CN108300801B - Molecular marker closely related to rape grain weight and silique length and application - Google Patents
Molecular marker closely related to rape grain weight and silique length and application Download PDFInfo
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Abstract
The invention relates to the field of rape breeding and molecular biology, in particular to a molecular marker closely related to rape grain weight and silique length and application thereof. A molecular marker closely related to the grain weight and the length of a pod of rape is characterized in that the molecular marker is a molecular marker of a gene BnaA.ARF18a, and the nucleotide sequence is shown as SEQ ID No. 1. The molecular marker solves the problems that the grain weight and the silique length in the conventional breeding method can only be identified in the later period, so that the breeding period is long, the method is easily influenced by the environment and the selection efficiency is low, the breeding efficiency of the rape is improved, the grain weight and the silique length of single plants of different varieties of rapes can be quickly and reliably identified in the rape seedling period, the breeding efficiency of the rape is improved, and the breeding period of the rape is greatly shortened.
Description
Technical Field
The invention relates to the field of rape breeding and molecular biology, in particular to a molecular marker closely related to rape grain weight and silique length and application thereof.
Background
Cabbage type rape is an important oil crop in China, not only edible oil is provided for human, but also feed rich in protein can be provided, so that the improvement of rape yield is always the primary goal of breeders. Seed size (weight) and silique length are important components of oilseed rape yield, and hundreds of grain weight and silique length QTLs have been found in oilseed rape and the like over the last two decades, but the genetic and molecular regulatory networks for these QTLs are unclear.
Seed size/weight is considered one of the most important traits, and as a product of sexual reproduction of higher plants, seeds are not only the most important form of plant propagation, but also the most important source of food on which humans rely for survival. The rape seeds are not only storage organs of oil and protein, but also the most main harvest substances of the rape, and the research on the size/weight of the seeds is of great significance. (1) The total yield of rape plants is mainly composed of three main factors, namely the number of pods per plant, the number of kernels per pod and the weight of kernels, wherein the heritability of the weight of the kernels is highest, the influence of the environment is minimum, the rape plants are an important economic trait of the rape plants, and the yield of the rape plants is positively correlated (Clarke and Simpson, 1978; Butruille et al, 1999; Shi et al, 2009). (2) Seed weight also affects traits such as oil content and protein content of seeds (Morgan et al, 1998; Adamskia et al, 2009; Lioneton et al, 2004). (3) Large seeds have better germination capacity than small seeds, and plants grown from large seeds have more environmental suitability than plants grown from small seeds. Therefore, the elucidation of the genetic basis of rape grain weight formation is beneficial to the yield improvement and quality improvement of rape.
Seed size/weight is one of the important yield traits of plants. In angiosperms, mature seeds, including the embryo, endosperm and seed coat, develop from the fertilized egg, fertilized polar nucleus and integument, respectively. The formation of grain weight in crops is a process of cell division to increase cell number, cell elongation to increase volume and photosynthetic product accumulation, and constant enrichment of the contents (Egli et al, 1989; Vibrio, 1995; Guo Wen, 1997; Liberway, 1998; Lina, 2015). In many plants, dry matter accumulation of cotyledons or endosperm is directly related to cell number and grain size (Virginia, 1995; Guaran, 1997; Liberway, 1998; Brocklehurst et al, 1978; Herzog et al, 1982; Guldan et al, 1985; Reddy et al, 1983; Zhang Dashon, 1998; Weilingji, 2004; Lina, 2015). To date, several signaling pathways have been reported to control seed size/weight by affecting endosperm or maternal growth, including: IKU pathway, ubiquitin-proteasome pathway, G-protein signaling pathway, mitogen-activated protein kinase signaling pathway, plant hormones and transcription factors (Li et al, 2016).
In recent years, with the development of molecular marker technology, the QTL positioning of thousand seed weight of rape has made great progress. Quijuda et al (2006) identified 3 QTLs for grain weight in 2-point 4-population experiments at 2 years (N7, N17 and N19), but none of the QTLs were repeatedly detectable in multiple populations. Udall et al (2006) identified 6, 4 and 5 QTLs in the Hua-DH, SYN-DH and one test cross population, respectively, and found that QTLs located in the N14 linkage group could be stably inherited in different populations and environments. Shi et al (2009) identified 159 thousand grain weight QTLs in 10 natural environments using a TNDH population and an RC-F2 population, but only 4 major QTLs and only one QTL-qsw.a7-2 could be detected in 10 environments. Fan et al (Fan et al.2010) constructed a DH population and an F2 population for thousand kernel weight QTL mapping. Of the 9 thousand-grain-weight QTLs, two major QTLs-TSWA 07a and TSWA07b could be detected repeatedly in different years, accounting for 27.6-37.9% of phenotypic variation, and the two major QTLs could be detected simultaneously in the DH population and the F2 population, confirming the important role of the QTL on thousand-grain weight. Yang et al (2012) mapped QTLs with 9 grain weights using a population of recombinant inbred lines containing 186 lines, in which a major QTL-cqSWA9 located on chromosome A09 explained the phenotypic variation of 28.2%, and the QTL and the major QTL-cqSLA9 controlling the length of silique were located in the same interval, suggesting that the interval may contain genes controlling both thousand-grain weight and silique length, or that there are genes affecting both thousand-grain weight and silique length.
Although studies have identified a collection of QTLs that affect grain weight and silique length at present, there are fewer functional molecular markers for rape grain weight and silique length and no markers that can distinguish between high grain weight in rape seeds have been found.
Disclosure of Invention
The molecular marker solves the problems that the grain weight and the silique length in the conventional breeding method can only be identified in the later period, so that the breeding period is long, the environmental influence is easy to cause, and the selection efficiency is low, and the breeding efficiency of the rape is improved.
A molecular marker closely related to the grain weight and the length of the silique of rape is characterized in that the molecular marker is a molecular marker of a gene BnaA.ARF18a, and the nucleotide sequence is a nucleotide sequence shown in SEQ ID No.1 or a nucleotide sequence of an amino acid sequence which is replaced, deleted or added with one or more nucleotides and has the same or similar codes and the same function.
Further, the gene bnaa. arf18a contains 9 SNPs, and the initiation codon ATG is defined as +1, wherein 6 SNPs are located in exon regions +186, +193, +262, +1045, +1303, +1345, and 3 SNPs are located in intron regions +59, +931, +1429, and the 9 SNPs constitute three haplotypes of Hap a, Hap B, and Hap C in different individual DNAs, the nucleotide sequence of Hap a type is shown in SEQ ID No.1, the nucleotide sequence of Hap B type is shown in SEQ ID No.2, and the nucleotide sequence of Hap C type is shown in SEQ ID No. 3.
Of the three haplotypes, the thousand grain weight (3.02. + -. 0.29g) and the silique length (5.09. + -. 0.35cm) of the material containing Hap A were the lowest, the largest was the material containing Hap C (4.05. + -. 0.46 g; 7.37. + -. 1.81cm) and the material containing Hap B was at an intermediate level (3.44. + -. 0.42 g; 5.38. + -. 0.36 cm).
Further, the molecular markers are primer pairs designed based on SNPs that differ between the three haplotypes.
Further, the primer pair is BnaA. ARF18a-186 or/and BnaA. ARF18a-262, and the primer pair
The nucleotide sequence of the forward primer of BnaA.ARF18a-186 is shown as SEQ ID No.4, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 5; the nucleotide sequence of the forward primer of BnaA. ARF18a-262 is shown as SEQ ID No.6, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 7.
The application of the molecular marker in rape breeding is also the protection scope of the invention, and the rape breeding comprises breeding methods such as molecular marker-assisted selective breeding, gene polymerization breeding, transgenic breeding and the like.
The invention also provides a method for applying the molecular marker in rape breeding, which comprises the following steps:
(1) establishing a PCR amplification system: PCR amplification is carried out by adopting any one or two pairs of primer pairs BnaA.ARF18a-186 and BnaA.ARF18a-262 and taking the DNA of the Brassica napus individual plant as a template.
(2) Carrying out enzyme digestion on the PCR amplified product:
if a primer pair BnaA. ARF18a-186 is used for amplification, the amplification product is subjected to EcoRV enzyme digestion and electrophoretic separation, and a band with the length of 211bp cannot be observed, the single plant is a high-grain-weight and silique single plant, and the single plant is not a high-grain-weight and silique single plant if a band with the length of 211bp is observed.
If a primer pair BnaA. ARF18a-262 is used for amplification, an amplification product is subjected to BsmAI enzyme digestion and electrically separated, and a 125 bp-long band is observed, the single plant is a low-grain-weight and silique single plant, and if the 125 bp-long band cannot be observed, the single plant is not a low-grain-weight and silique single plant.
If primer pairs BnaA.ARF18a-186 and BnaA.ARF18a-262 are respectively used for PCR amplification, the products amplified by the BnaA.ARF18a-186 are cut by EcoRV enzyme by the primers, the products amplified by the BnaA.ARF18a-262 are cut by BsmaI enzyme by the primers and are respectively electrophoretically separated, a band with the length of 211bp cannot be observed in the products cut by the EcoRV enzyme, and the single strain is a high-grain-weight and silique single strain; a band with the length of 125bp is observed in a BsmAI enzyme digestion product, and the single plant is a low grain weight and silique single plant; a211 bp band was observed in the EcoRV digested product and no 125bp band was observed in the BsmAI digested product, then the individual was of medium grain weight and medium length siliques.
The breeder can predict, screen or identify the grain weight and the silique length of a single plant according to needs, and selects DNA of any one or more pairs of the primer pairs to carry out amplification and enzyme digestion, thereby obtaining the grain weight and the silique length of the single plant.
The invention has the beneficial effects that:
the molecular marker of the invention can rapidly and reliably identify the grain weight and the silique length of single plants of different varieties of rapes in the seedling stage of the rapes, improve the breeding efficiency of the rapes and greatly shorten the breeding period of the rapes; by adopting the molecular marker for identification and selection, the grain weight and the silique length of the population can be respectively improved by 17 percent and 34 percent, and the workload of later-stage test and identification is reduced by more than 33 percent.
Drawings
FIG. 1 is the results of the correlation analysis of the grain weight (top) and silique length (bottom) of Brassica napus, where the abscissa is the physical distance of the chromosomes and the ordinate is the-log of each SNP site10(P) number, dotted horizontal line represents significant-log10(P) the value, thousand kernel weight 6.02, silique length 6.3;
fig. 2 is a diagram showing the results of haplotype analysis of bnaa. arf18a in the natural population, wherein (a) the structure of the bnaa. arf18a gene and three different haplotypes consisting of SNPs sites are shown, and gray boxes and black horizontal lines represent exons and introns, respectively. (b) Phenotypic differences in thousand kernel weight and silique length between the three different haplotypes, asterisks indicate significance of differences between Hap B and Hap C and Hap a,. p.ltoreq.0.05,. p.ltoreq.0.001;
figure 3 is a development of two pairs of dCAPS markers in bnaa. arf18a, the dCAPS marker sequences are shown in boxes, the numbers in brackets indicate the position of the gene sequence, the blue letters indicate SNPs between different haplotypes, and the red letters indicate the bases that were altered during the design of the dCAPS marker to introduce a cleavage site.
FIG. 4 is a schematic diagram showing the detection of dCAPS markers in materials of different grain weights and pod lengths, wherein 1-5 are the Hap A materials in the natural population, 6-10 are the Hap B materials in the natural population, and 11-15 are the Hap C materials in the natural population.
Detailed Description
The following is a specific embodiment of the process of the invention, but it is not intended to limit the process of the invention, and any variations that do not depart from the gist of the invention are intended to be within the scope of the invention.
Example 1:
molecular markers BnaA.ARF18a-186 and BnaA.ARF18a-262 closely related to the grain weight and the length of the pod of rape are obtained by the following method:
in this example, 157 parts of rape varieties with different genetic sources and large variation in grain weight and silique length are taken as examples to construct a natural population, and the method for obtaining molecular markers closely related to the grain weight and the silique length of rape is described in detail as follows:
(one) population construction:
157 natural populations composed of rape varieties with different genetic sources and large variation in grain weight and silique length (1.75-5.71g/4.02-10.62cm) are taken as research materials (collected worldwide).
(II) measuring the grain weight and the silique length:
(1) planting plant materials in the field, wherein each plant line has 3 rows, each row has 8-10 plants, and the random block design is adopted; sowing seeds in a seedbed in the Yangtze river basin in 9 months generally, and transplanting the seeds to a field in the last ten days of 10 months; performing field management by a conventional method until seeds are mature (5 months and early in the next year);
(2) at the mature stage, 5 individuals are randomly selected from each strain, and 20 pods with no obvious difference are sequentially taken from the bottom of the main inflorescence upwards, so that each strain has 100 pods.
Measuring length: the siliques of rape are not perfectly straight, and most of them are bent. Taking a long cotton thread, aligning the bottommost end (without a stem) of a first silique with the starting point of the cotton thread, extending the cotton thread along the direction of the silique forward until the front end of the beak of the silique is the end point of the cotton thread, namely, only taking the length of the silique body, recording the value from the starting point to the end point of the cotton thread, wherein the value is the length value of a single silique, and taking the average value of 100 siliques is the length (cm) of the silique of each variety.
Measuring the thousand seed weight: and (3) putting the seeds harvested from each strain in an oven for drying, randomly counting 1000 seeds from the seeds, weighing the seeds, repeating the weighing for 3 times, and taking the average value of the 3 measurements to obtain the grain weight (g) of each variety.
(III) DNA extraction
For all plant research materials, young leaves are collected in the seedling stage, and the genomic DNA is extracted by adopting the experimental step of 'CTAB method for extracting plant genomic DNA'.
(1) Collecting appropriate amount of tender leaves, and adding liquid N2Grinding into powder, and placing 0.4g into a 2mL centrifuge tube (precooling at-20 ℃).
(2) Preheating 2% CTAB (sterilizing) to 70 deg.C, adding 700 μ L into centrifuge tube containing leaf powder (optionally adding 14 μ L of beta-mercaptoethanol to prevent oxidation), and mixing (preventing freeze thawing).
(3) Immediately placing in 65 deg.C water bath for 45min, and reversing 1 time every 5 min;
(4) an equal volume (600. mu.L) of chloroform/isoamyl alcohol (24:1) was added thereto, and the mixture was inverted several times until the lower liquid phase became dark green. Centrifuge at 12000g for 5 minutes.
(5) The supernatant was taken and repeated 4 times (optionally omitted).
(6) mu.L of the supernatant was put into a new 2mL centrifuge tube, and 1mL of 95% ethanol and 20. mu.L of 10M NH4AC were added, mixed, and left at room temperature for 10 min.
(7) Centrifuging at 12000g for 10min, removing supernatant, washing precipitate with 75% anhydrous ethanol, and naturally drying for about 30 min.
(8) Add 50. mu.L of 0.5TE or sterile water (20. mu.g/RNase), stand overnight at 4 ℃ and after the DNA is dissolved, measure the DNA concentration and quality.
(IV) genome Re-sequencing
The DNA samples were sent to Peking Baimaike for sequencing. The experimental procedures are performed according to standard protocols provided by Illumina corporation, and include sample quality detection, library construction, library quality detection, library sequencing and the like. After the sample genome DNA is qualified, fragmenting the DNA by using a mechanical breaking method (ultrasonic), then carrying out fragment purification, end repairing, adding A at the 3' end, connecting a sequencing joint, carrying out fragment size selection by using agarose gel electrophoresis on the fragmented DNA to carry out PCR amplification to form a sequencing library, carrying out library quality inspection on the built library, sequencing the library qualified by quality inspection by using Illumina HiSeqTM 4000, wherein the sequencing length is PE125, and the sequencing depth is 5.
And filtering the original data obtained by sequencing to obtain clear Reads. Clear reads were aligned with the rape reference genome Brassica _ napus.annotation _ v5(http:// www.genoscope.cns.fr/branched/data /) using BWA (http:// bio-bw. sourceforce. net /) software; SNP and Indel detection are carried out by using GATK software, and the specific process refers to BestPractice of a GATK official website:
https:// www.broadinstitute.org/gatk/guide/best-primers. Deletion >0.6 sites, sites with deletion less than 0.6 were filled with the software beacon v4.1(https:// failure. washington. edu/browsing/beacon. html. download). Finally, sites with a heterozygosity rate greater than 0.25 and a Minimum Allele Frequency (MAF) less than 0.05 were removed, and the remaining sites were used for whole genome association analysis.
(V) correlation analysis
GWAS analysis (Team et al, 2014) is carried out by utilizing an mrMLM v1.3 software package of R language, a mixed linear model of Q + K is adopted to detect the associated sites of the traits and the markers, the threshold value of the mrMLM is set to be P < -log10(0.05/me) (me is the number of effective markers, and the specific number refers to a software analysis method), and the threshold value of the mrMLM is set to be LOD > 3. The results of rMLM are shown in Manhattan plots and Q-Q plots by the R software package qqman (https:// cran.r-project. org/web/packages/qqman /). An interval with significant association of grain weight and silique length was detected at 27.78Mb-28.61Mb of chromosome a09 (panel one).
(VI) candidate Gene prediction
The significant SNP positions were extended up and down by 100kb each or in the same haplotype region (r) as the selection and significant SNP2Interval > 0.5), we define such interval as a candidate association interval. Candidate genes were predicted in this interval with reference to the following conditions: 1) referencing results of reported QTL mapping; 2) SNPs fall directly inside genes and cause amino acid changes; 3) known functions related to traits on brassica napus or arabidopsis reference genomesThe gene of (1). One gene, BnaA. ARF18a (Liu et al, 2015), which has been reported to be related to grain weight and silique length of oilseed rape was selected according to the above method.
(VII) conversion and detection of functional markers
The inside of the BnaA. ARF18.a gene contains 9 SNPs in total, 3 SNPs are located in an intron, 6 SNPs are located in an exon, and only 2 SNPs cause amino acid changes; the 9 SNPs can form 3 different types of haplotypes (Haplotype), Hap A, Hap B and Hap C (FIG. two) among different materials. Of the three haplotypes, the thousand grain weight (3.02. + -. 0.29g) and the silique length (5.09. + -. 0.35cm) of the material containing Hap A were the lowest, the largest was the material containing Hap C (4.05. + -. 0.46 g; 7.37. + -. 1.81cm) and the material containing Hap B was at an intermediate level (3.44. + -. 0.42 g; 5.38. + -. 0.36 cm). Two pairs of dCAPS markers are designed according to SNP information different between three haplotypes (primer design is that a new restriction enzyme action site can be introduced by combining an SNP site on the basis of CAPS markers by introducing mismatched bases in amplification primers according to the principle of dCAPS markers to generate polymorphism similar to the CAPS markers; here, we use an online website dCAPS Finder 2.0(http:// helix.wustl.edu/dCAPS/dcaps.html): the forward primer sequence of BnaA. ARF18a-186 is 5'-ATGGCGAATGTAGATGGAGAT-3', and the reverse primer sequence is 5'-CTCTGTGTACAGCTGATCTTGATAT-3'; ARF18a-262 forward primer sequence 5'-GTTGTTTGTTTGATTTTCAGGTT-3', reverse primer sequence 5'-GACCCTGAGGGAAGTAGAAAAGTCT-3' (figure three).
According to the results of haplotyping in the natural population, 5 different lines (1-5: Hap A, 6-10: Hap B, 11-15: Hap C) were selected for each haplotype and PCR amplification was carried out using two pairs of dCAPS markers, and the PCR products were digested with the corresponding restriction enzymes. The results of 3% agarose gel electrophoresis show: as the experiment imagined, EcoRV can cut the BnaA. ARF18a-186PCR product of materials No. 11-15 but can not cut the PCR product of materials No. 1-10; BsmAI is capable of cleaving the BnaA. ARF18a-262PCR product of materials No. 6-15 but not the PCR product of materials No. 1-5 (FIG. four). By selection of two pairs of dCAPS markers, one can select the optimal haplotype for thousand kernel weight and silique length.
Example two:
the application of the molecular marker closely related to the grain weight and the silique length of the rape in the high-yield breeding of the rape comprises the following steps:
the method is characterized in that European winter variety Express (low grain weight silique) and Chinese semi-winter variety House7 (high grain weight silique) are used as parents, a DH separation population is constructed as a research material, and the detection range is further expanded by using two dCAPS markers, namely BnaA.ARF18a-186 and BnaA.ARF18a-262, and the detection method comprises the following steps:
(1) DH colony and parent DNA are extracted by the same method as in the second embodiment.
(2) The haplotype types of the parents of the DH group are firstly detected, and the PCR process is carried out by using the following primers:
the forward primer sequence of BnaA. ARF18a-186 is 5'-ATGGCGAATGTAGATGGAGAT-3', and the reverse primer sequence is 5'-CTCTGTGTACAGCTGATCTTGATAT-3';
ARF18a-262 forward primer sequence was 5'-GTTGTTTGTTTGATTTTCAGGTT-3', reverse primer sequence was 5'-GACCCTGAGGGAAGTAGAAAAGTCT-3'
(3) And (3) PCR reaction system: the total volume is 25 mu L, and the specific components are as follows:
(4) PCR amplification procedure: 5min at 94 ℃, 45s at [94 ℃, 45s at 55 ℃, 1min at 72 ℃ and x 35 cycle, 10min at 72 ℃. After the operation is finished, the product is stored at 4 ℃.
(5) Enzyme digestion of PCR amplification products: each 10. mu.l was treated with 2.5U of the corresponding restriction enzymes EcoRV and BsmAI at 37 ℃ for 1-16 h.
(6) After the 2 enzyme digestion products are separated by 3 percent agarose gel electrophoresis, the Express only has a strip with 211bp and a strip with 125bp, which indicates that the Express can not be digested by EcoRV and BsmAI, namely the Express is a monomer type A (hap A); house7 shows a 188bp (23bp too small to be seen) and a 98bp (27bp too small to be seen) band, indicating that House7 can be cut by EcoRV and BsmAI, i.e., House7 is haplotype C (hap C). Then only two haplotypes, hapa and Hap C, are present in the DH population, and only one primer can be used to distinguish between different phenotypes of material.
(7) The same method is adopted to carry out detection in a DH population, and the results show that: the number of lines in the DH population where the allele (Hap A) is derived from the parent 'Express' is substantially equal to the number of lines in the DH population where the allele (Hap C) is derived from the parent 'House 7'; all lines containing Hap C have mean values of thousand kernel weight and silique length of 3.72 +/-0.14 g and 6.21 +/-0.42 cm respectively, which are obviously higher than the mean values of all lines containing Hap A (3.22 +/-0.1 g and 5.13 +/-0.25 cm); both thousand grain weight and silique length have significant difference (P is less than or equal to 0.001). By selecting the individual plant containing Hap C, the high-yield rape variety can be further and quickly cultivated.
The above results fully demonstrate that these two dCAPS markers can indeed be used for the prediction, identification and screening of oilseed rape grain weight and silique length.
SEQUENCE LISTING
<110> university of southwest
<120> molecular marker closely related to rape grain weight and silique length and application
<130> 20180420
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 2622
<212> DNA
<213> Brassica napus
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aggttcttat caagatcagc tgtacacaga gctatggaaa gcctgtgcag gtccattagt 240
ggaggttcct cttgttggag aaagagtttt ctacttccct cagggtcaca tggaacaagt 300
atgtcttttt taccaaatta atcttttttt tttttaaact ccaattctct ttctgaattg 360
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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 (3)
1. A method for the prediction, identification and screening of rape grain weight and silique length, comprising the steps of:
(1) establishing a PCR amplification system: using a primer pairBnaA.ARF18a-186, performing PCR amplification by taking DNA of the Brassica napus single plant as a template;BnaA.ARF18athe nucleotide sequence of the forward primer of-186 is shown as SEQ ID No.4, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 5;
(2) enzyme digestion and electrophoresis of PCR amplification products: performing PCR amplification on the product obtained in the step (1)EcoWhen the single plant is subjected to RV enzyme digestion and electrophoretic separation, if a band with the length of 211bp cannot be observed, the single plant is a high-grain-weight and silique single plant, and if a band with the length of 211bp is observed, the single plant is not a high-grain-weight and silique single plant.
2. A method for the prediction, identification and screening of rape grain weight and silique length, comprising the steps of:
(1) establishing a PCR amplification system: using a primer pairBnaA.ARF18a-262, performing PCR amplification by using DNA of the Brassica napus single plant as a template;BnaA.ARF18athe nucleotide sequence of the forward primer of-262 is shown as SEQ ID No.6, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 7;
(2) enzyme digestion and electrophoresis of PCR amplification products: performing PCR amplification on the product obtained in the step (1)BsmAI enzyme digestion, electrophoretic separation, if 125b can be observedThe p-length band indicates that the plant is a low grain weight and silique plant, and if a 125 bp-length band is not observed, the plant is not a low grain weight and silique plant.
3. A method for the prediction, identification and screening of rape grain weight and silique length, comprising the steps of:
(1) establishing a PCR amplification system: using a primer pairBnaA.ARF18a-186 andBnaA.ARF18a-262, respectively performing PCR amplification by using DNA of a single cabbage type rape plant as a template;BnaA.ARF18athe nucleotide sequence of the forward primer of-186 is shown as SEQ ID No.4, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 5;BnaA.ARF18athe nucleotide sequence of the forward primer of-262 is shown as SEQ ID No.6, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 7;
(2) enzyme digestion and electrophoresis of PCR amplification products: to pairBnaA.ARF18a-186 product of amplification UsingEcoRV enzyme digestion, onBnaA.ARF18a-262 using the amplified productBsmAI digestion, separation by electrophoresis, inEcoA 211bp long band cannot be observed in the product obtained by the enzyme digestion of RV, and the single plant is a high grain weight and silique single plant; in thatBsmA 125bp long band is observed in the product of AI enzyme digestion, and the single plant is a single plant of low grain weight and silique; in thatEcoA211 bp band was observed in the RV-digested product andBsmif a 125bp band is not observed in the AI digestion product, the individual is a medium grain weight and medium length silique.
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| 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 |
| CN110184373B (en) * | 2019-04-16 | 2020-06-05 | 中国农业科学院油料作物研究所 | Molecular marker associated with thousand seed weight of rape and application thereof |
| CN115029465B (en) * | 2022-01-27 | 2023-03-24 | 淮阴师范学院 | KASP and dCAPS markers coseparated with rape seed secondary dormancy main effect QTL 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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| CN106317211A (en) * | 2015-07-02 | 2017-01-11 | 中国农业科学院油料作物研究所 | Rape grain weight related gene ARF 18 and application thereof |
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