CN113736910A - Linkage molecular marker of peanut single plant pod number main effect QTL site qPN7 and application thereof - Google Patents

Linkage molecular marker of peanut single plant pod number main effect QTL site qPN7 and application thereof Download PDF

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CN113736910A
CN113736910A CN202111185756.4A CN202111185756A CN113736910A CN 113736910 A CN113736910 A CN 113736910A CN 202111185756 A CN202111185756 A CN 202111185756A CN 113736910 A CN113736910 A CN 113736910A
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张胜忠
陈静
胡晓辉
苗华荣
王菲菲
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Abstract

The invention discloses a major QTL (quantitative trait locus) site for pod number of single peanut plantqPN7The linked molecular marker and the application thereof. The linkage molecular Marker is positioned on the 7 th chromosome of the peanut genome and comprises a molecular Marker4240, a molecular Marker4243 and a molecular Marker 4251. The nucleotide sequence of the molecular Marker4240 is shown as SEQ ID No. 1; the nucleotide sequence of the Marker4243 is shown as SEQ ID No. 3; the nucleotide sequence of the Marker4251 is shown as SEQ ID No. 5. The linked molecular markers are obtained using a primer composition. The invention discloses a peanut single plant pod number main effect QTL locusqPN7And a linkage molecular marker and an identification method thereof, which are suitable for the pod number gene of a single peanut plantPositioning and molecular marker assisted selective breeding.

Description

Linkage molecular marker of peanut single plant pod number main effect QTL site qPN7 and application thereof
Technical Field
The present invention belongs to peanut fieldThe technical field of seed genetic breeding, in particular to a major QTL (quantitative trait locus) site of pod number of single peanut plantqPN7The linked molecular marker and the application thereof.
Background
Peanut (A)Arachis hypogeaL.) is one of five oil crops all over the world, and is also an important source of edible oil and protein in China. In recent years, with the increase of economy and population in China, the consumption demand of peanuts is in a rigid growth situation, and the self-supply capacity of peanuts in China faces a great challenge. The method improves the unit yield of the peanuts by means of variety improvement, and is a main way for improving the total yield of the peanuts and meeting the balance of supply and demand of the market on the premise of farmland resource constraint.
The pod number of a single plant is one of the yield traits of peanuts, belongs to quantitative traits and is easily influenced by the environment. In traditional breeding, the effect of progeny selection only by virtue of phenotype is poor, and the breeding efficiency is often low. By constructing a high-density genetic linkage map, positioning and controlling the gene/QTL locus of the character, developing linkage molecular markers and utilizing the molecular markers for auxiliary selection, the prediction, verification and aggregation of the target character can be effectively realized, and the breeding efficiency is remarkably improved.
In recent years, researchers at home and abroad carry out genetic research on various peanut yield traits, and detect a batch of QTL for controlling peanut pod size, pod weight, seed kernel size, seed kernel weight and other traits. However, positioning research aiming at the pod number of a single peanut plant is rare at present, and reports of relevant main effect QTLs and linked molecular markers are lacked. Therefore, screening a stably expressible main QTL site for pod number of a single peanut plant has important significance for accelerating the map-based cloning of pod number genes of the single peanut plant and cultivating a new peanut variety with excellent pod number characters of the single plant.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a major QTL (quantitative trait locus) site for pod number of single peanut plantqPN7The linked molecular marker and the application thereof.
In order to realize the purpose, the invention adopts the technical scheme that:
the invention provides a peanut single plant pod number main effect QTL locusqPN7The linked molecular markers of (a) above (b),the linkage molecular marker and the peanut single plant pod number main effect QTL locusqPN7Is co-located on the 7 th chromosome of the peanut genome and comprises a molecular Marker4240, a molecular Marker4243 and a molecular Marker 4251.
Further, the nucleotide sequence of the molecular Marker4240 is shown in SEQ ID No. 1; the nucleotide sequence of the Marker4243 is shown as SEQ ID No. 3; the nucleotide sequence of the Marker4251 is shown as SEQ ID No. 5.
Furthermore, the polymorphism of the molecular Marker4240 is A/G, and the nucleotide sequence with polymorphism difference with the polymorphism is shown as SEQ ID No. 2; the polymorphism of the molecular Marker4243 is T/C, and the nucleotide sequence with polymorphism difference with the polymorphism is shown as SEQ ID No. 4; the polymorphism of the molecular Marker4251 is C/T, and the nucleotide sequence with polymorphism difference with the polymorphism is shown as SEQ ID No. 6.
The invention also provides a primer composition, which comprises 6 primers, and the nucleotide sequences of the primers are respectively shown as SEQ ID No. 7-SEQ ID No. 12.
Further, the primer composition comprises a primer of an amplification molecule Marker4240 with the nucleotide sequences shown as SEQ ID No.7 and SEQ ID No.8, a primer of an amplification molecule Marker4243 with the nucleotide sequences shown as SEQ ID No.9 and SEQ ID No.10, and a primer of an amplification molecule Marker4251 with the nucleotide sequences shown as SEQ ID No.11 and SEQ ID No. 12.
The invention also provides a pod number main effect QTL site of a single peanut plantqPN7The identification method of (2), said identification method comprising the steps of:
extracting genome DNA of the peanut material to be identified, carrying out PCR amplification on the template by using the primer composition by using the genome DNA as the template, carrying out sequencing analysis on an amplification product, and judging the polymorphism of the linkage molecular marker according to a sequencing result.
Further, according to the sequencing result of the peanut material to be identified, if the linked molecular marker has polymorphism, namely the nucleotide sequence of the linked molecular marker is the same as that of the strain 6-13, judging that the pod number of a single plant of the peanut material is more than or equal to 20; and if the linked molecular marker does not have polymorphism, namely the nucleotide sequence of the linked molecular marker is the same as the variety Huayu No. 36, judging that the number of the single-plant pods of the peanut material is less than 20.
Furthermore, the 801 th base of the strain 6-13 in the molecular Marker4240 is G, and the 801 th base of the strain variety No. 36 in the molecular Marker4240 is A; the 801 th base of the strain 6-13 on the molecular Marker4243 is C, and the 801 th base of the strain variety No. 36 on the molecular Marker4243 is T; the 801 th base of the strain 6-13 on the molecular Marker4251 is T, and the 801 th base of the strain 36 on the molecular Marker4251 is C.
Further, the reaction procedure of the PCR amplification is as follows: 5 min at 94 ℃; 35 cycles of 98 ℃ for 10 s, 60 ℃ for 30 s and 68 ℃ for 1 min; the extension was terminated at 68 ℃ for 10 min and stored at 4 ℃.
Further, the reaction system of PCR amplification is 50 μ L, specifically comprises 2 μ L of 10-20 ng DNA template, 1.5 μ L of each primer pair, 10 μ L of dNTP, 25 μ L of KOD buffer solution, 1 μ L of KOD polymerase, and ddH2O 9 μL。
The invention also provides a peanut single plant pod number main effect QTL locusqPN7Application of regulating and controlling pod number characters of single peanut plant, wherein the pod number main effect QTL site of the single peanut plantqPN7The physical position of the gene (a) is between 208645-1146586 base positions of chromosome 7 of a peanut genome, and the gene (b) can be obtained by detecting primers shown in SEQ ID No. 7-SEQ ID No. 12; the major QTL site of the pod number of a single peanut plantqPN7The synergistic allele of (A) is from peanut line 6-13, and can increase the pod number of a single peanut plant.
The invention also provides application of the linkage molecular marker or the primer composition in breeding high-single-plant pod number peanut varieties or strains.
The invention also provides application of the linkage molecular marker or the primer composition in peanut molecular breeding, cultivation of transgenic peanuts or improvement of peanut germplasm resources.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention discloses a peanut single plant pod number main effect QTL locus with synergistic allele from cultivated peanut strain 6-13 for the first timeqPN7And the gene type of the gene is located between the 7 th chromosome 208645-1146586 base positions of the peanut genome of the cultivated species, and the different gene types have different regulation and control functions on the pod number of each peanut plant, so that the high utilization value is realized for breeding peanut varieties or strains with high individual pod number.
2. The invention firstly screens and discloses a major QTL (quantitative trait locus) site of pod number of single peanut plantqPN7The three linked molecular markers Marker4240, Marker4243 and Marker4251, as well as the molecular Marker primer pair and the molecular Marker method for amplifying the linked molecular markers can be effectively applied to the gene map location cloning of the pod number of a single peanut plant, assist in the selection of progeny with the pod number character of the single peanut plant, are beneficial to improving the breeding efficiency and accelerate the genetic improvement of the pod number character of the single peanut plant.
Drawings
FIG. 1 shows the number of pods of a single peanut plant of the present inventionqPN7Location on chromosome 7, where the vertical bars represent peanut chromosome 7 and the short horizontal lines on the vertical bars represent molecular markers; number of pods of single peanut plantqPN7The linked 3 molecules are marked as Marker4251, Marker4240 and Marker 4243; the rectangle on the right side of the vertical column represents the major QTL locus detected in 4 different environmentsqPN7The confidence interval of (c).
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Test materials: peanut variety Huayu No. 36 as female parent and peanut variety 6-13 as male parent, and making them pass through a hybridization processObtaining a Recombinant Inbred Line (RIL) group F containing 181 families by a grain-borne method9(2019) and F10In the generation (2020), the number of the individual pods of the female parent flower breeding No. 36 is 15-20, the number of the individual pods of the male parent 6-13 is 24-28, and both can be purchased from peanut research institute in Shandong province.
1. And (3) phenotype identification: the test materials are planted in test bases of Dongying city (37.31 degrees N, 118.62 degrees E) and Qingdao city (36.82 degrees N, 120.51 degrees E) in 2019; planted in test bases of tabacco city (37.18 degrees N, 121.51 degrees E) and Weihai city (37.18 degrees N, 121.99 degrees E) in 2020; the planting mode is ridging single-row single-grain film mulching planting, the ridge distance is 0.3 m, 10 plants are planted in each row, the plant distance is 0.2 m, random block design is carried out, and 3 times of repetition are set. The test base has even fertility, is managed in the conventional field, and can prevent and control the plant diseases and insect pests in time. Mature pods of the middle 8 individual plants were harvested in each row and the number of mature pods per plant was counted.
The results show that the variation ranges of the pod number of each individual plant of the RIL population under 4 different environments (2019 Dongying, 2019 Qingdao, 2020 Sutai and 2020 Wissian) are respectively 6.08-58.16, 5.00-57.40, 7.11-42.10 and 5.74-41.51, and the population shows super-genetic (Table 1), which indicates that the population is suitable for carrying out QTL analysis on the pod number character of each individual plant of peanut.
TABLE 1 RIL population Individual pod number phenotype data in 4 environments
Figure 446530DEST_PATH_IMAGE002
2. Construction of a genetic map: the genomic DNA of variety No. 36, variety No. 6-13 and 181 RIL families was extracted using a Tiangen DNA extraction kit.
SNP typing: constructing a sequencing library according to the construction process of the SLAF library, and using Illumina HiSeq after the quality of the library is qualifiedTMSequencing with 2500 platform, wherein the sequencing depth of parent is more than 51 x, the sequencing depth of RIL family is more than 16 x, sequencing quality evaluation is carried out on the original data after off-line, and high quality reads are compared to peanut reference genome (https:// www.peanutbase.org/data/v 2/Arachis/hypogaea/genes/Tifrunn)Gnm1.kyv3 /). Detection of SNPs is mainly achieved using the GATK software toolkit. The SLAF tags are filtered according to the following rules: (1) filtering tags that were sequenced to a depth of less than 10 x in the parent; (2) filtering the partially separated labels; (3) label with filter integrity less than 70%. According to the RIL population type, the aa x bb type polymorphism SLAF tags are aligned to the peanut reference genome by BWA software (https:// www.peanutbase.org/data/v 2/Arachis/hypogaea/genes/tifrener. gnm1.KYV3 /). And filtering to obtain high-quality SNP for subsequent genetic map construction.
SSR typing: screening the variety No. 36 and lines 6-13 with polymorphic SSR markers by using published SSR markers (https:// www.peanutbase.org/search/marker), and then performing SSR typing on RIL families. The related PCR reaction system is as follows: mu.L of DNA template, 2. mu.L (1. mu.L each) of primer set (0.2. mu.M), 2 XTAQ PCR Master Mix 5. mu.L, ddH2O2. mu.L. The PCR reaction program involved was: 3 min at 94 ℃; 35 cycles of 98 ℃ for 1 min, 55 ℃ for 30 s and 72 ℃ for 90 s; extension was terminated at 72 ℃ for 10 min and stored at 4 ℃. And (3) carrying out electrophoretic analysis on the PCR amplification product by using 6% PAGE gel to obtain SSR typing data of the RIL family.
And (3) genetic map construction: high-quality SNP and SSR markers are distributed to different linkage groups based on paired MLOD values, and the SNP and SSR markers are sequenced and subjected to error-correcting marker typing by using a HighMap strategy. Obtaining the marking sequence of each linkage group by using MSTmap, correcting the error marking sequence by using SMOOTH algorithm, predicting the deletion marking genotype by using k-nearest neighbor algorithm, and estimating the genetic distance of the linkage group based on Kosambi mapping function.
Genetic map information: the gene totally comprises 3866 marker sites, is distributed in 20 linkage groups, covers the distance of a genetic map of 1,266.87 cM, and has the average distance between markers of 0.33 cM.
3. QTL positioning analysis: detecting a major QTL locus on the 7 th chromosome of a peanut cultivar reference genome by using the constructed genetic map and phenotype data of 4 environments and adopting a complete interval mapping method of QTL IiMapping V4.1 softwareqPN7(as shown in FIG. 1), the QTL can be stable in 4 environmentsThe LOD variation range of the fixed expression is 4.50-8.19, the contribution rate variation range is 12.76-16.92%, the synergistic allele comes from the strain 6-13, and the physical position is between the 7 th chromosome 208645-1146586 base positions of the genome of the cultivated peanut.
To further densify the map, major QTL sites were obtainedqPN7Closely linked molecular markers: a molecular Marker4240, a molecular Marker4243 and a molecular Marker 4251.
The physical position of the molecular Marker4240 is peanut reference genome Arahy.07:208645 bp (A/G), the upstream and downstream 800 bp sequence is shown as SEQ ID No.1, and the nucleotide sequence with polymorphism difference is shown as SEQ ID No. 2; the physical position of the molecular Marker4243 is peanut reference genome Arahy.07:811108 bp (T/C), the upstream and downstream 800 bp sequence is shown as SEQ ID No.3, and the nucleotide sequence with polymorphism difference is shown as SEQ ID No. 4; the physical position of the molecular Marker4251 is peanut reference genome Arahy.07:1146586 bp (C/T), the upstream and downstream 800 bp sequence is shown as SEQ ID No.5, and the nucleotide sequence with polymorphism difference is shown as SEQ ID No. 6.
Example 2
Utilizing the number of peanut pods as the major effective siteqPN7And 3 linked molecular marker primer pairs are used for carrying out PCR amplification on the genomic DNA of the RIL population, the variety No. 36 and the strains 6-13, and further judging the polymorphism of the RIL family in 3 molecular marker sequences.
Amplification of major QTL (quantitative trait locus) site of pod number of single peanut plantqPN7Primer pair of linked molecular Marker 4240:
a forward primer sequence 5'-ATTTCAAGGGGACACTCTGC-3' (shown as SEQ ID NO. 7);
the reverse primer sequence 5'-TGTCAAGTGGCCCGGTTTA-3' (shown as SEQ ID NO. 8).
The target base of the variety No. 36 flower culture in the molecular Marker4240 is A (the 801 th base shown in SEQ ID No. 1), and the target base of the strain 6-13 in the molecular Marker4240 is G (the 801 th base shown in SEQ ID No. 2).
Amplification of major QTL (quantitative trait locus) site of pod number of single peanut plantqPN7Introduction of linked molecular Marker4243Pairing:
a forward primer sequence 5'-ATAACAATCACCCTGATTTCG-3' (shown as SEQ ID NO. 9);
the reverse primer sequence 5'-CAACTCCAACTGCTGTCGTC-3' (shown as SEQ ID NO. 10).
The target base of the variety No. 36 flower culture in the molecular Marker4243 is T (the 801 th base shown in SEQ ID No. 3), and the target base of the strain 6-13 in the molecular Marker4243 is C (the 801 th base shown in SEQ ID No. 4).
Amplification of major QTL (quantitative trait locus) site of pod number of single peanut plantqPN7Primer pair of linked molecular Marker 4251:
a forward primer sequence 5'-ACTGAGTTTACCTACTGCTGAG-3' (shown as SEQ ID NO. 11);
the reverse primer sequence 5'-TAGTTGGACGAGGTTGTGAG-3' (shown in SEQ ID NO. 12).
The target base of variety No. 36 in the molecular Marker4251 is C (shown as the 801 th base in SEQ ID No. 5), and the target base of the strain 6-13 in the molecular Marker4251 is T (shown as the 801 th base in SEQ ID No. 6).
When the molecule is amplified for labeling, the reaction system of PCR amplification is 50 muL, and specifically comprises 2 muL (10-20 ng) of DNA template, 3 muL (1.5 muL each) of primer pair (0.2 muM), 10 muL of dNTP (50 muM), 25 muL of KOD buffer solution, 1 muL of KOD polymerase and ddH2O 9 μL。
The reaction program of the PCR amplification is 94 ℃ for 5 min; 35 cycles of 98 ℃ for 10 s, 60 ℃ for 30 s and 68 ℃ for 1 min; the extension was terminated at 68 ℃ for 10 min and stored at 4 ℃.
Sequencing the product after PCR amplification by using an Illumina HiSeq platform, and detecting the families of which the 3 molecular marker sequences are the same as the strain 6-13 through sequence comparison, wherein the families are marked as AA type families; the 3 molecular marker genotypes are all the same family as the variety No. 36, and are marked as aa family.
The results show that RIL (F)10) There were 33 families of the population of AA type, indicating that these families are inqPN7The alleles of the loci are identical to lines 6-13; 97 families were aa type, indicating that these families are inqPN7The position ofThe allele was identical to variety No. 36 (Table 2).
A total of 130 RIL families (33 AA types and 97 AA types) are planted in test bases of Dongying city (37.31 degrees N, 118.62 degrees E) and Qingdao city (36.82 degrees N, 120.51 degrees E) in 2020, the planting mode is ridging single-row single-seed film mulching planting, the ridge distance is 0.3 m, 10 plants are planted in each row, the plant distance is 0.2 m, and 3 times of repetition is set in random block design. The fertility of the test base is uniform, the field management is carried out conventionally, and the pest control is carried out in time. Mature pods of the middle 8 individual plants were harvested in each row and the number of mature pods per plant was counted.
The results showed (table 2) that the average pod numbers of AA family individual plants were 19.82 and 17.00, and the average pod numbers of AA family individual plants were 26.25 and 28.26; the number of the individual pods of the AA-type family and the AA-type family is very different, which indicates thatqPN7The different genotypes of the peanut single plant pod number marker have different regulation and control functions, and the 3 linked molecular markers have a good prediction function on the pod number character of the peanut single plant, so that the peanut single plant pod number marker is suitable for peanut molecular marker-assisted selective breeding.
TABLE 2 differencesqPN7Statistics of number of individual pods of genotype families
Figure 691566DEST_PATH_IMAGE004
Note: is represented inp<At the 0.01 level, there was a significant difference between the AA family and the AA family.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
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agcaactaaa tgtctgctga gaagcttttt ggaagtatgt tctattactc cattattttt 1500
catcttgagg tcattgttaa catgtccttt ttctttctgt attctttaat tattcttttc 1560
ttttgtaaat gaatattact tagttatttt cttgtgttct c 1601
<210> 2
<211> 1601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gatagccaag aagctagaaa tttagttgca tatatattat atagtgaagt taattggaaa 60
tggaagttat gggcatgtgt tgtgtgcttc taacggctaa gaaggaatgt tcgtattcca 120
tttcaagggg acactctgca ccaagtagga tcagatttta tgtgggcccc accataaggg 180
tgtgaaaatg tgatgaaaaa tctcatgaaa aaggatcatg tggttaaatt attagtgaca 240
aattatgctg cttacacagg tcacactcag atttagaaat aaactaccac aaaaaaataa 300
ttaaaagatg ttaagctaag aatcaaactt ctagaaacaa tttttgtaaa attaagtgct 360
tattgtttcc ctaacataat atcttcctaa acatgctata gccatgtgct tcaccaacag 420
gcaaagaaag tgttattata ttcttagttt tttctaatgt tagatgctct tctaactcaa 480
caaggattta ctaatataaa agcaattttg tagatgttac atgccctaat acaatgtttg 540
tagaaaaata gtagctgtaa acgagcaaca atgagatcat gagcaaattc ttttctttat 600
tacttttcat gagagaccat agttatgttg ggagcaacat ccatccggtt gtcaaacatt 660
tttctttttt tctgtttttt ttggtcaagc ggattggaca gtctccaatc ctaggcatta 720
cacccatgtt ttacacacct acacaacaca ctcacacact accatgggta ctatgggttc 780
ataaacaaca accaacctca gctggaattt gaacccggtg cacctccaag caaggcaaac 840
atagttgtca ctaaactaag tcttgcagtg caacattttt ctatttattc ctttttagta 900
gtttgttttt tttttttaaa tttacatgag gtctttttcg cgtaagttgt tgaacaaaca 960
ataagttatc taagaataaa taaaccgggc cacttgacaa cataaaaatc ctcttggagg 1020
aagtaaaata cataaccaga ttaaccaaac tcgactacaa gtatatatat atatatacat 1080
caacaataaa caaaacatat ggggcagtct ctcatgttgg aagcatacct gtttcaaaaa 1140
gcctgtacat tattggtatg caggatttga tttgtgattt gttttattga ctagtatact 1200
tcttcagtca aaaagatcag tgatgatagc gttatcccaa atggcacaca gttcctttat 1260
catgattttt ttgtggaagc taatattatg tcatcaaatg catattgtgg aagttctttt 1320
taactttaac aaggaaatgg cttttgcagg ctggtttgca agggatccta atattttggg 1380
catatactta tgcaagtacc atatatattt caacgcaatc catagctgat gattgttttc 1440
agcaactaaa tgtctgctga gaagcttttt ggaagtatgt tctattactc cattattttt 1500
catcttgagg tcattgttaa catgtccttt ttctttctgt attctttaat tattcttttc 1560
ttttgtaaat gaatattact tagttatttt cttgtgttct c 1601
<210> 3
<211> 1601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ataaaatata ttttttaatt attttgtatg gaataaaata tttttttaat ttctaaatta 60
ttattgacta tgtagagtat tttatttaaa atttgtgtac atgcatgtat ttacctaagt 120
cattagaaca ttacaaattt ttatagtact cctaacattt tttaagccat ttttttaaaa 180
ttattttgca tagaataaaa tattttttgt agtataattt aaaaaaattg ttaaaaaata 240
ttcatgagct attaaaaatg gacaaaagag tattgtatgg aattcaaatt gataactcat 300
taatatttta aagaagtctc gtaattaaat attttgttag ctttttttaa tgcatgaaat 360
aaaatatatt ttttaatttt taaattatta atttttttaa caattttttt aataagttat 420
taattttttt aataacaatc accctgattt cgaattagag ggtgtgtaca tgtgtatgta 480
attcgaatca aggtgattcg aagttcgaaa caacctaatt cgaattatat aaaaatgtgt 540
tttggttgat tcatgaatta atttttggtt tagcggattt gtgtaatatt ttgctcccct 600
tgccttaata tagtgatttg ccctatgttt aaatactaat tgaagaaaga gaaaaaaaaa 660
ggatggatat ttaaaatatc tcatcaaact tgttattttt caagatcaaa gaaagaagaa 720
gaacgacatt aagcgtaaaa cttcttttgc ccaactttaa ctcttagatg aaagggagcg 780
cactatagag gacgtttttt tgtttgtctt ttttttttgc tttctcttct attcttcatt 840
ggaatatatg caatgcacgg acagaaaaca tctgtaccca actgtatgct ctgcttaccc 900
caatcccaat ctataccctt ccttgataca acaaataaca ttttactgta gttaccatat 960
tctaatatcc aactatcaat acatattatg atcaagatct aataaatgtt tgttatttat 1020
taaccaaaat agcaacagcg gtaggatcaa attttcaaca acaaattctt cagaggggcc 1080
aaaaataatt atttcacccg aaacagcaaa ctcaagtact atagaacatg tagaatagta 1140
aaggtggaaa aaggaatata aaaatcaaca atatgagtta taaggttatg attcacctag 1200
ttccgtatca acttaaagat tatttggaat tgaaatgata acagaaaaaa ttcactctta 1260
aaattttcac tctaaaatat tgttgcgttg aattgggact gcggaagtca caattacaaa 1320
agctcaacta ctatttctta tattcttttt caaccttaac cattgtgtgt gacaacaaaa 1380
gtaaattcca gatataagtt caatgtataa acttttacga aaaagggtta aaaaatttta 1440
ataaattgtt acttaaaaag gcacacaggt gaatttgaaa ctcaatttgg ttgtgacaaa 1500
aaaaaaagac aaacagaaat cacgaaaata aaaaatagta aggtaacagg taacgacttc 1560
tcaagataca aaatgaggac gacagcagtt ggagttgaaa c 1601
<210> 4
<211> 1601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ataaaatata ttttttaatt attttgtatg gaataaaata tttttttaat ttctaaatta 60
ttattgacta tgtagagtat tttatttaaa atttgtgtac atgcatgtat ttacctaagt 120
cattagaaca ttacaaattt ttatagtact cctaacattt tttaagccat ttttttaaaa 180
ttattttgca tagaataaaa tattttttgt agtataattt aaaaaaattg ttaaaaaata 240
ttcatgagct attaaaaatg gacaaaagag tattgtatgg aattcaaatt gataactcat 300
taatatttta aagaagtctc gtaattaaat attttgttag ctttttttaa tgcatgaaat 360
aaaatatatt ttttaatttt taaattatta atttttttaa caattttttt aataagttat 420
taattttttt aataacaatc accctgattt cgaattagag ggtgtgtaca tgtgtatgta 480
attcgaatca aggtgattcg aagttcgaaa caacctaatt cgaattatat aaaaatgtgt 540
tttggttgat tcatgaatta atttttggtt tagcggattt gtgtaatatt ttgctcccct 600
tgccttaata tagtgatttg ccctatgttt aaatactaat tgaagaaaga gaaaaaaaaa 660
ggatggatat ttaaaatatc tcatcaaact tgttattttt caagatcaaa gaaagaagaa 720
gaacgacatt aagcgtaaaa cttcttttgc ccaactttaa ctcttagatg aaagggagcg 780
cactatagag gacgtttttt cgtttgtctt ttttttttgc tttctcttct attcttcatt 840
ggaatatatg caatgcacgg acagaaaaca tctgtaccca actgtatgct ctgcttaccc 900
caatcccaat ctataccctt ccttgataca acaaataaca ttttactgta gttaccatat 960
tctaatatcc aactatcaat acatattatg atcaagatct aataaatgtt tgttatttat 1020
taaccaaaat agcaacagcg gtaggatcaa attttcaaca acaaattctt cagaggggcc 1080
aaaaataatt atttcacccg aaacagcaaa ctcaagtact atagaacatg tagaatagta 1140
aaggtggaaa aaggaatata aaaatcaaca atatgagtta taaggttatg attcacctag 1200
ttccgtatca acttaaagat tatttggaat tgaaatgata acagaaaaaa ttcactctta 1260
aaattttcac tctaaaatat tgttgcgttg aattgggact gcggaagtca caattacaaa 1320
agctcaacta ctatttctta tattcttttt caaccttaac cattgtgtgt gacaacaaaa 1380
gtaaattcca gatataagtt caatgtataa acttttacga aaaagggtta aaaaatttta 1440
ataaattgtt acttaaaaag gcacacaggt gaatttgaaa ctcaatttgg ttgtgacaaa 1500
aaaaaaagac aaacagaaat cacgaaaata aaaaatagta aggtaacagg taacgacttc 1560
tcaagataca aaatgaggac gacagcagtt ggagttgaaa c 1601
<210> 5
<211> 1601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acaagttgaa aacagcaact atagctgacc aagcaaaaga caaacctatc tctaaaccgg 60
ccgaagacaa aatcagatgg gtaaaaaggt atcccgttga ttacaagatt aaatgaccat 120
atatgggtat ttattaccac cttctgctga tccatatttg ttttgctcaa cagaaactag 180
tggagacgcg ttgctcatct tgctacatta acaacttatt tacccaaggg gcaatgcgca 240
aaggcaggaa gaagttgctt acttaggatt agtttcaatg aattgaacta ccacattata 300
ctgagtttac ctactgctga gatgcttagg gatttgtttt tgttaaatta gaaagacttg 360
attccaagac atcttttgtt tttctacagt acaatgattt attggttatg ttacctaaaa 420
attatctatc tttttctata tattagaatc ccgaatttat caaggttttt tggtacagtt 480
ggcataattg tgcatgttga aataggtgac aactaaccat ccaccaatat agctaaactg 540
aacatcccat accttactat actgaacatc cagtttaact agataaaatt caaaacataa 600
caaccaagtg caacatagac aagtactaac gggtaactat ccatgcttag agtaaaaatg 660
aacatctgat tacctatggg aatgaacatc cactttggat cattaaaaat acttgtaaac 720
agtgtatata acaaacaact acgcaccgaa aaactataca gtgaagcatg tagaaataag 780
gtacaacttt attaaactat ctggtatatt acaaaataac caccaaatat actcgttatg 840
cattaagtat acacgaaata aaaatagtaa aatggtttgt tctaactaat gaaaaaaggg 900
ttcttgcaat taataacata tggacttttg caacagctgc cttagaacct tgaaactaac 960
atgaatccta aaaacacaga gatgagaaaa tatacaatct cacaacctcg tccaactaat 1020
cccacttttt gttgaaggag cttaacaaag atatgaaacc accggcttgt tcgggaccat 1080
tcaggccaga aacagcacaa tggtttatat cttgaagtgt gtgcggacga accacctgcc 1140
aaaaaaaaaa gaaaaagtcc aaatagttat gttctaggat agtactatac caaataaaca 1200
atgaacgttg agattaagcc aaagatatac cgggggtgaa ttcttttgtt ttctccgtct 1260
tgaattcttg atggacttct caagggcaga gtcaagcctc ttactctttg gttgcaaatg 1320
cagctttctt tgcataggtc acataaaagt catgcgccat ctgtaactga gcaaaccgca 1380
tttcaactct tggtatctcg tcttcttgta ggttaccatg atctggtaac tgtattttat 1440
attaatccaa aagaaaaatt aaataccatt agttatcaaa tcaaacatga acattataac 1500
tacacaaaag taagctattt tacaatacct catctccaag ctccttctct tcacttccaa 1560
catctgtaac atccgacaac tgcgtggcct aaaattccaa a 1601
<210> 6
<211> 1601
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
acaagttgaa aacagcaact atagctgacc aagcaaaaga caaacctatc tctaaaccgg 60
ccgaagacaa aatcagatgg gtaaaaaggt atcccgttga ttacaagatt aaatgaccat 120
atatgggtat ttattaccac cttctgctga tccatatttg ttttgctcaa cagaaactag 180
tggagacgcg ttgctcatct tgctacatta acaacttatt tacccaaggg gcaatgcgca 240
aaggcaggaa gaagttgctt acttaggatt agtttcaatg aattgaacta ccacattata 300
ctgagtttac ctactgctga gatgcttagg gatttgtttt tgttaaatta gaaagacttg 360
attccaagac atcttttgtt tttctacagt acaatgattt attggttatg ttacctaaaa 420
attatctatc tttttctata tattagaatc ccgaatttat caaggttttt tggtacagtt 480
ggcataattg tgcatgttga aataggtgac aactaaccat ccaccaatat agctaaactg 540
aacatcccat accttactat actgaacatc cagtttaact agataaaatt caaaacataa 600
caaccaagtg caacatagac aagtactaac gggtaactat ccatgcttag agtaaaaatg 660
aacatctgat tacctatggg aatgaacatc cactttggat cattaaaaat acttgtaaac 720
agtgtatata acaaacaact acgcaccgaa aaactataca gtgaagcatg tagaaataag 780
gtacaacttt attaaactat ttggtatatt acaaaataac caccaaatat actcgttatg 840
cattaagtat acacgaaata aaaatagtaa aatggtttgt tctaactaat gaaaaaaggg 900
ttcttgcaat taataacata tggacttttg caacagctgc cttagaacct tgaaactaac 960
atgaatccta aaaacacaga gatgagaaaa tatacaatct cacaacctcg tccaactaat 1020
cccacttttt gttgaaggag cttaacaaag atatgaaacc accggcttgt tcgggaccat 1080
tcaggccaga aacagcacaa tggtttatat cttgaagtgt gtgcggacga accacctgcc 1140
aaaaaaaaaa gaaaaagtcc aaatagttat gttctaggat agtactatac caaataaaca 1200
atgaacgttg agattaagcc aaagatatac cgggggtgaa ttcttttgtt ttctccgtct 1260
tgaattcttg atggacttct caagggcaga gtcaagcctc ttactctttg gttgcaaatg 1320
cagctttctt tgcataggtc acataaaagt catgcgccat ctgtaactga gcaaaccgca 1380
tttcaactct tggtatctcg tcttcttgta ggttaccatg atctggtaac tgtattttat 1440
attaatccaa aagaaaaatt aaataccatt agttatcaaa tcaaacatga acattataac 1500
tacacaaaag taagctattt tacaatacct catctccaag ctccttctct tcacttccaa 1560
catctgtaac atccgacaac tgcgtggcct aaaattccaa a 1601
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
atttcaaggg gacactctgc 20
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tgtcaagtgg cccggttta 19
<210> 9
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ataacaatca ccctgatttc g 21
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
caactccaac tgctgtcgtc 20
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
actgagttta cctactgctg ag 22
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tagttggacg aggttgtgag 20

Claims (10)

1. Major QTL (quantitative trait locus) site for pod number of single peanut plantqPN7The linkage molecular marker is characterized in that the linkage molecular marker and the major QTL locus of the pod number of a single peanut plantqPN7Is co-located on the 7 th chromosome of the peanut genome and comprises a molecular Marker4240, a molecular Marker4243 and a molecular Marker 4251.
2. The linked molecular Marker as claimed in claim 1, wherein the nucleotide sequence of the molecular Marker4240 is shown as SEQ ID No. 1; the nucleotide sequence of the Marker4243 is shown as SEQ ID No. 3; the nucleotide sequence of the Marker4251 is shown as SEQ ID No. 5.
3. The linked molecular Marker as claimed in claim 2, wherein the polymorphism of the molecular Marker4240 is A/G, and the nucleotide sequence with polymorphism difference is shown as SEQ ID No. 2; the polymorphism of the molecular Marker4243 is T/C, and the nucleotide sequence with polymorphism difference with the polymorphism is shown as SEQ ID No. 4; the polymorphism of the molecular Marker4251 is C/T, and the nucleotide sequence with polymorphism difference with the polymorphism is shown as SEQ ID No. 6.
4. A primer composition is characterized by comprising 6 primers, and the nucleotide sequences of the primers are shown as SEQ ID No. 7-SEQ ID No.12 respectively.
5. Major QTL (quantitative trait locus) site for pod number of single peanut plantqPN7The method for identification of (2), wherein the method for identification comprises the steps of:
extracting genome DNA of the peanut material to be identified, carrying out PCR amplification on the template by using the primer composition by using the genome DNA as the template, carrying out sequencing analysis on an amplification product, and judging the polymorphism of the linkage molecular marker according to a sequencing result.
6. The method of claim 5, wherein the number of pods per plant of peanut material is greater than or equal to 20 if the linked molecular markers are polymorphic based on the sequencing of the peanut material to be identified, i.e., the nucleotide sequence of the linked molecular markers is identical to that of lines 6-13; and if the linked molecular marker does not have polymorphism, namely the nucleotide sequence of the linked molecular marker is the same as the variety Huayu No. 36, judging that the number of the single-plant pods of the peanut material is less than 20.
7. According toThe method of claim 5, wherein the PCR amplification is performed by the following reaction sequence: 5 min at 94 ℃; 35 cycles of 98 ℃ for 10 s, 60 ℃ for 30 s and 68 ℃ for 1 min; terminating the extension at 68 ℃ for 10 min, and storing at 4 ℃; the reaction system for PCR amplification is 50 mu L, and specifically comprises 2 mu L of 10-20 ng DNA template, 1.5 mu L of each primer pair, 10 mu L of dNTP, 25 mu L of KOD buffer solution, 1 mu L of KOD polymerase and ddH2O 9 μL。
8. Major QTL (quantitative trait locus) site for pod number of single peanut plantqPN7The application of the QTL site in regulating and controlling the pod number characters of a single peanut plant is characterized in that the main QTL site of the pod number of the single peanut plantqPN7The physical position of the gene (a) is between 208645-1146586 base positions of chromosome 7 of a peanut genome, and the gene (b) can be obtained by detecting primers shown in SEQ ID No. 7-SEQ ID No. 12; the major QTL site of the pod number of a single peanut plantqPN7The synergistic allele of (A) is from peanut line 6-13, and can increase the pod number of a single peanut plant.
9. Use of the linked molecular marker of any one of claims 1-3 or the primer composition of claim 4 for breeding high single pod count peanut varieties or lines.
10. Use of the linked molecular marker of any one of claims 1-3 or the primer composition of claim 4 for peanut molecular breeding, breeding of transgenic peanuts, or improvement of peanut germplasm resources.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114438245A (en) * 2022-02-15 2022-05-06 中国农业科学院油料作物研究所 SNP molecular marker linked with peanut bacterial wilt-resistant major QTL locus and application thereof
CN117448474A (en) * 2023-06-29 2024-01-26 河南农业大学 InDel molecular marker related to peanut pod size and application thereof

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Publication number Priority date Publication date Assignee Title
US20080166699A1 (en) * 2006-05-25 2008-07-10 George James Baley Method to identify disease resistant quantitative trait loci in soybean and compositions thereof
CN112094937A (en) * 2020-09-27 2020-12-18 中国农业科学院油料作物研究所 SNP molecular marker related to pod and seed size on peanut A06 chromosome and application thereof
CN112626260A (en) * 2021-01-15 2021-04-09 中国农业科学院油料作物研究所 Molecular marker linked with peanut kernel weight major QTL (quantitative trait locus) and application thereof

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
US20080166699A1 (en) * 2006-05-25 2008-07-10 George James Baley Method to identify disease resistant quantitative trait loci in soybean and compositions thereof
CN107012246A (en) * 2006-05-25 2017-08-04 孟山都技术有限公司 The authentication method of soybean disease resistance quantitative trait locus and its composition
CN112094937A (en) * 2020-09-27 2020-12-18 中国农业科学院油料作物研究所 SNP molecular marker related to pod and seed size on peanut A06 chromosome and application thereof
CN112626260A (en) * 2021-01-15 2021-04-09 中国农业科学院油料作物研究所 Molecular marker linked with peanut kernel weight major QTL (quantitative trait locus) and application thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114438245A (en) * 2022-02-15 2022-05-06 中国农业科学院油料作物研究所 SNP molecular marker linked with peanut bacterial wilt-resistant major QTL locus and application thereof
CN114438245B (en) * 2022-02-15 2022-10-11 中国农业科学院油料作物研究所 SNP molecular marker linked with peanut bacterial wilt-resistant major QTL locus and application thereof
CN117448474A (en) * 2023-06-29 2024-01-26 河南农业大学 InDel molecular marker related to peanut pod size and application thereof
CN117448474B (en) * 2023-06-29 2024-04-09 河南农业大学 InDel molecular marker related to peanut pod size and application thereof

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