Characterization Of Microsatellite Repeat Sequences And Screening Of Microsatellite Markers Associated With Growth Traits In Culter Alburnus

The topmouth culter（Culter alburnus）,the largest fish in Culterinae,has important ecological and economic value.The production of C.alburnus has increased significantly in the past few decades.It is also one of the main fish species used for the stock enhancement of natural waters.The spawning amount of female C.alburnus is so high that a few parents can produce plenty of fry.Because of the time-sensitive of small fry,fewer parents are selected to mate in most breeding farms,which results in inbreeding depression.Microsatellite sequences,which are highly polymorphic codominant markers,are widely distributed in the genome of eukaryotes.They have been extensively used in molecular marker-assisted breeding and conservation genetics.To date,a few studies have analyzed the microsatellite repeats of C.alburnus,but the obtained information is inadequate for breeding.In this study,the magnetic-bead enrichment method was used to isolate the microsatellite sequences of C.alburnus.Microsatellite primers were designed,and the multiplex PCR system was used for paternity identification.The microsatellite sequence characteristics derived from the transcriptome and genome sequencing of C.alburnus were analyzed,and some microsatellite markers were verified.The relationship between growth traits and microsatellite markers on the basis of the growth hormone,insulin-like growth factor I,and growth hormone receptor genes was studied.The results of this study provide basic data for germplasm detection and novel breeding of C.alburnus.1.Microsatellite enrichment by magnetic-beads and paternity identification of C.alburnusTwo microsatellite mixed libraries for the caudal fin tissues of C.alburnus were established using the biotin magnetic-bead enrichment method with dinucleotide repeats（CA）₁₂ and（GA）₁₂,trinucleotide repeats（CAA）₈ and（GAA）₈ as the probes respectively.A total of 52 microsatellite sequences were obtained from the microsatellite enrichment libraries of dinucleotide（CA）₁₂ and（GA）₁₂,and 32 polymorphic microsatellite loci were isolated.Moreover,microsatellite sequences for 30 individuals out of the Wuxing（WX）population were analyzed.The number of alleles at each locus ranged from 3 to 12,with an average of 6.6.The observed heterozygosity and expected heterozygosity were 0.467–0.900 and 0.616–0.909,respectively.No linkage was detected among the loci after Bonferroni correction,and 3 loci did not comply with the Hardy-Weinberg equilibrium.A total of 159 microsatellite sequences were obtained from the microsatellite libraries enriched by the（CAA）₈ and（GAA）₈ probes.The sequence alignment analysis showed that 56 of 159 microsatellite sequences（35.2%）could be assigned to 3 microsatellite DNA families on the basis of the similarities of the microsatellite flanking region sequences.They shared regions of similarity ranging from 40 to 130 bp in length,with 96%sequence similarity.Furthermore,Family 1 had 39 microsatellite sequences containing CAA.Five microsatellites had similar flanking region sequences,but different number of microsatellite repeats while 9 sequences contained（CAA）₁₁ with different flanking region similarities.Moreover,Families 2and 3（7 and 10 sequences,respectively）were composed of GAA microsatellite sequences.According to the Repbase database,164 results were obtained from sequence alignment with 159 microsatellite sequences.If the PCR primers are located in conservative regions with high similarities,they will amplify the microsatellite sites of the same family and reduce detection efficiency.After excluding the sequences with high transposon alignment scores,38 pairs of microsatellite primers were designed,and 9 of them were polymorphic.Moreover,the number of alleles per locus was 4–15（average,6.6）in 30 samples of the WX breeding population.The observed heterozygosity and expected heterozygosity were 0.067–0.921 and0.616–0.909,respectively.Three groups of microsatellite multiplex PCR systems were established after redesigning the primers and optimizing primer concentration.Each group contained 4 microsatellite loci.The genetic relationships of 36 candidate parents and 136 offspring of 4 mating combinations were analyzed.All loci yielded a high amount of information content（polymorphic information content,PIC>0.5）.The parental exclusion probability（E-PP）was significantly correlated with the PIC value（P<0.01）.The analysis indicated that,when the number of candidate parents was less than 200,a higher allocation rate（>95%）could be obtained.In the real parent-offspring relationship analysis,99.26%of the offspring（135/136）were assigned to their parents with a high accuracy rate.The contribution rates of different parents to the offspring were significantly different（P<0.01）.2.Characteristics of microsatellite repeat sequences in the transcriptome of C.alburnusThe transcriptomes for the liver,muscle,kidney,brain,testis,and ovary of C.alburnus were sequenced using the Hi Seq2000 platform.A total of 118455 unigenes were obtained.MISA software was used to search for microsatellite repeats in the transcriptome,and a total of 30 360microsatellites were screened.Among 1–5 different base repeat types,the number of mononucleotide was the highest（total,18 017）,accounting for59.34%of the total number of microsatellites.The percentage of base repeat type sequences was 26.38%,dinucleotide;12.31%,trinucleotide;1.94%,tetranucleotide;and 0.04%,pentanucleotide.Among them,A,AC,AG,AT,ATC,AAT,AGC,AGG,AAC,and AAG were the top 10 microsatellite repeat categories.Eighty-seven sequences were selected from the transcriptome library for primer design,and 34 primer pairs were amplified stably.A total of 16 polymorphic microsatellite loci were obtained.Furthermore,these primers were used to analyze the genetic diversity of one wild population from Taihu Lake and 3 cultured populations from the Huzhou area（Wuxing,Changxing,and Linghu）.The number of alleles ranged from 3 to 10（average,5.25）.A certain bottleneck effect was detected among the 4 groups.The Changxing population showed a moderate degree of genetic differentiation with the other 3 populations,and a low degree of genetic differentiation was showed among them.Genetic variation among the 4 populations accounted for 94.09%,and the genetic variation within populations accounted for only 5.91%.3.Characteristics of microsatellite repeat sequences in C.alburnus genomeThe whole genome of C.alburnus was sequenced,assembled,and evaluated de novo,and the genome sequence of C.alburnus was obtained.The genome size was 1.055 Gb,and the sequencing coverage was 332 X.The lengths of contig N50 and scaffold N50 were 1.15 Mb and 1.17 Mb,respectively;4 584 conserved gene sets of Actinopterygii were selected as the reference.BUSCO was used to evaluate the genome integrity.A total of4 219 gene sets were completely matched,accounting for 92.03%of the total gene sets;this indicated that the genome quality obtained by assembling was good.The microsatellite repeats in the whole genome were searched using MISA software.A total of 772 276 microsatellites were screened,with a relative abundance of 732/Mb and relative density of 9 051 bp/Mb.The total length of microsatellites was 14.29 Mb,about 1.35%of the size of the whole genome.Among the 1–6 different base repeat types,the number of repeats for mononucleotide was the highest with a total of 416 303,which accounted for 53.91%of the total number of microsatellites.The percentages of the base repeat type sequences were 31.24%,dinucleotide;6.64%,trinucleotide;6.75%,tetranucleotide;1.26%,pentanucleotide;and 0.21%,hexanucleotide.Among them,A,AT,AC,AG,AAT,AGAT,C,ATCC,AAAT,and AAC were the top 10 sequence repeat types.4.Screening of microsatellite loci related to growth traits in C.alburnusThe growth hormone,insulin-like growth factor I,and growth hormone receptor are the major genes for fish development and growth.According to the transcriptomic and genomic data,fragments of these genes were amplified and verified using PCR.Seventeen polymorphic microsatellite loci were analyzed on the basis of the growth traits of 120 individuals bred in the same batch and cultured in the same pond.Six microsatellite loci were related to growth traits.4.1 Microsatellite loci in the flanking sequences of the growth hormone geneThe growth hormone gene（Cal-GH）,with 5 exons and 4 introns,spans5 966 bp.The lengths of the 5′-and 3′-flanking sequences are 2 282 kb and2 036 kb,respectively.（AAT）₈ and（TTC）₅T（TAA）₈ were located in the 5′-and 3′-flanking sequences,respectively.Furthermore,3 Cal-GH01 alleles（404,407,and 410 bp）and 6 genotypes were detected.The 407 bp allele was dominant,and 407/407 bp was the dominant genotype.The PIC of this microsatellite locus was 0.420（moderately polymorphic）,and the heterozygosity was 0.207 2.Moreover,a total of 6 alleles（366,375,378,381,384,and 387 bp）and 11 genotypes were identified for Cal-GH02.The381 bp allele was dominant,and 381/381 bp was the dominant genotype.The PIC and observed heterozygosity of the microsatellite locus were 0.450 and 0.466,respectively.This indicated that it was a moderately polymorphic locus.For microsatellite locus Cal-GH01,the associations between the polymorphisms of the loci and growth traits showed a trend for differences in body length and mass in individuals with different genotypes（P>0.05）.For microsatellite locus Cal-GH02,the percentage of 366/381-type individuals in the total samples was about 3.3%.The body length and body mass of individuals with 366/381-type were the highest;their mass was significantly higher than that of the other genotypes（P<0.05）.The 384/387-type individuals accounted for 2.5%of the total number of detected samples.Their body length and mass were the lowest among individuals with other genotypes.Their body length was significantly lower than that of the other genotypes（P<0.05）.4.2 Microsatellite loci in the insulin-like growth factor I geneIGF-I of C.alburnus has a total length of 14 567 bp,5 exons and 4introns,and 6 microsatellites.（GATG）₅AATAT（ATAG）₁₁ was present in the first intron.（CT）₈,（TTA）₅,（AC）₁₃,（TG）₁₂,and（ATT）₅ were located in the second intron.There were no polymorphisms in（TTA）₅ and（ATT）₅.The correlation analysis between genotypes and growth traits of the other four polymorphic microsatellite loci showed that the genotype of the microsatellite loci was not significantly associated with the growth traits（P>0.05）.4.3 Microsatellite loci in the growth hormone receptor genesBoth GHR1 and GHR2 are composed of 10 exons and 9 introns.Nine microsatellite sites in GHR1 were distributed in intron 1（3）,intron 2（5）,and intron 8（1）.One microsatellite locus（CT）₆ was located in exon 2,which encodes a signal peptide sequence.Moreover,7 microsatellite sites were polymorphic.The average number of alleles was 12.714,and the average number of effective alleles was 5.839.The expected heterozygosity and observed heterozygosity were 0.625 and 0.535,respectively.The PIC was0.608;2 loci showed low polymorphism（PIC<0.25）,and the other 5,high polymorphism（PIC>0.5）.Furthermore,the association analysis suggested that the body length and body mass of 257/259 individuals with the GHR1-3 locus were higher than those of the other genotypes.No significant differences in body mass were observed between 245/261 and 259/259（P>0.05）,but the body mass values were significantly higher than those of the other 5 genotypes（P<0.05）.GHR2 contains 4 microsatellite loci.（TG）₅ located in intron 1 and（TATC）₅（AT）₁₅（AC）₁₁（AT）₁₄（TG）₆ and（TA）₁₅ in intron 7 are highly polymorphic loci（PIC>0.5）.（GAAG）₅ in intron 6 is a moderately polymorphic locus（PIC=0.463）.The number of genotypes detected for the2 microsatellite loci in intron 7 was 50 and 61,respectively.These 2microsatellite loci showed good individual recognition potential.The association analysis showed that the 4 polymorphic microsatellite loci were correlated to growth traits.Nevertheless,association analysis of the body length and body mass and microsatellite Cal-GHR2-1 showed that the body length of type 341/349 samples was significantly lower than that of type345/345（P<0.05）.With respect to microsatellite locus Cal-GHR2-2,the body length and body mass of 201/205 individuals were significantly higher than those of 205/205 individuals（P<0.05）.With respect to microsatellite locus Cal-GHR2-3,the body length of 232/236 individuals was significantly lower than that of 236/238 individuals（P<0.05）.With respect to microsatellite locus Cal-GHR2-4,the body mass of 391/391 individuals was significantly higher than that of 391/393 individuals（P<0.05）.