Tandem Repeat Analysis Of Thinopyrum Bessarabicum And Development Of Oligonucleotide Probes For In Situ Hybridization

Fluorescence in situ hybridization(FISH)is a milestone technique in chromosome engineering.Thinopyrum bessarabicum(2n=2x=14,JJ)has strong salt tolerance and resists to multiple wheat diseases,which is an important germplasm for wheat(Triticum aestivum,2n=6x=42,AABBDD)breeding.However,due to the limits of the conventional FISH such as lack of enough plasmid probes,tedious procedure and expensive cost,current chromosome engineering gets a bottleneck.Oligonucleotide probe is an artificial single strand DNA fragment(20-60 nt)modified with a fluorophore,which is easy to design and synthesize,sensitive,cheap and stable.These advantages significantly improve FISH procedure and lead to production of a simple and efficient method for chromosome identification.Based on analysis of the 5S and 45S DNA sequences of 10 grass and 5 non-grass plants,12 oligonucleotides have been developed and formed two sets of general probes for identifying 5S and 45S rDNA loci of grass plants,where Grass-5S includes two 36 nt oligonucleotides,and Grass-45S includes ten 30～40 nt ones,which can be hybridized to the 5S and 45S loci in barley,wheat,sorghum,maize,rye,Triticum monococcum,Thinopyrum intermedium,Haynaldia villosa,Thinopyrum ponticum,and Th.bessarabicum.17 kinds of tandem repeats(71一 856 bp,occupying 0.01%～1.30%of the genome)were identified based on 4Gb ILLUMINA sequencing genomic sequences of Th.bessarabicum.These repeats were further developed into 39 oligonucleotide probes.Of them,21 from 8 repeats produced signals on Th.bessarabicum chromosomes and 27 from 12 repeats produced signals on Th.ponticum chromosomes.Tandem repeat BSCL135 produced signals on all seven Th.bessarabicum chromosomes.A new oligonucleotide probe multiplex#7(ONPM#7)was developed by integrating Grass-5S,BSCL135-1,BSCL135-2 and BSCL242-1 with the former ONPM#4.This new probe set produced rich and unique signals and allowed clear identification of all chromosomes in wheat and Th.bessarabicum.Based on the reference genome sequences of wheat A,B and D sub-genomes and the homoeology of homoeologous group 2,3 and 7,we developed 223 Intron-targeted(IT)molecular markers and 52 of them amplified Th.bessarabicum specific bands.Combining with cytological analysis,we identified three monosomic or multiple addition lines of 2J,3 J and 7J,which make up a set of Chinese Spring(CS)-Th.bessarabicum alien lines together with the previous CS-Th.bessarabicum substitution/addition lines 1J,4J,5J and 6J.Chromosomes of these lines together with Th.bessarabicum,CS and CS-Th.bessarabicum amphiploid were identified and compared by ONPM#7 FISH and sequential GISH(Genomic in situ hybridization),a high-resolution karyotype of Th.bessarabicum corresponding to the seven homoeologous groups of wheat were developed,where all seven chromosomes and 12 arms could be clearly distinguished by the signal combination of red and green.In addition,ONPM#7 produced additional and specific signals on wheat chromosomes 1A,4A,5A,6A,7A,4D,5D,6D and 7D,which make them better distinguishable.By ONPM#7 FISH and molecular marker analysis,three Thinopyrum bessarabicum intra chromosome translocations(T4JS·3JL,T3JS·4JL and T6JS·2JL)and one isochromosome(4JL·4JL)were clearly identified.This result proved that the high resolution ONPM#7 karyotype are able to accurately identify reciprocal translocations occurred between Th.bessarabicum chromosomes.Additionally,26 plants derived from four different CS-Th.bessarabicum alien chromosome lines were analyzed,34.6%of them contained chromosome aberrations,which included 6 deletions happened in 6 plants,4 translocations happened in 3 plants,where 3 deletions occurred at pericentromeric region,and 4 translocations were Robertsonian types.This indicated that spontaneous chromosome varaitions might frequently occur in wheat-Th.bessarabicum alien chromosome lines.In order to further simplify the oligonucleotide FISH procedure,a new oligonucleotide probe multiplex dye solution kit was developed for identifying CS and Th.bessarabicum chromosomes via direct dying with massive chromosome preparations,which saved procedures of chromosome dehydration,probe and slide denaturation,and reagents of formamide,salmon sperm and dextran sulfate.The dye solution could be recycled for no more than 10 times,which makes chromosome identification more convenient and cost efficient.