CN112680442A - Genome specific probe for peanut wild species and use method - Google Patents
Genome specific probe for peanut wild species and use method Download PDFInfo
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Images
Abstract
The invention relates to a peanut wild species genome specific probe and a using method thereof, wherein the peanut wild species is subjected to illumina double-end genome sequencing to obtain a genome sequence with the depth of 20 multiplied, and a peanut wild species specific repetitive sequence probe is designed through bioinformatics software analysis to obtain H genome specific probes Dard-127 and Pus-187, and a B genome specific probe Ipa-163. The genome specific probe is used for configuring a probe dye solution to dye a peanut chromosome, and the probe Pus-187 is found to be an H genome specific probe and can specifically identify H genome species; probe Dard-127 is an a.dardonoi specific probe, capable of specifically recognizing a.dardonoi; the probe Ipa-163 is a B and K genome specific probe, which indicates that the B and K genomes may have close relationship. The invention provides a cytological marker and a cytological method capable of effectively identifying peanut genome or specific wild species, and provides a new method and a theoretical basis for revealing genetic relationship between peanut wild species or genome, peanut interspecific hybridization research and wild germplasm utilization.
Description
Technical Field
The invention relates to a method for researching peanut cytogenetics, in particular to a method for designing and using a genome specific probe of a peanut wild species.
Background
The wild peanut species have high-quality characters such as rich biotic and abiotic stress resistance sources, high oil content and the like, and the resistance mechanism is different from that of the cultivated species peanut, so that the wild peanut species are important gene resources for improving the peanut cultivated species. Researches at home and abroad find that the peanut pests and diseases damaging peanuts are more than 120, and the yield loss is 5-100%; peanut wild species have high levels of resistance to many peanut diseases, insect pests and growth stresses, almost all peanut diseases and pests can find a source of resistance in peanut wild species, some wild species are immune to even some diseases and pests, such as a. diogoi and a. glabrata against peanut bud necrosis virus and tomato spotted wilt virus, a. kuhlmanii, a. duranenis and a. ipaensis against peanut cluster virus, a. magna and a. cardenaii against rust disease, etc., which are not found in peanut cultivars.
Interspecific hybridization is an important means for introducing excellent genes of wild peanut species into cultivars. However, species within the arachnid genome or between different genomes are subject to different degrees of reproductive segregation due to differences in chromosomal ploidy, genomic composition, etc., for example: the difficulty of introducing genes of different genome wild species into cultivars is different due to hybridization incompatibility, hybrid deaths, hybrid sterility and other hybridization obstacles. The research finds that the peanut cultivar (genome AABB) is almost compatible to hybridize with species in A, B, K, F, G isogenic groups of peanuts (section Arachis), and is easier to hybridize with species in a partially erect group (section Erectoides) with an E genome and species in a partially rhizome group (section Rhizomatosae) with an R genome; while crossing with species of the large root group having the C genome (section Caulorrhizae), the creeping group having the P genome (section procembentes), the trilobe group having the Te genome (section Trinectoides), the triple seed group having the T genome (section trisaeration), the peripheral group having the Ex genome (section Extranervosae), and the abnormal flower group having the H genome (section Heteranthae) was difficult to succeed. Therefore, the definition of the correlation among the peanut wild species gene groups is important for overcoming the incompatibility obstacle of peanut interspecific hybridization and efficiently utilizing excellent wild germplasm.
In the process of evolution of plant genomes, repeated sequences of the plant genomes are amplified and contracted to different degrees, the amplification and contraction cause the difference of the type and the content of the repeated sequences among species and in the species, and the difference can reflect the distance of relativity among the species. The tandem repeat sequence is a repeat sequence distributed in a cluster shape in a genome, and the amplification of the tandem repeat sequence provides a rich variation source for the evolution of the genome. Plant-specific tandem repeats, which are usually amplified only in related species or genomes and not in other genomes, are often used to determine relatedness between species or genomes.
The wild species of peanut undergoes a long evolution process, and different genome types such as A, B, K, F, G, C, P, T, Te, Ex and H are formed. However, no report is found on the current researches on genome-specific repetitive sequences of peanut wild species.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a peanut wild species genome specific probe and a using method thereof, wherein H genome species A.dardonoi and A.pusilla and B genome species A.ipaensis are taken as research objects, the peanut wild species genome specific probe is established through genome sequencing and bioinformatics analysis, and is applied to peanut wild species genetic relationship analysis, so that a new method and a theoretical basis are provided for peanut distant hybridization research and wild species utilization.
In order to achieve the purpose, the invention adopts the technical scheme that:
a genome specific probe of a peanut wild species comprises an H genome specific probe and a B genome specific probe; the H genome specific probe is Dard-127 and Pus-187, the B genome specific probe is Ipa-163, and the specific sequence is as follows:
probe Dard-127:
FAM-5′-TAAACTATGGTATTTTCATGAGTTTTGAGGCATGCCGGA-3′；
probes Pus-187:
TAMRA-5′-TCACTAGGCATATAATGCCACTCGATGGCGTTGAAACGCGGAGCT-3′；
probe Ipa-163:
TAMRA-5′-TAGGGTTTATGATTTAGGCTTTAGGGTTTGT-3′。
the design method of the peanut wild species genome specific probe comprises the following steps:
(a) firstly, carrying out illumina double-end genome sequencing on peanut species, wherein the sequencing depth is 20 times; then, filtering the original sequence of the genome sequencing by using Trimmomatic software, wherein the key parameters are as follows: ILLUMINALIP, TRIM _ HOME/adapters/TruSeq3-PE.fa:2:30:10, LEADING:5, TRAILING:5 SLIDINGFOW: 4:20 MINLENEN: 150 to obtain high-quality 'clean reads';
(b) using the peanut cultivar Tifrunner genome as a reference genome, constructing an index file by using bowtie2 software, aligning clean reads to the Tifrunner reference genome, and filtering the clean reads of which both ends are not aligned to the Tifrunner reference genome;
(c) randomly sampling 100 ten thousand sequences from the filtered clean reads by using a shuf command of Linux, and performing series-connection repeated sequence excavation on the clean reads obtained based on random sampling by using TAREAN software to excavate a series-connection repeated sequence set of high consistency;
(d) positioning the tandem repeat sequence to a peanut cultivar Tifrunner reference genome by using B2DSC, and excluding the highly enriched tandem repeat sequence in the cultivar; using NCBI-BLAST to eliminate possible gene sequences of the candidate tandem repeat sequence, and using the rest tandem repeat sequence for probe design;
(e) and (3) selecting the most conservative segment of the tandem repeat sequence, intercepting a 30-45bp sequence, and carrying out artificial synthesis modification on 5' -TAMRA or FAM to obtain the genome specific probe.
The Arachis species were H genome species A.dardonoi and A.pusilla, and the corresponding genome-specific probes were H genome-specific probes Dard-127 and Pus-187.
The peanut species is B genome species A. ipaensis, and the corresponding genome-specific probe is B genome-specific probe Ipa-163.
The application method of the peanut wild species genome specific probe comprises the following steps:
(a) obtaining diploid wild species root tip cell mitotic metaphase chromosomes with peanut A genome, peanut B genome, peanut E genome, peanut H genome and peanut K genome respectively by chromosome tabletting, freezing at-80 ℃, uncovering the chromosomes, dehydrating by absolute ethyl alcohol and air-drying;
(b) adding 40mL of 2 XSSC buffer solution, H genome specific probe dry powder or/and B genome specific probe dry powder and 5 microliter of DAPI mother liquor into a dye vat, preparing a wild species chromosome slide, placing the wild species chromosome slide into the dye vat for dyeing for 5 hours, and taking a picture by a fluorescence microscope; the H genome specific probe dry powder is 0.1OD Dard-127 and 0.1OD Pus-187, and the B genome specific probe dry powder is 0.1OD Ipa-163.
2 XSSC buffer from 0.3mol/L trisodium citrate C6H5Na3O7·2H2O and 3mol/L NaCl.
The DAPI mother liquor is DAPI staining solution with the concentration of 100 mu g/mL.
The diploid wild species of A genome, B genome, E genome, H genome and K genome are respectively A genome species A. duranensis, A species genome A. diogoi, B genome species A. ipaensis, E genome species A. paraguariensis, E genome species A. stenophylla, H genome species A. pusilla, H genome species A. dardonoi and K genome species A. batizocoi.
The invention has the beneficial effects that:
according to the invention, through carrying out the illumina double-end genome sequencing on the peanut wild species and utilizing the genome sequence and bioinformatics analysis, the specific probe markers of the genomes of the peanut wild species H and B are successfully developed, the cytological characteristics of the wild species are enriched, a design method of the specific probe of the genome of the peanut wild species is established, and a new effective method is provided for obtaining the specific cytological marker of the genome of the peanut wild species.
According to the invention, a plurality of peanut genome chromosomes are subjected to dyeing analysis by using newly designed peanut wild species H and B genome specific probes, and the probes Pus-187 are H genome specific probes and can specifically identify H genome species; probe Dard-127 is an a.dardonoi specific probe that specifically recognizes a.dardonoi; the probe Ipa-163 is a B and K genome specific probe, and confirms that the B and K genomes may have close relationship. The specific probe can be used for analyzing genomes of different peanut wild species, can effectively identify the peanut wild species or the genomes, determines the genetic relationship between the peanut wild species or the genomes, and provides a new method and a theoretical basis for peanut interspecific hybridization research and wild species utilization.
Drawings
FIG. 1 shows the results of staining signals with specific probes Pus-187 and Dard-127 on wild species A.duranensis (a, A genome), A.dioginis (B, A genome), A.ipaensis (c, B genome), A.paraguariensis (d, E genome), A.stenophylla (E, E genome), A.pusilla (f, H genome), A.dardonoi (g, H genome) and A.batizocoi (H, K genome).
In (a-h), the first column from left to right shows DAPI staining, the second column shows the distribution of Pus-187 probe red signals on the wild species chromosome, the third column shows the Dard-127 probe green signals on the wild species chromosome, and the fourth column shows the DAPI, Pus-187 and Dard-127 probe signal synthesis maps.
Fig. 2 shows the results of staining signals with specific probe Ipa-163 on wild species a. duranensis (a, a genome), a. diogio (B, a genome), a. ipaensis (c, B genome), a. paraguariensis (d, E genome), a. stenophylla (E, E genome), a. dardonoi (f, H genome) and a. batiocoi (g, K genome).
In (a-h), the first column from left to right shows DAPI staining, the second column shows the distribution of red signals of the Ipa-163 probe on the chromosomes of the wild species, and the third column shows the synthesis of DAPI and Ipa-163 probe signals.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1 design method of peanut wild species H genome specific probe
(a) Two H genome species A.dardonoi and A.pusilla were first subjected to illumina double-ended genome sequencing with a depth of 20X. Then, filtering the original sequence of the genome sequencing by using Trimmomatic (v0.38) software, wherein the key parameters are as follows:
ILLUMINALIP, TRIM _ HOME/adapters/TruSeq3-PE.fa:2:30:10, LEADING:5, TRAILING:5 SLIDINGFOW: 4:20 MINLENEN: 150, to obtain high quality "clean reads".
(b) Using the peanut cultivar Tifrunner genome (https:// www.peanutbase.org/download) as the reference genome, an index file was constructed with bowtie2(v2.3.5) software, then "clean reads" of A.dardonoi and A.pusilla were aligned to the Tifrunner reference genome, and "clean reads" with neither end aligned to the Tifrunner reference genome were filtered out.
(c) 100 ten thousand sequences are randomly sampled from the filtered clean reads by using a shuf command of Linux, and serial repeat sequences are excavated from the "clean reads" obtained by random sampling by using TAREAN software (http:// www.repeatexplorer.org) so as to excavate a serial repeat sequence set of "high confidence".
(d) Mapping the tandem repeat sequence to the peanut cultivar Tifrunner reference genome using B2DSC (http:// mcgb. uestc. edu. cn/B2DSC), excluding highly enriched tandem repeats in the cultivar; the potential gene sequences of candidate tandem repeats were excluded using NCBI-BLAST (https:// www.ncbi.nlm.nih.gov /), and the remaining tandem repeats were used for probe design.
(e) A section with the most conservative tandem repeat sequences of A.dardonoi and A.pusilla is selected, a 30-45bp sequence is cut out, and 5 '-TAMRA or 5' -FAM artificial synthesis modification is carried out to obtain H genome specific probes Dard-127 and Pus-187. Probe Dard-127:
FAM-5'-TAAACTATGGTATTTTCATGAGTTTTGAGGCATGCCGGA-3'; (SEQ ID NO: 1) Probe Pus-187:
TAMRA-5′-TCACTAGGCATATAATGCCACTCGATGGCGTTGAAACGCGGAGCT-3′(SEQ ID NO：2)。
example 2 design method of specific probe for genome B of wild peanut
(a) B genome species A. ipaensis is subjected to illumina double-end genome sequencing, and the sequencing depth is 20 x. Then, filtering the original sequence of the genome sequencing by using Trimmomatic (v0.38) software, wherein the key parameters are as follows: ILLUMINALIP, TRIM _ HOME/adapters/TruSeq3-PE.fa:2:30:10, LEADING:5, TRAILING:5 SLIDINGFOW: 4:20 MINLENEN: 150, to obtain high quality "clean reads".
(b) Using the peanut cultivar Tifrunner genome (https:// www.peanutbase.org/download) as the reference genome, an index file was constructed with bowtie2(v2.3.5) software, then "clean reads" from A.ipaensis were aligned to the Tifrunner reference genome, and "clean reads" that were not aligned on either end to the Tifrunner reference genome were filtered out.
(c) 100 ten thousand sequences are randomly sampled from the filtered clean reads by using a shuf command of Linux, and serial repeat sequences are excavated from the "clean reads" obtained by random sampling by using TAREAN software (http:// www.repeatexplorer.org) so as to excavate a serial repeat sequence set of "high confidence".
(d) Mapping the tandem repeat sequence to the peanut cultivar Tifrunner reference genome using B2DSC (http:// mcgb. uestc. edu. cn/B2DSC), excluding highly enriched tandem repeats in the cultivar; the potential gene sequences of candidate tandem repeats were excluded using NCBI-BLAST (https:// www.ncbi.nlm.nih.gov), and the remaining tandem repeats were used for probe design.
(e) And (3) selecting a segment with the most conservative A.ipaensis tandem repeat sequence, intercepting a sequence of 30-45bp, and carrying out artificial synthetic modification on 5' -TAMRA to obtain a B genome specific probe Ipa-163.
Probe Ipa-163: TAMRA-5'-TAGGGTTTATGATTTAGGCTTTAGGGTTTGT-3' (SEQ ID NO: 3). Example 3 method of Using a specific probe for H genome of wild species of peanut
First, by chromosome tabletting method, the wild species of flowers, A.duranensis (A genome), A.diotoi (A genome), A.ipaensis (B genome), A.paraguariensis (E genome), A.stenophylla (E genome), A.pusilla (H genome), A.dardonoi (H genome) and A.batizocoi (K genome), diploid root tip cells of the wild species were produced into mitotic metaphase, and the slide was removed by freezing at-80 ℃, dehydrated by absolute ethanol and air-dried.
Next, 40ml of 2 XSSC buffer (composed of 0.3M trisodium citrate C) was added to the dye vat6H5Na3O7·2H2O and 3M NaCl), 0.1OD Dard-127 and 0.1OD Pus-187 probes, and 5. mu.l of DAPI stock solution (100. mu.g/mL DAPI staining solution), the wild species chromosomes were stained in a staining jar for 5 hours, and photographed by a fluorescence microscope to obtain a stained image (FIG. 1).
As a result, both probes were found to have no staining signal on the chromosomes of a genomic species a.duranensis and a.diogoi, B genomic species a.ipaensis, E genomic species a.paraguariensis and a.stenophylla, and K genomic species a.batizocoi. Probes Pus-187 produced red signals (bright spots in the figure) on both H genome species A.pusilla and A.dardonoi chromosomes, indicating that the probes were H genome-specific probes that specifically recognized H genome species. Probe Dard-127 produced a green signal only on the a.dardonoi chromosome (bright spots in the figure), indicating that this probe is a specific probe for a.dardonoi from genome species H, and specifically recognizes a.dardonoi.
Example 4 method of Using a specific probe for genome B of wild species of peanut
First, by chromosome tabletting method, the wild species of flowers, A.duranensis (A genome), A.diotoi (A genome), A.ipaensis (B genome), A.paraguariensis (E genome), A.stenophylla (E genome), A.pusilla (H genome), A.dardonoi (H genome) and A.batizocoi (K genome), diploid root tip cells of the wild species were produced into mitotic metaphase, and the slide was removed by freezing at-80 ℃, dehydrated by absolute ethanol and air-dried.
Next, 40ml of 2 XSSC buffer (composed of 0.3M trisodium citrate C) was added to the dye vat6H5Na3O7·2H2O and 3M NaCl), 0.1OD of Ipa-163, and 5. mu.l of DAPI stock solution (100. mu.g/mL of DAPI staining solution), and the wild species chromosomes were stained in a staining jar for 5 hours by sectioning and photographed by a fluorescence microscope to obtain a stained image (FIG. 2).
As a result, both probes were found to have no staining signal on the chromosomes of the a genomic species a. duranensis and a. diogio, the E genomic species a. paraguariensis and a. stenophylla, the H genomic species a. pusilla and a. dardonoi. The probe Ipa-163 produces red signals (bright spots in the figure) only on chromosomes of the B genome species a. ipaensis and the K genome species a. batizocoi, indicating that the probe is a B and K genome specific probe, and suggesting that B may have a closer relationship to the K genome than to other genomes.
Sequence listing
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Claims (8)
1. A genome specific probe of a peanut wild species is characterized by comprising an H genome specific probe and a B genome specific probe; the H genome specific probe is Dard-127 and Pus-187, the B genome specific probe is Ipa-163, and the specific sequence is as follows:
probe Dard-127:
FAM-5′-TAAACTATGGTATTTTCATGAGTTTTGAGGCATGCCGGA-3′；
probes Pus-187:
TAMRA-5′-TCACTAGGCATATAATGCCACTCGATGGCGTTGAAACGCGGAGCT-3′；
probe Ipa-163: TAMRA-5'-TAGGGTTTATGATTTAGGCTTTAGGGTTTGT-3'.
2. The method for designing a genome-specific probe for a peanut wild species according to claim 1, comprising the steps of:
(a) firstly, carrying out illumina double-end genome sequencing on peanut species, wherein the sequencing depth is 20 times; then, filtering the original sequence of the genome sequencing by using Trimmomatic software, wherein the key parameters are as follows: ILLUMINALIP, TRIM _ HOME/adapters/TruSeq3-PE.fa:2:30:10, LEADING:5, TRAILING:5 SLIDINGFOW: 4:20 MINLENEN: 150 to obtain high-quality 'clean reads';
(b) using the peanut cultivar Tifrunner genome as a reference genome, constructing an index file by using bowtie2 software, aligning clean reads to the Tifrunner reference genome, and filtering the clean reads of which both ends are not aligned to the Tifrunner reference genome;
(c) randomly sampling 100 ten thousand sequences from the filtered clean reads by using a shuf command of Linux, and performing series-connection repeated sequence excavation on the clean reads obtained based on random sampling by using TAREAN software to excavate a series-connection repeated sequence set of high consistency;
(d) positioning the tandem repeat sequence to a peanut cultivar Tifrunner reference genome by using B2DSC, and excluding the highly enriched tandem repeat sequence in the cultivar; using NCBI-BLAST to eliminate possible gene sequences of the candidate tandem repeat sequence, and using the rest tandem repeat sequence for probe design;
(e) and (3) selecting the most conservative segment of the tandem repeat sequence, intercepting a 30-45bp sequence, and carrying out artificial synthesis modification on 5' -TAMRA or FAM to obtain the genome specific probe.
3. The method of claim 2, wherein the peanut species are H genome species A.dardonoi and A.pusilla, and the corresponding genome-specific probes are H genome-specific probes Dard-127 and Pus-187.
4. The method of claim 2, wherein the peanut genus species is genome B species a. ipaensis, and the corresponding genome-specific probe is genome B-specific probe Ipa-163.
5. A method of using the peanut wild species genome-specific probe of any one of claims 1-4, comprising the steps of:
(a) obtaining diploid wild species root tip cell mitotic metaphase chromosomes with peanut A genome, peanut B genome, peanut E genome, peanut H genome and peanut K genome respectively by chromosome tabletting, freezing at-80 ℃, uncovering the chromosomes, dehydrating by absolute ethyl alcohol and air-drying;
(b) adding 40mL of 2 XSSC buffer solution, H genome specific probe dry powder or/and B genome specific probe dry powder and 5 microliter of DAPI mother liquor into a dye vat, preparing a wild species chromosome slide, placing the wild species chromosome slide into the dye vat for dyeing for 5 hours, and taking a picture by a fluorescence microscope; the H genome specific probe dry powder is 0.1OD Dard-127 and 0.1OD Pus-187, and the B genome specific probe dry powder is 0.1OD Ipa-163.
6. The method of claim 5, wherein the 2 XSSC buffer is composed of 0.3mol/L trisodium citrate C6H5Na3O7·2H2O and 3mol/L NaCl.
7. The method for using a genome-specific probe for a peanut wild species according to claim 5, wherein the DAPI stock solution is a DAPI staining solution having a concentration of 100. mu.g/mL.
8. The method of claim 5, wherein the diploid A, B, E, H, and K genome species are respectively A.duranensis, A.diotoi, B.ipaensis, E.paraguariensis, E.stenophylla, H.pusilla, H.dardonoi, and K.batizocoi.
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