Variations In Pan-transcriptome And Genome Size In Tropical Maize (Zea Mays L.) And Their Applications

Maize is an important food,feed and fuel crop.Improvement on the reference genome is of importance for genomics and molecular breeding.Since the release of B73 reference genome in 2009,it has been playing an important role in maize gene function study and molecular breeding.However,as a temperate maize inbred line,we speculate that B73 lacks a lot of specific genes and transcripts existing in tropical maize inbred linesbased on the maize genome diversity and phenotypic differentiation.Several studies have showed a possible relationship between genome size and maize flowering time.However,the maize plants were usually tested in a single location.Few researchers used genomic features as a marker for flowering time.In view of huge diversity in maize genome,we found that the maize chips currently available,based on the B73 reference or a temperate maize germplasm,lacks some tropical specific markers when the existing maize chips are used for germplasm evaluation and marker development.Aiming at solving these problems,we choose 31 tropical inbred lines out of 79 resequenced maize inbred lines for bulked transcriptome sequencing by Pacbio system.Flow cytometry was used to measure maize genome size,and 180-bp knob abundance was calculated by resequencing data.Combining with flowering time,we analyzed the relationship of genome size and knob abundance with flowering.Based on resequencing data,we developed tropical-specific SNPs in tropical maize germplasms and included them in a newly developed 55 K SNP chip with improved genome coverage.The major results are as follows:1.Using Pacbio sequencing technology,we sequenced the transcriptome of 31 tropical maize inbred lines using their seedlings harvested in two weeks and shoot apical meristem in five weeks after planting.Analysis of the three libraries 1-2 Kb,2-3 Kb and 3-6 Kb,showed that their average lengths of insert reads were 1,511 bp,2,246 bp and 3,681 bp,respectively.Several issues associated with saturated pan-transcriptome were discussed.The density of chromosome distribution for the insert reads showed an evenly distribution on genome.After clustering and correction analysis and redundancy removal,35.63% reads could be mapped to the reference genome.2.By comparing with the reference,we annotated 16,121 genes,2,991 unknown functional genes and 944 fusion genes.By optimizing the gene structure,including 5' and 3' end of the extension,we obtained 41,136 sequences involving 9,932 genes.3.A significant difference in genome size between tropical and temperate inbred lines was revealed.We found a moderate positive correlation was found between genome size and 180-bp knob abundance,as determined by high-throughput sequencing 79 maize lines each at 10× depth.By assembling the reads that mapped to 180-bp knob sequences,we found that the top ten abundant 180-bp knob sequences were highly variable with only 50% of similarity.4.Genome size was associated with male and female flowering times for both tropical and temperate inbred lines across tropical and temperate environments.A genome-wide association study identified three QTL associated with genome size,of which two were novel while the other one was located nearby the known knobs K8L1 and K8L2.5.Based on our resequecing data from 79 maize inbred lines and the maize transcriptome data from 368 maize inbred lines,a new maize 55 K array was developed to improve the maize chips currently available.The new chip has several characteristics:(1)by using SNPs identified in tropical maize inbred lines which are not covered by B73 reference genome,the genome coverage of genetic diversity was improved and the chip can be used to detect for tropical-specific SNPs;(2)by training known heterotic group SNPs with support vector machine,SNPs which can distinguish different heterotic groups,particularlyheterotic groups A and B,were introduced into the new chip;(3)The new chip also included SNP markers for detecting transgenic events;(4)SNPs in maize classical genes were also included;(5)a set of core SNPs were selected from the existing chips.Taken together,our results can be used to explore genomic information that is specific in tropical maize germplasm,provide theory basis and technology support for the use of tropical germplasm in molecular breeding and germplasm evaluation.