MarieKubalakova, Pavla Suchankova ,Pavlina kavarova ,Jarmila Cihalkova, Jan Bartose,Nobuyoshi Watanabe, Sergio Lucretti, and Jaroslay DolezelAbstractIn durumwheat this study evaluate the potential of flow of cytometry for chromosomesorting .In histograme of inflorescence intensity (flow karyotypes) thechromosome consist of three peaks which is obtained after a DAPI-stainedanalysis.

Out of these three chromosome  peaks ,one peak represent the chrosome 3B ,thesecond  number small peak represented 1Aand 6A chromosome  and a third numberpeak which is large peak represented the remaining 11 chromosome. Afterfluorescence, the chrosome that are sorted onto microscope slide were identifyin situ hybridization (fish) with probes for GAA microsatellite , pSc119.2, andin Afa repeat.

For the first time in durum wheat the genomic distribution ofthese sequence  was determined and theirmolecular karyotype has been developed for wheat. In durum wheat flowkaryotyping in double –ditelosomic lines revaled that these lines facilitatingthe sorting of any arm of wheat A-genome and B –genome chrosome. The flowkaryotype of durum wheat is less complexed as compared to hexaploid wheat.

theless complex karyotype result better discrimination of telosome and highpurities in sorted fraction its ranging from 90 to 98 percent. By using flowcytometry ,we have revealed that large inserted libraries from DNA purified .chromosomal sorting study considerably expands the potential of flowcytogenetics for wheat genomics. This study has open the possibility ofsequencing the genome of durum wheat crop on arm of chrosome at a time.Durumwheat is originated from intergeneric hybridization and polyploidationinvolving two diploid species :T.urartu (2n = 2x = 14,AA genome) and B-genomediploid related ti Aegilops  speltoids (2n = 2x = 14,SSgenome). Durum wheat (triticum turgidum Desf. Var.

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durum) is an allotetraploidspecies with 2n = 4x = 28 (AABB genome). The T.aestivum which we also calledcultivated bread wheat arose from hybridization of T.turgidum (durum orpasta wheat) and Ae.tauschii only 8000 year ago, it was indicated by moleculargenetic data analysis. These two cultivated wheat specices of A genome and Bgenome have >99% similarities.

Durum wheat (triticum turgidum Desf.var.durum)  is an important cereal whose grains aredominantly used in food products such as pasta, couscous, and burghul. Durumwheat is not cultivated only in Mediterranean Basin, but it is also extensivelycultivated in Argentina, Australia, Canada, India, united States, and severalother countries.

Annual grain production of durum wheat is about 27.5 millionmatric ton, and >10% of the world wide wheat production. The durum wheatproduction is influence each year due to disease, pest, and environmentalproblem. There is great loss of yield due to these causes. It is expected thatspeed development of improved varities and creation of novel germplasm can beobtained by molecular techniques, not by use of classical approaches.

The genesare clustered in gene rich island where gene density is similar to that foundin small-genome species, it was indicated by recent genome analysis of wheat.Gene cloning which is based on recombination mapping is facilitated by thepresence of small  gene-rich island. itis originally considered to be difficult in polyploid species. Infact manylaboratories reported that there is successful map-based cloning in wheat.Thesuccess full map based cloning recreated the interest in construction ofphysical map, these maps based on large insert DNA libraries,thuough anchoringto high-resolution maps.

For durum wheat bacterial artificial chrosome libraryhad already prepared, because durum wheat has large size genome 13,000 Mbp,516,096 clones were required to achive a 5.1 coverage. library requiredtechnical demand to maintain and handling it due to its large size ,becauselibarary has unique genomic resource. Since the early 1990s for targetedanalysis of plant genome we uses the flow cytometry.The flow sorted chromosomeis used for physical mapping ,PCR with specific primers, targeted isolation ofmolecular marker and for integration of genetic and physical map.

For theprepration of subgenomic BAC library the chrosome sorted from hexaploid wheatwas used for it.BAC libary is specific for 1D, 4D and 6D chromosome, a BAClibrary specific for 3B and one for short arm of chromosome 1B.Flowcytometric purification of single chromosome and chromosome arm depend on useof cytogenetic shocks,such as telosomic and chromosome addition line in barleyrey and hexaploid wheat. In hexaploid wheat(2n=6x=42,AABBDD genome) a specificchrosome in each of genome could be compensate for loss of homologuschromosome.

In durum wheat there is possibility of substituting a D genomechrosome from the A or B genome, it was indicated by sears’ work.In chromosomesorting individual chromosome were isolated from various cultivar of durumwheat,from strain of T.turgidum and from series of double –ditelosomic line toshow feasibility of sorted individuals. In durum wheat a set of DNA propes wasused to develop the first molecular karyotype. Moleculer karyotype andprotocols provide important step toward development of BAC library in wheatarms of chrosome.RESULTSIt wasindicated by experiment that, in hexaploid wheat the protocol for cell cyclesynchronization was not suitable for durum wheat.

The root tip cells that arearrested with 2Mm HU recovered much later then expectations. So the recoverypattern that can approach in hexaploid wheat is 1.25 mM.

After release from HUblock the maximum activity (53% cells in mitosis) was observed after 5 hours.The timing of APM block that accumulate the synchronized cells in metaphase wasobtained after analysis of chromosome suspension of root tip that treated withAPM at various time interval during the discovery. After 5 hour when recoveredfrom HU and 2 hour APM treatment the flow karyotypes with least amount ofdebris background and highest resolution of chromosome peak was obtained. Nomodification of chromosome isolation protocol was needed like cell cycle synchronization.After fixation of 2% formaldehyde suspension of intact chromosome was obtained.

The analysis of DAPI stained chromosome suspension prepared from wheat  cv.Langdon has flow karyotype with two peakslabeled 2 and 3 and a peak represent chromosome 3B. For cerso and cultivar similarlyflow karyotype was also obtained.

The composite peak 1 of hexaploid wheatflowkaryotype  represented threechromosome of D genome and hence it absent in wheat  FIGURE 1: after the analysis of DAPI-stained flow karyotype obtained,chromosome suspensions prepared from durum wheat “Langdon” with a standardkaryotype and from three dDt lines (B–D)In flow karyotype of duram wheat there the aim of identifying thechromosome content of three peak present ,chromosomes were sorted ontomicroscopic and subjected to fish with probes for the Afa family repeate, GAAmicrosatellites, 5S rdna and pSc 119.2.The analysis shows that peak 2 shows orrepresented 2A, 3A, 4A ,5A, 7A chromosomes and all B geneome chromosome with theexception of chromosome 3B chromosome. Through observation we distingnish 3Bchromosome as a seprate peak could be sorted with high purity greater then 90percent in durum wheat.    Figure 2    Genomic distribution of repetitive DNAsequences obtained after FISH on flow-sorted chromosomes. Labeled DNA probeswere detected with either fluorescein (yellow-green) or Cy3 (red).

Chromosomeswere counterstained with either propidium iodide (red) By analysis of genomic distribution of setrepetive DNA sequence we have took advantage of performing FISH on large numberof sorted chromosome. From fig 2 it is giving Representation of florescent-labeling Patternswas obtained with probes  were similar tothe pattern that was observed for group A and B chromosomes of hexaploid wheat, the  same patteren was used for GAA satellite, alsofor Afa familyrepeat and for the pSc119.2.There are some unimportant or minor additional bands was seen in durum wheatfor the three DNA repeats.

For example, on the durum chromosomes 2A–7A, 2B, 4B,and 5B, the small bands of Afa repeats was located..But these bands weremissing in the hexaploid wheat chromosomes.

In fig 3 the diagram indicating thegenomic distribution of the durum wheat has four repeat sequences.In durumwheat the differences in fluorescent-labeling patterns may help in  identification of each of the 28 chromosomearms.Figer 3 Idiogram of the durum wheat Langdon showinggenomic distribution of four repetitive DNA sequences: GAA microsatellite(GAA), Afa familyrepeat (AFA), pSc119.2(119), and 5S rDNA (rDNA). Differences in labeling pattern identification ofeach.After the successful preparation or formationof suspensions of intact chromosome, it isit stimulated experiments to focused on thesorting o of the wheat A- and B-genome chromosomes could be easily seprated andsuitable for flow cytometry f single chromosome arms from dDt lines.After  the analysis of a coresponding set of sevendDt lines, it includes chromosome 1AS which has the lowestrelative DAPI fluorescence and the chromosome 3BL with the second- sortedhighest DAPI fluorescence (after 5BL),In figure 1 it is indicated that everyarm of chromosome of genome A and B genome could be sepratedand sorted. In figer 2 it showed that thechromosome arms could be sorted at very high purities ranging from 90 to 98%,themicroscopic analysis of sorted chromosome fractions it subject to FISH withprobes for GAA microsatellite and telomeric repeat.

when there is  replacement in a wild-type chromosome( whichoccour more frequently in poplution) with a pair of telosomes,its resultes notonly in the appearance of two additional peaks added on the flow karyotyperepresenting the two telosomes, but it also result in the change of thechromosome content , and change the presence of the original chromosome peaks.In figer 1 the peak II in dDt1A represented only the wild-type chromosome 6A,infiger 1 it is also indicated that the peak representing chromosome 3B wasabsent in dDt line As a 3B.From the strain ofT.tugidum var melanopus with15 chromosome pairs, this strain indicated theability to analyze chromosomes of T.turgidum var. durum alertus to analyze chromosome suspensions that prepared from a strain.

The resultfrom flow  karyotypes is similar to  those of double-ditelosomic lines of T. turgidum var. durum.In figure 4 itindicates that In addition to peaks II, III, and 3B, two peaks appeared to theleft of peak II .In figure 2 The analysis of fluorescence-labeling patternsidentified these chromosomes as 5BS and 5BL.

When chromosomes from these peakswere sorted onto microscope slides and is subjected to FISH with probes for GAAsatellite and telomeric repeat. Figer 4 Afterthe analysis of DAPI-stained chromosome suspension from strain of T turgidumvar its flowkaryotype obtained melanopus with2n = 30. Thekaryotype consists of two composite peaks representing by 1 and 2.