Chromatin Elimination And Introgression In Wheat Somatic Hybridization

Somatic hybridization has been suggested as a means of circumventing interspecific barriers to fertilization and has been successfully used in plant breeding. In our lab,studies on the somatic hybridization between wheat and many other genera of grasses have been processed for a long time,and somatic hybrid plants and their offsprings have been obtained from some combinations.When asymmetric somatic hybridization was applied to the bread wheat / tall wheatgrass combination,a number of high yielding and good quality introgression lines,carrying resistance to stripe rust, tolerance to salinity and drought have been generated.Besides,two fertile hybrids were obtained from symmetric somatic hybridization between bread wheat and tall wheatgrass,both of which were phenotypically similar to the tall wheatgrass parent. Although it is important for crop breeding,the mechanism of somatic hybridization is still unclear.Systemic investigation about chromatin elimination and introgression of donor in wheat somatic hybridization were progressed in this research.Time and fashion of chromatin elimination and introgression of donor were discovered. Influence of phylogenetic relationship of wheat with donor and radiation dose on chromatin elimination and introgression of donor were discussed.Besides,through symmetric somatic hybridization between wheat and two grasses(Poa annua L and Haynaldia villosa L),we investigated the complement of two types of calli of wheat in hybrids and the change of methylation in culture and somatic hybridization.Finally, somatic hybrids between wheat and tall wheatgrass were analyzed,which phenotype was tall wheatgrass-like.Chromosome elimination and introgression of wheat in hybrids were discovered.The sequence elimination of parents and change of HWM-GS in hybrids were also discovered.The main results of this research are listed as follows: 1.Fashions of chromatin elimination of donor in early stage of wheat somatic hybridizationAfter GISH analysis of somatic hybridization between wheat and four different donors(A.elongatum,H.villosa,P annua,Z.mays),we found that donor chromatin were mainly eliminated in interphase through micronucleus formation in early stage of wheat somatic hybridization.There were also other fashions of chromatin elimination of donor in early stage of wheat somatic hybridization,including micronucleus formation in anaphase,micronucleus formation in multi-nucleus and separation of parental nucleus.Besides,we found lagging chromosomes and chromosome bridges in somatic hybridization between wheat and H.villosa. Separation of parental nucleus were mainly occurred in remote somatic hybridization between wheat and donor,especially in the time of 5-7d after fusion.2.Chromatin elimination and introgression of donor in early stage of wheat somatic hybridizationAfter analysis of asymmetric somatic hybridization usingγ-ray between wheat and two grasses(P.annua and H.villosa),we found that the introgression frequency and elimination frequency of donor after fusion were all clearly higher in 5-7d than in 30d.The introgression frequency and elimination frequency of donor were lower than 10%in 30d.Besides,we analyzed asymmetric somatic hybridization using UV between wheat and four donors.The introgression frequency and elimination frequency of donor after fusion were also clearly higher in 5-7d than those in 12-15d. In conclusion,chromatin elimination and introgression of donor in early stage of wheat somatic hybridization were mainly occurred in 5-7d after fusion.3.Influence of phylogenetic relationship on chromatin elimination and introgression of donor in early stage of wheat somatic hybridization After analysis of somatic hybridization between wheat and four donors,which were different from wheat in phylogenetic relationship.We found that the introgression frequency of donor was clearly disparate in different somatic hybridization combinations between wheat and distinct donors.Compared to the combinations of P annua and Z.mays,which were remote to wheat,the introgression frequency was higher in the combinations of tall wheatgrass and H.villosa which were close to wheat.But elimination frequency of donor was lower in the combinations of H.villosa than that in the combinations of P annua,and Z.mays. Because of the larger polyploid of A.elongatum than H.villosa,the elimination frequency of donor in combinations of tall wheatgrass was higher than that in combinations of H.villosa.It was clear that chromatin elimination and introgression of donor in early stage of wheat somatic hybridization could be affected by the relationship of wheat and donor.When the donors had related relationship to wheat, the polyploidy of donor may also affect chromatin elimination and introgression of donor in early stage of wheat somatic hybridization.4.Influence of radiation dose on chromatin elimination and introgression of donor in early stage of wheat somatic hybridization Compared analysis of asymmetric somatic hybridization between wheat and four grasses,we found that radiation dose was related to chromatin elimination and introgression of donor in early stage of wheat somatic hybridization.Under a certain range of radiation dose,introgression of donor increased following the increasing radiation dosage,while chromatin elimination of donor decreased following the increasing radiation dosage.When radiation dose was higher than the range, elimination of donor increased abnormally following the increasing radiation dose.5.Effect of different relationship between wheat and donor on asymmetric degree of hybrids in symmetric somatic hybridization Through symmetric somatic hybridization between P.annua and two different wheat callus(cha9 and 176) which were captured from the same wheat(Jinan177),we get wheat / P.annua asymmetric somatic hybrids and hybrid plants.Compared with somatic hybrids of wheat with H.villosa,spot chromatin of P.annua were diffused in wheat chromosomes in wheat / P.annua hybrids,while intact chromosomes or chromosome segments of H.villosa were existed in wheat / H.villosa hybrids.The asymmetric degree of wheat / P.annua somatic hybrids was much higher than wheat/ H.villosa somatic hybrids.It was clear that the remote donor could get high asymmetric hybrids in wheat symmetric somatic hybridization. 6.Regeneration of somatic hybrids with complementation of parental chromatinUsing the special NORs sequence of 185-5.85-265rDNA as probe,FISH analysis was processed in the parents and somatic hybrids.We found that the distribution of NORs were different in the two wheat callus.Five chromosomes of Jinan 177 which could detect the NORs signals were detected in cha9,while just two chromosomes of Jinan 177 were detected in 176.Besides,special signals of NORs were detected in cha9 and 176 separately,which were caused by culture.These special signals were all detected in a wheat / P annua somatic hybrids(16H),and directly proved the complement of cha9 and 176 in wheat somatic hybrids.The phenotype and SSR analysis of hybrids also illuminated this complement.MSAP analysis of wheat callus and wheat / P annua somatic hybrids(4H) showed that there were clear changes of methylation.Compared with Jinan 177,there were many changes of methylation in cha9 and 176,which were caused by culture. Besides,there were also changes of methylation in hybrids between callus and plants. Both of these showed that changes of methylation were related to the length of culture time.Compared with Jinan 177,callus and plants of somatic hybrids had different changes of methylation.Some changes of methylation in callus were recovered, including 5.88%of methylation and 3.35%of demethylation.7.Chromosomes elimination and introgression of wheat in symmetric somatic hybridsChromosome analysis of the wheat/tall wheatgrass somatic hybrids showed that the number of hybrids were similar to tall wheatgrass(Agropyron elongatum(Host) Neviski).Some lagging chromosomes at telophaseâ… were observed,as chromosome bridges at anaphaseâ… and micronuclei at telophaseâ…¡.After GISH analysis of the PMC of R0 hybrids,we found the existence of wheat genomes and the asynchronism division in some hybrid cells of R0 plants,In the meiosis of some R₂ individuals,GISH was able to characterize the inheritance of introgressions in the hybrid genome.This analysis showed that all complete wheat chromosomes were eliminated,but that segments were introgressed into tall wheatgrass chromosomes.Of the 745 fragments scored in AFLP analysis,305 were specific to tall wheatgrass,and 267 to wheat.XI and CU carried a different set of wheat-specific and tall wheatgrass-specific fragments.They shared 256 tall wheatgrass-specific fragments but differed with respect to 27 fragments,of which 19 were carried by CU and eight by XI.Of the 13 wheat-specific fragments present in the R₂ lines,nine were present in both CU and XI,three was only present in XI and one in CU.The 13 wheat-specific fragments were detected by six of the 13 primer combinations.There were also 17 novel bands in each R₂ hybrids and another two were only present in XI.All of these showed that both wheat and tall wheatgrass sequences were lost or recombined.The HMW-GS profile of direct and later generation progeny of the two R₂ lines differed from that of tall wheatgrass by the absence of three subunits,which were replaced by three subunits not present in either the tall wheatgrass or the bread wheat parent.