Preliminary Study Of The Genetic And Environmental Effects On Gene Expression Of Larix Sibirica

Larix sibirica occupies the western,central,and southwest of eastern Siberia,eastern Kazakhstan,northern Mongolia,and northwestern China.Larix sibirica is the dominant tree species in the coniferous forest of these areas and plays important roles in the carbon cycle,water cycle and energy cycle of the ecosystem,and provides high quality wood material.Global warming is changing the phenology and secondary growth of Larix sibirica.In this thesis,I study the genetic variation and gene expression profiles in response to climate changes and achieved the following:the preservation of samples(for RNA extraction)at room temperature,the improvement of the RNA-seq library preparation method,the construction of the full-length transcriptome by third generation sequencing ISO-seq,the exploration of the effects of genetic and environmental factors on gene expression of populations from 12 locations,and the mining of rhythmicity of gene expression in Larix sibirica.The main results of this thesis are:(1)To find a method to preserve plant tissues in field sampling without liquid nitrogen,I evaluated the preservation of plant leaves with three home-made buffers.The results showed that Arabidopsis leaves could be preserved in all three buffers without severely degrading RNA.For polyphenol-rich plant tissues,only CTAB buffer is suitable for sample preservation and RNA isolation.(2)Taking advantage of the template switching properties of reverse transcriptase,I developed a simple 3’ m RNA-seq library preparation method that focused on the 3’ terminal of m RNA.By incorporating sample barcodes at the reverse transcription step,multiple samples can be pooled and processed together during library preparation,reducing on hand times,and enable up to 96 libraries to be prepared in one day.Also,unique molecular identifier(UMI)can be used to remove PCR duplicate more accurately.Another m RNA-seq library preparation method using Tn5 transposase was also tested.Tn5 was able to cut DNA/RNA hybrid double strand,this property circumvents the use of d UTP and UDG enzyme,making the library preparation protocol easier and cheaper.(3)By integrating genome sequences and third generation sequencing of transcriptomes,genes expressed actively in Larix sibirica leaves are annotated.Gene expression profiling of Larix sibirica collected from six locations arranged in 500 km revealed that environmental factors contribute more to differential gene expression than genetic variation.Individuals can be distinguished by the SNP variants called from transcriptome and polulations further away have greater genetic variations.(4)Because it is impossible to collect samples at the same time of a day in the wild field,and since many genes oscillate throughout the day according to the studies of model plant,its necessary to eliminate the effects of factors such as rhythmic oscillations on gene expression studies.To identify the circadian genes,we collect samples from three trees every 2 hours for a total of 2 days,and identified 1855 circadian rhythmic genes.These data provide a reference for the gene expression profiles of Larix sibirica in the future studiesThis study achieved the preservation of plant RNA at room temperature;designed a simple method for RNA-seq library preparation;found that environmental factors,in particular temperature,have more effects on the gene expression of Larix sibirica compared to genetic variation;and identified circadian rhythmic genes of Larix sibirica.These results revealed how Larix sibirica respond to environmental changes at the molecular level,and provide simple and efficient experimental methods and reliable data for future studies.