Pan-genome Analysis Reveals Transposon Variation Driven Genome Evolution In Cotton

Transposons have been recognized as an essential factor accelerating plant genome evolution and resulting in cotton genome changes and phenotypic diversity,which provides more opportunities to study the evolution and adaptability of cotton.However,the previous reference genome research based on a single reference genome is not widely representative,and the emergence of pan-genome can make a comprehensive comparison of the cotton genome,which can comprehensively understand the transposon variation and evolution in cotton.In this study,we used 344 accessions to construct a pan-genome for Gossypium arboreum（A₂）,a putative diploid progenitor of the allotetraploid cotton G.hirsutum（AD₁）,which can enrich the gene pool of cotton.We found that the Gypsy type of retrotransposon plays an important role in gene loss and gain in G.arboreum.By pan-genome comparison,we have revealed differences in transposon amplification in diploid and tetraploid cotton,which provides new insights into the activity of transposons in cotton.The main results are as follows:1.We collected the resequencing data of 344 representative diploid cotton accessions,based on the"map-to-pan"strategy,we identified the non-reference sequence and combined it with the existing reference genome,a pan-genomic sequence of 2,166 Mb and 57,257 pan-genes were obtained,and the 88.1%,3.1%,8.6%and 0.3%of the genes were identified as core,softcore,shell,and cloud genes.By constructing the distribution landscape of G.arboreum pan-genes in the population,we found 645 favorable genes and103 unfavorable genes that were optimized in the adaptive evolution of G.arboreum in China.2.We conducted the pan-genome comparison to identify the PAV by comparing the genomes of diploid and tetraploid cotton,excluding the interference of about 60MB of intraspecific variation.We found that 1324 genes were included in the intraspecific PAV,which was thought to be lost during polyploidization in studies based only on the reference genome,deviations based on the reference genome comparison were corrected.Further analysis of genome-specific K-mers,it is clear that the PAV between diploid and tetraploid is derived from the new sequence after species differentiation or the sequence loss,we found that the A₂,A_t,and D_tsubgenomes undergo genome expansion after polyploidized,while D5 is conserved.3.according to the age classification of 47257 pan-genes,we found that 15.6%of the species-specific new genes appeared only in the pan-genes,and the new genes evolved faster than the old genes.The G.arboreum has a high content of Gypsy LTR,which result in a higher frequency of occurrences near genes compared to the Copia LTR.We found that the 6217 genes contain TE domain,of these,3938（63.3%）genes were caused by the Gypsy insertion and were mainly concentrated in the species-specific genes（2206 genes）.At the same time,using the pan-gene dataset,we analyzed the single-copy gene loss and amplification of diploid and tetraploid cotton and found that the loss of 330 single-copy genes was caused by TE insertion,the effect of transposon on the gene evolution of cotton was clarified.4.By constructing the pan-TE library,we compared the differences in the diploid and tetraploid cotton and found that the amplification of the diploid and tetraploid cotton showed inconsistent evolutionary directions after the polyploidization,the LTR concerted proliferation in the tetraploid cotton,resulting in the genome expansion of the tetraploid,and the invasion of LTR retrotransposons from the A_tsubgenome expands the size of the D_tsubgenome,thus the genomic amplification of A_tis less than A₂,resulting in an opposite trend of genome size evolution in the two subgenomes of tetraploid cotton.In conclusion,we have discovered the role of LTR retrotransposons in the evolution of the cotton genome by pan-genome,which provides new insights into the evolution of polyploid cotton.