Development And Genetic Analysis Of Three Nullisomic Lines Of C Chromosome In Brassica Napus

The nullisomics lines losing a pair of chromosomes,can be used to map genes and genetic markers,and determine the corresponding relationship between linkage group and chromosome,have been recognized as rare but important genetic stocks in crop genetic improvement.The allotetraploid oilseed rape（Brassica napus,2n=38,AⁿAⁿCⁿCⁿ）,derived from the natural hybridization between diploid ancestor B.rapa（AA,2n=20）and B.oleracea（CC,2n=18）,is one of most important oil crops in the world.Although there is close relatedness between constituted A and C subgenomes in B.napus,rare nullisomic has been reported in B.napus.Herein,an aneuploidy population（BC₁F₂）of B.napus was developed through the interspecific cross and following backcross between B.napus“Oro”and restituted B.rapa“Oro”derived from B.napus“Oro”previously.Then the PCR amplification of C chromosome specific markers and fluorescence in situ hybridization were used to screen three nullisomic lines of C chromosome in B.napus first time,named NC1,NC2,and NC8.Subsquently,the phenomics,chromosome behavior,and gene expression patterns of these nullisomic lines were deciphered in detailly.The main results are as follows:The B.napus“Oro”was crossed with RBR Oro to generate the sesquidiploid hybrids（F₁,AⁿAⁿCⁿ,2n=29）.Because of the unbalanced C genome,these F₁hybrids can give rise to aneuploidy gametes.Then these hybrids were backcrossed to paternal“Oro”to obtain a total of 163offsprings（BC₁F₁）.These BC₁F₁plants were selfed with artificial supplementary pollination to obtain a BC₁F₂population with about ten thousand seedlings.After screening 3,296 plants of BC₁F₂population using the PCR amplification of C chromosome specific markers,counting chromosome numer,and FISH,three types of nullisomic of C chromosomes were developed（NC1,NC2,and NC8）.Compared to the parental“Oro”,the NC1 has the leaves with obvious burrs,particular in vein,indicating chromosome C1 harbors gene（s）that restrain the burrs development;the NC2 shows a obvious dwarf plant architecture and earlier blooming time of which is two months earlier than parental“Oro”,suggesting that chromosome C2 harbors the key gene（s）that affect the plant architecture and flowering time;the NC8 shows a slightly later blooming time and multiramose plant architecture.A dominat of pollen mother cells（PMCs）of 18 bivalents at diakinesis and a segragation of18:18 at meiosis anaphase I was observed in three nollisomic lines.However,some PMCs with one to two lagging chromosomes were observed.Out of 50 selected PMCs,16 PMCs（32%）of NC1 harbored the lagging chromosomes in NC1,while only five and two PMCs of NC2（10%）and NC8（4%）were observed.After checking the characteristics and chromosome number of these offsprings of three nullisomic,they are consistent with their parent,indicating these nullisomic lines can inherit in a stable manner.Transcriptome data of three nullisomic lines were obtained by RNA sequencing,which not only confirmed the results of missing chromosomes according to the reads distribution,but also revealed various degrees of dysregulated expression genes（trans-effects）from the balanced chromosomes（unaltered disomic chromosomes）in comparion of parent B.napus“Oro”.The NC2 shows the most widespreadly gene expressed perturbation,for 11,769（17.54%,the ratio of trans-effects DEGs to the total genes ruling of the genes on missing chromosomme）differetially expressed genes（DEGs）derived from trans-effects are detected,including 6,523（9.72%）up-regulated DEGs and 5,246（7.82%）down-regulated DEGs.A total of 9,688（14.53%）trans-effects DEGs are identified in NC8,comprising of 5,265（7.89%）up-regulated DEGs and4,423（6.63%）down-regulated DEGs.The NC1 shows the lowest gene expressed perturbation,for 6,382（9.31%）are detected,including 3,306（4.82%）up-regulated DEGs and 3,067（4.49%）down-regulated DEGs.Intriguingly,among these trans-effects DEGs,significant dominant up-regulated DEGs（χ2 test,p<0.01）are detected in all nullisomic lines.Moreover,the up-regulated DEGs from chromosome A1 which shows high homology with chromosome C1 are overrepresent（13.15%of total trans-effects DEGs）,implying that the genes which are located on homeologous chromosome A1 enhance their gene expression to compensate the absence of chromosome C1（homeologous genes expressed compensation）.However,no obvious homeologous genes expressed compensation is detected in NC2 and NC8 lines.Among these trans-effects DEGs,898 DEGs are co-regulated in three comparisons,occupying 14.07%of NC1,7.63%of NC2,9.27%of NC8,indicating missing different chromosomes result in diverse gene expressed perturbation.After checking the gene function of these co-regulated DEGs,these genes mainly involve in“binding”and“catalytic andactivity”and“cell part”in the GO.And these genes mainly involve in"biosynthesis of other Secondary metabolites","carbohydrate metabolism"and"Amino acid metabolism"in KEGG pathway.These nullisomic lines,which,effectively reduce the complexity of B.napus genome are developed in B.napus first time except NC2.These nullisomic lines can be used to anchor some important agronomic traits and verify results of gene mapping,meanwhile,they are facilitated to detect the interation between A and C genome gene expression,which eventually facilitate the development of germplasm resources,genetic improvement,and evolution analysis in B.napus.