Study On Chromosome Behaviour Of The Progenies Of The First Backcross Generation Between Two Herbicide-resistant Transgenic Oilseed Rape And Wild Brassica Juncea

Oilseed rape(Bras.sica napus,AACC=38)is predominantly self-fertile,and produces a lot of pollens.Meanwhile,it has many relatives in a natural environment.Therefore,one of the concerns about releasing genetically modifiedherbicide-resistant(GMHR)oilseed rape is that herbicide-resistant(HR)transgenes from GMHRoilseed rape may escape to theirrelatives,especially weedy relatives,via spontaneous hybridization and introgression.If this happens,weeds with the HR trait might produce new problems for weed control.The first step for transgene escape is the spontaneous hybridization between transgenic crop with weedy relatives.Besides the initial crop-weed hybridization,the probability of introgression is determined by these processes(1)gene transmission and expression during the successive generations(2)effective gene introgression through recombination between genomes(3)production of fertile and fit offspring through successive backcross or selfings.Many reserches focus on frenquecy of gene flow,inheritance of transgene and fitness of hybrids between transgenic oilseed rape and relatives.However,tracking researches on chromosome constitution and genetic stability of hybrids are lacking.Among wild relatives,wild B.junces(AABB,2n=36)is the closest relative weed with oilseed rape(AACC,2n=38)in China.The inheritance of herbicide-resistant gene in progenies of backcross generation and fitness of these progenies were studied in previous researches.The results demonstrated thatthe surviving percentage of progenies usingglyphosate-resistant oilseed rape as parent were higher than that using glufosinate-resistant oilseed rapeas parent after herbicide selection.The progenies with glyphosate-resistant gene presented a fasterfitness improvement with increasing of self-pollination of progenies than that with with glufosinate-resistant gene.Until now,the chromosome constitution and stable process.of progenies of the backcross generations between two transgenic herbicide-tolerant oilseed rape and wild B.juncea has not yet researched.In current reseach,the first to fourth progeny of the first backcross generation(BC1mFR2-5and BC1pFR2-5,M means backcross progeny obtained with wild B.juncea as maternal plants.p meansbackcross progeny obtained with wild B.juncea as paternal plants.)of transgenic glyphosate-resistant oilseed rape(DS-Roughrider,Roundup Ready,event RT73,resistant gene located on A-genome)and wild B.juncea,the first to fifth progeny of the first backcross generation(BC1mFL2-6 and BC1pFL2-6)of transgenic glufosinate-resistant oilseed rape(Swallow,Liberty Link,event HCN92,resistant gene located on C-genome)and wild B.juncea were used as materials.The chromosomenumber,constitution and chromosome behavior during meiosis of these progenies were studied by methods of classic cytogenetic and BAC-FISH.The results may provide cytogeneticevidence for the potential differencesof gene flow from two herbicide-resistant transgenes herbicide-resistant oilseed to wild B.juncea.The main results are as follow:1.The progenies with glyphosate-resistant transgene(BC1FR)were mixopliod,and could be set into 2n,=36(AABB),similar with wild B.juncea.Theprogenies of BC1F2R-BC1F5R between glyphosate-resistant oilseed rape and wild B.juncea were mixopliod.The variation of chromosome numbers decreasedprogressively across the generation of self-pollination of BC1F1.With the increase of generations,the chromosome numbers of cells with2n=36chromosomes increased.The plants displayed a veryparticular chromosome number distributionwith a large proportion of cell in majority plants with 2n=36 chromosomes.BAC-FISH observations showed that genomic constitution of cell with 36 chromosomes was 20A+16B through observing cell of BC1Fm5R and BC1Fp5R in three individual plants respectively,which was the same as wild B.juncea,2.The progenies with glufosinate-resistant transgene(BC1FL)were mixopliod,and could be set into 2n=37(AABB+1C),which were monosomic addition lines.The progenies of BC1F2L-BC1F5L between glufosinate-resistant oilseed rape and wild B.juncea were also mixopliod.The variation of chromosome numbers decreasedprogressively across the generation of self-pollination of BC1F1L.With the increase of generations,the chromosome numbers of cells with2n=37chromosomes increasedgradually.The plants displayed a veryparticular chromosome number distributionwith a large proportion of cell in majority plants with 2n=37 chromosomes.BAC-FISH observations showed that the cell with 37 chromosomes was 20A+16B+1C through observing cells in five and seven individual plants of BC1mF5L and BC1pF5L,respectively.Two cells(1.83%of the observed cells)with 37 chromosomes were 20A+15B+2C in one plants of BC1pF5L.The fourth progenies with glufosinate-resistant gene carried at least one additional oilseed rape C-chromosome.The progenies of BC1F5L weremonosomic addition lines.3.Observation on chromosome abnormalbehavior duringmeiosisThe abnormal meiosis was observed in pollen mother cell(PMC)of both the fourth progenies with glyphosate-resistant gene and with glufosinate resistant gene.The lagging chromosomeswere observed in metaphase I and II.These lagging chromosomesdid not pair normally and were not arranged on the metaphase plate.The lagging chromosomes and chromatid bridgein anaphase I and II were frequently observed.Some lagging chromosomesfailed to enter the polar region where the other chromosomes started to compact,these lagging chromosomes in anaphase I and II formed micronucleiin telophase I and II,which were recorded in telophase I and II at a low frequency.Although the fourth progenies with glyphosate and glufosinate resistant gene showed consistent abnormal behaviors,the chromosome pairing in the fourth progenies with glufosinate-resistant gene was much highly irregular than that with glyphosate-resistant gene.Meiotic indexes of BC1F5R plants were between 94.97 to 99.5,indicating considerably genetic stability.Meiotic indexes of BC1F5Lwere between 61.84 to87.86,indicating considerably genetic instability,except for one plant of BC1pF5L,which showed 91.3 meiotic index.4.The silique length and seed number per silique of progeniesThe silique lenghth and seed number per silique of BC1F(2-5)R and BC1F(2-6)Lplants were tested.The results showed thatseed number per silique of the same progenies varied widely.In general,the progenies presented a much strongerfertility improvement across the self-pollination generation.With the increase of generations,the average silique length of BC1F2-5R was similar to that of wild B.juncea.The average seed number per silique of BC1F2R was 3.6 and 3.03,significant lower than that of wild B.juncea,while BC1F3-5R were10.82to17.12,which were similar to that of wild B.juncea.The averagesilique length of BC1F2L was significant lower than that of wild B.juncea,while BC1F3L-BC1F6L were were similar to that of wild B.juncea.The average seed number per silique of BC1mF2L-BC1mF4L andBC1pF2-6L wassignificantly lower than that of wild B.juncea.However,thethe BC1mF5L and BC1mF6L were14.75 and 14.67,similar with that of wild B.juncea.Therefore,thefecundity ofprogenies with glyphosate-resistant oilseed rape as parent wasrestoredfaster than those with glufosinate-resistant oilseed rape as parent.