Analysis Of Genetic Characteristics Of Taihu Bream And Construction Of High-density SNP Genetic Linkage Map

In this research,the Culter alburnus after two generations of population selection and two generations of hetero gynogenesis induced were used as the female parent,and the Megalobrama terminalis after three generations of population selection were used as the male parent.The hybrid offspring were obtained through distant hybridization.The new varieties named C.alburnus?×M.terminalis??GS-02-001-2017?was approved by the National Commission for Examination and Approval of Original and Improved Aquatic Species.The hybrid fish combines the excellent characteristics of its parents,such as fast growth,high feed conversion rate,moderate size and strong stress resistance.At present,the research on the genetic breeding of fish interspecies hybrids is quite limited at home and abroad,but the research on the genetic improvement of C.alburnus?×M.terminalis?is even rarely reported.Therefore,in this paper,the morphological characteristics and GH gene of C.alburnus?×M.terminalis?were analyzed,and then the high-density genetic linkage map was constructed and QTL mapping of growth related traits were resealed.Accumulated the genetic background knowledge of the fish,which laid a solid foundation for analyzing the sub network regulation mechanism of candidate genes and SNPs markers.It will play an important role in the genetic improvement of the fish in the future,which will also provide a theoretical basis for the sustainable development of C.alburnus?×M.terminalis?.The research results were shown as follows:1.Morphological characteristics analysis of C.alburnus?×M.terminalis?The morphology of C.alburnus?×M.terminalis??orthogonal F₁??backcross F₁?C.alburnus?×M.terminalis???C.alburnus and M.terminalis was observed and multivariate statistical analysis was carried out on the proportion parameters of countable traits,quantifiable traits and frame traits in four populations.The results show that:?1?the body shapes of the C.alburnus?×M.terminalis?and the backcross F₁were longer and flat,which were similar to those of C.alburnus,but the body height was higher than that of C.alburnus.The intestines of C.alburnus?×M.terminalis?were about 1.88 times of body length,and that of backcross F₁was about 2.05 times of body length,which was similar to that of C.alburnus.There were three rows of teeth?2.4.4/5.4.2 or 2.4.5/4.4.2?in the lower pharynx of C.alburnus?×M.terminalis?and the teeth were hook like and similar to the female parent;There were three rows of teeth?2.4.4/5.4.2 or 2.4.5/4.4.2?in the lower pharynx of backcross F₁and the shape of teeth similar to the male parent.?2?The number of lateral line scales and scales above lateral line of C.alburnus?×M.terminalis?and backcross F₁were between their parents?P<0.05?,and the number of scales below lateral line?branched fins of pectoral fin?first gill rakes?outside?were significantly higher than their parents?P<0.05?.Although the number of branched fins of hip fin was similar in the four groups,there were also significant differences?P<0.05?.The number of first gill rakes?inside?of Megalobrama terminalis were significantly higher than others?P<0.05?.The average cross coefficient of 14 countable characters was 87.70,which indicated that the countable characters of C.alburnus?×M.terminalis?preferred to their male parents;the average cross coefficient of 14 countable characters was-9.76,which indicated that the countable characters of backcross F₁preferred to their female parents.?3?The results of five principal component analysis showed that:the contribution rate of total variance among different populations was 49.271%,10.680%,10.512%,7.091%,4.409%respectively,and the cumulative contribution rate was 81.963%.?4?The results of discriminant analysis of all 29 variables show that:the discriminant accuracy of parents and C.alburnus?×M.terminalis?was 100%,and that of backcross F₁was 96.7%.One sample was misjudged as C.alburnus?×M.terminalis?,and the comprehensive discriminant accuracy was 99.1%.?5?The proportion parameters of 9 measurable characters and 20 frame data of the parents and their hybrid progenies were clustered by the shortest Euclidean distance.The results showed that the genetic relationship between crossbred progeny was close,and the distance between the crossbred progeny and Culter alburnus was closer than Megalobrama terminalis.So the main morphological characteristics of the backcross progeny were close to Culter alburnus.2.Comparative analysis of GH gene between C.alburnus?×M.terminalis?and its parents and F₂generationUsing polymerase chain reaction?PCR?and T-A clone technology,a gene encoding of M.terminalis,C.alburnus?×M.terminalis?and F₂generation growth hormone?GH?including complete open reading frame was cloned.The gene of three species of fish respectively spanned 6009 bp?6042 bp and 6058 bp and transcription unit respectively spanned 2049bp?2014bp and 2045bp.There were comprised five exons,four introns,5?UTR and 3?UTR and GH gene gene of three species of fish encoding sequences of amino acids were 633bp and encoding 210 amino acids.Sequence comparison showed that the amino acids encoded by the two heterotopia spots in exon 5 of C.alburnus?×M.terminalis?changed;In intron 1 and intron 2,C.alburnus?×M.terminalis?is mainly inherited from his father,and in intron 3 and intron 4,the fish is mainly inherited from his mother.Homology comparison found that C.alburnus?×M.terminalis?had high sequence identity with parents and M.amblycephala,followed by other fish of Cypriniformes and purpose of Siluriformes homology is obviously higher than that of Perciformes.Phylogenetic analysis showed that C.alburnus?×M.terminalis?and Cypriniformes,Siluriformes were grouped within the same branch with a bootstrap of 100;The Epinephelus bruneus in Perciformes formed one branch separately and the other three fish formed another branch.3.Construction of high density genetic linkage map and QTL mapping of growth related traits in C.alburnus?×M.terminalis?Firstly,the F₂generation of C.alburnus?×M.terminalis?population was proved to be suitable for the construction of genetic map by cluster analysis of SNP,phylogenetic tree construction and principal component analysis;Then,the high-density genetic linkage map was constructed by using 2b-RAD technique in the C.alburnus?×M.terminalis?.The total length of the map was 1825.52 c M,and the average marker interval was 0.76 c M.The comparative genomic map showed that there were more homologous markers mapped to the genome of Megalobrama amblycephala than to Danio rerio and Cyprinus carpio.Based on the genetic map,27QTLs related to growth traits were detected in 9 linkage groups,and the contribution value of explained phenotypic variance?PVE?ranged from 9.4%to 24%,and the LOD score ranged from 2.15 to 5.96.Among the six growth-related traits?body length?total length?body weight?body height?head length and caudal peduncle length?,body weight related QTL?LOD=5.4?was the most significant,mapped to LG1,LG2,LG6 and LG18 respectively.Using P<0.01 and P<0.05 as the threshold of difference significance,the correlation analysis of single SNP loci of the six growth-related traits showed that under the two threshold of difference significance,the most SNP loci were detected by weight traits,which indicated that the economic character is closely related to the rapid growth of fish.