CN103947538A - Method for positioning two non-allelic genes for controlling same character of plant - Google Patents

Abstract

The invention relates to a method for locating two non-allelic genes controlling the same trait in a plant. The specific steps are as follows: 1) Select a plant with a certain trait, assuming that the trait is controlled by two non-allelic genes A and B, After A and B are mutated, the trait becomes a mutant trait; 2) The wild type and the mutant are crossed to obtain F1, and the genotype of F1 is AaBb, which is the same as the wild type, which is a normal trait; 3) After selfing of F1, the F2 segregation population is obtained , there are 9 genotypes in total; 4) F1 is backcrossed with mutants, and the BC1F1 segregation populations produced have 4 genotypes in total; 5) selfing of several BC1F1 individual plants with normal traits is obtained to obtain respective BC1F2 segregation populations; the present invention The beneficial effects are: this method combines the advantages of the two traditional positioning methods, and can accurately and quickly locate the two non-allelic genes controlling the same trait in the BC1F2 generation, and is especially suitable for self-bred plants that are not easy to handle. .

Description

A method for mapping two non-allelic genes controlling the same trait in plants

technical field

The invention relates to the field of biotechnology, in particular to a method for positioning two non-allelic genes controlling the same trait in plants.

Background technique

The two non-allelic genes controlling the same trait are mostly produced by genome doubling or gene duplication, and are most common in allotetraploid plants, such as common tobacco, Brassica napus, durum wheat, etc., and occasionally in diploid plants, Such as rice, Arabidopsis and so on. Because their biological functions are very similar and there is functional complementarity between them, a single gene mutation cannot cause the corresponding recessive phenotype. Molecular markers are currently the most important and reliable means of gene location, and play a very important role in the location of a single gene and map-based cloning. However, in terms of locating the two non-allelic genes that control the same trait in plants, the current popular methods are either too rough or take too long.

In terms of locating the two non-allelic genes that control the same trait in plants, there are mainly two methods according to the segregating population used: one is to use the F2 or BC1F1 segregating population, and use the above two genes as quantitative trait loci (QTLs). trait loci, QTLs) for positioning, this method can quickly obtain the positioning results due to the quicker acquisition of segregation populations (wild type and mutant hybridization and then self-crossing or backcrossing with mutants once), but the positioning accuracy is relatively low. Low, and cannot fine-map individual genes. The genes mapped by this method include two rice fertility restoration genes Rf3 and Rf(u) and two durum wheat yellow-green leaf genes ygld1 and ygld2; the other is the method of Advanced Backcross , first separate the two non-allelic genes into different backcross individual plants, and then locate them respectively. This method has high precision and can be used for map-based cloning of genes, but it takes a long time because the positioning usually starts after the third generation of backcross (BC3). The genes mapped by this method include a male sterility gene ms2 in Brassica napus and a three-loculed silique gene mc1 in Indian mustard.

Contents of the invention

Aiming at the defects existing in the prior art, the purpose of the present invention is to provide a method for locating two non-allelic genes controlling the same trait in plants.

The present invention adopts following technical scheme:

A method for locating two non-allelic genes controlling the same trait in a plant, the specific steps are as follows:

1) Select a plant with a certain trait, assuming that the trait is controlled by two non-allelic genes A and B, and the trait becomes a mutant trait after A and B are mutated, then the genotype of the wild type is AABB, which shows a normal trait , and the genotype of the mutant is aabb, showing mutation traits;

2) F1 is obtained by crossing the wild type and the mutant, and the genotype of F1 is AaBb, which behaves the same as the wild type and is a normal trait;

3) The F2 segregation population is obtained after F1 self-crossing, and there are 9 genotypes AABB, AABb, AAbb, AaBB, AaBb, Aabb, aaBB, aaBb, aabb, of which aabb accounts for 1/16, so the normal and mutant traits of the F2 population The separation ratio is 15:1;

4) Backcross F1 with mutants, and the resulting BC1F1 segregation population has four genotypes AaBb, Aabb, aaBb, and aabb, each accounting for 1/4, so the segregation ratio of normal traits and mutant traits of the BC1F1 population is 3:1 ;

5) Take several BC1F1 single plants with normal traits and self-cross to obtain respective BC1F2 segregation populations;

Since the genotype of 1/3 of BC1F1 normal traits is AaBb, the BC1F2 segregation population produced by self-crossing is the same as F1, with 9 genotypes, and the segregation ratio of normal traits and mutant traits is 15:1;

Another 1/3 of the BC1F1 individual plant genotype is Aabb, then the BC1F2 segregation population produced by self-crossing has three genotypes AAbb, Aabb, and aabb, of which aabb accounts for 1/4, so the segregation ratio of normal traits and mutant traits The expression is 3:1; since the segregation of normal traits and mutant traits is only related to the segregation of A genotype, containing A is a normal trait, and not containing A is a mutant trait, so this BC1F2 population is a population related to the segregation of the A gene. Use molecular markers to locate the A gene;

The genotype of the remaining 1/3 BC1F1 single plant is aaBb, then the BC1F2 segregation population produced by self-crossing has three genotypes aaBB, aaBb, and aabb, of which aabb accounts for 1/4, so the segregation ratio of normal traits and mutant traits The expression is 3:1; since the segregation of normal traits and mutant traits is only related to the segregation of the B genotype, B is a normal trait, and no B is a mutant trait, so this BC1F2 population is a population related to the segregation of the B gene. Molecular markers can be used to locate the B gene;

A method for locating two non-allelic genes that control the color of common tobacco stalks by using the above method, the specific steps are as follows:

1) The base of the stem of the normal Zhongyan 100 is green, and the base of the stem of the mutant is white. The hybrid F1 was obtained by crossing the mutant with a tobacco variety Honghua Dajinyuan with green stems, and F1 was obtained by selfing F1 Segregation population, F1 was backcrossed with Honghua Dajinyuan at the same time to obtain BC1F1 segregation population. Statistics and Chi-square test were carried out on the number of plants with green and white stalks in F2 and BC1F1 populations, and it was found that the green-white segregation ratios were 15:1 and 3:1, respectively, which indicated that the trait of white stalks was controlled by two recessive genes , we name them c and d respectively, then their corresponding dominant genes are C and D respectively;

2) From the BC1F1 segregation population in step 1, 9 individual plants with green stalks were selected for selfing to obtain respective BC1F2 segregation populations, numbered 1-9 respectively; The number of plants was counted and chi-squared tested, and it was found that the green-white separation ratio of the 4 populations from Nos. 1 to 4 was 15:1, while the green-white separation ratio of the other 5 populations from Nos. 5 to 9 was 3:1, which indicated that The genotype of the previous generation BC1F1 individual plant corresponding to the fifth population is Ccdd or ccDd, which can be used to locate the c gene or d gene;

3) Gene mapping using tobacco SSR molecular markers

According to the SSR marker linkage map M of common tobacco, 1376 pairs of SSR primers were selected for PCR on the genomic DNA of the mutant and Honghua Dajinyuan. For the SSR marker linkage map M of common tobacco and 1376 pairs of SSR primers, references: Bindler, G., Plieske, J., Bakaher, N., Gunduz, I., Ivanov, N., Van der Hoeven, R., Ganal, M. and Donini, P. (2011) A high density genetic map of tobacco (Nicotiana tabacum L .) obtained from large scale microsatellite marker development. Theoretical and Applied Genetics, 123, 219-230.

PCR products were separated by 6% polyacrylamide gel electrophoresis, and a total of 183 pairs of polymorphic primers were obtained between the mutant and Dajinyuan safflower, and these primers were used to pair the 10 green stems of the No. 5 BC1F2 population Genomic DNA of a single stalk and 10 white stalks was amplified and detected by electrophoresis, and it was found that the primer PT61414 on the 5th linkage group was only 2 of the 10 white stalks, that is, recessive individuals Exchange occurred, but no exchange was observed in 10 green stalk individuals. We further used this primer to detect the remaining 41 recessive individuals in No. 5 population, and finally found 6 out of 51 recessive individuals. exchanged individual plants, which indicated that the primer was linked to the control gene in No. 5 population, we assumed that the gene was c;

4) Using the same method as step 3, extract the genomic DNA of 10 green stalk individual plants and 10 white stalk individual plants in No. , the control gene in population No. 7 is linked, but it is not linked with the control gene in population No. The genotype of the previous generation BC1F1 individual plant corresponding to the number population should be ccDd;

183 pairs of polymorphic SSR primers were used to amplify and electrophoresis the genomic DNA of 11 green stalk individual plants and 10 white stalk individual plants of the No. There were only 3 individuals with white stalks, but no exchange was observed in 11 individuals with green stalks. We further used this primer to detect the remaining 38 recessive individuals in the population, and finally A total of 4 exchanged individuals were found from 48 recessive individuals, which indicated that the primer was linked to the d gene; in this way, we located two non-allelic genes controlling the white stalk trait of common tobacco, which were located in the common tobacco The 5th and 24th SSR markers are on the linkage group.

The method for locating two non-allelic genes controlling the same trait in plants as described above is used for locating two non-allelic genes controlling the same trait in allotetraploid plants.

The above-mentioned allotetraploid plants are common tobacco, Brassica napus, and durum wheat.

The beneficial effects of the present invention are:

This method combines the advantages of the two traditional mapping methods, and can accurately and quickly locate the two non-allelic genes controlling the same trait in the BC1F2 generation, and is especially suitable for self-bred plants that are difficult to handle.

Description of drawings

The present invention has following accompanying drawing:

Fig. 1 is the gel electrophoresis figure after the genomic DNA of 10 green stalk individual plants and 10 white stalk individual plants of No. 5 BC1F2 population in Example 2 are amplified by tobacco SSR primer PT61414; the first one on the left The strip is Dajinyuan safflower, the second strip is the mutant, the 3rd-12th is 10 individual plants with green stalks, and the 13th-22nd is 10 individual plants with white stalks; asterisks indicate exchanged individual plants ;

Fig. 2 is the gel electrophoresis figure after the genomic DNA of 11 green stalk individual plants and 10 white stalk individual plants of No. 8 population were amplified by tobacco SSR primer PT53716 in Example 2; the first one on the left The strip is Dajinyuan safflower, the second strip is the mutant, the 3rd-13th is 11 individual plants with green stalks, and the 14th-23rd is 10 individual plants with white stalks; asterisks indicate exchanged individual plants .

Detailed ways

The present invention will be described in further detail below in conjunction with the accompanying drawings.

Example 1

A method for locating two non-allelic genes controlling the same trait in a plant, the specific steps are as follows:

1) Select a plant with a certain trait, assuming that the trait is controlled by two non-allelic genes A and B, and the trait becomes a mutant trait after A and B are mutated, then the genotype of the wild type is AABB, which shows a normal trait , and the genotype of the mutant is aabb, showing mutation traits;

2) F1 is obtained by crossing the wild type and the mutant, and the genotype of F1 is AaBb, which behaves the same as the wild type and is a normal trait;

3) The F2 segregation population is obtained after F1 self-crossing, and there are 9 genotypes AABB, AABb, AAbb, AaBB, AaBb, Aabb, aaBB, aaBb, aabb, of which aabb accounts for 1/16, so the normal and mutant traits of the F2 population The separation ratio is 15:1;

4) Backcross F1 with mutants, and the resulting BC1F1 segregation population has four genotypes AaBb, Aabb, aaBb, and aabb, each accounting for 1/4, so the segregation ratio of normal traits and mutant traits of the BC1F1 population is 3:1 ;

5) Take several BC1F1 single plants with normal traits and self-cross to obtain respective BC1F2 segregation populations;

Since the genotype of 1/3 of BC1F1 normal traits is AaBb, the BC1F2 segregation population produced by self-crossing is the same as F1, with 9 genotypes, and the segregation ratio of normal traits and mutant traits is 15:1;

Another 1/3 of the BC1F1 individual plant genotype is Aabb, then the BC1F2 segregation population produced by self-crossing has three genotypes AAbb, Aabb, and aabb, of which aabb accounts for 1/4, so the segregation ratio of normal traits and mutant traits The expression is 3:1; since the segregation of normal traits and mutant traits is only related to the segregation of A genotype, containing A is a normal trait, and not containing A is a mutant trait, so this BC1F2 population is a population related to the segregation of the A gene. Use molecular markers to locate the A gene;

The genotype of the remaining 1/3 BC1F1 single plant is aaBb, then the BC1F2 segregation population produced by self-crossing has three genotypes aaBB, aaBb, and aabb, of which aabb accounts for 1/4, so the segregation ratio of normal traits and mutant traits The expression is 3:1; since the segregation of normal traits and mutant traits is only related to the segregation of the B genotype, B is a normal trait, and no B is a mutant trait, so this BC1F2 population is a population related to the segregation of the B gene. Molecular markers can be used to locate the B gene;

Example 2

A method for locating two non-allelic genes that control the color of common tobacco stalks by using the above method, the specific steps are as follows:

1) The base of the stem of the normal Zhongyan 100 is green, and the base of the stem of the mutant is white. The hybrid F1 was obtained by crossing the mutant with a tobacco variety Honghua Dajinyuan with green stems, and F1 was obtained by selfing F1 Segregation population, F1 was backcrossed with Honghua Dajinyuan at the same time to obtain BC1F1 segregation population. Statistics and Chi-square test were carried out on the number of plants with green and white stalks in F2 and BC1F1 populations, and it was found that the green-white segregation ratios were 15:1 and 3:1, respectively, which indicated that the trait of white stalks was controlled by two recessive genes , we name them c and d respectively, then their corresponding dominant genes are C and D respectively;

Table 1 Phenotypic statistics of F2 and BC1F1 segregation populations in step 1

separate groups Total number of plants number of green stems Number of white stems Separation ratio chi-square value F2 285 273 12 22.75:1 2.023 BC1F1 134 106 28 3.79:1 1.204

2) From the BC1F1 segregation population in step 1, 9 individual plants with green stalks were selected for selfing to obtain respective BC1F2 segregation populations, numbered 1-9 respectively; The number of plants was counted and chi-squared tested, and it was found that the green-white separation ratio of the 4 populations from Nos. 1 to 4 was 15:1, while the green-white separation ratio of the other 5 populations from Nos. 5 to 9 was 3:1, which indicated that The genotype of the previous generation BC1F1 individual plant corresponding to the fifth population is Ccdd or ccDd, which can be used to locate the c gene or d gene;

Table 2 BC1F1 segregation population 9 green stalks single plant selfing statistics and Chi-square test,

serial number Total number of plants number of green stems Number of white stems Separation ratio chi-square value 1 93 88 5 17.60:1 0.121 2 94 89 5 17.80:1 0.139 3 134 128 6 21.33:1 0.718 4 91 87 4 21.75:1 0.534 5 241 190 51 3.73:1 1.893 6 130 101 29 3.48:1 0.503 7 91 73 18 4.06:1 1.322 8 235 187 48 3.90:1 2.623 9 97 80 17 4.71:1 2.890

3) Gene mapping using tobacco SSR molecular markers

According to the linkage map M of common tobacco SSR markers, 1376 pairs of SSR primers were selected for PCR on the genomic DNA of the mutant and Honghua Dajinyuan. The PCR products were separated by 6% polyacrylamide gel electrophoresis, and a total of 183 pairs were obtained in the mutant The polymorphic primers between Dajinyuan and Honghua Dajinyuan were used to amplify and electrophoresis detect the genomic DNA of 10 green stalk individuals and 10 white stalk individuals of No. 5 BC1F2 population, It was found that the primer PT61414 on the 5th linkage group exchanged only 2 of the 10 white stalk individuals, that is, recessive individuals, but no exchange was observed in the 10 green stalk individuals. We further used The primer detected the remaining 41 recessive individuals in the No. 5 population, and finally found 6 exchanged individuals from the 51 recessive individuals, which indicated that the primer was linked to the control gene in the No. 5 population. We Assume the gene is c;

4) Using the same method as step 3, extract the genomic DNA of 10 green stalk individual plants and 10 white stalk individual plants in No. , the control gene in population No. 7 is linked, but it is not linked with the control gene in population No. The genotype of the previous generation BC1F1 individual plant corresponding to the number population should be ccDd;

Table 3 The statistics of the amplified band patterns of tobacco SSR primer PT61414 in 10 green stalk individual plants and 10 white stalk individual plants in No. 6-9 population

183 pairs of polymorphic SSR primers were used to amplify and electrophoresis the genomic DNA of 11 green stalk individual plants and 10 white stalk individual plants of the No. There were only 3 individuals with white stalks, but no exchange was observed in 11 individuals with green stalks. We further used this primer to detect the remaining 38 recessive individuals in the population, and finally A total of 4 exchanged individual plants were found from 48 recessive individual plants, which indicated that the primer was linked to the d gene; thus, we located two non-allelic genes controlling the white stalk trait of common tobacco, which were located in the common tobacco The 5th and 24th SSR markers are on the linkage group.

Claims (5)

1. two nonallelic methods of locating the same proterties of plant control, is characterized in that concrete step is as follows:

1) choose the plant with a certain proterties, suppose that this proterties is controlled by 2 non-allelic genes A and B, after A and B sudden change, this proterties becomes mutant character, the genotype of wild type is AABB so, show as normal proterties, and the genotype of mutant is aabb, shows as mutant character;

2) wild type and mutant hybridization are obtained to F1, the genotype of F1 is AaBb, shows same wild type, is normal proterties;

3) after F1 selfing, obtain F2 segregation population, have 9 kinds of frequency of genotypes AA BB, AABb, AAbb, AaBB, AaBb, Aabb, aaBB, aaBb, aabb, wherein aabb accounts for 1/16, so the ratio that separates of the normal proterties of F2 colony and mutant character shows as 15:1;

4) F1 is backcrossed with mutant, the BC1F1 segregation population of generation has 4 kinds of genotype AaBb, Aabb, aaBb, aabb, respectively accounts for 1/4, so the normal proterties of BC1F1 colony separates with mutant character than showing as 3:1;

5) get the BC1F1 individual plant selfing of some normal proterties, obtain BC1F2 segregation population separately;

The individual plant genotype of the normal proterties of BC1F1 due to 1/3 is AaBb, and the BC1F2 segregation population that its selfing produces is so identical with F1, has 9 kinds of genotype, and the ratio that separates of normal proterties and mutant character shows as 15:1;

Separately having the green individual plant genotype of 1/3 BC1F1 is Aabb, and the BC1F2 segregation population that its selfing produces so has 3 kinds of frequency of genotypes AA bb, Aabb, aabb, and wherein aabb accounts for 1/4, so the ratio that separates of normal proterties and mutant character shows as 3:1; Because separating of normal proterties and mutant character is only relevant to the genotypic separation of A, containing A is normal proterties, and not containing A is mutant character, therefore this BC1F2 colony is the colony about A Gene Isolation, A gene is positioned with molecular labeling;

The green individual plant genotype of BC1F1 of residue 1/3 is aaBb,, the BC1F2 segregation population that its selfing produces so has 3 kinds of genotype aaBB, aaBb, aabb, and wherein aabb accounts for 1/4, so the ratio that separates of normal proterties and mutant character shows as 3:1; Because separating of normal proterties and mutant character is only relevant to the genotypic separation of B, containing B is normal proterties, is not mutant character containing B, therefore this BC1F2 colony is the colony about B Gene Isolation, can position B gene with molecular labeling.

2. two non-allelic genes that utilize the common tobacco stem stalk of the method positioning control color described in claim 1, is characterized in that concrete step is as follows:

1) the stem culm base of normal Zhongyan-100 is green, the stem culm base of mutant is white in color, with mutant and stem stalk be the green large gold dollar hybridization of tobacco bred safflower, obtain hybrid F1, F1 selfing obtains F2 segregation population, and F1 backcrosses and obtains BC1F1 segregation population with the large gold dollar of safflower simultaneously.Stem stalk green and white individual plant number in F2 and BC1F1 colony have been carried out to statistics and Chi-square Test, find green white separation than meeting respectively 15:1 and 3:1, this shows that white stem stalk proterties is subject to 2 recessive gene controls, we are respectively by its called after c and d, and the dominant gene of their correspondences is respectively C and D so;

2) in the BC1F1 segregation population from step 1, chosen the individual plant selfing of 9 strain stem stalk greens, obtained BC1F2 segregation population separately, be numbered respectively No. 1-9; Stem stalk green and white individual plant number in these colonies have been carried out to statistics and Chi-square Test, the green white separation ratio of finding 4 colonies of No. 1-4 meets 15:1, and the green white separation ratio of 5 colonies of No. 5-9 meets 3:1 in addition, this genotype that shows previous generation BC1F1 individual plant corresponding to the 5th colony is Ccdd or ccDd, can be used for c gene or d gene to position;

3) adopt tobacco SSR molecular labeling to carry out gene location

According to common tobacco SSR mark linkage map M, the genomic DNA of choosing 1376 pairs of SSR primer pair mutant and the large gold dollar of safflower carries out PCR, PCR product separates through 6% polyacrylamide gel electrophoresis, obtain altogether 183 pairs of primers that have polymorphism between mutant and the large gold dollar of safflower, increase and electrophoresis detection with 10 green stem stalk individual plants of No. 5 BC1F2 colony of these primer pairs and the genomic DNA of 10 white stem stalk individual plants, find that primer PT61414 in the 5th linkage group is at 10 white stem stalk individual plants, be in recessive individual plant, to only have 2 individual plants to exchange, and in 10 green stem stalk individual plants, do not observe exchange, we have further detected remaining 41 recessive individual plants in No. 5 colony with this primer, finally from 51 recessive individual plants, find altogether 6 exchange individual plants, this shows that the controlling gene in this primer and No. 5 colony is chain, we suppose that this gene is c,

4) adopt the method identical with step 3, extract the genomic DNA of each 10 green stem stalk individual plants and 10 white stem stalk individual plants in 6-9 BC1F2 colony, detect with PT61414, find that the controlling gene in this primer and the 6th, No. 7 colonies is chain, but not with the 8th, No. 9 colonies in controlling gene chain, the genotype of previous generation BC1F1 individual plant corresponding to such 5-7 colony is Ccdd, and the genotype of previous generation BC1F1 individual plant corresponding to the 8th, No. 9 colonies should be ccDd;

Increase and electrophoresis detection with 11 green stem stalk individual plants of No. 8 colony of 183 pairs of polymorphism SSR primer pairs and the genomic DNA of 10 white stem stalk individual plants, find that primer PT53716 in the 24th linkage group is at 10 white stem stalk individual plants, only have 3 individual plants to exchange, and in 11 green stem stalk individual plants, do not observe exchange, we further detect remaining 38 recessive individual plants in Liao Gai colony with this primer, finally from 48 recessive individual plants, find altogether 4 exchange individual plants, this shows this primer and d gene linkage; Like this, we have located respectively 2 non-allelic genes controlling common tobacco white stem stalk proterties, and they lay respectively in common tobacco the 5th and 24SSR mark linkage group.

3. two non-allelic genes methods of the same proterties of location according to claim 1 plant control, is characterized in that the method controls two non-allelic genes of same proterties for locating plant.

4. two non-allelic genes methods of the same proterties of location plant control according to claim 3, is characterized in that described plant is allotetraploid plant.

5. two non-allelic genes methods of the same proterties of location plant control according to claim 4, is characterized in that described allotetraploid plant is common tobacco, cabbage type rape, durum wheat.