotype type for detecting DH group parents first carries out PCR processes using following primer：
The forward primer sequence of BnaA.ARF18a-186 is 5 '-ATGGCGAATGTAGATGGAGAT-3 ', reverse primer sequence
It is classified as 5 '-CTCTGTGTACAGCTGATCTTGATAT-3 '；
The forward primer sequence of BnaA.ARF18a-262 is 5 '-GTTGTTTGTTTGATTTTCAGGTT-3 ', reverse primer
Sequence is 5 '-GACCCTGAGGGAAGTAGAAAAGTCT-3 '
(3) PCR reaction systems：Total volume is 25 μ L, and specific ingredient is as follows：
(4) PCR amplification program：94 DEG C of 5min, [94 DEG C of 45s, 55 DEG C of 45s, 72 DEG C of 1min] × 35 are recycled, 72 DEG C
10min.It is preserved under the conditions of 4 DEG C after operation.
(5) digestion of pcr amplification product：Every 10 μ l are with restriction enzyme EcoRV and BsmAI corresponding 2.5U in 37 DEG C of items
1-16h is handled under part.
For (6) 2 kinds of digestion products after the separation of 3% agarose gel electrophoresis, it is 211bp and one that Express, which only occurs one,
Item is the band of 125bp, illustrates that Express cannot be haplotype A (Hap A) by EcoRV and BsmAI digestions, i.e. Express；
The band that House7 appearance one is 188bp (23bp is too small not to be seen) and one is 98bp (27bp is too small not to be seen), explanation
House7 can be haplotype C (Hap C) by EcoRV and BsmAI digestions, i.e. House7.The DH groups so got by the two
In only two kinds of haplotypes of Hap A and Hap C, only isophenic material can be differentiated with a wherein primer.
(7) it is detected in DH groups using same method, as a result, it has been found that：DH groups allelic (Hap A)
The strain number for coming from parent ' Express ' is substantially equal to DH groups allelic (Hap C) and comes from parent
The strain number of ' House7 '；All its mass of 1000 kernel of the strain containing Hap C and Pod length mean value are respectively 3.72 ± 0.14g
With 6.21 ± 0.42cm, it is significantly higher than the strain mean value (3.22 ± 0.1g and 5.13 ± 0.25cm) of all A containing Hap；The two thousand
All there is significant difference (P≤0.001) in grain weight and Pod length.It, can be further fast by selecting the single plant containing Hap C
Speed cultivates high yield rape variety.
Result above absolutely proves, the two dCAPS labels can be used for really rape grain weight and Pod length prediction,
Identification and screening.
SEQUENCE LISTING
<110>Southwestern University
<120>The molecular labeling and application that a kind of and rape grain weight and Pod length are closely related
<130> 20180420
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 2622
<212> DNA
<213> Brassica napus
<400> 1
atggcgaatg tagatggaga tgattccaga agttctttcc caagtgagtc aaagtttctt 60
tttttgttgt cttcaccatt gactctgttt tcttgctctg tattagctca aagtttacat 120
cttttttctt ctttttttgt tgtgtctcta aaatgtaact gtgttgtttg tttgattttc 180
aggttcttat caagatcagc tgtacacaga gctatggaaa gcctgtgcag gtccattagt 240
ggaggttcct cttgttggag aaagagtttt ctacttccct cagggtcaca tggaacaagt 300
atgtcttttt taccaaatta atcttttttt tttttaaact ccaattctct ttctgaattg 360
tttttttttt tttttttttt gcagcttgtg gcctcaacta atcaaggaat tgaatcagag 420
aaaatacctg attttaaact tcctcccaag atactctgtc aagttcttag tgtgatgtta 480
aaggtataaa ctttgatcat aacccttgct tgtcctcccc ttcttgtgaa ttcacctgaa 540
tctttttttg atgtataggc agagcatgac acagatgaag tctacgctca gatcacatta 600
aaaccagagg aagatgtaag ttctgtaaag tttgagcttg ttaatttgat gagaaagtga 660
tttttgagtt ttttttttgt ttcttgcagc aaagtgaacc tacaagtctt gatccaccaa 720
ttgttgaacc aacaaagcaa atgttccact cctttgtaaa gattctaacc gcttcagaca 780
caagcactca tggtggattc tctgttcttc gtaaacacgc cactgaatgc ttgcctgcct 840
tggacatgac acaagctatt cctactcaag aacttgtgac tagagatctt catgggtttg 900
agtggaggtt taagcatatt ttcagaggta attaactcta aaatcttgta atatattttg 960
gactaaatga gacttgagag ttgttgtttt ttttttgggt ttaggacaac ctaggaggca 1020
tttgcttact acaggctgga gtacatttgt ttcctcaaaa agacttgtag ctggagatgc 1080
ttttgtgttc ttgaggtacc actacttttc ctgaaacttt actagtcttg ttttagtttt 1140
aatgaaattc atttgtgttt gtaggggtga gaatggagat ttaagagttg gagtgaggcg 1200
tttagctagg catcagaaca ccatgcctgc ttcagttatc tctagtcaga gcatgcattt 1260
aggagtcctt gctacagctt ctcatgctgt gaacacccaa actatgtttc ttgtgtttta 1320
caagcctagg taagtgtgta catgatactc tttgctatat tcatcatagg acttagatta 1380
tttaagatta aaagctgatg tattttcagg ataagccaat tcatagtaag tgtgaacaag 1440
tatatggaag ctatgaagca tggtttctct cttggtacaa gattcaggat gaggtttgaa 1500
ggagaagagt ctcctgagag aatgtaagat tctttattta ttttatgatc attagttaac 1560
tgttcttgat tctgaatggt tgagttttct tttagattta ccggtactat tgtgggaatt 1620
ggagatttat cttcacaatg gccagcttct acatggagat cattgcaggt tcagttaacc 1680
attcagtgtt aaccattgca gcttctacat gtaaaccttc tttcttcctc ttttttaggt 1740
ccaatgggat gagccaacaa cagttcagag accagacaaa gtctcaccat gggagattga 1800
gcctttcttg ccatcttccc cagcttcaac accttctcaa caatcacaac ccaaaagcaa 1860
aaggtcaaaa cccattgaat catcaagttt gagtccaggt caagctagtt tcttaggcgt 1920
ccaagctgag cctcctcctc ctcctgcgag tagttgctat aggttgtttg gatttgatct 1980
cacaagcaat cctccagctc caatacctcc agacaagcaa ccgatggata cttctgaagc 2040
tgccaagtgt caagacccca tcactccaag ctcagttaat gagccaaaga agcaacaaac 2100
atcaaggact cgaaccaaag taatcatctt ccatcacttt atcacttcat ataaaatatt 2160
acaaagttct gaaatgttgt tttcttttaa aaggtgcaaa tgcaagggat agctgttggt 2220
cgtgcggtag atttaacgct gttgaaatca tatgatgaac tgattaagga gcttgaggag 2280
atgtttgaga tccaaggaca gcttcttccc cgagataaat ggatcgttgt cttcactgat 2340
gatgaaggtg acatgatgct tgctggagat gatccatgga agtaagtaaa taaatattca 2400
cagagtagta gtaaaccgtt tacggttcag ttaaaatcta ttaaaccgaa gtaaaccgga 2460
actggcttga caaagtaatt attgaatgtt ttgtttttgt ggtgcagtga gttttgtaag 2520
atggcgaaga agatatttat atattcaagc gatgaggtta agaaaatgac aaggagaatg 2580
aagagttctt cttcgttaga gaatgaagca agcatggatt aa 2622
<210> 2
<211> 2622
<212> DNA
<213> Brassica napus
<400> 2
atggcgaatg tagatggaga tgattccaga agttctttcc caagtgagtc aaagtttctt 60
tttttgttgt cttcaccatt gactctgttt tcttgctctg tattagctca aagtttacat 120
cttttttctt ctttttttgt tgtgtctcta aaatgtaact gtgttgtttg tttgattttc 180
aggttcttat caagatcagc tgtacacaga gctatggaaa gcctgtgcag gtccattagt 240
ggaggttcct cttgttggag agagagtttt ctacttccct cagggtcaca tggaacaagt 300
atgtcttttt taccaaatta atcttttttt tttttaaact ccaattctct ttctgaattg 360
tttttttttt tttttttttt gcagcttgtg gcctcaacta atcaaggaat tgaatcagag 420
aaaatacctg attttaaact tcctcccaag atactctgtc aagttcttag tgtgatgtta 480
aaggtataaa ctttgatcat aacccttgct tgtcctcccc ttcttgtgaa ttcacctgaa 540
tctttttttg atgtataggc agagcatgac acagatgaag tctacgctca gatcacatta 600
aaaccagagg aagatgtaag ttctgtaaag tttgagcttg ttaatttgat gagaaagtga 660
tttttgagtt ttttttttgt ttcttgcagc aaagtgaacc tacaagtctt gatccaccaa 720
ttgttgaacc aacaaagcaa atgttccact cctttgtaaa gattctaacc gcttcagaca 780
caagcactca tggtggattc tctgttcttc gtaaacacgc cactgaatgc ttgcctgcct 840
tggacatgac acaagctatt cctactcaag aacttgtgac tagagatctt catgggtttg 900
agtggaggtt taagcatatt ttcagaggta attaactcta aaatcttgta atatattttg 960
gactaaatga gacttgagag ttgttgtttt ttttttgggt ttaggacaac ctaggaggca 1020
tttgcttact acaggctgga gtacatttgt ttcctcaaaa agacttgtag ctggagatgc 1080
ttttgtgttc ttgaggtacc actacttttc ctgaaacttt actagtcttg ttttagtttt 1140
aatgaaattc atttgtgttt gtaggggtga gaatggagat ttaagagttg gagtgaggcg 1200
tttagctagg catcagaaca ccatgcctgc ttcagttatc tctagtcaga gcatgcattt 1260
aggagtcctt gctacagctt ctcatgctgt gaacacccaa actatgtttc ttgtgtttta 1320
caagcctagg taagtgtgta catgatactc tttgctatat tcatcatagg acttagatta 1380
tttaagatta aaagctgatg tattttcagg ataagccaat tcatagtaag tgtgaacaag 1440
tatatggaag ctatgaagca tggtttctct cttggtacaa gattcaggat gaggtttgaa 1500
ggagaagagt ctcctgagag aatgtaagat tctttattta ttttatgatc attagttaac 1560
tgttcttgat tctgaatggt tgagttttct tttagattta ccggtactat tgtgggaatt 1620
ggagatttat cttcacaatg gccagcttct acatggagat cattgcaggt tcagttaacc 1680
attcagtgtt aaccattgca gcttctacat gtaaaccttc tttcttcctc ttttttaggt 1740
ccaatgggat gagccaacaa cagttcagag accagacaaa gtctcaccat gggagattga 1800
gcctttcttg ccatcttccc cagcttcaac accttctcaa caatcacaac ccaaaagcaa 1860
aaggtcaaaa cccattgaat catcaagttt gagtccaggt caagctagtt tcttaggcgt 1920
ccaagctgag cctcctcctc ctcctgcgag tagttgctat aggttgtttg gatttgatct 1980
cacaagcaat cctccagctc caatacctcc agacaagcaa ccgatggata cttctgaagc 2040
tgccaagtgt caagacccca tcactccaag ctcagttaat gagccaaaga agcaacaaac 2100
atcaaggact cgaaccaaag taatcatctt ccatcacttt atcacttcat ataaaatatt 2160
acaaagttct gaaatgttgt tttcttttaa aaggtgcaaa tgcaagggat agctgttggt 2220
cgtgcggtag atttaacgct gttgaaatca tatgatgaac tgattaagga gcttgaggag 2280
atgtttgaga tccaaggaca gcttcttccc cgagataaat ggatcgttgt cttcactgat 2340
gatgaaggtg acatgatgct tgctggagat gatccatgga agtaagtaaa taaatattca 2400
cagagtagta gtaaaccgtt tacggttcag ttaaaatcta ttaaaccgaa gtaaaccgga 2460
actggcttga caaagtaatt attgaatgtt ttgtttttgt ggtgcagtga gttttgtaag 2520
atggcgaaga agatatttat atattcaagc gatgaggtta agaaaatgac aaggagaatg 2580
aagagttctt cttcgttaga gaatgaagca agcatggatt aa 2622
<210> 3
<211> 2622
<212> DNA
<213> Brassica napus
<400> 3
atggcgaatg tagatggaga tgattccaga agttctttcc caagtgagtc aaagtttcct 60
tttttgttgt cttcaccatt gactctgttt tcttgctctg tattagctca aagtttacat 120
cttttttctt ctttttttgt tgtgtctcta aaatgtaact gtgttgtttg tttgattttc 180
aggttgttat caggatcagc tgtacacaga gctatggaaa gcctgtgcag gtccattagt 240
ggaggttcct cttgttggag agagagtttt ctacttccct cagggtcaca tggaacaagt 300
atgtcttttt taccaaatta atcttttttt tttttaaact ccaattctct ttctgaattg 360
tttttttttt tttttttttt gcagcttgtg gcctcaacta atcaaggaat tgaatcagag 420
aaaatacctg attttaaact tcctcccaag atactctgtc aagttcttag tgtgatgtta 480
aaggtataaa ctttgatcat aacccttgct tgtcctcccc ttcttgtgaa ttcacctgaa 540
tctttttttg atgtataggc agagcatgac acagatgaag tctacgctca gatcacatta 600
aaaccagagg aagatgtaag ttctgtaaag tttgagcttg ttaatttgat gagaaagtga 660
tttttgagtt ttttttttgt ttcttgcagc aaagtgaacc tacaagtctt gatccaccaa 720
ttgttgaacc aacaaagcaa atgttccact cctttgtaaa gattctaacc gcttcagaca 780
caagcactca tggtggattc tctgttcttc gtaaacacgc cactgaatgc ttgcctgcct 840
tggacatgac acaagctatt cctactcaag aacttgtgac tagagatctt catgggtttg 900
agtggaggtt taagcatatt ttcagaggta tttaactcta aaatcttgta atatattttg 960
gactaaatga gacttgagag ttgttgtttt ttttttgggt ttaggacaac ctaggaggca 1020
tttgcttact acaggctgga gtacctttgt ttcctcaaaa agacttgtag ctggagatgc 1080
ttttgtgttc ttgaggtacc actacttttc ctgaaacttt actagtcttg ttttagtttt 1140
aatgaaattc atttgtgttt gtaggggtga gaatggagat ttaagagttg gagtgaggcg 1200
tttagctagg catcagaaca ccatgcctgc ttcagttatc tctagtcaga gcatgcattt 1260
aggagtcctt gctacagctt ctcatgctgt gaacacccaa acaatgtttc ttgtgtttta 1320
caagcctagg taagtgtgta catgctactc tttgctatat tcatcatagg acttagatta 1380
tttaagatta aaagctgatg tattttcagg ataagccaat tcatagtagg tgtgaacaag 1440
tatatggaag ctatgaagca tggtttctct cttggtacaa gattcaggat gaggtttgaa 1500
ggagaagagt ctcctgagag aatgtaagat tctttattta ttttatgatc attagttaac 1560
tgttcttgat tctgaatggt tgagttttct tttagattta ccggtactat tgtgggaatt 1620
ggagatttat cttcacaatg gccagcttct acatggagat cattgcaggt tcagttaacc 1680
attcagtgtt aaccattgca gcttctacat gtaaaccttc tttcttcctc ttttttaggt 1740
ccaatgggat gagccaacaa cagttcagag accagacaaa gtctcaccat gggagattga 1800
gcctttcttg ccatcttccc cagcttcaac accttctcaa caatcacaac ccaaaagcaa 1860
aaggtcaaaa cccattgaat catcaagttt gagtccaggt caagctagtt tcttaggcgt 1920
ccaagctgag cctcctcctc ctcctgcgag tagttgctat aggttgtttg gatttgatct 1980
cacaagcaat cctccagctc caatacctcc agacaagcaa ccgatggata cttctgaagc 2040
tgccaagtgt caagacccca tcactccaag ctcagttaat gagccaaaga agcaacaaac 2100
atcaaggact cgaaccaaag taatcatctt ccatcacttt atcacttcat ataaaatatt 2160
acaaagttct gaaatgttgt tttcttttaa aaggtgcaaa tgcaagggat agctgttggt 2220
cgtgcggtag atttaacgct gttgaaatca tatgatgaac tgattaagga gcttgaggag 2280
atgtttgaga tccaaggaca gcttcttccc cgagataaat ggatcgttgt cttcactgat 2340
gatgaaggtg acatgatgct tgctggagat gatccatgga agtaagtaaa taaatattca 2400
cagagtagta gtaaaccgtt tacggttcag ttaaaatcta ttaaaccgaa gtaaaccgga 2460
actggcttga caaagtaatt attgaatgtt ttgtttttgt ggtgcagtga gttttgtaag 2520
atggcgaaga agatatttat atattcaagc gatgaggtta agaaaatgac aaggagaatg 2580
aagagttctt cttcgttaga gaatgaagca agcatggatt aa 2622
<210> 4
<211> 21
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 4
atggcgaatg tagatggaga t 21
<210> 5
<211> 25
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 5
ctctgtgtac agctgatctt gatat 25
<210> 6
<211> 23
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 6
gttgtttgtt tgattttcag gtt 23
<210> 7
<211> 25
<212> DNA
<213> artificial
<220>
<223> artificial
<400> 7
gaccctgagg gaagtagaaa agtct 25
Claims (10)
1. a kind of molecular labeling being closely related with rape grain weight and Pod length, which is characterized in that the molecular labeling is pair
The molecular labeling of gene BnaA.ARF18a, the nucleotide sequence that gene BnaA.ARF18a has are shown in SEQ ID No.1
Nucleotide sequence or the sequence through replacement, missing or increase one or more nucleotide, and coding is same or similar, and have phase
The nucleotide sequence of the amino acid sequence of congenerous.
2. molecular labeling as described in claim 1, which is characterized in that gene BnaA.ARF18a contains 9 SNP, initiation codon
Sub- ATG is defined as+1, wherein 6 SNP are located at exon region+186 ,+193 ,+262 ,+1045 ,+1303 ,+1345,3
A SNP, which is located at, to be included+59 ,+931 ,+1429,9 SNP of subregion and constitutes Hap A, Hap B and Hap in different single plant DNA
Tri- kinds of haplotypes of C, the nucleotide sequence of Hap A types is as shown in SEQ ID No.1, the nucleotide sequence such as SEQ ID of Hap Type Bs
Shown in No.2, the nucleotide sequence of Hap c-types is as shown in SEQ ID No.3.
3. molecular labeling as claimed in claim 2, which is characterized in that the molecular labeling is according to different between three kinds of haplotypes
SNPs design primer pair.
4. molecular labeling as claimed in claim 3, which is characterized in that the primer pair includes BnaA.ARF18a-186,
The nucleotide sequence of the forward primer of BnaA.ARF18a-186 as shown in SEQ ID No.4, the nucleotide sequence of reverse primer such as
Shown in SEQ ID No.5.
5. molecular labeling as claimed in claim 3, which is characterized in that the primer pair includes BnaA.ARF18a-262,
The nucleotide sequence of the forward primer of BnaA.ARF18a-262 as shown in SEQ ID No.6, the nucleotide sequence of reverse primer such as
Shown in SEQ ID No.7.
6. molecular labeling as claimed in claim 4, which is characterized in that the primer pair further includes BnaA.ARF18a-186.
7. molecular labeling described in claim 4 includes the following steps in the application process of rapeseed breeding：
(1) PCR amplification system is established：Using primer pair BnaA.ARF18a-186, using cabbage type rape single plant DNA as template, into
Row PCR amplification；
(2) digestion of pcr amplification product and electrophoresis：EcoRV digestions are carried out to the product of step (1) PCR amplification, are separated by electrophoresis,
Not it is observed that the band of 211bp length, then the single plant is high grain weight and siliqua single plant, observes the band of 211bp length,
Then the single plant is not high grain weight and siliqua single plant.
8. application process of the molecular labeling described in claim 5 in rapeseed breeding, includes the following steps：
(1) PCR amplification system is established：Using primer pair BnaA.ARF18a-262, using cabbage type rape single plant DNA as template, into
Row PCR amplification；
(2) digestion of pcr amplification product and electrophoresis：BsmAI digestions are carried out to the product of step (1) PCR amplification, are separated by electrophoresis,
If it is observed that the band of 125bp length, which is low grain weight and silicle single plant, if not it is observed that 125bp length
Band, then the single plant be not low grain weight and silicle single plant.
9. application process of the molecular labeling described in claim 6 in rapeseed breeding, includes the following steps：
(1) PCR amplification system is established：Using primer pair BnaA.ARF18a-186 and BnaA.ARF18a-262, with Wild cabbage type oil
Menu strain DNA is template, carries out PCR amplification respectively；
(2) digestion of pcr amplification product and electrophoresis：EcoRV digestions are used to the product of BnaA.ARF18a-186 amplifications, it is right
The product of BnaA.ARF18a-262 amplifications uses BsmAI digestions, is separated by electrophoresis, cannot be seen in the product of EcoRV digestions respectively
The band of 211bp length is observed, which is high grain weight and siliqua single plant；125bp is observed in the product of BsmAI digestions
The band of length, then the single plant is low grain weight and silicle single plant；Observe 211 bands in the product of EcoRV digestions, and
Not it is observed that the band of 125bp, then the single plant is the list of medium grain weight and moderate-length silique in the product of BsmAI digestions
Strain.
10. claim 1-5 any one of them molecular labelings are applied in rapeseed breeding.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110157829A (en) * | 2019-04-16 | 2019-08-23 | 中国农业科学院油料作物研究所 | A kind of and the associated molecular marker SNP A9-5 of rape mass of 1000 kernel and application |
| CN110184373A (en) * | 2019-04-16 | 2019-08-30 | 中国农业科学院油料作物研究所 | A kind of and the associated molecular labeling of rape mass of 1000 kernel and application |
| CN115029465A (en) * | 2022-01-27 | 2022-09-09 | 淮阴师范学院 | KASP and dCAPS markers cosegregating with rape seed secondary dormancy major QTL and application thereof |
| CN116064900A (en) * | 2022-09-05 | 2023-05-05 | 中国农业科学院油料作物研究所 | SNP molecular marker related to angle of inclination of rape plant type pod and application thereof |
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| CN107164502A (en) * | 2017-06-15 | 2017-09-15 | 西南大学 | The molecular labeling being closely related and its application are whether there is with cabbage leaves epidermal hair |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157829A (en) * | 2019-04-16 | 2019-08-23 | 中国农业科学院油料作物研究所 | A kind of and the associated molecular marker SNP A9-5 of rape mass of 1000 kernel and application |
| CN110184373A (en) * | 2019-04-16 | 2019-08-30 | 中国农业科学院油料作物研究所 | A kind of and the associated molecular labeling of rape mass of 1000 kernel and application |
| CN110157829B (en) * | 2019-04-16 | 2022-03-15 | 中国农业科学院油料作物研究所 | Molecular marker SNPA9-5 associated with thousand seed weight of rape and application |
| CN115029465A (en) * | 2022-01-27 | 2022-09-09 | 淮阴师范学院 | KASP and dCAPS markers cosegregating with rape seed secondary dormancy major QTL and application thereof |
| CN116064900A (en) * | 2022-09-05 | 2023-05-05 | 中国农业科学院油料作物研究所 | SNP molecular marker related to angle of inclination of rape plant type pod and application thereof |
| CN116064900B (en) * | 2022-09-05 | 2024-01-02 | 中国农业科学院油料作物研究所 | SNP molecular marker related to angle of inclination of rape plant type pod and application thereof |
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| Publication number | Publication date |
|---|---|
| CN108300801B (en) | 2021-11-16 |
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