Development And Application Of Wheat,Thinopyrum Bessarabicum And Peanut Oligonucleotide And Multiplex Probes For Fluorescence In Situ Hybridization

Wheat(Triticum aestivum,2n=6x=42,AABBDD)are important food crops of our country.Thinopyrum bessarabicum(2n?2x=14,JJ)confers high salinity tolerance and multiple disease resistance and represents an important resource for wheat improvement.Chromosome engineering is an effective way to improve wheat.However,the limited number of plasmid probes,tedious procedures and expensive cost of traditional fluorescence in situ hybridization(FISH)hinder the efficiency of chromosome engineering.The single strand oligonucleotide(SSON)probe is a kind of synthetic DNA or RNA fragments of 20?60 nt in length which can be directly modified with different fluorescent dyes.In comparison with traditional probes,SSONs are easy and versatile to design,more sensitive and cost efficiency.Chromosome painting based on SSON probes thus greatly simplify the traditional FISH procedure,which not only saved the laborious procedure of plasmid introduction,propagation,extraction,labeling or PCR amplification,but also saved the denaturing procedure of both probe and chromosome preparations for some SSONs,representing a new generation of chromosome identification technology in a simple,economic and high efficient way.(1)In this study,through probe length and concentration effect comparison,SSONs carried from 29?59 nt are proved to be more efficient for chromosome painting than SSONs with 14?15 nt in low concentrations.A method for developing SSONs using known repeats and genomic sequences of wheat chromosome 3B and Th.bessarabicum chromosome arm 4JL was thus established.Based on Wang(2013)'s first development of SSONs derived from repeat pSc119.2 and pAsl in wheat,16 new SSONs probes were successfully develop.ed which included four(DP4J24903,DP4J27982,DP4J31304 and DP4J28086)were J genome-specific and one(3K-5)was chromosome 3B-specific.These SSON probes could be used for both FISH and non-denatured FISH(ND-FISH),but the FISH signals are richer and more stable than ND-FISH.More interestingly,SSON probes(GAA)10,(AAC)10 and pSc119.2-1 can invade chromosome DNAs in a very low concentrations(as low as 0.01 ng/?l,0.01 ng/?l and 0.2 ng/?l,respectively)and non-denatured FISH conditions,thus a new kind of recycled chromosome dyes named as SSON dyes were thus developed and used for chromosome painting in wheat and rye,which provides a high throughput tool for chromosome automatic analysis and might be very useful for chromosome sorting based on different fluorescence signals in different chromosomes.(2)According to the characteristics of single SSON signals,four multiplexes were developed to identify chromosomes of wheat,rye and Th.bessarabicum.Among them,multiplex#1 and multiplex#4 showed richer and more stable and clear signals,which could identify all genomes and chromosomes of the three species based on the color and signal density differentiation.Using the multiplex#1 and multiplex#4,high resolution karyotypes of Chinese Spring(CS)and Th.bessarabicum were developed and used to identify the genetic diversity of cultivated wheat and chromosomal compositions of CS-Th.bessarabicum alien chromosome lines.By using multiplex#1 and DP4J27982,three CS-Th.bessarabicum disomic substitution lines and five CS-Th.bessarabicum translocations,and chromosome homoeology of CS and Th.bessarabicum were identified.Using multiplex#4,chromosome polymorphism of 8 cultivars(lines)could be clearly identified by once FISH and found most variations in B genome chromosomes,followed by A and D genome chromosomes,the wheat-rye translocation T1RS·1BL and obvious deletion of 7B can be directly distinguished after only once FISH.(3)Peanut(Arachis hypogaea,2n?4x=40,AABB)is one of the most important oil and cash crops in our country.However,chromosome engineering in peant has many difficulties mainly because of being lacking of high resolution karyotypes.In this study,using SSR,telomeric repeats and 4 Gb sequences of A.duranensis,seven peanut SSON and two multiplexes#5 and 6 probes with signals distributed all over the chromosomes of peanut were identified and developed.Higher resolution karyotypes of 9 Arachis species were developed by sequential FISH using SSONs,45S rDNA,5S rDNA,A.duranensis and A.ipaensis genomic DNA probes.Based on these karyotypes,obvious structural variations such as heterogeneous distribution of 45S rDNA in homologous chromosomes,inversion and repetitive sequence amplification were seen in these species,which revealed complex chromosome rearrangement occurred during peanut evolution.(4)Peanut genetic research is relatively weak,thus the genetic map,sequence map and the actual chromosomes of peanut still do not correspond well.To realize the relationship of sequence map and actual chromosomes of peanut,a library 6A-1 containing 20,000 SSONs(42?48 nt)specific for the sequenced and assembled chromosome A6 was designed in the 0-8.5 Mb region of A6 distal ends of A.duranensis.After probe preparation,FISH and sequential FISH combined with 45S rDNA,SSONs DP-1 and DP-5,the library 6A-1 produced clear signals in the cytological karyotype chromosome A3,thus chromosome A6 in sequence map might correspond to the actual chromosome A3.This result indicated that it's possible to establish clear correspondence between peanut chromosome genome sequence and actual chromosomes by chromosome painting using such SSONs developed in this study.