Pan-Genome Assembly Of Maize Founder Inbred Lines And Analysis Of Heterosis Mechanism

Heterosis refers to the phenomenon that progeny of diverse varieties of a species or crosses between species exhibit greater biomass,speed of development,stress resistance,viability than both parents.Heterosis plays a key role in the process of maize genetic improvement.In the first half of the 20th century,the successful cultivation and application of maize hybrids led to a breakthrough increase in maize yield.However,the genetic mechanism of heterosis is complex,and the genetic variation between different maize inbred lines is abundant.At present,the formation and evolution of maize heterosis remain obscure.This study was based on Pac Bio third-generation sequencing technology,Bio Nano optical map and Illumina sequencing technology to assemble and annotate the high-quality genomes of twelve key founder inbred lines（FILs）,which were widely used for hybrid maize breeding worldwide.In addition to the published B73 and Mo17 genomes,the pan-genome of maize FILs was constructed to provide a more complete genome for maize genetic improvement.In addition,this study revealed the correlation between better-parent heterosis of the F₁hybrids with the genetic variation between the parental lines through correlation analysis and further explored the genetic variation and genes related to maize heterosis and heterotic group formation,in order to analyze the genetic mechanism of maize heterosis.The main conclusions of this study were as follows:（1）The average genome size of the 12 inbred lines was 2.25 GB,with an average of 44,554 genes annotated,and the average proportion of repetitive sequences in the genome was 84.68%.Combined with BUSCO evaluation,alignment results of Illumina sequencing reads,LAI value and syntenic analysis with the published genomes,it is confirmed that this study has constructed twelve high-quality and high-integrity genome of maize founder inbred lines.（2）The results of pan-genome analysis showed that with the increase of the number of inbred lines,the number of pan-genome gene families was increasing,but the number of core gene families was decreasing.When the number of genomes reached 12,the trend of the number of pan-genome and core gene family changes tended to be smooth.Clustering results showed that there were 43,518 gene families in the pan-genome,including 18,525（43.75%）core gene families,9,976（22.92%）softcore gene families,14,992（34.45%）dispensable gene families,and 25（0.06%）private gene families.The expression of core,softcore and dispensable genes decreased in genome,while the non-synonymous mutation rate increased,which indicated that the stability of these four genes decreased sequentially in genome.（3）The results of genetic variation detection showed that the average number of SNPs and In Dels in 91 pair-wise comparisons of the 14 assemblies was 8,489,166 and1,223,484 respectively.The average length of SVs and PAVs was 97,841,839 bp and14,712,962 bp respectively.The average length of non-syntenic region and the average number of large effect genes was 939,841,102bp and 7,390,respectively.At the same time,12 high-quality Mb SVs were identified in the non-syntenic region.The results of syntenic analysis showed that the average syntenic length among the 14 genomes was 1,209,723,365 bp,which account for 56.31%of the whole genome.These results indicated that there were abundant genetic variations between the parental genomes.（4）eQTL mapping and association analysis clarified that structural variations can regulate gene expression.Combined with haplotype analysis,we discovered the excellent haplotype Hap-0/363 of the gene Zm LOX3 related to maize ear rot resistance.Meanwhile,we functionally validated the candidate genes Zm00001d006055 and Zm00001d011140 and their excellent haplotypes related to the tassel branch number and ear height,respectively,which provided certain genetic basis for the improvement of important agronomic traits in maize.Also,the frequency distribution of functional variants of Zm LOX3,Zm CCT9,VGT1,ZCN8,KRN4,KNR6,Zm GDIαand Zm Trxh in different heterotic populations indicated that genetic variation was the basis of genomic and phenotypic differentiation in different heterotic group.（5）Correlation analysis between genetic variations and GYPP heterosis index BPH revealed that the length of syntenic regions showed significant negative correlation with GYPP＿BPH（p=2.09e-03,r=-0.320）.In addition,SNPs and small In Dels in the syntenic regions only showed weak correlation with GYPP＿BPH and the correlation did not reach the significant difference level.In contrast,non-syntenic length,SV＿Number and PAV＿Number all showed significant positive correlation with GYPP＿BPH.Among them,SVs number showed the strongest correlation with GYPP＿BPH（p=2.17e-03,r=0.319）.The SVs could be further divided into three types.Among them,the Insertion＿Deletion showed the strongest correlation with GYPP＿BPH.It indicated that SVs played an important role in the process of heterosis formation in maize.（6）Haplotype analysis and heterosis effect analysis of diallel cross population F₁s showed that the genetic variations among Hap-Ye478,Hap-SL17 and Hap-Jing92haplotypes in ZmACO2 gene can the regulated the formation of heterosis in maize yield.The genetic variation among Hap-TSPT,Hap-NSS and Hap-SS haplotypes of ZAR1gene also regulated the formation of heterosis in maize yield.In addition,the results showed that the natural variation in both ZAR1 and ZmACO2 contributed to yield heterosis in an overdominance fashion.The results of this study preliminarily expound the genetic mechanism of maize heterosis groups and heterosis formation and provide new insights into the molecular basis of heterosis in maize,further more providing a solid theoretical basis and genetic resources for the breeding of breakthrough maize hybrids.