Study Of The Non-coding RNA Regulation In Heat Tolerance,Morphological Characteristics And Taxonomicand In Populus Qiongdaoensis

Populus qiongdaoensis T.Hong et P.Luo is a unique tropical plant of Populus and a key protected plant in Hainan Province,China.However,the number of natural populations in P.qiongdaoensis is declining and easily leads to inbreeding,which greatly threatens growth and reproduction.The protection and research in P.qiongdaoensis appear urgent.Studying the natural survival status,morphological characteristics,taxonomic status,and the molecular regulation mechanism under heat stress in P.qiongdaoensis is the basis for conservation and breeding.Therefore,this study obtained the survival status of the P.qiongdaoensis trees in the wild through investigating.An off-site preservation nursery was constructed through breeding young seedlings in P.qiongdaoensis.The morphological characteristics of the P.qiongdaoensis trees were obtained by measuring and recording.Sequence information such as the simple repeat sequence marker（SSR）of orthologous genes,nuclear genes（nr DNA）,chloroplast genes（cp DNA）,and micro RNA（mi RNA）were obtained by sequencing to analyze the taxonomic status in P.qiongdaoensis.The non-coding RNA response mechanism in P.qiongdaoensis was studied by 40℃stress treatment.（1）The investigated results showed that the distribution areas of the P.qiongdaoensis trees includes the Zizai forestry farm of Baisha County and Hongshui forestry farm of Changjiang County,Hainan Hainan Province.The altitude of the distribution area is600-900 m,the annual average temperature is 22-25℃,and the maximum and minimum temperatures are 37℃and 2℃.The relative humidity is about 75%,and the total annual rainfall is 1500-2500 mm.According to the preliminary investigation results of the two distribution areas,there are about 200 individuals in P.qiongdaoensis retained in the wild with the diameter at breast height is 0.5-1.5 m and the average height of the P.qiongdaoensis tree is 20-40 m.We have constructed an off-site preservation of P.qiongdaoensis by collecting seeds.A conservation nursery of 200 P.qiongdaoensis plants was constructed in Danzhou City,Hainan Province through seedling breeding,which realized the off-site preservation of the P.qiongdaoensis trees.The morphological characteristics of the P.qiongdaoensis trees were measured and statistics,and the results showed that the leaves of the P.qiongdaoensis are 5-16 cm in length,4-12 cm in width.The length of the petiole is 3-8 cm.Blooming time is from December to January of the following year.The male inflorescence is 5-12 cm in length,the female inflorescence is5-11 cm.The stigma is 2-lobed,the ovary is ovoid,containing 3-5 ovules.The infructescence is 14 cm in length,it splits into two petals when the fruit is mature.After the seeds have matured and fall on the soil,they can germinate in 1-2 days under suitable conditions.（2）The analysis results of morphological characteristics showed that the P.qiongdaoensis and Leuce are closely related.Two pairs of polymorphic SSR primers were obtained by the transcriptome data of P.qiongdaoensis.Then,a total of 14 different length sequences were obtained from 11 Populus species by capillary electrophoresis,and the polymorphic information content（PIC）was 0.7254.The cluster analysis results of the SSR sequence showed that the P.qiongdaoensis and Leuce are closely related.Two nr DNA,two cp DNA and four mi RNA sequences were obtained by cloning and sequencing,and the genetic distance of the SSR sequences showed that the P.qiongdaoensis and Leuce are closely related（0.010,0.017,and 0.038,respectively）.（3）A total of 88161 full-length transcripts were obtained as reference sequences using the Pac Bio Sequel II sequencing platforms.A total of 95794107 small RNA reads with a length of 18-30 nt were obtained by small RNA sequencing.A total of 38415 m RNAs,928long non-coding RNAs（lnc RNAs）,and 216 mi RNAs were obtained after annotation.Under the dual criteria of|log₂fold-change|≥1 and P-value<0.05,1690 m RNAs,26lnc RNAs,and 15 mi RNAs were found significantly differentially expressed under heat stress.Furthermore,563 and 595 genes were predicted as target genes of 14 mi RNAs and26 lnc RNAs by biometric calculation.Functional annotation analysis of these genes showed they were related to cell membrane stability,plant hormone signal transduction,and antioxidation caused by plant heat stress.A mi RNA–lnc RNA–m RNA interaction network was constructed through co-expression relationships,and a key interaction network consisted of mi R1444d,mi R482a.1,mi R530a,lnc HSP18.2,HSP18.1,and HSP18.2 were discovered,indicating that mi RNAs can negatively regulate lnc RNAs and m RNAs under heat stress in P.qiongdaoensis.In this study,the wild individuals of the P.qiongdaoensis trees were investigated,and an off-site preservation nursery with the seedling were constructed.The morphological characteristics of the P.qiongdaoensis trees were measured and analyzed.The sequences of the SSR,nr DNA,cp DNA,and mi RNA were used to analyze the relationship and found that P.qiongdaoensis have a close relationship with Leuce.The data analysis of the transcriptome and small RNA sequencing reveals the non-coding RNA regulatory network under heat stress in P.qiongdaoensis.The above results will provide a theoretical foundation for P.qiongdaoensis to the protection,development,and utilization.