CN108588263B - Method for positioning and cloning double recessive genes for controlling same character in plant - Google Patents
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Abstract
The invention discloses a method for positioning and cloning double recessive genes for controlling the same character in a plant. The method for positioning and cloning the double recessive genes for controlling the same character in the plant can quickly construct a positioning population with separated target characters from common conventional materials, does not need to use non-conventional materials (such as a DH system), can finely position and map-clone the genes in BC1F1 generation, has the advantages of simplicity, quickness and high efficiency, and can be widely used for finely positioning and map-cloning the double recessive genes for controlling the same character.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for positioning and cloning double recessive genes for controlling the same character in a plant.
Background
The double recessive genes controlling the same trait are mostly generated by genome duplication or gene replication, are most common in heterotetraploid plants, such as common tobacco, brassica napus, durum wheat and the like, and occasionally occur in diploid plants, such as rice, corn and the like. Due to the completely redundant biological functions of such genes, mutation of a single gene cannot lead to the corresponding recessive phenotype.
Molecular markers are the most important and reliable means for the current gene localization, and in the aspect of utilizing the molecular markers to localize double recessive genes for controlling the same character, two methods are mainly used at present according to the used segregation population: one is to use F2 or BC1F1 to isolate groups and locate 2 genes as Quantitative Trait Loci (QTLs). The method can quickly obtain a positioning result because the separated population is obtained quickly, but has low positioning precision and is not generally used for fine positioning and map cloning of genes. The genes located by this method include 2 rice fertility restorer genes Rf3, Rf (u), and 2 durum wheat yellow-green leaf genes ygld1, ygld2, etc. (Li et al 2013; Yao et al 1997). The other method is to use a multi-generation backcross method, firstly separate 2 dominant alleles into different single plants through backcross, then prepare a single gene separated population through hybridization with a recessive homozygous mutant, and then respectively position the single gene separated population. The method has high precision and is commonly used for fine positioning and map-based cloning of genes; but it takes a long time because the localization is usually performed at the backcross high generation. The genes located by this method include 1 male sterility gene ms2 of Brassica napus and 1 gene mc1 of Brassica juncea (Lei et al 2007; Xu et al 2013).
Based on the current situation, a new gene positioning method combining the advantages (simplicity, rapidness and high efficiency) of the two methods is developed, so that the fine positioning process and the map-based cloning process of the double recessive genes are undoubtedly accelerated, and the functional genomics research and the molecular marker-assisted breeding of important polyploid crops are facilitated.
Disclosure of Invention
The technical problem to be solved by the invention is how to rapidly locate and clone the double recessive genes controlling the same character in the plant.
In order to solve the above technical problems, the present invention provides a method for mapping and cloning a double recessive gene controlling the same trait in a plant.
The method for locating and cloning the double recessive gene for controlling the same character in the plant comprises the following steps:
(1) hybridizing the mutant plant with a wild plant to obtain an F1 generation plant, and hybridizing the F1 generation plant with the mutant plant to obtain a BC1F1 segregation population; said mutant plant has a phenotype A and said wild type plant has a phenotype B, said phenotype A and said phenotype B being the same trait and being opposite phenotypes;
(2) respectively carrying out primary positioning on double recessive genes controlling the same character by using molecular markers to respectively obtain primary positions of the double recessive genes in a genome;
(3) selecting one gene locus, developing a molecular marker according to the primary position of the gene locus, and finely positioning the gene locus based on a mutant type single plant which has the same phenotype as the mutant plant in the BC1F1 segregation population to obtain a fine positioning interval of the gene locus;
(4) comparing the sequence difference of the wild plant and the mutant plant in the fine positioning interval to obtain a candidate gene A;
(5) comparing and analyzing the genome sequences of the wild plant and the mutant plant to obtain a homologous gene of the candidate gene A, namely a candidate gene B; the candidate gene A and the candidate gene B are double recessive genes for controlling the same character.
In the above method, in the above (1), in order to increase the polymorphism between parents and facilitate more efficient development of molecular markers for gene fine targeting, the mutant plant may be replaced with a plant variety having the same phenotype and genotype as the mutant plant. In a specific embodiment of the invention, the burley tobacco variety TN90 was used in place of the white-stem mutant plant ws 1.
In the above method, the primary localization method in (2) is a conventional method in the prior art, and a molecular marker disclosed in the prior art can be used, specifically, a method in the literature "Mapping of two white step genes in branched common bacteria tobacaco (Nicotiana tabacum L.)".
In the above methods, in the (2) and (3), the molecular marker may be an SNP marker or an SSR marker. In practical application, in view of the characteristics of low use cost and high detection efficiency of SSR markers, the markers are preferentially used for gene localization, and if SSR markers cannot be developed in a fine localization interval, SNP markers are considered, are relatively easy to develop due to wider distribution in a genome, but have high use cost and are not suitable for screening (fine localization) of oversized groups. In a particular embodiment of the invention, the molecular marker is in particular an SSR marker.
In the above method, in the (3), the fine positioning method includes the steps of:
(3-1) searching a simple repetitive sequence capable of designing an SSR primer according to the preliminary position of the gene locus, and designing an SSR primer according to the simple repetitive sequence and the upstream and downstream sequences thereof;
(3-2) detecting the mutant plant and the wild plant by using the SSR primers, and selecting SSR primers with polymorphism;
(3-3) detecting the mutant type single-plant small population by using the SSR primer with polymorphism, and positioning the gene locus in an interval A;
the small mutant single plant population is a small part of the small mutant single plant population;
the mutant individual population is a population of plants in the isolated population of BC1F1 having the same phenotype as the mutant plant;
(3-4) searching a simple repetitive sequence capable of designing an SSR primer in the interval A, designing an SSR primer according to the simple repetitive sequence and upstream and downstream sequences of the simple repetitive sequence, detecting the residual mutant type single plant population by using the SSR primer, and positioning the gene locus in an interval B, namely the fine positioning interval;
the residual mutant individual plant population is a population consisting of residual plants except the mutant individual plant small population in the mutant individual plant population.
In the above methods, in the above (3-1) and (3-4), a simple repetitive sequence for which an SSR primer can be designed is searched using the SSRH software.
In the above method, in the above (3-1) and (3-4), SSR primers are designed based on the simple repeat sequence and the 150bp sequence upstream and downstream thereof.
In the above method, in the step (3-2), the method for selecting an SSR primer having polymorphism is to select an SSR primer that has a different amplification band type in the mutant plant and the wild-type plant.
In the above-mentioned methods, in (3-3), the method of detecting a small population of mutant individuals using the SSR primers having polymorphisms to locate the gene site in the interval A is a conventional method in the prior art, and specifically, the method is described in the literature "Mapping of two white step genes in transgenic common tobacaco (Nicotiana tabacum L.)".
In the above method, the plant may be tobacco.
In the method, the mutant plant is white-stem mutant plant ws1 or burley tobacco variety TN 90; the wild plant is a safflower large golden element.
In the above method, the candidate gene A is ws1b gene; the candidate gene B is a ws1a gene. The nucleotide sequence of the ws1b gene is a sequence 1 in a sequence table; the ws1a gene is shown as sequence 2 in the sequence table.
The invention provides a new method for positioning and cloning double recessive genes for controlling the same character in a plant, which can quickly construct a positioning population for separating a target character from common conventional materials, and has the main advantages that the gene can be finely positioned and subjected to map-based cloning in BC1F1 generation without using non-conventional materials (such as DH system) compared with the method in Edwards et al.
Drawings
FIG. 1 compares the initial position and the fine position of ws1a and ws1 b.
FIG. 2 shows the phenotypes of Honghuadajinyuan (HD) and TN90(TN) at the seedling stage and the adult stage.
FIG. 3 shows the amplification of markers S5 and S7 in a partial BC1F1 recessive individual (white stem) (recombinant individual).
FIG. 4 is a fine mapping and map cloning of ws1a and ws1 b.
FIG. 5 is a comparison of the phenotype of the genetic complementation plants of WS1A and WS1B with that of the control.
FIG. 6 shows transmission electron microscope observation of chloroplast of Honghuadajinyuan (HD) and TN90 (TN).
FIG. 7 shows the amplification of primers specific for WS1A, WS1a, WS1B and WS1b in 22 Burley tobacco varieties and 24 green-stem tobacco varieties.
FIG. 8 shows the amplification of primers specific to WS1A, WS1a, WS1B and WS1b in a portion of the BC1F1 strain and the genotyping of the green stem strain.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the quantitative tests in the following examples, three replicates were set up and the results averaged.
Example 1 localization and mapping of genes ws1a and ws1b
First, initial localization of genes ws1a and ws1b
1. A chlorophyll-deficient white stem mutant white stem1(ws1) was selected from EMS-mutagenized Nicotiana tabacum 100 (ZY). Genetic analysis was carried out by the method of allelic testing (see, for example, Mapping of two white stem genes in transgenic common tobacaco (Nicotiana tabacum L.)), in the following manner: hybridizing the white-stem mutant ws1 with a burley tobacco variety to obtain F1 generation, wherein the F1 generation is the same as the parent and the female parent; and then selfing the F1 generation to obtain selfed progeny, wherein the selfed progeny has no separation of characters. Genetic analysis shows that the white stem character is controlled by 2 recessive cell nuclear genes ws1a and ws1b, and is allelic with the white stem gene of burley tobacco, an important type of cultivation in nicotiana tabacum.
2. A tobacco SSR marker is used for primary localization of two genes ws1a and ws1b (see the following documents: Mapping of two white stem genes in a branched common tobacam L.) by crossing a white stem mutant (ws1) with a red-flower large gold element (HD) of a green-stem tobacco variety to obtain a hybrid F1 generation, then backcrossing the hybrid F1 generation with TN90 to obtain a BC1F1 segregating population, then selfing a green stem single strain in the BC1F1 segregating population to obtain a BC1F2 segregating population, obtaining a population with a green: white segregation ratio of 3:1 by phenotypic identification (in a particular population, only the ws1a or the ws1b gene is segregated separately, FIG. 1), one of the populations is selected for screening SSR markers linked to the white stem mutant phenotype, the genes linked to the marker is selected for the SSR marker linked to the marker a, and the population is selected for the remaining populations where the white stem marker is linked to the SSR marker b, finally positioning ws1a in a 12cM interval bounded by SSR markers PT54006 and PT51778 in the 5 th linkage group and positioning ws1b in a 17.12cM interval bounded by SSR markers PT53716 and TM11187 in the 24 th linkage group; and based on several evidences such as genetics and molecular marker maps, it is presumed that ws1a and ws1b are homologous genes.
Second, fine mapping and map-based cloning of genes ws1a and ws1b
1. Because the experimental materials for genetic analysis and gene localization in the step one belong to the same type of common tobacco, and the polymorphism of the SSR marker is lower in the same type of common tobacco and higher in different types of common tobacco, in order to improve the polymorphism of the SSR marker and facilitate the following gene fine localization and map-based cloning, the invention selects a common tobacco burley tobacco variety TN90(TN) to replace a white stem mutant to carry out the following tests: firstly, a burley tobacco variety TN90(TN) is hybridized with a green stem tobacco variety safflower Hongda (HD) to obtain a hybrid F1. The hybrid F1 generation was then backcrossed with TN90, resulting in a BC1F1 segregating population (FIGS. 1 and 2). The BC1F1 population had a total of 8500 strains, of which 2151 was identified as white stems (recessive) with a segregation ratio of 3:1(χ)2 0.050.424, less than critical value 3.841).
2. Since ws1a is located within a 12cM interval bounded by SSR markers PT54006 and PT51778 in the 5 th linkage group, ws1b is located within a 17.12cM interval bounded by SSR markers PT53716 and TM11187 in the 24 th linkage group. By aligning the SSR primer sequences provided in the document "A high specificity genetic map of tobaco (Nicotiana tabacum L.) amplified from large scale chromosome marker expression" with the genome sequence of safflower large gold (HD), it was shown that ws1b is located between chromosome 19, 64.73Mb and 70.45Mb, whereas the two SSR markers of ws1a, being located on different scalafolds respectively, temporarily failed to confirm the specific chromosomal location of ws1 a.
3. Fine positioning of ws1b is performed. The method comprises the following specific steps: (1) simple repeats of SSR primers can be designed by Searching for the region between chromosome 64.73Mb and 70.45Mb of Honghuadajinyuan 19 using the SSRH Software (described in the following documents: Development of a Local Searching Software for SSR Sites); (2) the obtained SSR simple repetitive sequence and the upstream and downstream 150bp sequences thereof are used for designing SSR primers and detecting the polymorphism between TN90 and safflower large gold; (3) polymorphic markers were used to detect white stem individuals (recessive) in the BC1F1 population. All experimental techniques used in fine Mapping, including genomic DNA preparation, PCR amplification, polyacrylamide gel electrophoresis and silver staining, were performed as described in the literature "Mapping of two white step genes in biochemical common to tobaco (Nicotiana tabacum L.)".
The SSR markers designed by the invention are 88 in total, 10 (S1-S10) have polymorphism between TN90 and safflower large gold, and the electrophoresis bands of the amplification products are clear. First, a small population of 282 individuals (here, the small population is a small part of 2151 white stalk individuals in the BC1F1 population in step 1 and contains 282 white stalk individuals) was tested by using the 10 polymorphic markers, and the markers were found to be closely linked to the white stalk mutant phenotype (fig. 3). Preliminarily locating ws1b within a 910kb interval based on genetic recombination between the SSR marker and ws1 b; subsequently, 6 polymorphic SSR markers (S11-S16) were developed in this interval and further localized in a 220kb interval using the remaining population (the remaining population refers to the remaining white stalk individuals of the 2151 white stalk individuals in step 1 except the small population consisting of 282 white stalk individuals as described above) (FIG. 4). The interval has 5 prediction genes, and a gene coding for metalloprotease (zinc metalloprotease) is preliminarily determined as a candidate gene of ws1b by sequence difference alignment between TN90 and safflower large gold. The ws1b gene has 10 exons and 9 introns, and a single base T insertion located in exon 9 results in a frame shift mutation of the gene (FIG. 4). The nucleotide sequence of the ws1b gene is shown in sequence 1.
4. Since ws1a and ws1b are presumed to be homologous genes, and the ws1b sequence was subsequently searched as query sequence in the safflower macrogold genomic sequence, it was found that another gene highly homologous to the ws1b candidate gene and encoding the same metalloprotease has a sequence difference between TN90 and safflower macrogold. The gene is located on chromosome 18 of safflower large gold, and also has 10 exons and 9 introns, and the deletion of 8 bases located on exon 2 results in frame shift mutation of the gene (figure 4), which is a candidate gene of ws1a, and the nucleotide sequence of the gene is shown as sequence 2.
5. The 8-base deletion described in step 4 was designed as a co-dominant marker M-a, and 470 white stalk individuals (recessive) were detected (here, 470 white stalk individuals (recessive) are part of the 2151 white stalk individuals in the BC1F1 segregating population described above), and the results show that: this marker was fully linked to the white stem mutant phenotype, indicating that ws1a was indeed located in the region where its candidate gene was located (FIG. 4). All the marker information used in fine positioning is shown in table 1.
TABLE 1 molecular markers for the fine mapping and map-based cloning of ws1a and ws1b
| Marking | Chromosome | Physical location (Mb) | Forward primer sequence | Reverse primer sequence |
| S1 | Chr.19 | 66.34 | TATGATTCTCCTTTTATTCCTA | TGCGGTCCACTCCACTGA |
| S2 | Chr.19 | 66.91 | GTGGCAACTAAATGAAAAAAGA | TTAGATATTCAACATCCTCCTT |
| S3 | Chr.19 | 67.49 | GTTCTATATTTTCAAACAGTGTG | TGACAACCTCAATAAGCCAC |
| S4 | Chr.19 | 67.92 | TCTTATTCTCTTACAACACTCTG | GTAGACAAGCGTAATGAGGA |
| S5 | Chr.19 | 68.10 | GATGTGTTTCTTTTGCTCTTTAT | AGTCTGAGATTATACTGGGTTG |
| S6 | Chr.19 | 68.48 | AGTTGAATATGAACCTATACAAAT | GATAGTGAAGAAAAATGTGAAAAT |
| S7 | Chr.19 | 69.01 | GGTACAGCGGGGAAAGATA | AAACCTGCAATTACAAGTCAAA |
| S8 | Chr.19 | 69.38 | TTTTTCCCTACCGATTCTCTAC | TGTTGCTTCTTCACACACATTA |
| S9 | Chr.19 | 69.80 | CCACTGTTTAAGCAACTTTAGATA | ACACCATATAAAATGATTGTGAAG |
| S10 | Chr.19 | 69.92 | AAACAAAACCGAACCAAACC | GAACGGACGCTAATTCTCAA |
| S11 | Chr.19 | 68.20 | GTAAAAGATTGATTAAGATTTAGAC | GAGAATTGAAATTATGAGATTATC |
| S12 | Chr.19 | 68.62 | AAAGGGCACTCCCGAATAT | ATGCTTGTAAATCAAATGATGATG |
| S13 | Chr.19 | 68.66 | TCGTGTAGGTTTAATAAAGGAG | ACAAAAGGAAAGAGGGAAAC |
| S14 | Chr.19 | 68.79 | CGGACATTGATAAGTTGTAGAT | TCCATACGACTGAATAATAGGT |
| S15 | Chr.19 | 68.83 | AAACGAAATAAATAAAGGAAAGAA | GGGCATAAAAGTCGATCAATAT |
| S16 | Chr.19 | 68.88 | TCTTACCACCATTGTGTAGGA | CAAGTGAGCGTCAGTATTTTC |
| M-a | Chr.18 | 13.25 | ACCTGTTCATGGTGGAAGAG | CTGCGTGGTTGACGAGTTC |
Example 2 application of metalloprotease WS1A or WS1B in regulating and controlling plant chloroplast metabolism
First, obtaining transgenic WS1A tobacco and transgenic WS1B tobacco
1. Construction of recombinant vectors
(1) Cloning of genes WS1A and WS1B
(1-1) extracting RNA of the leaves of the Honghuadajinyuan, and carrying out reverse transcription to obtain cDNA. RNA Extraction and reverse transcription were performed using the MiniBEST plant RNA Extraction Kit (Code No.9769) and the PrimeScript II 1st Strand cDNA Synthesis Kit (Code No.6210), from TaKaRa, respectively. And (3) taking the cDNA as a template, and adopting a primer CW-1F/CW-1R for amplification to obtain a PCR product.
(1-2) PCR products were subjected to TA cloning (Mighty TA-cloning Kit (TaKaRa Co., Code No.6028)) and sequencing to identify WS1A and WS1B, respectively. The WS1A gene sequence is shown as sequence 3, and the WS1B gene sequence is shown as sequence 4.
(2) Construction of expression vectors
(2-1) double cleavage of pCAMBIA1300-35S (pCAMBIA1300-35S described in "Loose Plant Architecture1, an INDETERMINATE DOMAIN Protein Involuted in Shoot G promoter, Regulates Plant Architecture in Rice") with restriction endonucleases PstI and EcoRI to obtain a 310bp fragment containing the NOS transcription terminator and ligating it into pCAMBIA1300 vector to form an intermediate vector pCAMBIA 1300-NOS;
(2-2) amplifying the WS1A gene using a primer ST-3F/ST-1R using the WS1A plasmid containing the correct sequence as a template, and inserting the amplified gene into an intermediate vector pCAMBIA1300-NOS using an In-Fusion HD Cloning Kit (TaKaRa Co.) to obtain pCAMBIA1300-WS 1A-NOS;
using WS1B plasmid with correct sequencing as a template, adopting a primer ST-3F/ST-1R to amplify WS1B gene, and inserting the amplified gene into an intermediate vector pCAMBIA1300-NOS by using an In-Fusion HD Cloning Kit of TaKaRa company to obtain pCAMBIA1300-WS 1B-NOS;
(2-3) taking safflower large-gold genomic DNA as a template, and respectively amplifying by using two pairs of primers SP-2F/SP-2R and TP-2F/TP-2R to respectively obtain promoters of WS1A and WS 1B;
(2-4) inserting the promoter of WS1A into pCAMBIA1300-WS1A-NOS using In-Fusion HD Cloning Kit of TaKaRa to obtain expression vector pWS1A: WS 1A;
the promoter of WS1B was inserted into pCAMBIA1300-WS1B-NOS using In-Fusion HD Cloning Kit (TaKaRa Co.) to obtain expression vector pWS1B: WS 1B;
(2-5) amplifying the WS1A gene by using a WS1A plasmid containing correct sequencing as a template and adopting a primer ST-1F/ST-1R; the WS1A gene was inserted into pCAMBIA1300-35S expression vector using In-Fusion HD Cloning Kit (Code No.639648) of TaKaRa Co., Ltd to obtain expression vector p35S: WS 1A;
amplifying the WS1B gene by using a primer ST-1F/ST-1R by taking the WS1B plasmid containing correct sequencing as a template; the WS1B gene was inserted into pCAMBIA1300-35S expression vector using In-Fusion HD Cloning Kit (Code No.639648) of TaKaRa to obtain expression vector p35S: WS 1B.
The expression vector pWS1A: WS1A and the expression vector pWS1B: WS1B are both expression vectors of which own promoters drive WS1A and WS 1B; the expression vector p35S: WS1A and the expression vector p35S: WS1B are both expression vectors of which CaMV35S strong promoter drives WS1A and WS 1B. The expression vector pWS1A: WS1A and the expression vector p35S: WS1A both express WS1A protein shown in a sequence 5; the expression vector pWS1B: WS1B and the expression vector p35S: WS1B both express the WS1B protein shown in the sequence 6.
All the primer information used for constructing the vectors described above is shown in Table 2.
TABLE 2 primers for WS1A and WS1B genetic complementary vector construction and cDNA amplification
2. Acquisition of transgenic tobacco plants
(1) The four vectors p35S: WS1A, p35S: WS1B, pWS1A: WS1A and pWS1B: WS1B are transferred into an agrobacterium strain LBA4404 (Bai ao Bai Si special chemical reagent, Inc. in Qingdao, the code number BC301-01) by electric shock, and white rib tobacco variety TN90 is transformed according to the method in the document "High-throughput generation of an activation-tagged mutant library for functional genetic analysis in tobaca", so as to obtain a transferred p35S: WS1A tobacco strain, a transferred p35S: WS1B tobacco strain, a transferred pWS1A: WS1A tobacco strain and a transferred pWS1B: 1B tobacco strain respectively.
(2) PCR identification
PCR identification was carried out using the primers in Table 3 for the p35S: WS1A, p35S: WS1B, pWS1A: WS1A and pWS1B: WS1B tobacco respectively.
TABLE 3 primer pairs for the identification of genetically complementary transgenic plants WS1A and WS1B
Note: a: p35S: WS 1A; b: p35S: WS 1B; c: pWS1A: WS 1A; d: pWS1B WS1B
Through PCR detection and sequencing analysis, more than 20 transgenic tobacco plants are obtained from each vector.
Second, the phenotype of transgenic WS1A tobacco and transgenic WS1B tobacco
The phenotype of the tobacco plant of the p35S: WS1A, the tobacco plant of the p35S: WS1B, the tobacco plant of the pWS1A: WS1A, the tobacco plant of the pWS1B: WS1B and the white rib tobacco variety TN90 is observed.
The results show that: compared with white stalks of TN90, the p35S: WS1A tobacco strain, the p35S: WS1B tobacco strain, the pWS1A: WS1A tobacco strain and the pWS1B: WS1B tobacco strain are recovered to green stalks which are the same as the wild type safflower large golden dollars. The above results indicate that WS1A and WS1B indeed control the white stalk trait of TN90, and either of them can restore it to wild type green stalk (FIG. 5).
Subcellular localization of tris, WS1A and WS1B
1. On-line analysis of the subcellular localization of WS1A and WS1B was performed using Predotar server (https:// urgi. versales. inra. fr/Predotar /).
The results show that: both WS1A and WS1B are localized in the plastids (chloroplasts).
2. To further analyze the effects of WS1A and WS1B in chloroplasts, mid-leaf samples of TN90 and safflower macrogol at the initial flowering stage were prepared by referring to the method in "alternate Chloroplast Development and Delayed front mutation used by Mutations in a Zinc Metalloprotease at the flowering stage of Tomato", and the ultrastructure of chloroplasts was observed by using a Hitachi H-7650 type transmission electron microscope.
The results show that: the thylakoid membrane of TN90 chloroplast was severely damaged, with few basal lamina layers and only a few basal lamina layers, in sharp contrast to intact thylakoid membranes of Honghuadajinyuan (FIG. 6). The results show that WS1A and WS1B influence the development of chloroplast by controlling the formation of chloroplast thylakoid membrane, thereby regulating the metabolism of chlorophyll.
Example 3 molecular markers Co-segregating with the Burley tobacco control Gene
Primer design for WS1A, WS1a, WS1B and WS1b genotype identification
1. Downloading WS1A and WS1B whole gene sequences from a safflower large gold genome database, performing sequence comparison by using an online analysis tool MUSCLE (https:// www.ebi.ac.uk/Tools/msa/MUSCLE /) to obtain the sequence difference between WS1A and WS1B, and intercepting the 200-bp and 400-bp genome sequences upstream and downstream of the mutation sites of WS1a and WS1b for later use;
2. due to the high homology of sequences of WS1A and WS1B, specific PCR primers containing gene mutation sites are respectively designed by utilizing the sequence difference between the sequences:
(1) for WS1A and WS1a, a pair of specific primers 1325-2F/1325-1R (i.e. M-a) are designed based on the 8-base length difference between the two, PCR products are separated by polyacrylamide gel electrophoresis, the ones with the fragment size similar to safflower large gold element are considered to contain WS1A, the ones with the fragment size similar to TN90 are considered to contain WS1a, and the ones with the fragment size similar to safflower large gold element and the fragment size similar to TN90 are considered to be WS1A/WS1a hybrid,
in practical application, whether the tobacco to be tested contains the WS1A gene, the WS1a gene, the WS1A gene and the WS1a gene can be determined according to the following method:
if the amplification product size of the 1325-2F/1325-1R primer is 209bp, the tobacco to be detected contains the WS1A gene;
if the amplification product size of the 1325-2F/1325-1R primer is 201bp, the tobacco to be detected contains the ws1a gene;
if the amplification product size of the 1325-2F/1325-1R primer is 209bp and 201bp, the tobacco to be detected contains the WS1A gene and the WS1a gene.
To verify that WS1A and WS1a were identified as correct, PCR sequencing was performed using another pair of specific primers 1325-1F/1325-1R for further confirmation.
(2) For WS1B and WS1B, the difference in single gene length between them could not be clearly shown by polyacrylamide gel electrophoresis, as in ACT-PCR in the references "A simple and effective method for CRISPR/Cas9-induced mutant screening", a reverse primer BW-N4R was designed to pair with the forward specific primer B-N1F for specific amplification of WS1B, and a reverse primer BM-N3R was designed to pair with the forward specific primer B-N1F for specific amplification of WS 1B. The optimal amplification conditions of the WS1B and WS1b specific primers are both obtained by searching the large gold elements of the safflower and the TN90 by a temperature gradient PCR method, and the conditions are determined according to the fact that the specifically amplified gene bands are clear, and the corresponding alleles have no bands or very weak bands.
In practical application, whether the tobacco to be tested contains the WS1B gene, the WS1b gene, the WS1B gene and the WS1b gene can be determined according to the following method:
if the BW-N4R/B-N1F primer has no amplified band or weak amplified band and the amplification product size of the BM-N3R/B-N1F primer is 251bp, the tobacco to be detected contains the ws1B gene;
if the amplification product size of the BW-N4R/B-N1F primer is 250bp, and the BM-N3R/B-N1F primer has no amplification band or weak amplification band, the tobacco to be detected contains the WS1B gene;
if the amplification product size of the BW-N4R/B-N1F primer is 250bp, and the amplification product size of the BM-N3R/B-N1F primer is 251bp, the tobacco to be detected contains a WS1B gene and a WS1B gene;
to verify that WS1B and WS1b were identified as correct, PCR sequencing was performed using another pair of specific primers 6877-1F/6877-2R for further confirmation.
The sequences of the relevant primers and the PCR amplification conditions are shown in Table 4.
TABLE 4 primers for the genotyping of WS1A, WS1a, WS1B and WS1b
Application of primers for identifying WS1A, WS1a, WS1B and WS1b genotypes
1. And (3) carrying out genotype identification on 22 burley tobacco varieties and 24 green-stem tobacco varieties (tables 5 and 6) provided by a national tobacco germplasm resource library (http:// www.ycsjk.com.cn /) by using the specific primers in the step one.
The results show that: all burley tobacco varieties contained homozygous WS1a gene and WS1b gene, while all green-stem tobacco varieties contained homozygous WS1A gene and WS1B gene (fig. 7).
Table 5, 22 burley tobacco germplasm resources for the genotyping of WS1A, WS1a, WS1B and WS1b
Table 6, 24 Nicotiana tabacum germplasm resources for the genotyping of WS1A, WS1a, WS1B and WS1b
| Variety of (IV) C | Color of stem |
| Adcock | Green colour |
| Cekpka | Green colour |
| Connecticat-S98 | Green colour |
| Greece Basma | Green colour |
| Havana No. 1 | Green colour |
| K326 | Green colour |
| KARABAGLAR izmir | Green colour |
| Katerini A | |
| Kutsaga | |
| 110 | Green colour |
| Maden | Green colour |
| Samsun | Green colour |
| Saribaglar | Green colour |
| Wisconsin 38 | Green colour |
| Xanthi NN | Green colour |
| An 88-2 | Green colour |
| Baila cigarette | |
| Hainan | |
| 10 | Green colour |
| Leaf apex of Salix cheilophila | Green colour |
| Construct Heheng one number | Green colour |
| Reclamation of agricultural crops | Green colour |
| Shao Huang I Hao | Green colour |
| Iron blue 3 | Green colour |
| Medium cigarette 100 | Green colour |
| Build ripples No. 2 | Green colour |
2. To further verify the effectiveness of these specific primers, the genotypes of 376 individuals in the BC1F1 population, generated by the hybridization and backcross of TN90 with safflower macrogol, were identified.
As a result, 114 white stem individuals all contained the homozygous WS1a gene and WS1b gene, while 262 green stem individuals had three genotypes in total, respectively WS1Aws1a WS1Bws1b, WS1Aws1aWS1Bws1b and WS1aWS1a WS1Bws1b, the number of the individual plants was 76, 88 and 98 (FIG. 8), and the separation ratio was 1:1:1(χ 1: 1) (FIG. 8)2 0.052.779, less than critical value 5.991).
The results show that the molecular marker co-separated from the burley tobacco control gene and designed in the step one can quickly and accurately identify the tobacco genotype and the stalk character in molecular breeding.
Sequence listing
<110> tobacco institute of Chinese academy of agricultural sciences
<120> a method for locating and cloning a double recessive gene controlling the same trait in a plant
<160> 67
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1645
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgggaacgc taacgagctg cagtttcagc tcaatgaata taaggttccg tttgaatcct 60
ccagttaatt acactttcag tcgaagaatc caattgaaga gaatgtccaa acggaatttc 120
ggtcgattga ttattaggtg tagtagcggt agtagtggca atggcagtag caatgacagt 180
ggtagcagta gcgatgggaa attggaaaag gattcttcaa atttggctac agttactgaa 240
gaaaccactg aagaaaggaa cggcggcggt ggcgccagcg gtgtggaaaa tgattcggat 300
gattctccgg tgtcaatttc ttccagacca acaatatcca ctgttggatc aacttataat 360
aatttccaag tagattcttt taagttgatg gaacttcttg gaccagaaaa ggttgatccc 420
agtgatgtga agttcattaa ggaaaagtta tttggctact ctactttttg ggtgactaaa 480
gaagaaccat ttggagatct tggagagggc attcttttcc ttgggaatct tagaggaaag 540
agggaggatg tttttgccaa acttcagagt cagttatcag aaattatggg tgataagtac 600
aacctgttca tggtggagga acctaattca gaggggccag acccgcgtgg tgggcccaga 660
gtcagctttg gtatgctgcg gaaagaagtt tctgaaccag gtccaacaac tctctggcaa 720
tatgtaattg cttttctgtt gttccttctc actattggtt cctctgtgga gctaggaatt 780
gcatctcaga taactcgcct tcctcctgag gtagttaagt actttactga tccaaatgca 840
attgaaccac cagatatgca gcttttatta ccgtttgtgg attctgcttt accgttggca 900
tatggtgtgc tgggtgtgca gttatttcat gaaattgggc attttctggc tgcatttcca 960
aggaatgtga aattaagcat tcctttcttt attccaaaca tcactcttgg aagctttgga 1020
gcaatcactc agttcaaatc tattcttccc gatcgcaaag caaaggtaga catttctctt 1080
gcgggtcctt ttgctggtgc tgcattgtct tcttccatgt ttgcggttgg cctgttactc 1140
tcatccaatc ctgctgctgc tggagagttg gttcaggttc ctagcacact tttccagggc 1200
tctttgcttc tcgggcttat tagcagagcc actcttggtt atggagcaat gcatggtgca 1260
atggtttcaa tccatcctct tgtgatagct ggctggtgtg gcttgactac atcggctttt 1320
aatatgctgc cagttggatg tcttgatggt gggagagctg tgcagggagc ctttgggaaa 1380
ggatcactta ttggttttgg tttggcgaca tacacacttc tgggcttggg cgtgcttggt 1440
ggacctcttg tcacttcctt ggggattgta tgtgcttata tgtcagagga caccggagaa 1500
accatgcttg aatgatgtaa cagaggtcgg aaattggaga aaagcagctc ttggtgtggc 1560
tatattcctt gttgtattga ctcttcttcc tgtatgggat gaacttgcag aagaactagg 1620
tataggtctt gtaaccagct tttga 1645
<210> 2
<211> 1627
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atgggaacgc taacgagctg cagtttcagc acaatgaata taaggttccg tttgaatcct 60
ccagttaatc acagtttcag tcgaagaatc caattgaaga gaatgtccaa acggaatttc 120
ggtagattga ttattaggtg tagtagtgga aatggcagta gcaataacaa tggcagcagt 180
agcgatggga aattggaaaa ggattcttca aatttagcta cagttactga agaaaccact 240
gaagaaagga acggcggcgg tggcgccagc ggtgtggaaa atgattccga ggattctccg 300
gtgtcaattt cttccagacc aacaatatcc acggttggat caacttataa taatttccaa 360
gtagattctt ttaagttgat ggaacttctt ggaccagaaa aggttgatcc cagtgatgtg 420
aagataatta aggaaaagtt atttggctac tctacttttt gggtgactaa agaagaacca 480
tttggagatc ttggagaggg cattcttttc cttgggaatc ttagaggaaa gagggaggat 540
gtttttgcca aacttcagag tcagttatca gaaattatgg gtgataagta caacctgttc 600
atggtggaag agcctaactc tggacccacg tggtgggccc agagttagct ttggtatgct 660
gcggaaagaa gtttctgaac caggtccaac aactctctgg caatatgtaa ttgcttttct 720
gttgttcctt ctcacaattg gttcctctgt ggagctagga attgcatctc agataactcg 780
ccttcctcct gaggtagtta agtactttac ggatccaaat gcaattgaac caccagatat 840
gcagctttta ctaccgtttg tggattctgc tataccactg gcatatggtg tgttgggcgt 900
gcagttattt catgaaattg ggcattttct ggctgcgttt ccaaggaatg tgaaattaag 960
cattcctttc tttattccaa acatcactct tggaagcttt ggagcaatca ctcagttcaa 1020
atctattctt cctgatcgaa aagcaaaggt agatatttcg cttgtgggtc cttttgctgg 1080
tgctgcattg tcttcttcaa tgtttgcggt tggcctgtta ctctcatcca atcctgctgc 1140
ttctggagag ttggttcagg ttcctagcac acttttccag ggatctttgc ttcttgggct 1200
tattagcaga gccactcttg gttatggagc aatgcatgga gcaatggttt caatccatcc 1260
tcttgtgatt gctggctggt gtggtttgac tacgtcggct tttaatatgc taccagttgg 1320
atgtcttgat ggtgggagag ctgtgcaggg agcctttggg aaaggatcac ttattggttt 1380
tggtttggcg acatacacac ttctgggctt gggcgtgctt ggtggacctc tgtcacttcc 1440
ttggggatta tatgtgctta tatgtcagag gacaccagag aaaccatgct tgaacgatgt 1500
aacagaggtc ggaacttgga gaaaagcagc tcttggtgtg gctatattcc ttgtagtatt 1560
gactcttctt cctgtatggg atgaacttgc agaagaacta ggtataggtc ttgtaaccag 1620
cttttga 1627
<210> 3
<211> 1635
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgggaacgc taacgagctg cagtttcagc acaatgaata taaggttccg tttgaatcct 60
ccagttaatc acagtttcag tcgaagaatc caattgaaga gaatgtccaa acggaatttc 120
ggtagattga ttattaggtg tagtagtgga aatggcagta gcaataacaa tggcagcagt 180
agcgatggga aattggaaaa ggattcttca aatttagcta cagttactga agaaaccact 240
gaagaaagga acggcggcgg tggcgccagc ggtgtggaaa atgattccga ggattctccg 300
gtgtcaattt cttccagacc aacaatatcc acggttggat caacttataa taatttccaa 360
gtagattctt ttaagttgat ggaacttctt ggaccagaaa aggttgatcc cagtgatgtg 420
aagataatta aggaaaagtt atttggctac tctacttttt gggtgactaa agaagaacca 480
tttggagatc ttggagaggg cattcttttc cttgggaatc ttagaggaaa gagggaggat 540
gtttttgcca aacttcagag tcagttatca gaaattatgg gtgataagta caacctgttc 600
atggtggaag agcctaattc agaggggcca gacccacgtg gtgggcccag agttagcttt 660
ggtatgctgc ggaaagaagt ttctgaacca ggtccaacaa ctctctggca atatgtaatt 720
gcttttctgt tgttccttct cacaattggt tcctctgtgg agctaggaat tgcatctcag 780
ataactcgcc ttcctcctga ggtagttaag tactttacgg atccaaatgc aattgaacca 840
ccagatatgc agcttttact accgtttgtg gattctgcta taccactggc atatggtgtg 900
ttgggcgtgc agttatttca tgaaattggg cattttctgg ctgcgtttcc aaggaatgtg 960
aaattaagca ttcctttctt tattccaaac atcactcttg gaagctttgg agcaatcact 1020
cagttcaaat ctattcttcc tgatcgaaaa gcaaaggtag atatttcgct tgtgggtcct 1080
tttgctggtg ctgcattgtc ttcttcaatg tttgcggttg gcctgttact ctcatccaat 1140
cctgctgctt ctggagagtt ggttcaggtt cctagcacac ttttccaggg atctttgctt 1200
cttgggctta ttagcagagc cactcttggt tatggagcaa tgcatggagc aatggtttca 1260
atccatcctc ttgtgattgc tggctggtgt ggtttgacta cgtcggcttt taatatgcta 1320
ccagttggat gtcttgatgg tgggagagct gtgcagggag cctttgggaa aggatcactt 1380
attggttttg gtttggcgac atacacactt ctgggcttgg gcgtgcttgg tggacctctg 1440
tcacttcctt ggggattata tgtgcttata tgtcagagga caccagagaa accatgcttg 1500
aacgatgtaa cagaggtcgg aacttggaga aaagcagctc ttggtgtggc tatattcctt 1560
gtagtattga ctcttcttcc tgtatgggat gaacttgcag aagaactagg tataggtctt 1620
gtaaccagct tttga 1635
<210> 4
<211> 1644
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
atgggaacgc taacgagctg cagtttcagc tcaatgaata taaggttccg tttgaatcct 60
ccagttaatt acactttcag tcgaagaatc caattgaaga gaatgtccaa acggaatttc 120
ggtcgattga ttattaggtg tagtagcggt agtagtggca atggcagtag caatgacagt 180
ggtagcagta gcgatgggaa attggaaaag gattcttcaa atttggctac agttactgaa 240
gaaaccactg aagaaaggaa cggcggcggt ggcgccagcg gtgtggaaaa tgattcggat 300
gattctccgg tgtcaatttc ttccagacca acaatatcca ctgttggatc aacttataat 360
aatttccaag tagattcttt taagttgatg gaacttcttg gaccagaaaa ggttgatccc 420
agtgatgtga agttcattaa ggaaaagtta tttggctact ctactttttg ggtgactaaa 480
gaagaaccat ttggagatct tggagagggc attcttttcc ttgggaatct tagaggaaag 540
agggaggatg tttttgccaa acttcagagt cagttatcag aaattatggg tgataagtac 600
aacctgttca tggtggagga acctaattca gaggggccag acccgcgtgg tgggcccaga 660
gtcagctttg gtatgctgcg gaaagaagtt tctgaaccag gtccaacaac tctctggcaa 720
tatgtaattg cttttctgtt gttccttctc actattggtt cctctgtgga gctaggaatt 780
gcatctcaga taactcgcct tcctcctgag gtagttaagt actttactga tccaaatgca 840
attgaaccac cagatatgca gcttttatta ccgtttgtgg attctgcttt accgttggca 900
tatggtgtgc tgggtgtgca gttatttcat gaaattgggc attttctggc tgcatttcca 960
aggaatgtga aattaagcat tcctttcttt attccaaaca tcactcttgg aagctttgga 1020
gcaatcactc agttcaaatc tattcttccc gatcgcaaag caaaggtaga catttctctt 1080
gcgggtcctt ttgctggtgc tgcattgtct tcttccatgt ttgcggttgg cctgttactc 1140
tcatccaatc ctgctgctgc tggagagttg gttcaggttc ctagcacact tttccagggc 1200
tctttgcttc tcgggcttat tagcagagcc actcttggtt atggagcaat gcatggtgca 1260
atggtttcaa tccatcctct tgtgatagct ggctggtgtg gcttgactac atcggctttt 1320
aatatgctgc cagttggatg tcttgatggt gggagagctg tgcagggagc ctttgggaaa 1380
ggatcactta ttggttttgg tttggcgaca tacacacttc tgggcttggg cgtgcttggt 1440
ggacctctgt cacttccttg gggattgtat gtgcttatat gtcagaggac accggagaaa 1500
ccatgcttga atgatgtaac agaggtcgga aattggagaa aagcagctct tggtgtggct 1560
atattccttg ttgtattgac tcttcttcct gtatgggatg aacttgcaga agaactaggt 1620
ataggtcttg taaccagctt ttga 1644
<210> 5
<211> 544
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Gly Thr Leu Thr Ser Cys Ser Phe Ser Thr Met Asn Ile Arg Phe
1 5 10 15
Arg Leu Asn Pro Pro Val Asn His Ser Phe Ser Arg Arg Ile Gln Leu
20 25 30
Lys Arg Met Ser Lys Arg Asn Phe Gly Arg Leu Ile Ile Arg Cys Ser
35 40 45
Ser Gly Asn Gly Ser Ser Asn Asn Asn Gly Ser Ser Ser Asp Gly Lys
50 55 60
Leu Glu Lys Asp Ser Ser Asn Leu Ala Thr Val Thr Glu Glu Thr Thr
65 70 75 80
Glu Glu Arg Asn Gly Gly Gly Gly Ala Ser Gly Val Glu Asn Asp Ser
85 90 95
Glu Asp Ser Pro Val Ser Ile Ser Ser Arg Pro Thr Ile Ser Thr Val
100 105 110
Gly Ser Thr Tyr Asn Asn Phe Gln Val Asp Ser Phe Lys Leu Met Glu
115 120 125
Leu Leu Gly Pro Glu Lys Val Asp Pro Ser Asp Val Lys Ile Ile Lys
130 135 140
Glu Lys Leu Phe Gly Tyr Ser Thr Phe Trp Val Thr Lys Glu Glu Pro
145 150 155 160
Phe Gly Asp Leu Gly Glu Gly Ile Leu Phe Leu Gly Asn Leu Arg Gly
165 170 175
Lys Arg Glu Asp Val Phe Ala Lys Leu Gln Ser Gln Leu Ser Glu Ile
180 185 190
Met Gly Asp Lys Tyr Asn Leu Phe Met Val Glu Glu Pro Asn Ser Glu
195 200 205
Gly Pro Asp Pro Arg Gly Gly Pro Arg Val Ser Phe Gly Met Leu Arg
210 215 220
Lys Glu Val Ser Glu Pro Gly Pro Thr Thr Leu Trp Gln Tyr Val Ile
225 230 235 240
Ala Phe Leu Leu Phe Leu Leu Thr Ile Gly Ser Ser Val Glu Leu Gly
245 250 255
Ile Ala Ser Gln Ile Thr Arg Leu Pro Pro Glu Val Val Lys Tyr Phe
260 265 270
Thr Asp Pro Asn Ala Ile Glu Pro Pro Asp Met Gln Leu Leu Leu Pro
275 280 285
Phe Val Asp Ser Ala Ile Pro Leu Ala Tyr Gly Val Leu Gly Val Gln
290 295 300
Leu Phe His Glu Ile Gly His Phe Leu Ala Ala Phe Pro Arg Asn Val
305 310 315 320
Lys Leu Ser Ile Pro Phe Phe Ile Pro Asn Ile Thr Leu Gly Ser Phe
325 330 335
Gly Ala Ile Thr Gln Phe Lys Ser Ile Leu Pro Asp Arg Lys Ala Lys
340 345 350
Val Asp Ile Ser Leu Val Gly Pro Phe Ala Gly Ala Ala Leu Ser Ser
355 360 365
Ser Met Phe Ala Val Gly Leu Leu Leu Ser Ser Asn Pro Ala Ala Ser
370 375 380
Gly Glu Leu Val Gln Val Pro Ser Thr Leu Phe Gln Gly Ser Leu Leu
385 390 395 400
Leu Gly Leu Ile Ser Arg Ala Thr Leu Gly Tyr Gly Ala Met His Gly
405 410 415
Ala Met Val Ser Ile His Pro Leu Val Ile Ala Gly Trp Cys Gly Leu
420 425 430
Thr Thr Ser Ala Phe Asn Met Leu Pro Val Gly Cys Leu Asp Gly Gly
435 440 445
Arg Ala Val Gln Gly Ala Phe Gly Lys Gly Ser Leu Ile Gly Phe Gly
450 455 460
Leu Ala Thr Tyr Thr Leu Leu Gly Leu Gly Val Leu Gly Gly Pro Leu
465 470 475 480
Ser Leu Pro Trp Gly Leu Tyr Val Leu Ile Cys Gln Arg Thr Pro Glu
485 490 495
Lys Pro Cys Leu Asn Asp Val Thr Glu Val Gly Thr Trp Arg Lys Ala
500 505 510
Ala Leu Gly Val Ala Ile Phe Leu Val Val Leu Thr Leu Leu Pro Val
515 520 525
Trp Asp Glu Leu Ala Glu Glu Leu Gly Ile Gly Leu Val Thr Ser Phe
530 535 540
<210> 6
<211> 547
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Met Gly Thr Leu Thr Ser Cys Ser Phe Ser Ser Met Asn Ile Arg Phe
1 5 10 15
Arg Leu Asn Pro Pro Val Asn Tyr Thr Phe Ser Arg Arg Ile Gln Leu
20 25 30
Lys Arg Met Ser Lys Arg Asn Phe Gly Arg Leu Ile Ile Arg Cys Ser
35 40 45
Ser Gly Ser Ser Gly Asn Gly Ser Ser Asn Asp Ser Gly Ser Ser Ser
50 55 60
Asp Gly Lys Leu Glu Lys Asp Ser Ser Asn Leu Ala Thr Val Thr Glu
65 70 75 80
Glu Thr Thr Glu Glu Arg Asn Gly Gly Gly Gly Ala Ser Gly Val Glu
85 90 95
Asn Asp Ser Asp Asp Ser Pro Val Ser Ile Ser Ser Arg Pro Thr Ile
100 105 110
Ser Thr Val Gly Ser Thr Tyr Asn Asn Phe Gln Val Asp Ser Phe Lys
115 120 125
Leu Met Glu Leu Leu Gly Pro Glu Lys Val Asp Pro Ser Asp Val Lys
130 135 140
Phe Ile Lys Glu Lys Leu Phe Gly Tyr Ser Thr Phe Trp Val Thr Lys
145 150 155 160
Glu Glu Pro Phe Gly Asp Leu Gly Glu Gly Ile Leu Phe Leu Gly Asn
165 170 175
Leu Arg Gly Lys Arg Glu Asp Val Phe Ala Lys Leu Gln Ser Gln Leu
180 185 190
Ser Glu Ile Met Gly Asp Lys Tyr Asn Leu Phe Met Val Glu Glu Pro
195 200 205
Asn Ser Glu Gly Pro Asp Pro Arg Gly Gly Pro Arg Val Ser Phe Gly
210 215 220
Met Leu Arg Lys Glu Val Ser Glu Pro Gly Pro Thr Thr Leu Trp Gln
225 230 235 240
Tyr Val Ile Ala Phe Leu Leu Phe Leu Leu Thr Ile Gly Ser Ser Val
245 250 255
Glu Leu Gly Ile Ala Ser Gln Ile Thr Arg Leu Pro Pro Glu Val Val
260 265 270
Lys Tyr Phe Thr Asp Pro Asn Ala Ile Glu Pro Pro Asp Met Gln Leu
275 280 285
Leu Leu Pro Phe Val Asp Ser Ala Leu Pro Leu Ala Tyr Gly Val Leu
290 295 300
Gly Val Gln Leu Phe His Glu Ile Gly His Phe Leu Ala Ala Phe Pro
305 310 315 320
Arg Asn Val Lys Leu Ser Ile Pro Phe Phe Ile Pro Asn Ile Thr Leu
325 330 335
Gly Ser Phe Gly Ala Ile Thr Gln Phe Lys Ser Ile Leu Pro Asp Arg
340 345 350
Lys Ala Lys Val Asp Ile Ser Leu Ala Gly Pro Phe Ala Gly Ala Ala
355 360 365
Leu Ser Ser Ser Met Phe Ala Val Gly Leu Leu Leu Ser Ser Asn Pro
370 375 380
Ala Ala Ala Gly Glu Leu Val Gln Val Pro Ser Thr Leu Phe Gln Gly
385 390 395 400
Ser Leu Leu Leu Gly Leu Ile Ser Arg Ala Thr Leu Gly Tyr Gly Ala
405 410 415
Met His Gly Ala Met Val Ser Ile His Pro Leu Val Ile Ala Gly Trp
420 425 430
Cys Gly Leu Thr Thr Ser Ala Phe Asn Met Leu Pro Val Gly Cys Leu
435 440 445
Asp Gly Gly Arg Ala Val Gln Gly Ala Phe Gly Lys Gly Ser Leu Ile
450 455 460
Gly Phe Gly Leu Ala Thr Tyr Thr Leu Leu Gly Leu Gly Val Leu Gly
465 470 475 480
Gly Pro Leu Ser Leu Pro Trp Gly Leu Tyr Val Leu Ile Cys Gln Arg
485 490 495
Thr Pro Glu Lys Pro Cys Leu Asn Asp Val Thr Glu Val Gly Asn Trp
500 505 510
Arg Lys Ala Ala Leu Gly Val Ala Ile Phe Leu Val Val Leu Thr Leu
515 520 525
Leu Pro Val Trp Asp Glu Leu Ala Glu Glu Leu Gly Ile Gly Leu Val
530 535 540
Thr Ser Phe
545
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acctgttcat ggtggaagag 20
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ctgcgtggtt gacgagttc 19
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
caatcgttgt ccagtgtcta tttg 24
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ccccaaggaa gtgacagagg 20
<210> 11
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
caatcgttgt ccagtgtcta tttg 24
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
ccccaaggaa gtgacaagag 20
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tatgattctc cttttattcc ta 22
<210> 14
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
tgcggtccac tccactga 18
<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gtggcaacta aatgaaaaaa ga 22
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ttagatattc aacatcctcc tt 22
<210> 17
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gttctatatt ttcaaacagt gtg 23
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
tgacaacctc aataagccac 20
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
tcttattctc ttacaacact ctg 23
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
gtagacaagc gtaatgagga 20
<210> 21
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gatgtgtttc ttttgctctt tat 23
<210> 22
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
agtctgagat tatactgggt tg 22
<210> 23
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
agttgaatat gaacctatac aaat 24
<210> 24
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gatagtgaag aaaaatgtga aaat 24
<210> 25
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
ggtacagcgg ggaaagata 19
<210> 26
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aaacctgcaa ttacaagtca aa 22
<210> 27
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tttttcccta ccgattctct ac 22
<210> 28
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tgttgcttct tcacacacat ta 22
<210> 29
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ccactgttta agcaacttta gata 24
<210> 30
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
acaccatata aaatgattgt gaag 24
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
aaacaaaacc gaaccaaacc 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
gaacggacgc taattctcaa 20
<210> 33
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
gtaaaagatt gattaagatt tagac 25
<210> 34
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
gagaattgaa attatgagat tatc 24
<210> 35
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
aaagggcact cccgaatat 19
<210> 36
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
atgcttgtaa atcaaatgat gatg 24
<210> 37
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tcgtgtaggt ttaataaagg ag 22
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
acaaaaggaa agagggaaac 20
<210> 39
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
cggacattga taagttgtag at 22
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
<210> 41
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
aaacgaaata aataaaggaa agaa 24
<210> 42
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
gggcataaaa gtcgatcaat at 22
<210> 43
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
tcttaccacc attgtgtagg a 21
<210> 44
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
caagtgagcg tcagtatttt c 21
<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
acctgttcat ggtggaagag 20
<210> 46
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
ctgcgtggtt gacgagttc 19
<210> 47
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
gggactgcag gtcgacatgg gaacgctaac gagctg 36
<210> 48
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
ggaaattcga gctcggtacc tcaaaagctg gttacaagac ctatac 46
<210> 49
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
ccaagcttgc atgcctgcag atgggaacgc taacgagctg 40
<210> 50
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
ccaagcttgc atgcctgcag ctagaagaat ctaaataaat aagaagc 47
<210> 51
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
tcattgtgct gaaactgcag ctcgttagcg ttcccattgt ga 42
<210> 52
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
ccaagcttgc atgcctgcag attactccac tgtgattcta aaagc 45
<210> 53
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
tcattgagct gaaactgcag ctcgttagcg ttcccattgt ga 42
<210> 54
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
atgctcwtac tcaaacctca cc 22
<210> 55
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
cgtacagtca aaagatcagt atga 24
<210> 56
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
cggtcggcat ctactctatt 20
<210> 57
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
<210> 58
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
atgctcwtac tcaaacctca cc 22
<210> 59
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
ggtawagcag aatccacaaa cgg 23
<210> 60
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
<210> 61
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
catacaggaa gaagagtcaa tact 24
<210> 62
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
atcgcaaagc aaaggtagac 20
<210> 63
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
catacaggaa gaagagtcaa taca 24
<210> 64
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
tgatcccagt gatgtgaaga ta 22
<210> 65
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
ctgcgtggtt gacgagttc 19
<210> 66
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ccagtgtcta tttgttccct c 21
<210> 67
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
cattcaagca tggtttctcc 20
Claims (3)
1. A method for locating and cloning a double recessive gene controlling the same trait in a plant comprising the steps of:
(1) hybridizing the mutant plant with a wild plant to obtain an F1 generation plant, and hybridizing the F1 generation plant with the mutant plant to obtain a BC1F1 segregation population; said mutant plant has a phenotype A and said wild type plant has a phenotype B, said phenotype A and said phenotype B being the same trait and being opposite phenotypes;
(2) respectively carrying out primary positioning on double recessive genes controlling the same character by using molecular markers to respectively obtain primary positions of the double recessive genes in a genome;
(3) selecting one gene locus, developing a molecular marker according to the primary position of the gene locus, and finely positioning the gene locus based on a mutant type single plant which has the same phenotype as the mutant plant in the BC1F1 segregation population to obtain a fine positioning interval of the gene locus;
(4) comparing the sequence difference of the wild plant and the mutant plant in the fine positioning interval to obtain a candidate gene A;
(5) comparing and analyzing the genome sequences of the wild plant and the mutant plant to obtain a homologous gene of the candidate gene A, namely a candidate gene B; the candidate gene A and the candidate gene B are double recessive genes for controlling the same character;
in the (3), the fine positioning method includes the steps of:
(3-1) searching a simple repetitive sequence capable of designing an SSR primer according to the preliminary position of the gene locus, and designing an SSR primer according to the simple repetitive sequence and the upstream and downstream sequences thereof;
(3-2) detecting the mutant plant and the wild plant by using the SSR primers, and selecting SSR primers with polymorphism;
(3-3) detecting the mutant type single-plant small population by using the SSR primer with polymorphism, and positioning the gene locus in an interval A;
the small mutant single plant population is a small part of the small mutant single plant population;
the mutant individual population is a population of plants in the isolated population of BC1F1 having the same phenotype as the mutant plant;
(3-4) searching a simple repetitive sequence capable of designing an SSR primer in the interval A, designing an SSR primer according to the simple repetitive sequence and upstream and downstream sequences of the simple repetitive sequence, detecting the residual mutant type single plant population by using the SSR primer, and positioning the gene locus in an interval B, namely the fine positioning interval;
the residual mutant type single plant population is a population consisting of residual plants except the mutant type single plant small population in the mutant type single plant population;
the plant is tobacco;
the mutant plant is a white stem mutant plantws1Or burley tobacco variety TN 90; the wild typeThe plant is Honghuadajinyuan;
the candidate gene A isws1bA gene; the candidate gene B isws1aA gene.
2. The method of claim 1, wherein: the SSR primers can be designed by searching simple repetitive sequences by using SSRH software.
3. The method of claim 1, wherein: and designing SSR primers according to the simple repetitive sequence and the upstream and downstream 150bp sequences.
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Citations (2)
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|---|---|---|---|---|
| CN101874120A (en) * | 2007-03-26 | 2010-10-27 | 解码遗传学私营有限责任公司 | Genetic variants on chr2 and chr16 as markers for use in breast cancer risk assessment, diagnosis, prognosis and treatment |
| CN103947538A (en) * | 2014-04-28 | 2014-07-30 | 中国农业科学院烟草研究所 | Method for positioning two non-allelic genes for controlling same character of plant |
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2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101874120A (en) * | 2007-03-26 | 2010-10-27 | 解码遗传学私营有限责任公司 | Genetic variants on chr2 and chr16 as markers for use in breast cancer risk assessment, diagnosis, prognosis and treatment |
| CN103947538A (en) * | 2014-04-28 | 2014-07-30 | 中国农业科学院烟草研究所 | Method for positioning two non-allelic genes for controlling same character of plant |
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| Mapping of two white stem genes in tetraploid common tobacco (Nicotiana tabacum L.);Qingzhang Wu等;《Mol Breeding》;20140517;第34卷;第1065–1074页 * |
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