CN100410392C - Auxiliary method for screening high-oil-content soybean varieties and dedicated primer therefor - Google Patents
Auxiliary method for screening high-oil-content soybean varieties and dedicated primer therefor Download PDFInfo
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- CN100410392C CN100410392C CNB2006100762509A CN200610076250A CN100410392C CN 100410392 C CN100410392 C CN 100410392C CN B2006100762509 A CNB2006100762509 A CN B2006100762509A CN 200610076250 A CN200610076250 A CN 200610076250A CN 100410392 C CN100410392 C CN 100410392C
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Abstract
The present invention discloses an auxiliary method for sieving high-oil soybean varieties and a special primer of the method. The auxiliary method for sieving the high-oil soybean varieties uses genomic DNA of soybeans to be tested as a template, and a pair of primers composed of a nucleotide sequence of a sequence 1 and a nucleotide sequence of a sequence 2 in a sequence list are used for carrying out PCR amplification. If 450 to 500 bp of DNA segments are obtained in the soybeans to be tested, the soybeans to be tested are candidate high-oil soybean varieties.
Description
Technical field
The present invention relates to the method and the primer special thereof of a kind of assisting sifting high-oil-content soybean varieties in the genetically engineered field.
Background technology
Most of economical character of soybean is that in such polygene system, each gene pairs objective trait only shows little effect effect, does not have tangible dominance, and phenotype is subjected to environmental influence very big by the quantitative character of polygene or QTL control.Thereby adopt traditional breeding approach that these proterties are selected very big difficulty.Molecular marker assisted selection provides new research thinking for the improvement of quantitative character.(Paterson A H such as Paterson, Landon E S, Hewitt J D, et al.Resolution of quantitativeinto Mendelian factors by using acomplete RFLP linkage map.Nature, 1988,335:721-726) think the foundation of High Density Molecular genetic map, make and cover will the control the size minor-polygene of proterties of complete genomic molecule marker and resolve into the Mendelian inheritance factor, and navigate on the karyomit(e) at molecule marker place, and then study according to classical hereditary pattern.This becomes possibility with regard to making to utilize with the closely linked molecule marker of QTL to the improvement that these QTL shift to reach these quantitative characters.
Little satellite is to be core unit's multiple dna sequence dna of repeatedly connecting with a few Nucleotide, is that a kind of simple sequence repeats that (simple sequence repeats, SSR), both sides generally are conserved sequences.Because it has polymorphism height, codominance, detects with PCR and characteristics such as the result is reliable and stable easily, is a kind of very good molecule marker therefore.
Oil content is important quality trait of soybean and quantitative character, utilize molecular marking technique seek with high oil base be emphasis in the Position Research of soybean quantitative character because of chain molecule marker always.
Summary of the invention
The method and the primer special thereof that the purpose of this invention is to provide a kind of assisting sifting high-oil-content soybean varieties.
The primer that is used for the assisting sifting high-oil-content soybean varieties provided by the present invention, name is called Satt160, be made up of forward primer and reverse primer, described forward primer has the nucleotide sequence of sequence 1 in the sequence table, and described reverse primer has the nucleotide sequence of sequence 2 in the sequence table.
The method of assisting sifting high-oil-content soybean varieties provided by the present invention, be that genomic dna with soybean to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 1 and sequence 2 to carry out pcr amplification, obtain the dna fragmentation of 450-500bp as soybean to be measured, then this soybean to be measured is candidate's high-oil-content soybean varieties.
Experiment showed, that the total DNA with 24 soybean varieties strains is a template, to carry out pcr amplification with the Satt160 primer, the coincidence rate of banding pattern that amplification obtains and the banding pattern of high oily parent Hei Nong 45 is 83.33%.Illustrate that the Satt160 primer can be used for the assisted Selection of high-oil-content soybean.The method of assisting sifting high-oil-content soybean varieties of the present invention will play a significant role in the breeding of high-oil-content soybean, for the seed selection high-oil-content soybean provides a kind of system of selection efficiently.
Description of drawings
Fig. 1 is a part SSR primer polymorphism The selection result
Fig. 2 is the pcr amplification result of satt160 primer in 120 individual plants
Fig. 3 is the molecular genetic linkage map of 6 linkage groups
Fig. 4 is the pcr amplification result of satt160 primer in 24 soybean varieties strains and parent
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Soybean varieties strain used among the following embodiment all can obtain from commercial channels.
Embodiment 1, with the high oil base of soybean because of the acquisition of chain molecule marker
1, the screening of polymorphism SSR primer
Breathing out friendship 99-5448-4 with the high-protein soybean strain is that male parent and high-oil-content soybean varieties are deceived farming 45 as maternal, the preparing hybrid combination.Hybrid F
1For the seed chamber planting.F
2Plant in the experiment field for seed, long 3 meters of cell row, 10 centimetres of spacing in the rows obtain F
2: 3Family, totally 120 individual plants try segregating population as confession.By field evaluation and indoor evaluation parent and colony's economical character are investigated.
With reference to Cregan (Cregan P B, Jarvik T.An integrated genetic linkage map ofthe soybean genome.Crop Sci, 1999,35 (5): " the public collection of illustrative plates of soybean " 1464-1490) selected the SSR primer, one of standard of selecting is that the seat is evenly distributed, the 2nd, and gene diversity degree height.Select the basic sequence that covers complete genomic 239 pairs of SSR primers (seeing Table 1) the SSR primer of soybean to obtain in soybean database SoyBase, station address is
Http:// www.soybase.com/
The employed 239 pairs of SSR primers of this experiment of table 1
Be about 2cm from handing over 99-5448-4 and Hei Nong 45 to get size for examination individual plant, two stock plant Kazakhstan respectively for 120 strains the examination segregating population
2Light green blade, place the 1.5ml centrifuge tube, in centrifuge tube, add a small amount of liquid nitrogen, freeze dried blade is smashed to pieces (can directly blade be smashed to pieces during no liquid nitrogen) rapidly with glass stick or stainless steel awl with glass stick, standby.DNA extraction employing SDS method (Guan Rongxia, normal Ru Zhen, Qiu Lijuan. be used for the rapid extraction of the soy bean DNA of ssr analysis. soybean science, 2003,21 (1): 73-74).The SDS extracting solution that adds 400 μ l preheatings in the above-mentioned 1.5ml centrifuge tube that lay-by material is housed (contains 100mmol/L Tris-HCl pH8.5,50mmol/L EDTA pH8.0,500mmol/L NaCl, 2%SDS), 65 ℃ of water-bath 30min, the cooling back adds equal-volume phenol/chloroform (1: 1), fully mixing leaves standstill 10min, the centrifugal 5min of 1000rpm, get supernatant liquor in another new centrifuge tube, add 2 times of centrifugal 2min of volume dehydrated alcohol 6000rpm, abandon supernatant, wash the DNA precipitation once with 70% ethanol, air-dry, add the dissolving of 300 μ l ultrapure waters.
PCR is reflected on the pcr amplification instrument ABI-9700 and carries out.Contain 50-100ng genomic dna template in the 20ul reaction system, 1 * PCR damping fluid, 1.25mM MgCl
2, 0.2mM dNTP, 0.2 μ M SSR primer and 1U Tag archaeal dna polymerase.The PCR response procedures is: 95 ℃ of pre-sex change of 4min earlier; 95 ℃ of 30s again, 47 ℃ of 30s, 72 ℃ of 30s, 35 circulations.Behind 72 ℃ of 10min, in 4 ℃ of preservations.Amplified production separates with sequencing gel (6% polyacrylamide, 8mol/L urea), and silver-colored dyeing technique detects.Detected result shows that all 251 pairs of SSR primers all amplify stable product, and wherein 48 pairs of SSR primers have polymorphism, and the polymorphism primer frequency is 25.1% (Fig. 1), and these 48 pairs of SSR primers are (seeing Table 2).
Table 248 produces the SSR primer of polymorphism pair between the parent
| sat_003 | satt149 | satt193 | satt252 | satt323 | satt485 |
| satt038 | satt150 | satt196 | satt255 | satt336 | satt488 |
| satt046 | satt160 | satt201 | satt259 | satt343 | satt536 |
| satt064 | satt160 | satt204 | satt260 | satt362 | satt551 |
| satt084 | satt167 | satt226 | satt262 | satt388 | satt573 |
| satt121 | satt179 | satt233 | satt269 | satt423 | satt590 |
| satt123 | satt187 | satt239 | satt289 | satt427 | satt592 |
| satt131 | satt191 | satt247 | satt308 | satt440 | satt594 |
2, the compartment analysis of polymorphism SSR mark
With these 48 pairs of polymorphism primers one by one at F
2Launch in the segregating population (120 individual plants), the gained amplified production carries out silver and dyes detection behind electrophoresis, banding pattern and record data that statistics silver dyes.Through x
2Check wherein has 41 SSR to be marked at and is 1: 2: 1 segregation ratio in the colony, accounts for 85.42%, meets codominant marker's hereditary feature, and the pcr amplification result of satt160 primer in 120 individual plants in these 41 SSR marks as shown in Figure 2; There are 7 SSR marks to present inclined to one side separation, 5 black farmings 45 of SSR mark deflection, 2 SSR mark deflections are breathed out and are handed over 00-5448-4, the ratio of separating primer partially is 14.58%, with (Keim such as Keim, P., B.W.Diers, T.C.Olson.and R.C.Shoemaker.1990.RFLP mapping in soybean:Associationbetween marker loci and variation in quantitative traits.Genetics126:735-742.) report is 13.3% close, but is lower than (Zhang Deshui, Dong Wei such as Zhang Deshui, the Huidong prestige, Chen Shouyi, ZhuanBing Chang. make up genome molecule mark linkage map frame diagram with the hybrid F2 colony between the cultivated soybean and wild soybean. Science Bulletin, 1997,42 (2): 1326-1330) Bao Dao (Wu Xiaolei such as 25.0% and Wu Xiaolei, Wang Yongjun, He Chaoying, Chen Shouyi, Gai Junyi, Wang Xuechen. the qtl analysis of soybean Main Agronomic Characters. Acta Genetica Sinica, 2001a, 28 (10): 947-955) 21.7% of report.The heterozygosis rate of each mark is between 39.58%～59.90%, and is average 49.74%, meets F substantially
2The hereditary feature of colony.The inclined to one side separation phenomenon ubiquity of mark is compared with existing report in the segregating population, and the inclined to one side separation marking ratio of this research colony is less, (Wu Xiaolei such as this and Wu Xiaolei, Wang Yongjun, He Chaoying, Chen Shouyi, Gai Junyi, Wang Xuechen. the qtl analysis of soybean Main Agronomic Characters. Acta Genetica Sinica, 2001a, 28 (10): result 947-955) is similar, they think that also from the inclined to one side separation case in dissimilar molecule marker sites, the inclined to one side segregation ratio in SSR site is minimum.
3, SSR construction of genetic atlas
Utilize 48 polymorphism SSR marks of screening, in segregating population, individuality is carried out the SSR seat carry out the allelic variation statistics, the allelic variation identical with the male parent banding pattern is recorded as " A ", the allelic variation identical with maternal banding pattern is recorded as " B ", the heterozygosis banding pattern is recorded as " H ", and the data disappearance is recorded as "-".Application software MAPMAKER/EXPV3.0 (Lander etc., 1987) carries out the structure of collection of illustrative plates.Utilize " Group " to order linkage analysis and the grouping of carrying out between mark (LOD=3.0), the linked marker number sorts less than 8 use " Compare " order, and reference numerals sorts more than or equal to 8 the Ripple order of will reusing.Utilize QTL Cartographer1.1 software to carry out composite interval mapping, LR value 11.5 (LOD value 2.5) is estimated for threshold value positions with effect QTL.The result obtains a molecular genetic linkage map (Fig. 3) that comprises 6 linkage groups, SSR reference numerals on the collection of illustrative plates is 18, does not have chain SSR to be labeled as 30, and the genome length that whole collection of illustrative plates covers is 306cM, mean length is 17cM, the localized requirement of accord with Q TL.And with public collection of illustrative plates (Cregan P B, Jarvik T.An integrated genetic linkage map of the soybean genome.CropSci, 1999,35 (5): 1464-1490) compare, the result shows that 6 linkage groups fully can be with on the linkage group in the public collection of illustrative plates be corresponding.Among Fig. 3, the genetic distance between two SSR marks that the numeral in the linkage group (LG) is adjacent or QTL and the adjacent SSR mark.
Utilize Mapmaker QTL1.1 to carry out QTL location, located the QTL of a control soybean oil content on linkage group F, this QTL is positioned at this interval of satt160-satt193, with the genetic distance of satt160 be 26.0cM, its genetic contribution is 23.2%.
Wherein, the primer sequence of Satt160 is: forward primer: TCC CAC ACA GTT TTC ATA TAA TATA, reverse primer: CAT CAA AAG TTT ATA ACG TGT AGA T.
Embodiment 2, utilize satt160 primer assisting sifting high-oil-content soybean varieties
Experiment material comprises that male parent high-protein soybean strain is breathed out friendship 99-5448-4, maternal high-oil-content soybean varieties is deceived 24 materials in farming 45 and the table 1.
Table 3 is used for the soybean varieties strain of molecular marker assisted selection
| Sequence number | Title | Proterties | Sequence number | Title | Proterties |
| 1 2 3 4 5 6 7 8 9 10 11 12 | Black farming 35 black farming 43 black farming 44 black farming 45 black farming 47 black agricultural 48 Hobbit close rich 25 and close rich 42 and close rich 43 and cultivate farming 4 and cultivate farming 18 | The high innage innage of high protein high protein produces the high innage of high protein and produces high innage innage innage oil | 13 14 15 16 17 18 19 20 21 22 23 24 | The beans 14 M9 M12 M14 of the Liao Dynasty exhale and hand over 251 to exhale and hand among tender rich 17 K97-126 of 03-286 32 7,628 5,186 7622 | High innage innage innage innage innage innage innage innage innage produces the high yield high yield |
The seed of above-mentioned soybean material is got simple grain and is worn into bean powder, places the 1.5ml centrifuge tube respectively, and 4 ℃ of preservations are extracted genomic dna according to the method for embodiment 1.
Breathe out the molecular marker assisted selection of handing over 24 materials in 99-5448-4, the black farming 45 of maternal high-oil-content soybean varieties and the table 3 with male parent high-protein soybean strain, concrete grammar is as follows: utilize primer satt160, according to the method for embodiment 1 DNA of these 26 cultivars and strains is carried out pcr amplification, electrophoresis detection, the result shows that 12 materials are arranged (deceives farming 44, black farming 45 (with P as shown in Figure 4
2With), Hobbit, close rich 25, close rich 43, cultivate farming 4, cultivate farming 18, the Liao Dynasty's beans 14, M14, tender rich 17, in 32,5186) have high oily parent Hei Nong 45 the dna fragmentation that increases out, detect coincidence rate and reached 50%, have only two materials (to close rich 25 in the wherein detected high oil variety, 5186) not the high oil variety strain, coincidence rate in detected high oil variety has reached 83.33%, illustrate with the chain high oil base of this mark of satt160 because of in these 10 parts of materials, finding simultaneously, utilize molecule marker to carry out the purpose of assisted Selection thereby reached.Among Fig. 4, P
1Hand over 99-5448-4, P for breathing out
2Be the kind in black farming 45,1 to 24 correspondence table 3.
Sequence table
<160>2
<210>1
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>1
tcccacacag ttttcatata atata
<210>2
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>2
catcaaaagt ttataacgtg tagat
Claims (1)
1. the method for an assisting sifting high-oil-content soybean varieties, genomic dna with soybean to be measured is a template, use a pair of primer of forming by the nucleotide sequence of the nucleotide sequence of sequence in the sequence table 1 and sequence 2 to carry out pcr amplification, when pcr amplification obtains the dna fragmentation of 450-500bp, then this soybean to be measured is candidate's high-oil-content soybean varieties.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000018963A1 (en) * | 1998-10-01 | 2000-04-06 | Monsanto Company | Methods for breeding for and screening of soybean plants with enhanced yields, and soybean plants with enhanced yields |
| CN1757728A (en) * | 2004-10-10 | 2006-04-12 | 东北农业大学 | Quantitative character gene site related to soybean oil content |
| CN1757729A (en) * | 2004-10-10 | 2006-04-12 | 东北农业大学 | Quantitative character gene site related to soybean oil content |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000018963A1 (en) * | 1998-10-01 | 2000-04-06 | Monsanto Company | Methods for breeding for and screening of soybean plants with enhanced yields, and soybean plants with enhanced yields |
| CN1757728A (en) * | 2004-10-10 | 2006-04-12 | 东北农业大学 | Quantitative character gene site related to soybean oil content |
| CN1757729A (en) * | 2004-10-10 | 2006-04-12 | 东北农业大学 | Quantitative character gene site related to soybean oil content |
Non-Patent Citations (6)
| Title |
|---|
| integration of simple sequence repeat(SSR)markers into amolecular linkage map of common bean(phaseolus vulgaris L). K.Yu et al.The journal of Heredity,Vol.91 No.6. 2000 * |
| Length polymorphisms of simple sequence repeat DNAinsoybean. Akkaya m s et al.the Genetics society of america,No.132. 1992 * |
| Microsatellite fingerprinting and mapping of soybean. Gregan p b et al.Meth Mol Cell Bio,No.5. 1994 * |
| PCR-amplified microsatellites as markers in plant genetics. Morgante m et al.the Plant Journal,Vol.3 No.1. 1993 * |
| 大豆遗传图谱的构建和若干农艺性状的QTL定位分析. 杨喆等.植物遗传资源学报，第5卷第4期. 2004 * |
| 微卫星分子标记在大豆中的应用. 吴俊江.农业系统科学与综合研究，第21卷第1期. 2005 * |
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