CN101368181A - Numerator mark concatenated with rice purple pericladium gene PSH1 (t), acquiring method and application thereof - Google Patents
Numerator mark concatenated with rice purple pericladium gene PSH1 (t), acquiring method and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker of rice purple leaf sheath gene PSH1(t) linkage, and an acquired method and an application thereof, which belong to the field of molecular genetics. The rice non purple leaf sheath variety fragrant japonica 9407 and the purple leaf sheath variety R151 and the F2 which is obtained by hybridization are extracted with isolated population individual DNA. The two parents receives the PCR product polymorphism screening by utilizing 360 pairs of SSR molecular markers, then the screening of the molecular markers of purple leaf sheath gene linkage is carried out sequentially to a F2 mixing pool with purple leaf sheath DNA, a F2 mixing pool without purple leaf sheath DNA and each of the individuals of a F2 population, and the molecular genetic map of the purple leaf sheath gene PSH1(t) is constructed on the first chromosome. The selection efficiency of purple leaf sheath rice is greatly improved through the molecular markers of the major genes of purple leaf sheaths to test whether purple leaf sheath variety R151 and derived varieties (lines) thereof contain the genes of the purple leaf sheaths. The invention is specialized in the screening of the rice purple leaf sheath genes, and is used for the study of cloning the purple leaf sheath gene PSH1 (t).
Description
Technical field
The present invention relates to molecule marker and preparation method and the application chain, belong to the molecular genetics field with rice purple leaf sheath gene PSH 1 (t).
Background technology
Paddy rice is the important food crop of China.Form
Shape plays an important role in the genetic improvement of paddy rice and map construction process.In at present traditional breeding process, the breeder still serves as a mark the morphological characters that is easy to discern and instructs breeding.Along with the development of hybrid rice in China, the purity of hybrid rice seed was identified become more and more important in recent years.Select both to show obviously to be different from positive evergreen leaf in seedling stage, and robust plant, to the little leaf color marker proterties of yield effect such as purple leaf, purple leaf sheath, pale green leaf, import to sterile line, recover system, utilize the leaf color marker to help to detect false cross-fertilize seed in seedling stage, to be a kind of simple and effective method (Cao Liyong etc., 1999 of improving seed purity of hybrid rice; Shen Shengquan etc., 2004).
Studies show that purple leaf sheath has very important potential advantages in the evaluation as the important economical character of paddy rice cross-fertilize seed seedling stage.Heredity foundation about the leaf sheath gene is studied at different segregating populations, and the result shows that the gene of controlling purple leaf sheath proterties has one or two main effect QTL control (Song Guoqing etc., 1994; Huang Biguang and Lu Yongquan, 2002; Qiu Zhenguo, 2006).Bing etc. (2006) utilize recombinant inbred lines located 3 with the relevant QTL of rice purple leaf sheath proterties, lay respectively on paddy rice the 1st, 6,10 karyomit(e)s, on the 1st karyomit(e) between SSR mark RM129 and RM5, but the genetic distance of the locus at RM129 and RM5 two marks and purple leaf sheath gene place report not.Also do not set up the molecular genetic linkage map of rice purple leaf sheath PSH1 (t) gene at present.
The inventor utilizes holy rice 301 of rice varieties and IRBB60 hybridization, and selfing continuously, utilizes single seed descent, has developed a recombinant inbred lines that comprises 210 familys, and the strain frenulum that is numbered RI51 in this colony has purple leaf sheath proterties.The F that utilizes fragrant round-grained rice 9407 of no purple leaf sheath rice varieties and purple leaf sheath kind RI51 hybridization to produce
2Segregating population, its purple leaf sheath gene PSH 1 (t) that has is positioned, set up the molecular genetic linkage map of PSH1 (t) gene, find with the closely linked molecule marker of PSH1 (t) gene basis on, determine that by the amplification of closely linked SSR mark purple leaf sheath gene is to exist with homozygotic state or with heterozygous state, just can easily carry out seed selection research.(Marker-assisted selection, MAS) technology is on purpose carried out the transformation of purple leaf sheath gene, cultivates the sterile line that has purple leaf sheath gene and recovers system, accelerates the process of breeding by molecular marker assisted selection.And can be used for the clone of purple leaf sheath gene PSH 1 (t).The present invention can be used to instruct the seed selection research of purple leaf sheath rice varieties, and chain with it molecule marker is accurately to select with the individual plant that isozygotys or heterozygous state exists to carrying purple leaf sheath gene, improves breeding efficiency greatly.The structure of purple leaf sheath gene PSH 1 (t) molecular genetic linkage map provides good basis for this gene of map based cloning simultaneously.
Summary of the invention
The objective of the invention is: filter out several and rice purple leaf sheath gene PSH 1 (t) close linkage and the molecule marker based on PCR not affected by environment, just can identify in seedling stage that to carry purple leaf sheath gene be with the individual plant that isozygotys or heterozygous state exists, accelerate purple leaf sheath trait molecular marker assistant breeding, improve the efficient of selecting greatly.The structure of purple leaf sheath gene PSH 1 (t) molecular genetic linkage map can accelerate to clone purple leaf sheath gene simultaneously.
With the chain molecule marker of rice purple leaf sheath gene PSH 1 (t), it is characterized in that,
Use labeled primer RM7202
The left end primer sequence is: AACGTGGAGGCTCCTTTTTC
The right-hand member primer sequence is: TTGCTATTAGTTGGTGGGGC
Labeled primer RM7318
The left end primer sequence is: CCATAGCTGCGTGATTCTCC
The right-hand member primer sequence is: AGATGAAACTGGCACGTGTG
Labeled primer RM3475
The left end primer sequence is: GTCGGTTTGCCTAGTTGAGC
The right-hand member primer sequence is: TTCCTCGGTGTATGGGTCTC
Labeled primer RM488
The left end primer sequence is: CAGCTAGGGTTTTGAGGCTG
The right-hand member primer sequence is: TAGCAACAACCAGCGTATGC
Labeled primer RM3143
The left end primer sequence is: AGCCTGGATAAGATGGTTCG
The right-hand member primer sequence is: CGAGAAGACCCAGTTTCTGC
Labeled primer RM8260
The left end primer sequence is: AATCTAACGTTTGACTATCCATC
The right-hand member primer sequence is: TCTACCAGTACTCCCTTCACC
Labeled primer RM246
The left end primer sequence is: GAGCTCCATCAGCCATTCAG
The right-hand member primer sequence is: CTGAGTGCTGCTGCGACT
Labeled primer RM128
The left end primer sequence is: AGCTTGGGTGATTTCTTGGAAGCG
The right-hand member primer sequence is: ACGACGAGGAGTCGCCGTGCAG
The amplified fragments size that the amplifying rice genomic dna obtains is about 157bp, 103bp, 150bp, 177bp, 98bp, 192bp, 116bp and 157bp, be molecule marker RM7202 mark, RM7318 mark, RM3475 mark, RM488 mark, RM3143 mark, RM8260 mark, RM246 mark and the RM128 mark chain with rice purple leaf sheath gene PSH 1 (t), mark M488 wherein, RM8260, RM3475 is positioned at PSH1 (t) near a side of galianconism, is respectively 7.1cM, 5.8cM and 2.0cM with the genetic distance of PSH1 (t); Mark RM7202, RM7318, RM3143, RM246 and RM128 are positioned at the opposite side of PSH1 (t), be respectively 1.1cM, 1.4cM with the genetic distance of PSH1 (t), 4.9cM, 5.8cM and 18.9cM are respectively RM3475 and RM7202 from the nearest both sides mark of PSH1 (t) gene.
Above-mentioned and the chain molecule marker of rice purple leaf sheath gene PSH 1 (t) obtain by the following method:
(1) utilizes holy rice 301 (Shandong Rice Research Institute's improved variety of rice varieties, country's seed bank all has preservation, can externally provide) and rice varieties IRBB60 (public material, country's seed bank all has preservation, can externally provide) hybridization, and selfing continuously, utilize single seed descent, developed a recombinant inbred lines that comprises 210 familys, the strain frenulum that is numbered RI51 in this colony has purple leaf sheath proterties;
(2) utilize the fragrant round-grained rice 9407 of no purple leaf sheath kind and purple leaf sheath kind RI51 (public material, national seed bank all has preservation, can externally provide) hybridization, F
1The seed selfing produces F
2, extract the fragrant round-grained rice 9407 of paddy rice, RI51 and F thereof with the CTAB method
1, F
2The individual plant DNA of segregating population;
(3) adopt 360 pairs of SSR primers that evenly cover rice genome to parent RI51 and fragrant round-grained rice 9407 and F thereof
1Carry out pcr amplification, carry out the polymorphism screening of primer; The polymorphism primer that filters out is then successively to F
2Purple leaf sheath and no purple leaf sheath DNA mixing pit are arranged, F
2The molecule marker of the purple leaf sheath gene linkage of the individual screening of each of colony;
(4) filter out 2 couples of polymorphism primer RM7202 earlier on the 1st karyomit(e), RM7318 carries out mark and encrypts in this zone, filter out 8 couples of polymorphism primer RM488 altogether, RM8260, RM3475, RM7202, RM7318, RM3143, RM246, RM128; To 50 F with purple leaf sheath of nothing of recessive character
2The segregating population individual plant is analyzed, and proves conclusively these 8 pairs of SSR primers and PSH1 (t) gene linkage;
(5) utilize the F of these 8 pairs of primers to 420 segregating population individual plants
2Analyze, obtain colony's genotype data;
(6) according to chain exchange rule, utilize colony's genotype data to make up the genetic map of paddy rice PSH1 (t) gene, adopt software MAP MAKER/EXP3.0, with the Kosambi function recombination value is converted to genetic distance (cM); The purple leaf sheath gene PSH 1 of dominance (t) is positioned on the 1st karyomit(e), and SSR mark M488, RM8260, RM3475 are positioned at the side of PSH1 (t) near galianconism, is respectively 7.1cM, 5.8cM and 2.0cM with the genetic distance of PSH1 (t); SSR mark RM7202, RM7318, RM3143, RM246 and RM128 are positioned at the opposite side of PSH1 (t), are respectively 1.1cM, 1.4cM, 4.9cM, 5.8cM and 18.9cM with the genetic distance of PSH1 (t); Be respectively RM3475 and RM7202 from the nearest both sides mark of PSH1 (t) gene.
The chain molecule marker of the present invention and rice purple leaf sheath gene PSH 1 (t) is exclusively used in the seed selection of rice purple leaf sheath kind and the utilization of germ plasm resource, and can be used for cloning purple leaf sheath gene.
The molecule marking method that has the purple leaf sheath gene PSH 1 (t) of purple leaf sheath proterties rice varieties RI51 provided by the present invention has the following advantages:
(1) mark is stable, and is not affected by environment.Utilize the SSR mark to locate the purple leaf sheath gene PSH 1 (t) of paddy rice RI51 in the world first by the present invention, made up the molecular genetic linkage map of purple leaf sheath gene.The parent who does not have the fragrant round-grained rice 9407 of purple leaf sheath kind * purple leaf sheath kind RI51 combination, F
1And F
2Segregating population part individual plant was taken a sample in different growing in 2007, detected with these marks, and the mark performance is stable, not affected by environment.
(2) clear and definite by the localized key-gene seat position of molecule marker of the present invention, it is convenient to identify.By detecting and the chain molecule marker of this locus, can predict that purple leaf sheath gene is with the individual plant that isozygotys or the heterozygous state genotype exists, accelerate purple leaf sheath trait molecular marker assistant breeding, improve the efficient of selecting greatly.It is easy to detect fast, and is not affected by environment.
(3) the assistant breeding select target is clear and definite, saves cost.In hybridisation rice is produced, because sterile line or recovery system are unstable, cause impurity of seeds, be one of principal element that influences hybrid purity always.The transformation of purple leaf sheath gene in sterile line or recovery system, is cultivated the sterile line that has purple leaf sheath proterties or recovered system, both can carry out artificial impurity elimination in seedling stage.Therefore utilize with the closely linked molecule marker of purple leaf sheath gene and carry out assistant breeding, judge that purple leaf sheath gene is to exist with isozygotying with heterozygosis, be chosen in the excellent proterties individual plant that has that isozygotys on the purple leaf sheath locus exactly, and can targetedly purple leaf sheath gene be selected in the laboratory to obtain, improve the efficient of selecting greatly.
(4) be used to clone the research of purple leaf sheath gene PSH 1 (t).The prerequisite of the purple leaf sheath gene PSH 1 of map based cloning (t) is to obtain the closely linked molecule marker of PSH1 (t) gene.Molecule marker RM3475 and RM7202 are present and the most closely linked molecule marker of PSH1 (t) gene.
Description of drawings
Fig. 1 is the position of the purple leaf sheath gene PSH 1 (t) of rice varieties RI51 on the 1st karyomit(e).
Fig. 2 is that mark RM7318 is not to there being the fragrant round-grained rice 9407 * RI51F of purple leaf sheath kind
2For the molecular marker assisted selection electrophoretogram
P1, fragrant round-grained rice 9407; P2, RI51; 1-20, F
2Separate individual plant.
Fig. 3 is that mark RM7202 is not to there being the fragrant round-grained rice 9407 * RI51 of purple leaf sheath kind F
2For the molecular marker assisted selection electrophoretogram
P1, fragrant round-grained rice 9407; P2, RI51; 1-20, F
2Separate individual plant.
Embodiment
(1) fragrant round-grained rice 9407 of no purple leaf sheath kind and purple leaf sheath kind RI51 hybridization F
2The establishment of colony and phenotypic evaluation
Utilized holy rice 301 and rice varieties IRBB60 hybridization in (1) 2000 year to 2005, and selfing continuously, utilize single seed descent, developed a recombinant inbred lines that comprises 210 familys, the strain frenulum that is numbered RI51 in this colony has purple leaf sheath proterties.
Do not have the fragrant round-grained rice 9407 * RI51 hybridization of purple leaf sheath kind in (2) 2006 years, the end of the year in 2006 F
1Seed send the Sanya, Hainan Island to add generation, and selfing produces F
2Seed.2007 with F
2Segregating population is seeded in Shandong Rice Research Institute experimental farm, sowing on May 1, rice transplanting on June 20, individual plant transplanting.20 days extracting F of rice transplanting
2The individual plant DNA of segregating population, and the leaf sheath proterties of investigation individual plant divide purple leaf sheath and greenery sheath two kinds of phenotypes.
(2) fragrant round-grained rice 9407 F of RI51/
2The molecular marker analysis of colony
(1) extracts fragrant round-grained rice 9407/RI51 F with the CTAB method
2Each individual plant DNA.
(2) ssr analysis is with reference to the method for Panaud et al. (1996).20 μ l PCR reaction systems are: template DNA 10ng/ μ l, and 0.15 μ l 10mM dNTPs, the Taq enzyme of 1.5 units, 2 μ l, 10 * PCRbuffer (contains MgCl
2), 2uM forward and the reverse primer of 1.5 μ l, reaction volume 20 μ l.The PCR response procedures is: DNA94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 1min then, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, and last 72 ℃ are extended 5min.Increase on the PTC-200PCR instrument, amplified production carries out separation electrophoresis on 6% polyacrylamide gel, and silver dyes the observation that develops the color of method with reference to (2002) such as Li Wentao, record experimental result.
(3) select to have the DNA balanced mix of purple leaf sheath proterties 30 strains, constitute purple leaf sheath mixing pit, select the DNA balanced mix of no purple leaf sheath proterties 30 strains, constitute no purple leaf sheath mixing pit.Select the primer that performance has polymorphism in two mixing pits, 50 recessive individual plants that do not have purple leaf sheath proterties are increased, analyze according to phenotype and genotype, further the primer and the purple leaf sheath gene PSH 1 (t) of proof performance polymorphism in two mixing pits are chain.Utilize then identify with the chain SSR mark of purple leaf sheath gene PSH 1 (t) to F
2420 individual plants of segregating population increase, and obtain F
2The genotype data of each individual plant of colony.
(4) according to the genetic distance of rice genome genetic map, select 360 pairs of SSR molecule markers, guarantee to select a mark within 5cM, selected mark evenly covers whole rice genome.Utilize these marks at first to above-mentioned parent and F thereof
1Carry out the polymorphism screening, then successively to F
2Purple leaf sheath and no purple leaf sheath DNA mixing pit are arranged, and 50 recessive individual plants that do not have purple leaf sheath proterties are increased, judge mark and target gene are chain, utilize the positive mark who filters out then, to F
2Each individuality of colony carries out the genotype mark.The SSR primer sequence derives from http://www.gramene.org/ website.Gene engineering company limited is synthetic by match Parkson, Beijing.
(5) will compose with the individuality of the identical banding pattern of RI51 according to the requirement of Mapmaker is 1, composing with fragrant round-grained rice 9407 identical banding patterns is 3, and the individuality tax with heterozygosis banding pattern is 2, and what data lacked is designated as 0, utilize MAPMAKER/EXP3.0 software that 420 individual plants are analyzed, make up SSR mark genetic map.And utilize the Kosambi function to be converted to genetic distance (cM).
(3) result and analysis
(1) at F
2In the segregating population, investigated the phenotype of 760 individual plants, individual plant 574 strains of wherein purple leaf sheath proterties, individual plant 186 strains (having the greenery sheath) with no purple leaf sheath proterties, the individual plant segregation ratio with purple leaf sheath proterties and no purple leaf sheath proterties well meets 3:1 (χ
2=1.38＜χ
2 0.05,1=3.84), prove that purple leaf sheath proterties is a dominance, and by a pair of key-gene control.
(2) utilize 360 pairs of primers in the polymorphism screening between fragrant round-grained rice 9407 of parent and the RI51, have 156 pairs of SSR primers 9407 and RI51 between have polymorphism.Utilize these 156 pairs of polymorphism primers that the DNA mixing pit is increased, discovery is on the 1st karyomit(e), there are two mark RM7202 and RM7318 in the DNA mixing pit that purple leaf sheath proterties and no purple leaf sheath proterties are arranged, to show difference, further this regional primer is screened, screen 6 couples of primer RM488 again, RM8260, RM3143, RM3475, RM246, RM128 shows difference in purple leaf sheath and no purple leaf sheath proterties mixing pit.The sequence of these 8 pairs of SSR primers and amplified fragments size see Table 1.For further these 8 pairs of marks of proof and purple leaf sheath gene PSH 1 (t) are chain, utilize these 8 pairs of SSR primers to F
250 individual plants with greenery sheath recessive character in the segregating population increase, the genotypic segregation ratio example obviously departs from the 1:2:1 segregation ratio, and most genotype and phenotype be divided into from, prove that these 8 pairs of SSR marks and purple leaf sheath gene PSH 1 (t) are chain.Utilize these 8 pairs of primers to F then
2420 individual plants of segregating population picked at random increase.Genotype and phenotypic data input computer, utilize the MAPMAKER/EXP3.0 program to carry out linkage analysis, set up the molecular genetic linkage map of purple leaf sheath gene PSH 1 (t), be respectively 2.0cM and 1.1cM from two nearest molecule marker RM3475 and RM7202 distance.Therefore utilize the chain molecule marker of these and purple leaf sheath gene PSH 1 (t) can carry out the molecular breeding of the molecular marker assisted selection and the purple leaf sheath proterties of purple leaf sheath gene effectively, utilize big segregating population simultaneously, purple leaf sheath locus is carried out physical mapping, PSH1 (t) gene is defined in a littler interval, the clone who accelerates purple leaf sheath gene.
The sequence of table 1. labeled primer and amplified fragments size
The nucleotides sequence tabulation
＜110〉High and New Technology Research Center, Shandong Prov. Academy of Agriculture S
＜120〉with chain molecule marker and preparation method and the application of rice purple leaf sheath gene PSH 1 (t)
<140>200810014910x
<141>2008—03—31
<160>16
<210>1
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (0ryza sativa) genomic dna RM7202 left end primer
<400>1
aacgtggagg?ctcctttttc?20
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM7202 right-hand member primer
<400>2
ttgctattag?ttggtggggc?20
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM7318 left end primer
<400>3
ccatagctgc?gtgattctcc?20
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM7318 right-hand member primer
<400>4
agatgaaact?ggcacgtgtg?20
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM3475 left end primer
<400>5
gtcggtttgc?ctagttgagc?20
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM3475 right-hand member primer
<400>6
ttcctcggtg?tatgggtctc?20
<210>7
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM488 left end primer
<400>7
cagctagggt?tttgaggctg?20
<210>8
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM488 right-hand member primer
<400>8
tagcaacaac?cagcgtatgc?20
<210>9
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM3143 left end primer
<400>9
agcctggata?agatggttcg?20
<210>10
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM3143 right-hand member primer
<400>10
cgagaagacc?cagtttctgc?20
<210>11
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM8260 left end primer
<400>11
aatctaacgt?ttgactatcc?atc?23
<210>12
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM8260 right-hand member primer
<400>12
tctaccagta?ctcccttcac?c?21
<210>13
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM246 left end primer
<400>13
gagctccatc?agccattcag?20
<210>14
<211>18
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM246 right-hand member primer
<400>14
ctgagtgctg?ctgcgact?18
<210>15
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM128 left end primer
<400>15
agcttgggtg?atttcttgga?agcg?24
<210>16
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉reaction requires design according to PCR, is used for amplifying rice (Oryza sativa) genomic dna RM128 right-hand member primer
<400>16
acgacgagga?gtcgccgtgc?ag?22
Claims (3)
1. with the chain molecule marker of rice purple leaf sheath gene PSH 1 (t), it is characterized in that,
Use labeled primer RM7202
The left end primer sequence is: AACGTGGAGGCTCCTTTTTC
The right-hand member primer sequence is: TTGCTATTAGTTGGTGGGGC
Labeled primer RM7318
The left end primer sequence is: CCATAGCTGCGTGATTCTCC
The right-hand member primer sequence is: AGATGAAACTGGCACGTGTG
Labeled primer RM3475
The left end primer sequence is: GTCGGTTTGCCTAGTTGAGC
The right-hand member primer sequence is: TTCCTCGGTGTATGGGTCTC
Labeled primer RM488
The left end primer sequence is: CAGCTAGGGTTTTGAGGCTG
The right-hand member primer sequence is: TAGCAACAACCAGCGTATGC
Labeled primer RM3143
The left end primer sequence is: AGCCTGGATAAGATGGTTCG
The right-hand member primer sequence is: CGAGAAGACCCAGTTTCTGC
Labeled primer RM8260
The left end primer sequence is: AATCTAACGTTTGACTATCCATC
The right-hand member primer sequence is: TCTACCAGTACTCCCTTCACC
Labeled primer RM246
The left end primer sequence is: GAGCTCCATCAGCCATTCAG
The right-hand member primer sequence is: CTGAGTGCTGCTGCGACT
Labeled primer RM128
The left end primer sequence is: AGCTTGGGTGATTTCTTGGAAGCG
The right-hand member primer sequence is: ACGACGAGGAGTCGCCGTGCAG
The polymorphic bands size that the amplifying rice genomic dna obtains is about 157bp, 103bp, 150bp, 177bp, 98bp, 192bp, 116bp and 157bp, be rice purple leaf sheath gene PSH 1 (t) chain molecule marker RM7202 mark, RM7318 mark, RM3475 mark, RM488 mark, RM3143 mark, RM8260 mark, RM246 mark and RM128 mark, mark M488 wherein, RM8260, RM3475 is positioned at PSH1 (t) near a side of galianconism, is respectively 7.1cM, 5.ScM and 2.0cM with the genetic distance of PSH1 (t); Mark RM7202, RM7318, RM3143, RM246 and RM128 are positioned at the opposite side of PSH1 (t), be respectively 1.1cM, 1.4cM with the genetic distance of PSH1 (t), 4.9cM, 5.8cM and 18.9cM are respectively RM3475 and RM7202 from the nearest both sides mark of PSH1 (t) gene.
2. the described and chain molecule marker of rice purple leaf sheath gene PSH 1 (t) of claim 1 is characterized in that, obtains by the following method:
(1) utilize holy rice 301 of rice varieties and IRBB60 hybridization, and selfing continuously, utilize single seed descent, developed a recombinant inbred lines that comprises 210 familys, the strain frenulum that is numbered RI51 in this colony has purple leaf sheath proterties;
(2) utilize the fragrant round-grained rice 9407 of no purple leaf sheath kind and purple leaf sheath kind RI51 hybridization, F
1The seed selfing produces F
2, extract the fragrant round-grained rice 9407 of paddy rice, RI51 and F thereof with the CTAB method
1, F
2The individual plant DNA of segregating population;
(3) adopt 360 pairs of SSR primers that evenly cover rice genome to fragrant round-grained rice 9407 and parent RI51 and F thereof
1Carry out pcr amplification, carry out the polymorphism screening of primer; The polymorphism primer that filters out is then successively to F
2Purple leaf sheath and no purple leaf sheath DNA mixing pit are arranged, F
2The molecule marker of the purple leaf sheath gene linkage of the individual screening of each of colony;
(4) filter out 2 couples of polymorphism primer RM7202 earlier on the 1st karyomit(e), RM7318 carries out mark and encrypts in this zone, filter out 8 couples of polymorphism primer RM488 altogether, RM8260, RM3475, RM7202, RM7318, RM3143, RM246, RM128; To 50 F with purple leaf sheath of nothing of recessive character
2The segregating population individual plant is analyzed, and proves conclusively these 8 pairs of SSR primers and PSH1 (t) gene linkage;
(5) utilize these 8 pairs of primers to 420 F
2The segregating population individual plant is analyzed, and obtains colony's genotype data;
(6) according to chain exchange rule, utilize colony's genotype data to make up the genetic map of paddy rice PSH1 (t) gene, adopt software MAP MAKER/EXP3.0, with the Kosambi function recombination value is converted to genetic distance (cM); The purple leaf sheath gene PSH 1 of dominance (t) is positioned on the 1st karyomit(e), and SSR mark M488, RM8260, RM3475 are positioned at the side of PSH1 (t) near galianconism, is respectively 7.1cM, 5.8cM and 2.0cM with the genetic distance of PSH1 (t); SSR mark RM7202, RM7318, RM3143, RM246 and RM128 are positioned at the opposite side of PSH1 (t), are respectively 1.1cM, 1.4cM, 4.9cM, 5.8cM and 18.9cM with the genetic distance of PSH1 (t); Be respectively RM3475 and RM7202 from the nearest both sides mark of PSH1 (t) gene.
3. the described and chain molecule marker of rice purple leaf sheath gene PSH 1 (t) of claim 1 is characterized in that, is exclusively used in the seed selection of rice purple leaf sheath kind and the utilization of germ plasm resource, and can be used for cloning purple leaf sheath PSH1 (t) gene.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899502A (en) * | 2010-03-24 | 2010-12-01 | 宁夏农林科学院 | Molecular marking method and application of rice scent gene |
| CN104762395A (en) * | 2015-04-10 | 2015-07-08 | 西北农林科技大学 | Molecular marker screening method for qualitative trait genes with complementary action |
| CN105525026A (en) * | 2016-02-19 | 2016-04-27 | 山东省农业科学院生物技术研究中心 | SSR molecular markers tightly linked with rice purple leaf sheath QTL and application |
| CN109652577A (en) * | 2016-02-04 | 2019-04-19 | 山东省农业科学院生物技术研究中心 | SSR molecular marker L08 and application with the high bar QTL close linkage of rice |
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2008
- 2008-03-31 CN CNA200810014910XA patent/CN101368181A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899502A (en) * | 2010-03-24 | 2010-12-01 | 宁夏农林科学院 | Molecular marking method and application of rice scent gene |
| CN104762395A (en) * | 2015-04-10 | 2015-07-08 | 西北农林科技大学 | Molecular marker screening method for qualitative trait genes with complementary action |
| CN104762395B (en) * | 2015-04-10 | 2017-12-12 | 西北农林科技大学 | For the method for screening molecular markers of complementation qualitative trait gene |
| CN109652577A (en) * | 2016-02-04 | 2019-04-19 | 山东省农业科学院生物技术研究中心 | SSR molecular marker L08 and application with the high bar QTL close linkage of rice |
| CN105525026A (en) * | 2016-02-19 | 2016-04-27 | 山东省农业科学院生物技术研究中心 | SSR molecular markers tightly linked with rice purple leaf sheath QTL and application |
| CN105525026B (en) * | 2016-02-19 | 2019-03-15 | 山东省农业科学院生物技术研究中心 | With the SSR molecular marker and application of rice purple leaf sheath QTL close linkage |
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