CN113557956B - Construction method of peanut genetic population - Google Patents
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Abstract
The invention discloses a construction method of a peanut genetic population, belonging to the technical field of genetic population construction. The method for constructing the peanut genetic population comprises the steps of hybridizing the two peanuts with FAD2 genotype difference, and detecting F by competitive Allele Specific PCR (KASP)1Replacing FAD2 genotype of the seed kernels, screening the heterozygous seed kernels with FAD2 genotype, and removing the homozygous seed kernels with FAD2 genotype; observation and detection F1Single seed sowing harvest F2Whether the target phenotype of the generation has segregated, for F2And (3) carrying out FAD2 genotype detection on strains with unobvious phenotype to eliminate homozygous strains, and then constructing related genetic populations through selfing or backcrossing. The method of the invention can simply and efficiently screen out true hybrids and construct target character effective segregation population.
Description
Technical Field
The invention belongs to the technical field of genetic population construction, and particularly relates to a construction method of a peanut genetic population.
Background
Sexual hybridization is an important way for crop breeding and is an indispensable method for constructing genetic groups. For self-pollinated crops, artificial hybridization needs two stages of female parent emasculation and artificial pollination. During peanut hybridization, non-pollinated flowers need to be removed before and after hybridization to avoid contamination of non-hybridized needles. However, in the actual operation process, false hybrids with a certain proportion exist in the harvested hybrid fruits due to the reasons of incomplete removal of stamens of female parents, missing of picking flowers and the like. Therefore, screening true hybrids and rejecting false hybrids become important work after hybrid fruits are harvested. For a long time, breeders have observed F1And in F2If the generation has character segregation, the true hybrid is selected, but the selection is carried out on the premise that the two hybrid parents need obvious character differences which are easy to identify and the characters of the differences need to be F2Separation occurs. If a trait is determined by 2 pairs of the above genes, F2The population segregation ratio needs to reach a certain number before it can be visualized. Moreover, phenotypic testing of many traits, such as oil content, fatty acid content, disease resistance, stress resistance, etc., is burdensome and inaccurate. With the rapid development of molecular biotechnology, screening hybrid species by using molecular markers is becoming an effective method for identifying true hybrids. Such as using SSR markers, AhmITE markers, CAPS markers, KASP markers, and SNP markersThe identification of true hybrids in peanuts has been reported. The method firstly needs to carry out polymorphism screening on the hybrid parents, and the hybrid parents widely selected in production have the current situations of relatively close relationship and low polymorphism, so that the screening work is complicated and the efficiency is low.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to provide a method for constructing a peanut genetic population.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for constructing a peanut genetic population comprises the following steps:
(1) adopting two amphipathic peanuts with obvious target character difference and stable inheritance to carry out hybridization, and harvesting the hybrid pod F1The amphiphilic peanut has the FAD2 genotype difference of non-target traits besides the target traits;
(2) cutting F1Cotyledon at the far side of embryo end of the kernel substitute is used for extracting genome DNA, and the kernel with the cut is sealed by smearing the cut with paraffin;
(3) extracting DNA of the cut cotyledon, designing a Specific primer, detecting the genotype of FAD2 by adopting competitive Allele Specific PCR (Kompetitive Allele Specific PCR, KASP), screening the genotype of FAD2 into heterozygous kernels, and rejecting the genotype of FAD2 into homozygous kernels;
(4) the hybrid F obtained by screening in step (3)1Sowing in field, after mature, harvesting according to individual plant number F2Generation;
(5) observation and detection F2Whether separation of phenotype of interest occurred, for F2Representative, non-segregating strains, corresponding to F2Extracting DNA from leaves of the strain, detecting the genotype of FAD2 by adopting competitive allele specific PCR (KASP), and eliminating the genotype of FAD2 as a homozygous strain;
(6) f obtained by secondary screening2Performing single-seed sowing on the seeds, then continuously selfing for 5-6 generations according to a single-seed transmission method to construct a Recombinant Inbred Line (RIL) genetic population, or constructing through backcrossNear Isogenic Lines (NIL) genetic populations.
The existing FAD2 genotype difference is that the amphiphilic peanut contains at least one genotype difference of AhFAD2A/2a and AhFAD2B/2 b.
The specific primers are differential primers FAD2A-WT, FAD2a-M and a universal reverse primer FAD2A-R which are respectively designed aiming at a wild type locus and a mutation locus according to the difference of bases G/ASNP of an AhFAD2A/2a locus (448 th locus of AhFAD2 gene); according to the deletion/insertion difference of A bases of AhFAD2B/2b sites (442 th site of AhFAD2 gene), difference primers FAD2B-WT, FAD2b-M and a universal reverse primer FAD2B-R are designed aiming at a wild type site and a mutation site respectively. Primer sequences are shown in a table 1, and 5' end tailing sequences of wild type locus primers FAD2A-WT and FAD2B-WT are labeled by FAM fluorescence; the 5' end tailing sequences of the mutation site primers FAD2a-M and FAD2b-M are both marked by HEX fluorescence.
A method for constructing a genetic population of a recombinant inbred line aiming at the oil content phenotype of peanuts comprises the following steps:
(1) adopting two amphipathic peanuts with obvious peanut oil content character difference to carry out hybridization, and harvesting the hybrid pod F1The amphiphilic peanut has the FAD2 genotype difference of non-target traits besides the target traits;
(2) cutting F1Cotyledon at the far side of embryo end of the kernel substitute is used for extracting genome DNA, and the kernel with the cut is sealed by smearing the cut with paraffin;
(3) extracting DNA of the cut cotyledon, designing a specific primer, detecting the genotype of FAD2 by adopting competitive allele specific PCR (KASP), screening the genotype of FAD2 as heterozygous seed kernels, and rejecting the genotype of FAD2 as homozygous seed kernels;
(4) the hybrid F obtained by screening in step (3)1Sowing in field, after mature, harvesting according to individual plant number F2Generation;
(5) observation and detection F2Whether separation of phenotype of interest occurred, for F2Representative, non-segregating strains, corresponding to F2Extracting DNA from leaves of the strain, detecting the genotype of FAD2 by competitive allele specific PCR, and eliminatingThe genotype of FAD2 is homozygous;
(6) f obtained by secondary screening2And performing single-seed sowing on the seeds, and then continuously selfing for 5-6 generations according to a single-seed transmission method to construct a recombinant inbred line genetic population aiming at the oil content phenotype of the peanuts.
In a particular embodiment, the peanut parents are the high oil parent yuhua No. 14 and the low oil parent SPI 098.
A method for constructing a recombinant inbred genetic population aiming at a peanut pod length phenotype comprises the following steps:
(1) adopting two parents of peanuts with obvious pod length difference to carry out hybridization, and harvesting the hybrid pod F1The amphiphilic peanut has the FAD2 genotype difference of non-target traits besides the target traits;
(2) cutting F1Cotyledon at the far side of embryo end of the kernel substitute is used for extracting genome DNA, and the kernel with the cut is sealed by smearing the cut with paraffin;
(3) extracting DNA of the cut cotyledon, designing a specific primer, detecting the genotype of FAD2 by adopting competitive allele specific PCR (KASP), screening the genotype of FAD2 as heterozygous seed kernels, and rejecting the genotype of FAD2 as homozygous seed kernels;
(4) the hybrid F obtained by screening in step (3)1Sowing in field, after mature, harvesting according to individual plant number F2Generation;
(5) observation and detection F2Whether separation of phenotype of interest occurred, for F2Representative, non-segregating strains, corresponding to F2Extracting DNA from leaves of the strain, detecting the genotype of FAD2 by adopting competitive allele specific PCR (KASP), and eliminating the genotype of FAD2 as a homozygous strain;
(6) f obtained by secondary screening2And performing single-seed sowing on the seeds, and then continuously selfing for 5-6 generations according to a single-seed transmission method to construct a recombinant inbred line genetic population aiming at the pod length phenotype of the peanuts.
In a specific embodiment, the peanut parents are the large variety Luhua 11 and the small variety BC 54.
The technical scheme of the invention has the advantages that:
wherein, the high oleic acid molecular markers AhFAD2A/2a and AhFAD2B/2b are the most widely applied SNP markers in peanuts. The base G at 448nt of the coding region of the AhFAD2A gene is mutated into A (AhFAD2a), so that the coded amino acid is changed from aspartic acid to asparagine, and the activity of the AhFAD2A enzyme is greatly reduced; meanwhile, the insertion of 1A base (AhFAD2b) at 442nt of the coding region of the AhFAD2B gene leads to the premature termination of the encoded protein, thereby leading to the loss of the enzymatic activity of the AhFAD 2B. The peanut germplasm resources and varieties which are not improved do not contain AhFAD2b locus, and the very small part of the peanut germplasm resources and varieties contains AhFAD2a locus. Therefore, the hybridization between the currently ubiquitous non-high oleic acid material (AABB) and the bred high oleic acid material (AABB) can be performed at F1The AhFAD2a/2b site is used for true hybrid identification and selection, so that the true hybrid can be simply and efficiently screened out, and a target trait effective segregation population is constructed.
Drawings
FIG. 1 shows the sequence and genotype of high oil parent Yuhua 14 at AhFAD2A/2B gene locus;
FIG. 2 shows the sequence and genotype of the low oil parent SPI098 and the little fruit parent BC54 at the AhFAD2A/2B gene sites;
FIG. 3 is the oil content (%) phenotypic difference for the high oil parent Yuhua 14 and the low oil parent SPI 098;
FIG. 4 is F harvested after hybridization of Yuhua 14 with SPI0981The KASP detection result of the kernel at the AhFAD2A/2B gene sites (wherein, AaBb is a true hybrid obtained by successful hybridization, and AABB is a female parent inbred seed harvested by failed hybridization);
FIG. 5 is F1F harvested after selfing2The KASP detection result of the seeds at the AhFAD2B gene locus (the existence of real hybrid offspring (left) of three genotypes of BB, Bb and BB and the existence of false hybrid offspring (right) of the BB genotype only;
FIG. 6 shows the results of oil content (%) phenotypic identification of the RIL population (Yuhua 14X SPI 098);
FIG. 7 shows the sequence and genotype of the parental Luhua 11 with big fruit at AhFAD2A/2B gene sites;
FIG. 8 is a graphical representation of the pod length (mm) phenotypic difference between the parent Luhua 11 and the parent BC 54;
FIG. 9 is F harvested after crossing Luhua 11 with BC541Detecting the KASP detection result of grains at AhFAD2B/2b sites (wherein Bb is a true hybrid obtained by successful hybridization, and BB is a female parent selfed variety harvested by failed hybridization);
FIG. 10 shows the results of pod length (mm) phenotypic identification of RIL population (Luhua 11 × BC 54).
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
The present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
Parent strain: peanut high-oil parent Yuhua No. 14, the sites of FAD2 are AhFAD2A and AhFAD2B (the genotype is AABB); the peanut low-oil parent SPI098 has AhFAD2a and AhFAD2b at FAD2 sites (the genotypes are aabb), the genotype difference of the two FAD2 sites is shown in FIGS. 1 and 2, and the oil contents of Yuhua No. 14 and SPI098 are significantly different, as shown in FIG. 3.
The method for constructing the genetic population of the peanuts comprises the following steps:
1. artificial pollination and hybridization of peanut
High-oil parent Yuhua 14 (common oleic acid content) and low-oil parent SPI098 (high oleic acid content) are planted at intervals in the field. Manually removing the buds of the female parent Yuhua 14 for about 1 week by one week before the full-bloom stage (about 7 weeks after sowing); removing anthers in the buds of the female parent by using tweezers after the blooming period is 17: 00 pm every day to finish castration; pollen of a male parent SPI098 is taken before 8: 00 in the morning of the next day, and is smeared on pistil stigma of a flower bud after female parent castration, so that artificial hybridization pollination work is completed; after the hybridization lasts for about 10 days, the buds are manually removed for about 1 week; then, carrying out rope binding marking on the fruit needles which are born after hybridization; the marked pods are harvested after the pods have matured.
2. Peanut hybrid fruit genome DNA extraction
Removing the kernels after the pods are naturally dried to obtain F1Cutting the side of the distal embryonic endAdding cotyledon about 10mg into a 1.5mL centrifuge tube, grinding with a tissue grinder, extracting genome DNA according to the instruction of a plant genome DNA extraction kit of Beijing Tiangen Biotechnology Co., Ltd, dissolving in 50 μ L sterile water, and detecting the concentration and quality of the DNA with an ultraviolet spectrophotometer and gel electrophoresis; the kernel with the cut is sealed by smearing the cut with paraffin, specifically, the solid paraffin is melted at high temperature and then dripped to the cut until the solid paraffin is naturally solidified.
3. Primer design
According to the difference of bases G/A SNP of an AhFAD2A/2a locus (448 th locus of AhFAD2 gene), designing difference primers FAD2A-WT and FAD2a-M and a universal reverse primer FAD2A-R aiming at a wild type locus and a mutation locus respectively; according to the deletion/insertion difference of A bases of AhFAD2B/2b sites (442 th site of AhFAD2 gene), difference primers FAD2B-WT, FAD2b-M and a universal reverse primer FAD2B-R are designed aiming at a wild type site and a mutation site respectively. Primer sequences are shown in a table 1, and 5' end tailing sequences of wild type locus primers FAD2A-WT and FAD2B-WT are labeled by FAM fluorescence; the 5' end tailing sequences of the mutation site primers FAD2a-M and FAD2b-M are both marked by HEX fluorescence.
TABLE 1 primer sequences for KSAP labeling
4. KASP reaction
The KASP reaction was performed in an LGC SNpline XL water bath PCR instrument in a reaction system of 1. mu.L. Firstly, adding a proper amount (10-50 ng) of extracted F into a PCR reaction plate1Genomic DNA of cotyledon was centrifuged, dried at 65 ℃ for about 30min, and added with 0.5. mu.L Master mix, 0.013. mu.L of each forward and reverse primer (0.2. mu. mol/L), and deionized water to make up 1. mu.L. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 15 min; the first round of amplification was performed with denaturation at 94 ℃ for 20s, annealing at 68 ℃ for 1min, 10 Touch Down cycles (0.6 ℃ reduction per cycle); the second round was performed with denaturation at 94 ℃ for 20s, annealing at 62 ℃ for 1min, and 26 cycles. The detection of the G/A allelic difference of the AhFAD2A/2a adds a third cycle of 3 cycles of amplification under the conditions of 94 ℃ denaturation for 20s and 62 ℃ degradationFire extension was 1 min. Data reading and results analysis were performed using LGC SNPviewer software.
5. KASP identifies the genotype difference between AhFAD2A/2a and AhFAD2B/2b
F harvested after Yuhua 14 hybridization1The KASP detection results of the kernels at FAD2A and FAD2B sites are shown in figure 4, FAM fluorescence is marked at the 5' end of a primer at a wild type AhFAD2A site, HEX fluorescence is marked by a primer at a mutant site, when only FAM fluorescence is detected, the 448 th site is homozygous for G base, and the genotype is wild type AA; when FAM and HEX fluorescence exists at the same time, the gene is a heterozygous genotype Aa. Similarly, FAM fluorescence is marked at the 5' end of a wild type AhFAD2B locus primer, HEX fluorescence is marked at a mutant locus primer, when only FAM fluorescence is detected, the 442 th locus is homozygous for A base deletion, and the genotype is wild type BB; when FAM and HEX fluorescence exist simultaneously, the genotype is heterozygous Bb.
6. Screening true hybrid and rejecting false hybrid according to genotype identification result
The genotypes of Yuhua 14 at the AhFAD2A and AhFAD2B are AA and BB, and the SPI098 is AA and BB. Thus, F1The true hybrid genotype is AaBb. And screening samples showing heterozygosity at AhFAD2A and AhFAD2B gene sites according to the KASP detection result to obtain a true hybrid with the genotype of AaBb (figure 4). The screened true hybrid is bred in Hainan in the same year and harvested according to a single plant to obtain F2And (4) generation.
7、F2Phenotypic identification of surrogate targets and confirmation of FAD2 locus genotype
True hybrid F2The generation should have trait segregation at both the target trait and FAD2 locus. Each F is measured1F obtained from an individual plant2The oil content phenotype in the colony, and selecting and reserving the strain with the oil content with the segregation variation. For the strain with small oil content separation and biased to female parent Yuhua 14, taking the leaves of the strain to extract DNA, detecting the genotype of FAD2 by utilizing competitive allele specific PCR (KASP), if AhFAD2b locus exists in the group, the strain is a true hybrid progeny and is reserved; if AhFAD2b site is not present in the population, it is likely to be derived from F1The strain was rejected by false positive in generation-molecule detection (FIG. 5).
8. Construction of RIL populations Using Single seed-transfer method
Selecting and reserving the true hybrid progeny F after the secondary identification2And (3) seed, harvesting single plants after single-seed sowing, randomly planting 2-4 plants in each single plant, randomly selecting 1 plant during harvesting, carrying out self-crossing propagation for continuous 5-6 generations according to a single-seed-transmission method, and creating a recombinant inbred line RIL group.
7. Detection of target traits in RIL populations
After 6 generations of selfing, RIL colony containing 460 families is bred, sowed in Qingdao in 2020, and after harvest, Soxhlet extraction is adopted to determine the oil content of each family. The results are shown in FIG. 6, where the oil content phenotype of the population is continuously and normally distributed. Can be used as an effective population for positioning the oil content gene.
Example 2
Parent strain: the peanut large fruit variety Luhua 11 has FAD2 sites of AhFAD2a and AhFAD2B (genotype is aaBB), the peanut small fruit line BC54 is obtained by hybridizing Luhua 11 with Kangnong 1715 and backcrossing with female parent Luhua 11 for 4 times, the FAD2 sites are AhFAD2a and AhFAD2b (genotype is aaBB), the genotype difference of the FAD2 sites is shown in FIGS. 7 and 2, and the pod length of the Luhua 11 and BC54 is obviously different, as shown in FIG. 8.
A method for constructing a peanut genetic population comprises the following steps:
1. artificial pollination and hybridization of peanut
The large-fruit parent Luhua 11 (with common oleic acid content) and the small-fruit parent Luhua 11 and the Kangnong 1715 are planted at intervals in the field to generate a backcross progeny BC54 (with high oleic acid content). Manually removing buds of the female parent Luhua 11 for about 1 week before the full-bloom stage (about 7 weeks after sowing); removing anthers in the buds of the female parent by using tweezers after the blooming period is 17: 00 pm every day to finish castration; pollen of a male parent BC54 is taken before 8: 00 a.m. the next day, and is smeared on pistil stigma of a flower bud after female parent castration, so as to complete artificial hybridization pollination work; after the hybridization lasts for about 10 days, the buds are manually removed for about 1 week; then, carrying out rope binding marking on the fruit needles which are born after hybridization; the marked pods are harvested after the pods have matured.
2. Peanut hybrid fruit genome DNA extraction
Removing the kernels after the pods are naturally dried to obtain F1Cutting cotyledon of about 10mg at one side of the far embryo end, putting the cotyledon into a 1.5mL centrifuge tube, grinding the cotyledon by using a tissue grinder, extracting genome DNA according to the instruction of a plant genome DNA extraction kit of Beijing Tiangen Biotechnology Co., Ltd, dissolving the genome DNA in 50 mu L of sterile water, detecting the concentration and the quality of the DNA by using a spectrophotometer and gel electrophoresis, smearing a cut on the seed kernel with the cut with paraffin for sealing, specifically, dripping the melted solid paraffin at high temperature to the cut, and naturally solidifying the seed kernel.
3. Primer design
According to the deletion/insertion difference of A bases of AhFAD2B/2b sites (AhFAD2 gene 442 site), differential primers FAD2B-WT and FAD2b-M and a universal reverse primer FAD2B-R are designed aiming at a wild type site and a mutation site respectively. Primer sequences are shown in a table 1, and FAM fluorescence labeling is used for a tail sequence added at the 5' end of a wild type locus primer FAD 2B-WT; the 5' end tailing sequence of the mutation site primer FAD2b-M is marked by HEX fluorescence.
4. KASP reaction
The KASP reaction was performed in an LGC SNpline XL water bath PCR instrument in a reaction system of 1. mu.L. Firstly, adding a proper amount (10-50 ng) of genomic DNA into a PCR reaction plate, centrifuging, drying at 65 ℃ for about 30min, adding 0.5 mu L of Matser mix, respectively adding 0.013 mu L (0.2 mu mol/L) of forward and reverse primers, and complementing 1 mu L of deionized water. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 15 min; the first round of amplification was performed with denaturation at 94 ℃ for 20s, annealing at 68 ℃ for 1min, 10 Touch Down cycles (0.6 ℃ reduction per cycle); the second round was performed with denaturation at 94 ℃ for 20s, annealing at 62 ℃ for 1min, and 26 cycles. Data reading and results analysis were performed using LGC SNPviewer software.
5. KASP identifies the genotype difference of AhFAD2B/2b
The 5' end of a wild AhFAD2B locus primer is marked with FAM fluorescence, a mutant locus primer is marked with HEX fluorescence, when only FAM fluorescence is detected, the 442 th locus is homozygous for A base deletion, and the genotype is wild type BB; the presence of both FAM and HEX fluorescence was heterozygous genotype Bb (fig. 9).
6. Screening true hybrid and rejecting false hybrid according to genotype identification result
The genotypes of the Luhua 11 at the AhFAD2A and AhFAD2B are aaBB, and the BC54 is aaBB. Thus, F1The true hybrid genotype was aaBb (FIG. 9). And screening a sample showing heterozygosis at the AhFAD2B gene locus according to the KASP detection result to obtain a true hybrid with the genotype Bb. The screened true hybrid is bred in Hainan in the same year and harvested according to a single plant to obtain F2And (4) generation.
7、F2Phenotypic identification of surrogate targets and confirmation of FAD2 locus genotype
True hybrid F2The generation should have trait segregation at both the target trait and FAD2 locus. Each F is measured1F obtained from an individual plant2And (4) carrying out phenotype on pod length of the population, and selecting and reserving a strain with the pod length and the separation phenotype. For the strain with small pod length separation and biased to female parent Luhua 11, taking the leaves of the strain to extract DNA, detecting the genotype of FAD2 by utilizing competitive allele specific PCR (KASP), if AhFAD2b locus in the population is a true hybrid progeny, and reserving the progeny; if AhFAD2b site is not present in the population, it is likely to be derived from F1And (5) false positive of generation molecule detection, and eliminating the strain.
8. Construction of RIL populations Using Single seed-transfer method
Selecting and reserving the true hybrid progeny F after the secondary identification2And (3) seed, harvesting single plants after single-seed sowing, randomly planting 2-4 plants in each single plant, randomly selecting 1 plant during harvesting, carrying out self-crossing propagation for continuous 5-6 generations according to a single-seed-transmission method, and creating a recombinant inbred line RIL group.
9. Detection of target traits in RIL populations
After 6 generations of selfing, RIL group containing 520 families is bred, sowed in Qingdao in 2020, and pod length is measured after harvest. The results are shown in FIG. 10, where the pod length phenotype of this population is continuously normally distributed. Can be used as an effective group for positioning pod size genes.
The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
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Claims (7)
1. A method for constructing a peanut genetic population is characterized by comprising the following steps:
(1) adopting two amphipathic peanuts with obvious target character difference and stable inheritance to carry out hybridization, and harvesting the hybrid pod F1The amphiphilic peanut has the FAD2 genotype difference of non-target traits besides the target traits;
(2) cutting F1Cotyledon at the far side of embryo end of the kernel substitute is used for extracting genome DNA, and the kernel with the cut is sealed by smearing the cut with paraffin;
(3) extracting DNA of the cut cotyledon, designing a specific primer, detecting the genotype of FAD2 by adopting competitive allele specific PCR, screening the genotype of FAD2 as heterozygous seed kernels, and rejecting the genotype of FAD2 as homozygous seed kernels;
(4) the hybrid F obtained by screening in step (3)1Sowing in field, after mature, harvesting according to individual plant number F2Generation;
(5) observation and detection F2Whether separation of phenotype of interest occurred, for F2Representative, non-segregating strains, corresponding to F2Extracting DNA from leaves of the strain, detecting the genotype of FAD2 by competitive allele specific PCR, and eliminating the genotype of FAD2 as a homozygous strain;
(6) f obtained by secondary screening2And performing single-seed sowing on the seeds, and then continuously selfing for 5-6 generations according to a single-seed transmission method to construct a recombinant inbred line genetic population.
2. The method for constructing the genetic population of peanuts according to claim 1, wherein the existing FAD2 genotype difference is that the amphipathic peanuts containAhFAD2A/2a andAhat least one genotype difference in FAD2B/2 b.
3. The method for constructing the genetic population of peanuts according to claim 1, wherein the specific primer is shown as SEQ ID NO. 1-SEQ ID NO. 8.
4. A method for constructing a genetic population of a recombinant inbred line aiming at the oil content phenotype of peanuts is characterized by comprising the following steps:
(1) adopting two amphipathic peanuts with obvious peanut oil content character difference to carry out hybridization, and harvesting the hybrid pod F1The amphiphilic peanut has the FAD2 genotype difference of non-target traits besides the target traits;
(2) cutting F1Cotyledon at the far side of embryo end of the kernel substitute is used for extracting genome DNA, and the kernel with the cut is sealed by smearing the cut with paraffin;
(3) extracting DNA of the cut cotyledon, designing a specific primer, detecting the genotype of FAD2 by adopting competitive allele specific PCR, screening the genotype of FAD2 as heterozygous seed kernels, and rejecting the genotype of FAD2 as homozygous seed kernels;
(4) the hybrid F obtained by screening in step (3)1Sowing in field, after mature, harvesting according to individual plant number F2Generation;
(5) observation and detection F2Representative target phenotypeWhether or not separation occurred, for F2Representative, non-segregating strains, corresponding to F2Extracting DNA from leaves of the strain, detecting the genotype of FAD2 by competitive allele specific PCR, and eliminating the genotype of FAD2 as a homozygous strain;
(6) f obtained by secondary screening2And performing single-seed sowing on the seeds, and then continuously selfing for 5-6 generations according to a single-seed transmission method to construct a recombinant inbred line genetic population aiming at the oil content phenotype of the peanuts.
5. The method for constructing a genetic population of recombinant inbred lines for peanut oil content phenotype according to claim 4, wherein said existing FAD2 genotype difference is that the amphipathic peanut contains at least one genotype difference of AhFAD2A/2a and AhFAD2B/2 b.
6. A method for constructing a recombinant inbred genetic population for a peanut pod length phenotype, comprising the steps of:
(1) adopting two parents of peanuts with obvious peanut pod length character difference to carry out hybridization, and harvesting the hybrid pod F1The amphiphilic peanut has the FAD2 genotype difference of non-target traits besides the target traits;
(2) cutting F1Cotyledon at the far side of embryo end of the kernel substitute is used for extracting genome DNA, and the kernel with the cut is sealed by smearing the cut with paraffin;
(3) extracting DNA of the cut cotyledon, designing a specific primer, detecting the genotype of FAD2 by adopting competitive allele specific PCR, screening the genotype of FAD2 as heterozygous seed kernels, and rejecting the genotype of FAD2 as homozygous seed kernels;
(4) the hybrid F obtained by screening in step (3)1Sowing in field, after mature, harvesting according to individual plant number F2Generation;
(5) observation and detection F2Whether separation of phenotype of interest occurred, for F2Representative, non-segregating strains, corresponding to F2The leaves of the strain, DNA extracted and detected by competitive allele-specific PCRThe genotype of FAD2 is homozygous strain excluding the genotype of FAD 2;
(6) f obtained by secondary screening2And performing single-seed sowing on the seeds, and then continuously selfing for 5-6 generations according to a single-seed transmission method to construct a recombinant inbred line genetic population aiming at the pod length phenotype of the peanuts.
7. The method for constructing a genetic population of recombinant inbred lines for peanut pod length phenotypes according to claim 6, wherein the presence of the FAD2 genotype difference is that amphipathic peanuts contain at least one genotype difference between AhFAD2A/2a and AhFAD2B/2 b.
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| WO2017218969A1 (en) * | 2016-06-16 | 2017-12-21 | Nuseed Pty Ltd. | Inbred transgenic canola line ns-b50027-4 and seeds thereof |
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| CN105385778A (en) * | 2015-12-26 | 2016-03-09 | 山东省农业科学院生物技术研究中心 | Primers and method for detecting SNP gene typing of AhFAD2B genes of peanuts in high throughput |
| WO2017218969A1 (en) * | 2016-06-16 | 2017-12-21 | Nuseed Pty Ltd. | Inbred transgenic canola line ns-b50027-4 and seeds thereof |
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