CN108396075B - The application of molecular labeling pPGPseq2A5 relevant to peanut oil content - Google Patents
The application of molecular labeling pPGPseq2A5 relevant to peanut oil content Download PDFInfo
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Abstract
The present invention provides the application of molecular labeling pPGPseq2A5 relevant to peanut oil content, utilize the primer (SEQ ID NO:1-2) of molecular labeling pPGPseq2A5, PCR amplification is carried out to the complete genome DNA of A.duranensis wild-type peanut material to be identified, amplified production carries out electrophoresis detection, determines that the seed of peanut material to be measured has higher or lower oil content according to electrophoresis result.Peanut material is detected using the molecular labeling, the height of peanut seed oil content can be predicted in seedling stage, greatly accelerate the breeding process of peanut.
Description
Technical field
The invention belongs to molecular genetic breeding fields, specifically, being related to molecular labeling relevant to peanut oil content
The application of pPGPseq2A5.
Background technique
Peanut is the highly important oil crops in China, industrial crops and export crop, in China's agricultural production
It has a very important role.Every year, about the 60% of China's peanut total output accounts for China's domestic vegetable oil total amount for extracting oil
26% or so.According to measuring and calculating, the peanut raw material that extracts oil is every to improve 1 percentage point, is equivalent to 2 percentage points of output increased, processing benefit
It can be improved 7%.Currently, the oil content of the main peanut varieties in China is 45%~55%, and Average oil concentration 50.6%, cultivar
The narrow genetic diversity of peanut limits further increasing for its oil content.Jiang Huifang etc. contains 87 parts of wild-type peanut resources
Oil mass analysis shows: average value 55.82%, range of variation be 51.44%~62.9%；Wild-type peanut resource material oil content
No matter peak, average value or range of variation it is all bigger than cultigen peanut.Therefore, it will be controlled in wild-type peanut resource high
The channel genes of oil content are the effective ways for improving existing kind oil content into cultivation peanut varieties.But since peanut contains
Oil mass is more complicated quantitative character, affected by environment big, therefore measures oil content by conventional method, screens floorboard with high oil content
The effect of material is bad, accuracy is poor, efficiency is lower.
Molecular mark based on Molecular Marker Assisted Selection Technology is the importance of crop molecular breeding,
The individual containing target gene can be rapidly and efficiently obtained, be widely used in breeding and is obtained in certain characters
Success.SSR (Simple Sequence Repeats) label is one kind developed in recent years using specific primer PCR as base
The molecular marking technique of plinth, also referred to as microsatellite DNA (Microsatellite DNA) are a kind of (general by several nucleotide
Be 1~6) be repeat unit group at the tandem repetitive sequence up to tens nucleotide.Compared with other molecular labelings,
SSR marker has the advantage that (1) quantity is abundant, covers whole gene group, and the polymorphism of announcement is high；(2) there are more equipotential bases
The characteristic of cause, the information content provided are high；It (3) is in codominance with Mendelian fashion heredity.
Currently, with the exploitation of the molecular labeling of wild-type peanut oil content close linkage it is few, using less.
Summary of the invention
The object of the present invention is to provide the applications of molecular labeling pPGPseq2A5 relevant to peanut oil content.
In order to achieve the object of the present invention, the present invention provide molecular labeling pPGPseq2A5 relevant to peanut oil content with
Under any application:
I) identification and breeding floorboard with high oil content peanut varieties in application；
Ii) the application in floorboard with high oil content peanut varieties early screening；
Iii) the application in peanut molecular mark.
For it is above-mentioned i) and ii), using the peanut material to be measured of primer pair shown in SEQ ID NO:1-2 genomic DNA into
Row PCR amplification, amplified production carry out electrophoresis detection and determine peanut material to be measured if only there are the characteristic bands of 290bp size
Seed oil content with higher, if only there are the characteristic bands of 260bp size, determine peanut material to be measured seed tool
There is lower oil content.
Peanut of the present invention includes peanut wild species Arachis duranensis.
Molecular labeling pPGPseq2A5 of the present invention is prepared by the following:
(1) investigate and count peanut wild species --- the oil content of 6 parts of resources of A.duranensis, number and oil-containing
Amount is respectively as follows: WH4416 (51.2%), WH4417 (52.3%), WH4340 (59.4%), WH4396 (60.3%), WH4399
(60.1%) and WH4414 (59.5%)；
(2) with the leaf DNA of 6 parts of materials in CTAB method extraction step (1)；
(3) it selects and expands with the DNA in 40 pairs of SSR primer pair steps (2) in the linkage group of oil content related gene
Increase；
(4) polyacrylamide gel electrophoresis is carried out to the amplified production in step (3), counted 6 under different SSR primer amplifications
The amplified fragments size of part material；
(5) the SSR banding pattern comprehensive analysis that will be counted in the oil content result counted in step (1) and step (4), filters out
Banding pattern is more single, different in high and low oil content storeroom banding pattern, but banding pattern is identical in all floorboard with high oil content, while low
Banding pattern also identical SSR marker --- pPGPseq2A5 in oil content material.In the label, floorboard with high oil content material WH4340,
WH4396, WH4399, WH4414 institute amplified fragments size are 290bp, and are amplified in low oil content material WH4416, WH4417
Clip size be 260bp.
The SSR molecular marker pPGPseq2A5 is located at the position of peanut A8 chromosome 39.9Mb, and SSR sequence is
(TAA)19。
The present invention also provides the following any of the SSR molecular marker pPGPseq2A5 primer (SEQ ID NO:1-2)
Using:
I ') identification and breeding floorboard with high oil content peanut varieties in application；
Ii ') application in floorboard with high oil content peanut varieties early screening；
Iii ') preparation is for identifying and the detection reagent of breeding floorboard with high oil content peanut varieties or the application in kit.
For above-mentioned i ') and ii '), utilize the genomic DNA of the peanut material to be measured of primer pair shown in SEQ ID NO:1-2
PCR amplification is carried out, amplified production carries out electrophoresis detection and determines peanut material to be measured if only there are the characteristic bands of 290bp size
The seed of material oil content with higher determines the seed of peanut material to be measured if only there are the characteristic bands of 260bp size
With lower oil content.
Application above-mentioned, the reaction system of PCR amplification are 10 μ L, including Trans 2 × EcoTaq PCR SuperMix
10~20ng of DNA profiling of (containing dyestuff) 5 μ L, peanut material to be identified, 0.4 μM of upstream primer, 0.4 μM of downstream primer.
The condition of pcr amplification reaction are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, 65 DEG C of renaturation 45s, each cycle down
Low 1 DEG C, 72 DEG C of extension 45s, totally 10 recycle；94 DEG C of denaturation 30s, 55 DEG C of renaturation 45s, 72 DEG C of extension 45s, totally 30 recycle；
Last 72 DEG C of extensions 10min.
By above-mentioned technical proposal, the present invention at least have following advantages and the utility model has the advantages that
The present invention passes through to peanut wild species --- and 6 parts of resources of A.duranensis carry out SSR genetic analysis and oil-containing
It is fixed to measure, and has filtered out the SSR molecular marker pPGPseq2A5 with oil content close linkage, the peanut varieties for floorboard with high oil content
Marker assisted selection.The method of the present invention fast and easy, not affected by environment, purpose is stronger, and workload is small, more efficient,
It is at low cost, by the detection to label pPGPseq2A5, floorboard with high oil content peanut material can be identified and be filtered out in seedling stage, is saved significantly
About production cost and raising efficiency of selection.
Detailed description of the invention
Fig. 1 be the embodiment of the present invention 1 in molecular labeling pPGPseq2A56 wild-type peanut material WH4416, WH4417,
Amplified fragments in WH4340, WH4396, WH4399 and WH4414.
Amplification piece of the difference labeled primer in 6 parts of wild-type peanut materials when Fig. 2 is selection markers in the embodiment of the present invention 1
Section.Wherein, 1-6 is respectively peanut material WH4340, WH4396, WH4416, WH4399, WH4414, WH4417.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW,
Molecular Cloning:a Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
The acquisition of 1 SSR molecular marker pPGPseq2A5 of embodiment
(1) material to be tested: peanut wild species --- A.duranensis material, number and oil content are respectively WH4416
(51.2%), WH4417 (52.3%), WH4340 (59.4%), WH4396 (60.3%), WH4399 (60.1%) and WH4414
(59.5%).(2) leaf DNA of above-mentioned 6 parts of materials is extracted with CTAB method.
(3) it selects and expands with the DNA in 40 pairs of SSR primer pair steps (2) in the linkage group of oil content related gene
Increase.
(4) polyacrylamide gel electrophoresis is carried out to the amplified production in step (3), counted 6 under different SSR primer amplifications
The amplified fragments size of part material.
(5) the SSR banding pattern comprehensive analysis that will be counted in the oil content result counted in step (1) and step (4), filters out
Banding pattern is more single, different in high and low oil content storeroom banding pattern, but banding pattern is identical in all floorboard with high oil content, while low
Banding pattern also identical SSR marker --- pPGPseq2A5 in oil content material.In the label, floorboard with high oil content material WH4396,
WH4399, WH4340, WH4414 institute amplified fragments size are 290bp, and are amplified in low oil content material WH4416, WH4417
Clip size be 260bp (Fig. 1).
The information of 40 pairs of SSR primers is shown in Table 1:
The information (5 ' -3 ') of 1 40 pairs of SSR primers of table
| Primer | Upstream primer | Downstream primer |
| TC9F10 | ATCACAATCACAGCTCCAACAA | GGCAAGTCTAATCTCCTTTCCA |
| AHGS1285 | CATAGACAACAATCATAAAGGTTCAA | TTTGGCTAACAATTTGCTTCAA |
| TC6H03 | TCACAATCAGAGCTCCAACAA | CAGGTTCACCAGGAACGAGT |
| AHGS3201 | CTGTGAAGCAATGGTGCTGT | CAGGGAAATCATTGTCAGGG |
| AHGS1470 | GACACCGCTGGACAAGAAAG | ATCAAAGCAACAACGGGAAC |
| RN18H05 | AATCAAGCTCCAAGATTTCG | CTCAATGCAGAGATCCATGATA |
| AHS1969 | GCTAAACGACGAACCAAAGC | GCGGCTGAGTAGTGGAGTTT |
| TC22C01 | AGACCCTCAACTTCCAGAACTC | CAATGAAGGGCAAACATTATCA |
| RM17H09 | CCCATCACAGTTGGCATATCT | TGTAGTGGATTAGACAGCCCC |
| RM5G08 | ATAGTCCATGATAGCCCCATGT | TTACAACCGAATCTGCAAAGAC |
| TC23H09 | GGAACCAGCTTCACTTTCATCT | TATTCTAATTCCGGTTTCCCTG |
| AHGS2608 | ATCTGCTGTCCAGGAAGCTC | CGCATCAGTATGCCAAAAGA |
| AHGS3212 | CGAGATTGCGATACACCTCA | TCCCTCAACCTCTCAAATCG |
| AHS0661 | GCGATTGGGAGCTTTATTGA | ATGAACTTGGAGCAGAACGG |
| AHGS2151 | AGCGCTAGCAGAGTGTGTGA | GTCAGCAAACCAAATCCACC |
| AHGS1385 | TCCCTTTGCTTGTTACCACA | AATTTTGAAAGCGGGCATAA |
| AHGS1891 | CAGCAACCAGCAACTCAGAA | GCGAACTCTGCACACATCAT |
| AHGS0208 | TGTAATCCTTGCTGTGGTGG | GAAGCCTTCTCACGGTTCTG |
| pPGPseq2A5 | GGGAATAGCGAGATACATGTCAG | CAGGAGAGAAGGATTGTGCC |
| AHGS1578 | ACGGTTGTGAAGGGATCAAG | CCCGTATTTCTTTCACCCAA |
| AHS0116 | ACAAGACCACCACCATAGCC | CGGTTATGAGGGGGAAGATT |
| AHGS1884 | CGGACACATTCTTTCATCCC | TCTCCTTTGTTGTGACGACG |
| AHS2990 | TCTTCTTTTGGGTGTGCCTC | GGGTAAACCCTGTTGGTGTG |
| AHS1582 | TCAACATCACCATCACCACC | AATTCCAACAAATCACCCCA |
| AHGS2185 | TGCTTTCCCGTTACTTTTGG | CTGAAGGGTATGGTACAACCG |
| AHS0608 | CAGTTTGGCTTGGGACAGTT | CCTCTGATTCACGCCTCTTC |
| AHGS3171 | TGATATATTAAAGCATGCAACTAACAA | CCAAAGATGGAGCCATATGAA |
| AHGS2561 | CCTCTCCCTCTCTCCCTCTC | CCGTAATTCGTTTCCCTTCA |
| AHGS1312 | AAGCCCCAATGTTTTCACAG | TGATATCAATCCACACGATGG |
| AHS3040 | AGGCATTGCTAACACGGTTT | AAATCCTAGCTAGAAATAGGGGAAA |
| GM1628 | AAACGTGCTCTAGAAACATACAAAA | CAACACATGCAATGCAACAA |
| AHGS1644 | TCCTGCTAGCTCTCTATCCAGAA | GTGCGAACTGTGGAAATCCT |
| AHS0796 | CCCATCAGAGGGAAAGTTCA | AGAAGTGCACTGGTTGTCCC |
| TC9F04 | CCTAAACAACGACAAACACTCA | AAGCACAACACAGAACCCTAAA |
| AHGS1421 | TGAATGACCACCGACAAAGA | AAACGGAGTGAGAGAAGCCA |
| AHGS3227 | CCAGAGTTGTCTGCCTGTCA | GGTCAAAAACAGCACATTGG |
| AHGS1727 | ACCATCATCATGGCTGGTTG | TTCAGCTCAACAGTCGCATT |
| AHS0039 | AACACATCACTTTCCTTTTCCA | GGAATGAAGGTGTGTGAGCA |
| AHGS3163 | CAAATCTTACCCTACCAATTCCA | TAGCGGCCACCCTATATGAC |
| AHS2019 | GGAATTCTCTCAAGGCCTCC | ATAAGATGGAGTGTTCGCCG |
Amplification of the different SSR label primers in 6 parts of wild-type peanut materials is shown in Fig. 2.Figure it is seen that primer
To in the band of pPGPseq2A5 amplification, low oil content material WH4416, WH4417 banding pattern is consistent, and four parts of floorboard with high oil content materials
WH4340, WH4396, WH4399 are consistent with WH4414 banding pattern, and two kinds of banding patterns have more apparent difference；And except the primer pair with
Other outer 39 pairs of primer institute's amplified bands do not have the notable feature.
The application of the label of embodiment 2 pPGPseq2A5
Material to be tested: peanut wild species --- A.duranensis material, number WH4396, WH4398 extracts the above material
The complete genome DNA of material.
Genotype identification: PCR is carried out using complete genome DNA of the molecular labeling pPGPseq2A5 to WH4396 and WH4398
Amplification, amplified production detect clip size using polyacrylamide gel electrophoresis.
Phenotypic evaluation confirmation: the oil content of material to be detected is measured using nuclear magnetic resonance or Soxhlet extraction method.Compare base
Because type qualification result is found, the segment of 290bp is amplified in WH4396 (oil content 60.3%), and in WH4398 (oil content
52.6%) segment of 260bp is amplified in.
Therefore, molecular labeling pPGPseq2A5 can accurately distinguish peanut wild species --- high oil-containing in A.duranensis
Amount and low oil content material, floorboard with high oil content material amplify 290bp segment, and low oil content material amplifies 260bp segment.
Wherein, PCR reaction system is 10 μ L, including Trans 2 × EcoTaq PCR SuperMix (+dye) 5 μ L, gene
Group 10~20ng of DNA profiling, 0.4 μM of upstream primer, 0.4 μM of downstream primer.
PCR reaction condition are as follows: 94 DEG C of initial denaturation 3min；94 DEG C of denaturation 30s, (each circulation reduces by 1 to 65 DEG C of renaturation 45s
DEG C), 72 DEG C of extension 45s, totally 10 circulation；94 DEG C of denaturation 30s, 55 DEG C of renaturation 45s, 72 DEG C of extension 45s, totally 30 recycle；Most
72 DEG C of extension 10min afterwards, 4 DEG C of heat preservations.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or is improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Inst. of Oil Crops, Chinese Academy of Agriculture
<120>application of molecular labeling pPGPseq2A5 relevant to peanut oil content
<130> KHP181111966.4
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gggaatagcg agatacatgt cag 23
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
caggagagaa ggattgtgcc 20
Claims (3)
1. the following of primer for detecting molecular labeling pPGPseq2A5 relevant to wild species or peanut oil content any is answered
With:
I) identification and breeding floorboard with high oil content wild-type peanut kind in application；
Ii) the application in floorboard with high oil content wild-type peanut kind early screening；
For it is above-mentioned i) and ii), utilize the peanut material to be measured of primer pair shown in SEQ ID NO:1-2 genomic DNA carry out PCR
Amplification, amplified production carry out electrophoresis detection and determine the kind of peanut material to be measured if only there are the characteristic bands of 290bp size
Son oil content with higher, if only there are the characteristic bands of 260bp size, determine the seed of peanut material to be measured have compared with
Low oil content.
2. application according to claim 1, which is characterized in that the condition of pcr amplification reaction are as follows: 94 DEG C of initial denaturation 3min；
94 DEG C of denaturation 30s, 65 DEG C of renaturation 45s, each circulation reduce by 1 DEG C, 72 DEG C of extension 45s, totally 10 circulations；94 DEG C of denaturation 30s, 55
DEG C renaturation 45s, 72 DEG C of extension 45s, totally 30 circulations；Last 72 DEG C of extensions 10min.
3. application according to claim 1 or 2, which is characterized in that the peanut is peanut wild species Arachis
duranensis。
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| CN112080578B (en) * | 2020-09-24 | 2022-11-18 | 中国农业科学院油料作物研究所 | Molecular marker linked with major QTL (quantitative trait loci) of peanut oil content and application thereof |
| CN112094937B (en) * | 2020-09-27 | 2022-05-17 | 中国农业科学院油料作物研究所 | SNP molecular marker related to pod and seed size on peanut A06 chromosome and application thereof |
| CN112980993B (en) * | 2021-04-09 | 2022-04-08 | 中国农业科学院油料作物研究所 | SNP molecular marker linked with major QTL site qPSIIB10 for resisting aspergillus flavus infection of peanuts and application thereof |
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