Genome-wide Identification And Expressionprofiling Of The Bone Morphogenetic Proteins Gene Family,osteologicalontogeny In Larval And Juvenile Rachycentron Canadum

The skeletal system of fish plays an important role in supporting the body,protecting internal organs and coordinating life activities.The process of skeletal development is closely related to the process of fish body development.Cobia(Rachycentron canadum)is a fast-growing marine fish with breeding potential,but little is known about its skeletal development.In view of this,the morphological ossification characteristics of the skulls,vertebrae,and appendages of Cobia larvae [1–33 days post hatching(DPH)] were described using a car-tilage bone clearing and staining technique to provide information on the larval and juvenile organ structure development and functional adaption.On the same time,we documented skeletal deformities in tankreared Cobia juveniles(25DPH).Based on observations of cleared and double-stained specimens,the skeletal deformities were described.Using the existing genomic data of Cobia,the BMP genes were identified and analyzed.Finally,explore the BMP genes using Real-time Polymerase Chain Reaction(RT-PCR)technology changes in expression patterns in different days past hatching and tissues.1.Skeletal development in the larval and juveniles of CobiaThe results showed that the Dentary,Maxillary and premaxilla of the skull began ossification at 11 DPH.At 15 DPH,the Opercle,Preopercle,subopercle,Branchiostegal ray and articular began to ossify,while the Frontal,Dentary,Maxillary and premaxilla were ossified at 18 DPH.At 26 DPH,the Basihyal,Quadrate and Suborbital maxillawere ossified;most of the skull was completely ossified by 28 DPH.Ossification of the vertebrae began at 13 DPH from the anterior to the posterior.The dorsal rib and ventral rib began to ossify from the base to the outside at 17 and 20 DPH,and were completely ossified by 20 and 29 DPH.Ossification of the haemal arch began at 13 DPH from the to posterior the anterior,The neural arch began to ossify from ends to centre at 13 DPH.The haemal arch,haemal spine,neural arch,and neural spine from the base to the distal end were ossified.The ossification order of the appendages was from the pectoral fin to the caudal fin,dorsal fin,anal fin,and pelvic fin.The cleithrum of the pectoral fin was ossified at 12 DPH,while the coracoid and scapula were ossified at 20 DPH.The urostyle and hypural of the caudal fin began to ossify at 15 and 18 DPH.The dorsal fin and anal fin were ossified at 17 and 18 DPH,respectively.The pelvic fins were ossified at 18 DPH.The appendicular skeleton was fully ossified at 30 DPH.As a result,72 out of the 180 fish were deformed(with a deformity rate of 40.00%),and a total of 22 types of skeletal malformations were identified.These skeletal malformations were found on 25 days after hatching Cobia juveniles,the deformity rate from high to low was mainly manifested as Mikel’s cartilage deformity,epural deficiency,bifurcated haernal spines,basihyal deformity and epural fusion.Malformations were most common in the cranium and caudal fin,the highest incidence of skeletal deformities is Meckel’s cartilage deformity(12.22%)and epural deficiency(7.78%).All the fish with these skeletal deformities showed no visible morphological changes.In addition,a significant difference in the total length was found between normal fish and individuals with deformities(P<0.01).2.Genome-wide identification and Expression profiling of the bone morphogenetic proteins gene familyUsing the existing genomic data of Cobia,the BMP genes were identified through bioinformatics analysis tools such as NCBI,MEME and Tbtools etc.,19 BMP family genes(BMP1～BMP16)were identified in Cobia,which belong to five groups.The BMP are composed of 1170～3009 bp nucleotidesencoding 236～699 amino acid,and their isoelectric points are 5.26～10.The 19 BMP are unevenly distributedon 13 chromosomes.All 19 BMP genes structure of the same subgroup was similar.The results of q RT-PCR showed that the expression of BMP2 and BMP4 was significantly increased and the expression of BMP3 b was increased,the expression of BMP5 and BMP11 was significantly decreased at 11 DPH compared with 1 DPH.On the 18 DPH,expression of BMP2,BMP4,BMP5 and BMP16 was significantly increased,and the expression of BMP8 a,BMP11 was significantly decreased.The expression of BMP4,BMP9,BMP5 and BMP16 was significantly increased at 25 DPH.Expression of BMP4 and BMP16 was significantly increased,and the expression of BMP8 a was significantly decreased at 30 DPH.BMP1、BMP2、BMP3a、BMP4、BMP7b、BMP8a、BMP9、BMP16 was widely expressed in various tissues.The expression levels of Bmp3 b and BMP11 were the highest in Mikel’s cartilage,BMP4 was the highest expression levels in vertebrae and fin,Bmp8 a was the highest expression levels in scale.In summary,Cobia larvae were described using a car-tilage bone clearing and staining technique to provide information on the larval and juvenile skeletal development and skeletal deformities,our results provide insights into the skeletal developmental process and optimization of rearing conditions for Cobia.19 BMP family genes were characterized from the whole genome level in Cobia,and their gene expression patterns in larval and juvenile and various tissues provide a theoretical basis for the role of BMP genes in skeletal developmental process.