| The Quasipaa spinosa(Amphibia:Ranidae),a large unique frog species found mainly in southern China,such as Yunnan province,Guizhou province,Zhejiang province and so on.This species likes to inhabit near the edge of mountain stream500-1500 m above sea level with highly valued in Chinese markets for its medicinal and nutritional value.It is indicate that the amount of protein in the frog muscles reach to 18.55%-19.39% with a fairly low amount of fat.In order to provide more frogs for eating,the artificial breeding programs of Q.spinosa was launched in the1980 s,depending on the natural supply of tadpoles or adults.As a economically relevant traits,feeding behavior and food preference domestication determine production cost and profitability.Its demonstrated that the feeding habits of wild animals can be induced gradually for eating artificial compound diets.Hence,a taming growth pattern was build in order to clarify the growth performance,activities of digestive enzymes and antioxidant enzymes in Q.spinosa.Then,we constructed an expression profile of the Q.spinosa through the high-throughput sequencing and then obtained the SSR markers from the constructed library with the default settings.The results can provide reference value and theoretical basis for studying the molecular mechanism of breeding and subsequent feeding of frogs.1.Effects of feeding acclimation on growth and enzyme activity of Q.spinosaWith "to live driving method(tame)+ water drive method(consolidation)" training mode,after two months of domestication,there were significant differences in body weight,body length,head length,head width and gut length between the easy taming group and the non-taming group(P<0.05).Among them,the biggest change was in body weight,which increased by about 3 times in the easy taming group and decreased by 36.20% in the untaming group,showing a negative growth pattern.The contents of digestive enzymes(lipase,trypsin and amylase),antioxidant enzymes(superoxide dismutase,catalase)and malondialdehyde in easy taming group were compared with those in non-taming group.It was found that the activities of amylase in liver,kidney,foregut,midgut and hindgut in easy taming group were higher than those in non-taming group(P<0.05).The activity of lipase in the liver and midgut of the untamed group was significantly higher than that of the easy tamed group(P<0.05),and the activity in midgut was the highest.The activity of trypsin in liver,kidney,foregut,midgut and hindgut of the easy taming group was higher than that of the non-taming group(P<0.05)and had the highest intestinal activity before.The activity of SOD in the foregut,midgut and hindgut of the easy taming group was higher than that of the non-taming group(P<0.05).The activity of catalase in liver and kidney of easy taming group was higher than that of non-taming group(P<0.05).The content of malondialdehyde in the liver,kidney,foregut,midgut and hindgut of the non-tamed Q.spinosa was higher than that in the non-tamed Q.spinosa,but the difference was significant only in the liver and foregut(P<0.05).2.Transcriptome analysis related to feeding domestication of Q.spinosaA total of 78.47 GB of Clean Data were obtained by transcriptome sequencing of easy taming and difficult taming Q.spinosa.100,461 Unigenes were assembled from scratch,and 30,092 Unigenes were annotated,accounting for 29.9% of All Unigenes.These results provided a large number of sequences for the study of feeding mechanism of Q.spinosa.It was found that 70.34% of Unigene could be matched to the sequence of Nanorana parkeri,followed by Rana catesbeiana(8.13%),Xenopus laevis(7.45%)and Xenopus tropicalis(7.08%).Among them,there were 139 DEG,66 up-regulated genes and 73 down-regulated genes in the brain tissues of the easy taming group and the non-taming group.There were 1517 DEG genes,881up-regulated genes and 636 down-regulated genes in liver tissue.GO enrichment analysis was performed on the screened differentially expressed genes.It was found that in the category of biological processes,the enrichment trend of differential genes was different from that of the overall gene enrichment trend in the second term,such as behavior,growth,and rhythm.In the second term of different trends in the molecular function categories,there are signal transduction and antioxidant capacity,etc.,which reveals that the training difficulty of Q.spinosa may be closely related to the above biological processes.For training and training to feed DEG KEGG enrichment analysis,in the brain and liver,each 10 and feeding the most closely related to metabolic pathways,one of the common pathways are eight,in all the 12 pathway,Six pathways were consistent with other reports,and six pathways were speculated to be newly discovered feeding related pathways in Q.spinosa.These related genes and signaling pathways provide the genetic basis for studying the molecular mechanism of feeding habit formation in Q.spinosa.3.Development and validation analysis of SSR related to feeding habits of Q.spinosa32,726 microsatellite loci were found in 100,461 sequences,of which 17,308 contained SSR loci,and 10,843(62.65%)were annotated and could be used as priority candidate genes for the development of SSR markers.Among them,there were 17,233 SSR loci with SSR length over 12 bp,accounting for 59.37% of all SSR loci,indicating that Q.spinosa had moderately high fragment length polymorphism.After multiple layers of condition screening,127 food-related microsatellite loci that could successfully design the primers were finally screened out,and through the verification of agarose gel,39 pairs of primers amplified a single band,and the size was consistent with the expected product.These results provide data support for the further study of feeding domestication and resource conservation of Q.spinosa.In conclusion,Q.spinosa can feed on artificial compound diet after domestication.Compare easy breaking food and not easily breaking the spine chest the growth of the Q.spinosa,the different characteristics of enzyme activity and the transcriptome,the mining trainer food related functional genes and explore the path of food related training,to develop the food related microsatellite marker,for subsequent spine mass domestication and artificial compound feed of the Q.spinosa development provide theoretical basis and technical support,also for amphibians artificial domesticated breeding to provide technical guidance. |
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