Study On The Germplasm Characteristics And Sex Determination Mechanism Of Coilia Fish In The Yangtze Estuary

Coilia is a small and medium-sized fish species belonging to the order Clupeiformes and Engraulidea,with the coast from the western central Pacific to the Indian Ocean as its main distribution area.Coilia nasus is a river-sea migratory fish,which is mainly distributed in the Yellow Sea,Bohai Sea and the East China Sea in China.The annual output of C.nasus is the highest in the lower reaches of the Yangtze River and the Yangtze estuary.Coilia mystus is a semi-anadromous migratory fish,which is distributed in the Bohai Sea,the Yellow Sea,the East China Sea,the South China Sea and the Taiwan Province Sea.In this paper,the lactic dehydrogenase（LDH）and karyotype composition as well as sex-specific SNP molecular markers of C.nasus and C.mystus were tested and analyzed at the biochemical,cellular and molecular levels to describe the germplasm characteristics of the main Coilia species in the Yangtze estuary,aiming to provide the basic data and theoretical basis for the establishment of germplasm inspection standards of C.nasus and C.mystus,as well as the data reference and theoretical basis for the establishment of germplasm inspection standards of C.nasus and C.mystus,and also the technical support for the genetic breeding of C.nasus and C.mystus.The main research results are as follows:1.Study on LDH isoenzyme of C.nasus and C.mystusThe polyacrylamide gel electrophoresis（PAGE）was used to analyze the lactate dehydrogenase（LDH）of six tissues including heart,liver,kidney,eye,muscle and gill of C.nasus and C.mystus.The results showed that LDH was widely distributed in C.nasus and C.mystus,and had obvious tissue specificity.There were six LDH enzyme bands in six tissues of C.nasus,which were composed of LDH-A₄,LDH-A₃B,LDH-A₂B₂,LDH-AB₃,LDH-B₄,and LDH-C₄ enzyme bands.In addition to the LDH-C₄ enzyme band,the other five enzyme bands were expressed in various tissues,while the LDH-C₄ enzyme band formed by the combination of LDH-C sites was only expressed in liver tissue,which could be called"liver band".The six LDH enzyme bands in C.mystus tissues also shared six enzyme bands,which were expressed in all tissues,but only four enzyme bands could be detected in all tissues,and the expression and activity intensity of enzyme bands showed significant tissue differences.The interspecific differences of C.nasus and C.mystus could be revealed by the differences in LDH zymogram structures.There were differences between this study and previous studies on LDH in different tissues of C.nasus.The differences were mainly manifested as the total enzyme bands,the expression of LDH-C₄ enzyme bands and the expression of enzyme bands in muscle tissue.Through the comparative analysis of LDH among different populations of C.nasus,the above three points were verified.The total enzyme bands of LDH in C.nasus were six,and LDH-C₄enzyme bands were expressed and only in the liver.The enzyme bands in muscle were five,but not four,and the expression of LDH enzyme bands in C.nasus was subject to individual differences.According to the reference indexes selected by biochemical genetic markers,the LDH expression in the eye tissue of C.nasus was stable,strong and easy to obtain,which was suggested to be used as a biological genetic marker for identifying the germplasm characteristics of C.nasus.2.Karyotype analysis of coilia in the Yangtze EstuaryThe short-term in vitro culture of gill filament cells was used to prepare chromosome specimens and the karyotypes of C.nasus and C.mystus in the inland waters of Chongming Island in Shanghai and the coastal waters near Lingang were studied.The results showed that there was no difference in the number of chromosomes between male and female C.nasus and C.mystus.The number of chromosomes was 2n=48,both of which belonged to terminal centromeric chromosomes（T）,and the karyotype was 2n=48 t;Arm count（NF）=48 and the sex chromosome type was ZW/ZZ.The relative chromosome lengths of C.nasus males ranged from 2.98±0.24 to 5.67±0.34 and females from 2.32±0.30 to 5.67±0.22.The relative chromosome lengths of C.mystus males ranged from 2.66±0.27 to 5.54±0.25 and females from 2.31±0.33 to 5.60±0.36.The T test for the relative length of chromosomes of C.nasus and C.mystus showed that there was no significant difference in the relative length of chromosomes of female（P>0.05）and male（P<0.05）.Different from the previous studies in which the chromosome karyotypes of C.nasus and Coilia brachygnathus were ZO/ZZ type,we found an obvious"dot"chromosome in the metaphase mitotic phase of female chromosomes of C.nasus and C.mystus.After mode analysis,we found that the probability of distribution of this"dot"was more than 84.2%（240 metaphase mitotic phases）,confirming that it was an abnormal chromosome of Coilia species.3.Screening research on sex-specific SNP markers of C.nasus and C.mystusA simplified genomic sequencing and screening of sex-specific SNP markers were conducted on 40 C.nasus and C.mystus collected from the waters of Chongming Island,Yangtze estuary,Shanghai using sequencing genotyping（GBS）technology.The analysis results of DNA sample quality detection,data quality evaluation and data comparison indicated that high-quality genomic sequencing data were obtained from this sequencing.The preliminary screening results of gender-specific candidate sequences showed that a total of 115 marker candidate sequences（100♀,15♂）were obtained from C.nasus.No candidate sequences were selected for C.mystus.After in-depth screening of the candidate sequences（compared with the C.nasus genome annotation file,the average depth of sequencing was increased）,11 sex-specific SNP markers of C.nasus were obtained,all of which were female mutant base types and located on two chromosomes respectively,and the male genotype of C.nasus was confirmed to be homozygous and the female was confirmed to be heterozygous.Combined with the results of chromosome karyotype analysis,the sex chromosome type of C.nasus was confirmed to be ZW/ZZ,which corrected the mistake in the previous study of "ZO/ZZ" type.