Research And Application Of Peanut Oligo Probes Based On Whole Genome Sequence Analysis

Peanut(Arachis hypogaea L.,2n=4x=40,genome AABB)is an important oil and commercial crop in the world.However,narrow heredity basis and germplasm resources shortage are now puzzling most peanut breeders.Artificial mutagenesis is a critical method for quality enhancement of peanut,which can effectively create gene mutation and chromosomal alteration materials,as well as increase the genetic diversity of peanut species.However,due to lacking of cytological markers,the karyotype resolution of peanut is relatively low,which has restricted the effective identification of peanut chromosomal variation.Oligonucleotide-fluorescence in situ hybridization(Oligo-FISH)is a newly developed and cost-effective method for chromosome identification,for which probes can be designed based on DNA sequences,providing a new strategy for chromosome mutation identification in peanut.The newly released peanut genome assemblies provide reference sequences for the development of oligonucleotide probes.Therefore,this study developed oligonucleotide probes based on the tandem repeats(TRs),interspersed repeats(IRs),single copy sequences,and genome specific sequences of cultivated(Tifrunner)and wild peanut species genome sequences by methods of bioinformatics and cytogenetics.With the probes,we constructed a new Oligo-FISH karyotype of cultivated peanut,which could highly improve our ability of peanut chromosome recognition,help us realize chromosome segments visualization and reveal the genetic relationship between genomes.Meanwhile,we irradiated peanut plants with 60Co-γ to induce chromosomal variation materials,and identified them using the new Oligo-FISH karyotype.The main contents and results of the study are as follows:1.Construct a new karyotype of Oligo-FISH in peanut based on genome sequences.With the reference genome sequences of Tifrunner and its donor parent A(Arachis duranensis)and donor parent B(Arachis ipaensis),114 new oligonucleotide probes were developed after searching for repeat sequences.The results showed that all the probes have clear and stable FISH signals on chromosomes of peanut.The 114 probes were classified into 28 types according to their hybridization sites.Thus,28 representative probes were selected and their physical location were investigated:eight probes were located in the secondary constrictions,arm and terminal regions of the chromosomes;fifteen probes were located in the centromeres;four probes were specific for B genome and one was specific for B09 chromosome.Multiplex#3 and Multiplex#4 were selected through combining 6 newly developed probes with previously reported probes.Among them,Multiplex#3 contained FAM modified TIF-439,TIF-185-1,TIF-134-3 and TIF-165-3,while Multiplex#4 contained TAMRA-modified IPA-1162,IPA-1137,DP-1 and DP-5.A peanut karyotype with chromosome numbers corresponding to the genome sequence map was established for the first time,using the newly developed probe sets by sequential FISH/GISH(Genomic in situ hybridization)and computer electronic localization technology.The karyotype provides a basis for integrating the genome sequence information and cytological information.2.Develop the wild-type species specific oligonucleotide probes to enhance the recognition ability of their chromosomes.For wild-type peanut species without reference genome sequences,we got high confidence tandem repeats by carried out cluster analysis of genome resequencing reads,aligned them to cultivated peanut genomes,filtered out wild-type special sequences,and finally designed one H genome-specific probe and one species-specific probe.This provides effective probes for the identification of H genome species A.pusilla and A.dardonoi,and also provides a reference for the development of genome-specific probes for related species of other crops.Besides,we obtained 12 non-specific oligonucleotide probes from the reads of wild-type peanut species,of which 4 from A genome species,4 from E genome species,and 4 from H genome species.These probes could enrich the chromosome hybridization bands of wild species and provide new markers for the identification of exogenous chromosomes in subsequent distant hybrids.3.Invente an efficient Oligo-GISH staining technique to reveal the relationship between wild-type peanut species and sub-genomes of cultivated peanut.We performed a cluster analysis on the TRs found in the genome of Tifrunner,and obtained a cluster of TRs,in which exists TRs whose highly similar copies uniformly distributed in one of the peanut sub-genomes.Base on the complete sequences of TR arrays in the cluster,we designed four oligonucleotide probes and grouped them.to two probe cocktails,Multiplex#A and Multiplex#B,which can be used to stain and distinguish the A and B sub-genomes of peanut.Thus,we established an efficient Oligo-GISH staining technique that can replace the GISH using A.duranensis and A.ipaensis genomic DNA as the probe,greatly reducing the time and cost.When utilizing the probe cocktails in 14 wild species,we discovered that A.duranensis,A.diogoi,A.herzogii,A.villosa,A.microsperma,A.simpsonii,and A.duranensi-2 genomes were closely related to A sub-genome of peanut,and A.ipaensis,A.valida,A.batizocoi,and A.trinitensis genomes were closely related to B sub-genome of peanut.The A.stenophylla genome had higher homology relationship with both A and B sub-genomes of peanut,while the A.pusilla and A.dardonoi genomes were lower homology relationship with both A and B sub-genomes of peanut.Therefore,this technique can be successfully used for the analysis of the homology relationship between peanut sub-genomes and other genomes.4.Peanut chromosome segments visualization by FISH with single copy oligonucleotide probe libraries.Based on the reference genome of A.duranensis,two single-copy oligonucleotide probe libraries,L3A-1 and L4A-1,were designed to visualize the 1-8 Mb of chromosome A03 and 114-122 Mb of chromosome A04 by Oligo-FISH,targeting the terminus of A03 and A04,which are gene enrichment regions.In addition,FISH showed that,in Tifrunner,L3A-1 could generate hybridization signals in the centromeres of A01 and other chromosomes,which was inconsistent with the results of electronic localization of the probe library in A.duranensis.Further analysis of L3A-1 probe library and Tifrunner genome sequences revealed that four probes in L3A-1 are repetitive oligonucleotide probes,they had hybridization signals on both Tifrunner and A.duranensis chromosomes,suggesting that the A.duranensis genome sequence map which is used to design single copy oligonucleotide probe libraries may need to be further improved.5.Establish effective methods to create and identify peanut chromosome variations,and cultivate new materials for genetic research and breeding.We established an effective method to create and identify chromosome variations in peanut for the first time,by irradiating four red flowering plants with 60Co-γ rays and FISH/GISH technique.It was found that 1200 rad and 1600 rad can effectively induce peanut chromosome variation,but there was no significant difference in the number of pods and grains harvested after the two different doses of radiation,and the chromosome variation rate of 1600 rad dose was higher(25.7%)than 1200 rad(24.0%).Ninety-seven peanut materials with chromosomal structure.variation and chromosome number variation were obtained by this method,which promoted the genetic material exchange between some homologous chromosomes and non-homologous chromosomes in peanut.Furthermore,19 Silihong translocation lines and deletion lines was identified by new Oligo-FISH karyotype,which provided basis for chromosome physical mapping,gene mapping,and breeding of peanut.In conclusion,this study established an effective peanut oligonucleotide probe design method based on genome sequence and bioinformatics analysis,which could be a reference for the development of chromosome markers in other plants.A number of useful oligonucleotide probes were developed and an efficient probe staining technique was established,which improved the ability of peanut chromosome identification and provided support for the subsequent high-throughput identification of chromosome variation in peanut.In addition,a number of new peanut materials containing chromosomal variants were created and identified,which provided new resources for follow-up chromosome research and breeding of peanut.