| Pinus massoniana Lamb(Pinus massoniana Lamb)is one of the main lipid-production tree species in China.In recent years,due to the reduction of its forest size and lipid-production trees,pine resin production has been reduced;the classic reintroduction breeding strategy is used to genetically improve the high lipid-production of Pinus massoniana Lamb the required cycle is long,and it is difficult to meet the market's demand for high-yield and refined varieties of Pinus massoniana Lamb in a short time.Using genomic association analysis methods,by screening out molecular markers that are significantly associated with lipid-production traits,carrying out molecular marker-assisted selection studies is of great significance for rapid breeding of high-yieldlipid-production Pinus massoniana Lamb and shortening the breeding cycle.It is an effective way to discover the important genes controlling the target traits by deep mining the gene sequence where the significant association markers are located.It is of great significance for further exploration and ultimately revealing the genetic regulation mechanism of the mass production trait of Pinus massoniana Lamb.This study is based on 3 significant association molecular markers(1 SSR marker and 2 SNP markers)obtained from the candidate genome association analysis of Pinus massoniana Lamb lipid-production traits and simplified genome association analysis institute to carry out deep mining of related gene sequences.The main contents of the research include:(1)Based on high-throughput transcriptome sequencing results,trace the gene sequence of the SSR-PCZ90 marker,and analyze the mRNA sequence of the SSR-PCZ90 marker by bioinformatics software,including homologous sequence alignment,protein structure(including primary,secondary and tertiary structure)and prediction of functional genes;(2)Based on the sequence fragments of SNP-M181697 and SNP-M249970 obtained by simplified genome sequencing,two SNP positions The sequence of the point is extended,and more sequence information is obtained to lay the foundation for finally obtaining 2 marker-related genes.The main conclusions of this study are as follows:(1)The SSR-PCZ90 maker second-generation sequencing original sequence has the highest homology(over 90.95%)with TCHQD-type glutathione S-transferase(GST)of Pinus tabuliformis,Larix kaempferi,Picea glauca,and Pinus taeda,coverage varies.Its sequence length is 1636 bp,which can encode 264 amino acids;its molecular mass is 30910.65 Da,its theoretical isoelectric point is 9.27,it is weakly positively charged,unstable and easily decomposed,and its 65 th threonine is the most hydrophilic.Leucine position 153 is the most hydrophobic.There are two types of transmembrane helix structures between amino acids 183-200.There is no signal peptide and coiled-coil structure.It contains 21 phosphorylation sites and is mainly distributed in cytoplasm.The secondary structure contains 157 ?-helices,13 ?-turns,26 extension strands and 68 random coils.Tertiary structure contains the N-terminal domain and the C-terminal domain of the glutathione S-transferase family.SSR-PCZ90-labeled 3rd-generation sequencing-related sequences have the highest homology(over 91.52%)with TCHQD-type glutathione S-transferase(GST)of Pinus tabulaeformis,Larix kaempferi,Picea glauca,and Pinus taeda,coverage varies.Its sequence is 1222 bp long and can encode 183 amino acids;its molecular mass is 21429.71 Da,its theoretical isoelectric point is 9.45,it is weakly positively charged,unstable and easily decomposed,and its 117 th isoleucine is the most hydrophilic.Leucine at position 72 is the most hydrophobic.There are two types of transmembrane helix structures between amino acids 100-120.There is no signal peptide and coiled-coil structure.It contains 18 phosphorylation sites,mainly distributed in the cytoplasm.The secondary structure contains 122 ?-helices,12 ?-turns,12 extension strands and 37 random coils.Tertiary structure contains the C-terminal domain of the glutathione S-transferase family.Based on the basic characteristics of GST,it is speculated that it participates in the process of cellular immune defense,which increases terpene metabolites in response to external expression;or the genes involved in terpene metabolism linked to it,due to the chain relationship when they are highly expressed,make terpenes metabolites increase.The content of phosphorylation sites is high,speculating that it may carry phosphoric acid groups to provide the energy required by the body.(2)Excavate the sequence of SNP-M181697 and SNP-M249970,and amplify the middle unknown sequence of the SLAF tag where the two sites are located,respectively 127 bp and 178 bp in length.Further digging through chromosome walking continues,because the Pinus massoniana Lamb genome is large and the proportion of highly repetitive sequences is high,the mismatch rate of primer binding sites is high and it is impossible to continue digging.It is necessary to obtain more genomic information through whole genome sequencing and other studies before continuing to further study the genes related to the 2 SNP sites. |
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