Abstract Digital PCR is a new absolute quantitative method that does not rely on the external standard curves, and it shows broad application prospects in quantified detection of genetically modified organisms (GMOs), evaluation the exogenous gene copy number of GMOs and validation the certified values of GM reference materials. In this study, three digital PCR platforms including chamber digital PCR (cdPCR), droplet digital PCR (ddPCR) and 3D digital PCR (3D-dPCR) were used to quantify the copy number of exogenous fragments and endogenous gene phosphoenolpyruvate carboxylase (PEP) of GM canola (Brassica campestris) multi-targets plasmid reference molecule, and the certified value were determined by the mean copy number ratios of exo-gene/endo-gene. The results indicated that the certified values from three dPCR technology were close to the true value of 1 (copies/copies) and the relative standard deviations were (RSD) below 25%, The three dPCR all had stable reaction systems and good repeatability and showed high accuracy in value determination of plasmid DNA reference materials. Meanwhile, the plasmid value determination methods based on cdPCR, ddPCR and 3D-dPCR platforms provide a new model for validation of other reference materials' certified values.

Abstract Fertility performance of pig (Sus scrofa) is a main objective trait in pig genetic improvement, which is difficult to achieve great genetic progress with traditional breeding method due to its relative lower heritability. To investigate the performance of the restriction association site DNA sequencing (RAD-seq) technology on genomic prediction of the breeding value for fertility traits, 725 sows with records of total number born (TNB), number born alive (NBA) and litter weight (LW) from Shenzhen Agroi Pastoral Enterprises Co., Ltd. were collected. 618 sows in all of 725 sows with RAD-seq were sequenced and 79 725 genome-wide distributed SNPs after quality control were produced. To validate the efficiency of the RAD-seq on genomic selection, the 618 sows into two parts with 4∶1 ratio were divided, which served as the reference and validation population, respectively. Then, the predictive accuracy and biasness of three methods including best linear unbiased prediction (BLUP), genomic BLUP (GBLUP) and single-step genomic BLUP (SS-GBLUP) were studied, respectively, for validation population for which phenotypic records were masked. The results showed that GBLUP increased the prediction accuracy of breeding value from 0.109 (TNB), 0.067 (NBA) and 0.009 (LW) with BLUP to 0.220 (TNB), 0.184 (NBA) and 0.205 (LW), respectively, and SS-GBLUP performed similar prediction accuracy as GBLUP. Furthermore, GBLUP and SS-GBLUP improved the prediction biasness in comparison with BLUP. The results suggested RAD-seq based genomic selection method was effective in breeding value prediction for fertility traits. The exploration of the application of RAD-seq technology on pig genomic selection has theoretical and practical significance.

Abstract Psathyrostachys huashanica is an endemic species in China and possesses multi-resistance to biotic and abiotic stresses. In order to develop Ns genome-specific molecular markers of P. huashanica, random amplified polymorphic DNA (RAPD) analysis was performed on genome DNA of P. huashanica and wheat (Triticum aestivum) using 204 arbitrary primers. Primer OP-D15 and OP-F19 could specifically amplify fragments in P. huashanica, but not in China Spring (CS). The two Ns genome-specific sequences, named with Psh-D15 and Psh-F19, were isolated and characterized. Psh-D15 was 1 106 bp containing one copy of CAAAA motif, one pair of direct repeat as well as one pair of invert repeat. Psh-F19 was 1 344 bp long and two copies of CAAAA motif, direct repeats as well as invert repeats were also present in this sequence. The results of BLAST analysis indicated that Psh-D15 had 66% sequence identity to a long terminal repeat (LTR) Gypsy retrotransposon which was part of sequence of T. aestivum (AM932686.1), and Psh-F19 shared 78% homology with LTR Copia retrotransposon which was part of sequence of T. aestivum (HG670306.1). Thus, it could be inferred that both Psh-D15 and Psh-F19 were novel Ns genome-specific repeated sequences of P. huashanica belonging to Gypsy-like and Copia-like retrotransposons, respectively. Based on the sequence information, two pairs of sequence characterized amplified region (SCAR) primers were designed from the low-homologous regions of Psh-D15 and Psh-F19, respectively, and they were designated as Psh-D15 F/R and Psh-F19 F/R. These two pairs of SCAR primers could successfully amplify target bands 270 and 558 bp, respectively, only in P. huashanica, but not in the other genetic stocks, such as Aegilops, Dasypyrum, Leymus、Elytrigia, Agropyron, Hordeum, Secale, Triticum urartu, Triticum durum and T. aestivum. This result indicated that the two novel SCAR markers could reliably and easily distinguish the chromatin of P. huashanica, thus, they could be used as Ns genome-specific markers for monitoring genetic materials of P. huashanica, and designated as Psh-D15270 and Psh-F19558. The development of species-specific markers, Psh-D15270 and Psh-F19558, provides a new tool for characterizing wheat- P. huashanica alien genetic stocks.

Abstract Pandora neoaphidis, one of important Entomophthoromycota fungi, is a representative obligated aphid pathogenic fungi that can infect a variety of aphids and are widely distributed. Under suitable conditions, P. neoaphidis can cause a strong epidemic in the aphids in short term and plays a leading role in biological control of aphids. Here, the RNA-seq data of hyphae and primary conidia were carried out using second-generation sequencing platform Illumina Hiseq 2500, and the differentially expressed genes were assessed systematically. Totally, 90 639 contigs and 49 138 unigenes were detected among the RNA-seq data. Results based on Gene Ontology (GO) functional enrichment analysis showed that 49 138 unigenes were annotated to 47 function categories, covering 3 ontologies including molecular function, cellular component and biological processes. Besides, according to reads per kilobase per million of mapped reads(RPKM), a total of 3 555 genes expressed differentially, involving in 2 294 up-regulated genes and 1 261 down-regulated genes. Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis involved 22 metabolic pathways during the conidia stage, of which carotenoid biosynthesis and glycosaminoglycan degradation were significantly up-regulated against the hyphae stage. These basis data would provide a solid foundation for further excavation and analysis of functional genes related to the growth and development of P. neoaphidis.

Abstract The external characters of melon (Cucumis melo) fruit are the important agronomic characters which are closely related to the consumption habits. Discovery of the genes/loci affecting melon fruit character variation provides a basis for genetic improvement of exterior fruit quality. For this purpose, a total of 104 SSR markers distributed evenly across the melon chromosomes were used in the present study for identification the genotypes of a melon core collection, which was established using a stratified sampling method in our previous study. Five fruit characters of melon, fruit length (FL), fruit diameter (FD), fruit shape index (FS), flesh thickness (FT) and fruit fresh weight (FW) were determined during three years (from 2013 to 2015), and the genotype data of the core set was performed with genome-wide association study (GWAS). The results showed that a total of 456 alleles were detected in the melon core set, with polymorphism information index (PIC) of the markers ranged from 0.12~0.89 (mean=0.51). The core set had a high level of genetic diversity, all the Shannon's index of the five characters in the core set exceeded 2.0. Based on the Bayesian model, the melon core set could be separated into two subgroups, which were coincident with the subspecies classification of melon. This finding indicated that the melon core collection was suitable for association mapping. Totally, 42 loci (P＜0.01) associated with the five characters were detected by GWAS within three successive years, of which four loci appeared repeatedly under three environmental conditions and 21 loci appeared repeatedly under two environmental conditions. Most of the stably expressed loci were in agreement with the previous results from linkage analysis. There were 10, 5, 9, 8 and 10 loci associated with FL, FD, FS, FT and FW, respectively, with the maximum explanation of phenotypic variation (R2) loci of HNM36, HNM16, CMCCA145, HNM31 and HNM31, respectively. Ten loci were found to associate with two or more characters, indicating the existence of trait correlation or pleiotropism. The results will lay a basis for fine mapping of melon fruit character QTL, candidate gene discovery and molecular marker development.

Abstract The miR-17-92 cluster plays crucial roles in cell proliferation, differentiation, apoptosis, animal development and tumorigenesis in mammals. However, the roles and mechanisms of miR-17-92 cluster in chicken growth and development remain to be elucidated. To explore the roles and the mechanisms of miR-17-92 cluster in chicken growth and development, in this study, chicken miR-17-92 cluster was PCR amplified from chicken genomic DNA and ligated into pacAd5 miR-GFP Shuttle Vector to yield recombinant vector containing the chicken miR-17-92 cluster. The resultant vector pacAd5-miR-17-92 cluster and backbone plasmid pacAd5 9.2-100 were respectively linearized with PacⅠ, and then cotransfected into AD293 cells to generate the recombinant adenovirus expressing miR-17-92 cluster, named as rAd5-miR-17-92 cluster, by homologous recombination. The virus titer was determined by using TCID50 assay after the virus amplification. The recombinant adenovirus rAd5-miR-17-92 cluster was identified by PCR and sequencing. The recombinant adenovirus rAd5-miR-17-92 cluster and empty vector virus were inoculated into the immortalized chicken preadipocytes (ICPA), respectively, and the expression of miR-17-92 cluster member was detected by Real-time stem-loop RT-PCR. The results showed that the titers of both of recombinant adenovirus rAd5-miR-17-92 cluster and empty vector adenovirus rAd-control reached 1×109 pfu/ml. PCR identification showed a specific product of 879 bp was amplified from the recombinant adenovirus containing chicken miR-17-92 cluster, and sequencing result showed that the acquired sequence was identical to the expected sequence. Real-time stem-loop RT-PCR analysis showed the relative expression levels of miR-19a and miR-20a, two members of miR-17-92 cluster, were significantly increased to (2.2±0.2)-fold in ICPA cells infected by recombinant adenovirus when compared with those in cells infected with empty vector virus (P＜0.01). Taken together, these results suggested the recombinant adenovirus rAd5-miR-17-92 cluster was successfully prepared. Four human embryonic kidney epithelial cells (HEK) (293A, 293T, AD293 and HEK293), ICPA cells, chicken fibroblast (DF1), human epithelial cells (HaCaT) and sheep embryonic fibroblasts (SEF) were individually infected by rAd5-miR-17-92 cluster, and infection efficiency was detected by fluorescence microscopy. The results showed that the infection efficiency of rAd5-miR-17-92 cluster was different in different cell types, it was high in 293A, 293T, AD293 and HEK293, moderate in HaCaT and SEF, but Low in ICPA and DF1 cells. Taken together, these data suggested that recombinant adenovirus expressing miR-17-92 cluster was successfully prepared, which can contribute to further study on the roles and mechanisms of miR-17-92 cluster in chicken growth and development.

Abstract Rice (Oryza sativa) is originated from the tropics, but human is continuously exploring its suitable planting areas northward, thus, the selection of cold-tolerant rice cultivars is required. However, the studies on the mechanism of differentiation on cold tolerance among rice cultivars has not been documented. In the present study, three materials of rice germ from different climate regions in Liaoning, Jiangsu and Guangdong provinces in China were selected. Their cold-tolerance was comparatively determined by indexes of cold injury and chlorophyll florescent. Methylation sites of coding and promoter regions of ICE1 (inducer of CBF expression 1) in CBF(C-repeat binding factor) cold response transcription pathway were determined to elucidate molecular mechanism of cold differentiation in different regions using DNA methylation sequencing sulfite (bisulfite sequencing PCR, BSP). The results showed that the differences in methylation levels of ICE1 gene were only found at promoter regions(P＜0.05). Danjing-17 rice cultivar from Liaoning (LN004) had 2 cytosine methylations at ICE1 gene promoter region and the highest index of cold tolerance; Nanjing-5055 from Jiangsu (JS013) had 7 cytosine methylations, and the middle index. Yuexinzhan-2 from Guangdong (GD008) had 11 cytosine methylation and the the lowest index. qRT-PCR tests indicated that ICE1 showed significant positive correlation with downstream regulated expression of CBF1 (C-repeat DRE binding factor 1) and CBF3 (C-repeat DRE binding factor 3) on cold tolerance level (P＜0.05), but ICE1 showed significant negative relationship with the ICE1 methylation levels(P＜0.05). These indicated that cold tolerance among 3 rice cultivars may be adapted to local climate, and were regulated by CBF cold response transcription pathway, which was determined by the methylation of ICE1 gene. This study may provides theoretical basis for further explanation of the molecular mechanism of cold tolerance in rice.

Abstract Post-weaning diarrhea (PWD) is a very common infectious disease in pig (Sus scrofa) production, which has caused huge losses to the pig industry. MicroRNAs(miRNAs) play an important role in physiological and pathological processes by acting on the corresponding target genes. MiRNAs mainly perform complete or incomplete pairing with 3' UTR of target mRNA to degrade target mRNA or inhibit mRNA translation. Afterwards, the transcriptional expression of the target gene is regulated to affect the biological activity of the individual. Our previous studies have screened out miR-192 which may be important miRNAs for regulating Escherichia coli F18 infection in weaned piglets by high-throughput sequencing and verification test, and these studies have preliminarily screened out two key target genes discs large homolog 5 (DLG5) and activated leukocyte cell adhesion molecule (ALCAM) which regulate E. coli F18 infection. In this study, the dual-luciferase reporter system and Western blotting analysis were performed to explore whether DLG5 and ALCAM genes were under the specific regulation of miR-192. Recombinant plasmids which included miRNA target sites were constructed and were co-transfected with PRL-TK and miRNA-192 mimics, inhibitor or NC into 293 cells (miRNA-192 mimics and negative control). Cells were collected for detection of luciferase activity and protein levels after 24 h transfection. Then mRNA expression of two target genes in IPEC-J2 cells infected by E. coli was detected. The 3'UTR regions of 2 target genes were constructed into the dual-luciferase reporter vector. Then, this study successfully obtained luciferase reporter gene recombinant plasmids. The results of luciferase activity showed that miR-192 mimics could bind to binding sites of target genes and significantly inhibit luciferase activity of ALCAM and DLG5 3'UTR dual-luciferase reporter recombinant vector (P＜0.05), and miR-192 inhibitor could significantly enhance luciferase activity of ALCAM and DLG5 3'UTR dual-luciferase reporter recombinant vector (P＜0.05). Meanwhile, Western blotting analysis further indicated the target protein levels of miRNA-192 mimics group were significantly lower than the negative control group, which verified the preceding luciferase activity results. After being infected with three kinds of E. coli, the expression of 2 target genes significantly or extremely significantly increased. The results indicated that porcine mir-192 could inhibit the expression of DLG5 and ALCAM gene via targeting those 2 genes. MiR-192 and its targets played important regulatory function in replying the invasion of E. coli, maintaining stability of intestine and regulating immunization in weaned piglets. This study provides a theoretical and experimental evidence for participating in resistance to E. coli infection via porcine mir-192 targeting DLG5 and ALCAM gene, and lays the foundation for the further studies on the molecular mechanism of weaned piglets E. coli F18 diarrhea disease, thereby it can provide a theoretical basis for searching for the effective genetic markers of resistance to colibacillosis in pigs.

Abstract Heat shock transcription factors (Hsfs) are the central regulators of the heat shock responsive genes that encode Hsps, and can specifically bind to heat shock elements (HSE) in the upstream promoter region of heat shock protein genes to realize the regulation to these genes. There are at least 30 Hsf members in maize(Zea mays) Hsf family which includes 18 members of class A, among which there are 4 Hsfs of subclass A2. In our previous work, the structure, characteristics and regulating roles in thermotolerance and drought-stress tolerance of ZmHsf06 have been investigated. In present study, ZmHsf04 was cloned from maize young leaves treated by heat shock at 42 ℃ for 1 h using homologous cloning methods. The coding sequence (CDS) of ZmHsf04 was 1 074 bp encoding a protein of 357 amino acids. Protein sequence of ZmHsf04 contained DNA-binding domain (DBD) and other functional domains such as a nuclear localization signal(NLS)of KRKELEDTISKKRRR, NES(LAQQLGYL) and AHA(LKMFESGVLN). Homologous analysis showed that ZmHsf04 protein sequence shared 90%, 86% and 78% identities with the hypothetical protein SORBIDRAFT_01g021490(XP_002467215.1) of Sorghum bicolor, SiHsfA2c-like(XP_012698415.1) of Setaria italic and OsHsfA2c-like(XP_006661780.2) of Oryza brachyantha, respectively. qRT-PCR results showed that ZmHsf04 was expressed in multiple tissues and organs of maize. Compared with young leaf, young root, stem, functional leave, immature embryo and ear, transcriptional expression level of ZmHsf04 was the highest in pollens with the value of 16 times of the control. ZmHsf04 expression was up-regulated significantly by both 42 ℃ heat shock and abscisic acid (ABA), the highest value reached 340 times of the control, and the peak values appeared at 30 min and 24 h, respectively. Meanwhile, ZmHsf04 expression was up-regulated by both salicylic acid(SA) and H2O2, and the highest value was 12 times of the control, and was significantly lower than that treated by HS or ABA. If pretreated with SA or H2O2 and then heat shock (HS), the ZmHsf04 expression was similar to that treated with HS. Through transient reporter assay with onion (Allium cepa) epidermal cells, it was found that ZmHsf04 was located in nuclei. ZmHsf04 protein could be induced by Gal in yeast (Saccharomyces cerevisiae), and yeast overexpressing pYES2-ZmHsf04 showed stronger growth potential than the controls expressing pYES2 after HS, though yeast growth potential was decreased by HS. The results revealed that ZmHsf04 perhaps played a key role in regulating pollen development and the response to heat stress. These results will provide theoretical basis for analysis biological functions and regulating mechanism of ZmHsf04 further.

Abstract Photosystem Ⅱ subunit R (PsbR) is a small subunit protein in photosystemⅡ and plays important roles in photosynthetic physiology of plants. To investigate the function of PsbR in winter turnip rape (Brassica campestris), a strong cold-tolerance winter turnip rape cultivar "Longyou 7" was used as experimental materials in this study. The PsbR was separated and cloned from the leaf by differential display reverse transcription PCR(DDRT-PCR) and rapid-amplification of cDNA ends (RACE). The full length cDNA of PsbR was 570 bp and the open reading frame (ORF) encoding a protein of 138 amino acids was 417 bp. The accession number was KC894423 in GenBank. Its molecular mass was 14.39 kD and the isoelectric point (pI) was 9.42. The prediction of the second structures indicated that PsbR was a steady protein and had a transmembrane region and contained a conserved amino acid sequence. PsbR belonged to the PsbR superfamily, which presented high similarity with PsbR proteins from B. oleracea var. oleracea, E. salsugineum and B. juncea. The relative expression of PsbR gene under low temperature stress and the net photosynthetic rate (Pn)and efficiency of primary conversion of light energy of photosystemⅡ(Fv/Fm) in the natural temperature falling were determined with qRT-PCR technology, CIRAS-2 photosynthetic apparatus and FMS-2 pulse modulated fluorometer, respectively. The main results showed that as temperature fell, the expression of PsbR, Pn and Fv/Fm decreased. Therefore, the expression of the PsbR gene may be inhibited under low temperature stress and then decreased net photosynthetic rate and photochemical efficiency of PSⅡ. The results provide a theoretical basis for resolving the biological functions of PsbR and the cold-tolerance molecular mechanism of winter turnip rape.

Abstract Pig (Sus scrofa) is an important economic animal. Studies have found that the serum thyroid hormone levels of lean pigs are higher than those of obese pigs. MicroRNAs (miRNAs) play important roles in various biological processes. Despite a significant improvement in the identification of miRNAs in porcine tissues, the coverage of the porcine thyroid miRNAome is still scarce. In this study, castrated Jinhua (obese pigs) and Yorkshire pigs (lean pigs) at age of 120-days-old were used to delineate the expression profiles of miRNAs with high throughput sequencing. The number of raw reads of each transcriptome ranged from 11 to 12 million. After low quality sequences, polyA, sequences shorter than 18 or longer than 30 nucleotides, and repeated sequences were removed, the number of clean reads from each thyroid miRNA transcriptome decreased to 10~11 million. The small RNA length distribution showed that the most abundant species were 21~24 nucleotides, a typical size range for Dicer-derived products. Totally 293 known miRNAs were successfully identified in porcine thyroid gland. Besides, 60 novel microRNAs were predicted by using miREvo and mirdeap2 software. After expression levels of miRNAs were normalized to transcripts per million (TPM), a total of 15 miRNAs were found to be expressed at high levels (＞10 000), including ssc-miR-148a-3p, ssc-miR-30a-5p, ssc-miR-99a, ssc-miR-27b-3p, ssc-let-7f, ssc-miR-26a, ssc-let-7i, ssc-let-7g, ssc-miR-21, ssc-miR-7, ssc-miR-126-3p, ssc-miR-30d, ssc-miR-10a-5p, ssc-miR-143-3p and ssc-let-7a. This set of miRNAs may play important housekeeping roles in thyroid gland. DESeq was used to analyze the differentially expressed miRNAs in 2 pig breeds. In our study, 18 miRNAs were found with differential expression levels between Yorkshire and Jinhua pigs (P＜0.05), 6 miRNAs had a higher expression level in Yorkshire (ssc-miR-532-3p, ssc-miR-708-5p, ssc-miR-129a-5p, ssc-miR-708-3p, novel_7 and ssc-miR-362), whereas 12 miRNAs in Jinhua (ssc-miR-335, ssc-miR-7, novel_51, ssc-miR-153, ssc-miR-10a-3p, novel_36, ssc-miR-221-3p, ssc-miR-369, ssc-miR-452, novel_12, novel_11 and ssc-miR-221-5p). The expression level of 6 differentially expressed miRNAs was selected and verified by qRT-PCR. Consistent with the miRNA sequencing data, the qRT-PCR results of 6 randomly selected miRNAs showed a similar pattern of expression in 2 pig breeds, which further confirmed that our sequencing data was reliable. MiRanda was used to predict target genes of 18 differentially expressed miRNAs, and a total of 14 913 were identified as potential targets. Among them, thyroid peroxidase (TPO), solute carrier family 5 member 5 (SLC5A5), dual oxidase 2 (DUOX2) and dual oxidase maturation factor 2 (DUOXA2) were in involved the thyroid hormone synthesis. Furthermore, the predicted target genes were annotated in KEGG pathways to identify potential pathways that may be regulated by the differentially expressed miRNAs. The results indicated that target genes were involved in pathways like thyroid hormone synthesis pathway, endocytosis, calcium signaling pathway, peroxisome and lysosome which have been reported to be closely related with thyroid hormone regulation. This is the first study about miRNA profiles of porcine thyroid gland. The results provide information about miRNAs that are differentially expressed in thyroid gland of 2 different pig breeds with different levels of fat deposition, providing a better understanding of the possible roles of miRNAs in thyroid hormone regulation and the molecular mechanism of genetic difference between Yorkshire and Jinhua pigs.

Abstract Reproductive function is most easily damaged by heat stress in all physiological functions. Heat stress causes the oxidative stress in tissues and damages spermatogenesis, which can affect the reproduction efficiency of animals. The main purposes of this study were to investigate the effect of baicalin on testicular injury in mice (Mus musculus) and to further investigate the antioxidant mechanism of baicalin. The mice were divided into four groups as following, normal temperature control group (Group C), baicalin group (Group C+B), heat stress group (Group H) and heat stress plus baicalin group (Group H+B). Mice in Group C and Group H were given saline via intraperitoneal injection, and mice in Group C+B and Group H+B were injected intraperitoneally with baicalin (50 mg/kg) for 7 days. Then, group H and Group H+B were treated with heat stress (41 ℃) for 2 hours on day 8. The mice in treatment groups and control groups were killed immediately after heat stress, and the testes and epididymis were removed and weighed. Testicular and epididymal organ coefficients were calculated, pathological injuries were detected in testis tissue sections, and the levels of malondialdehyde (MDA) in the testis tissue were examined. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) were detected by using corresponding kits in testicular tissue. The cell apoptosis of testicular tissue was detected by terminal deoxynucleotidyl transferase assay (TdT-mediated dUTP, nick-end labeling, TUNEL). The expression of Fas/FasL protein in testis tissue was detected by Western blot. The results showed that the intercelluar space enlargement, seminiferous tube wall thinning, cell degeneration, vacuoles in cytoplasm, spermatognic cell apoptosis and shedding appeared in mice testicular tissues under heat stress. In addition, testicular and epididymal organ coefficients could be significantly reduced by heat stress (P＜0.05), while baicalin could alleviate this change induced by heat stress(P＜0.05). Moreover, compared with the heat stress group at 41 ℃, the contents of MDA in the heat stress with baicalin group significantly decreased, and the activities of SOD, CAT and GSH-Px increased significantly(P＜0.05). There was no significant difference between baicalin group and heat stress with baicalin group. Furthermore, the apoptotic rate of testis in heat treated group increased significantly compared with the control group (P＜0.01), while the apoptosis rate of testicular cells in heat stress plus baicalin group was significantly lower (P＜0.05) when compared with heat stress group. Besides, comparing with the control group, heat stress could significantly increase the expression of Fas (P＜0.05) and FasL (P＜0.01) protein in testes, but baicalin could significantly reduce the expression of Fas/FasL protein in the testis of mice induced by heat stress (P＜0.05). Overall, baicalin could reduce the apoptosis caused by oxidative damage in the testis of mice exposed to heat stress, and the mechanism of baicalin might be related to the increase of antioxidant enzyme activity and the inhibition of Fas/FasL protein expression to inhibit apoptosis. This study provides a theoretical basis for finding ways to alleviate the heat stress in animals.

Abstract Diacylglycerol acyltransferase 1(DGAT1), considered as an important candidate gene for milk fat traits, plays key roles in triglycerides synthesis. The Bos grunniens milk has rich nutrition and higher milk fat. However, there are few studies on the genetic mechanism of dairy quality traits, because it is restricted by distribution, local environment, economy and production condition. In the present study, the tissue distribution of Bos grunniens DGAT1 mRNA and the genotype polymorphisms in 3'-untranslated region (3' UTR) and exon 8 of yak DGAT1 gene were detected by quantitative real-time PCR (qRT-PCR) and single strand conformation polymorphism (SSCP), and the associations of different genotypes and alleles with milk quality traits of Gannan yak were analyzed. The results indicated that the basal expression level of DGAT1 were relatively high in mammary gland, longissimus dorsi, heart and subcutaneous fat, relatively moderate in kidney, small intestine, large intestine, but low in spleen. At the c.*197 T>C locus, animals with genotype FF had higher milk fat rate than those with EE and EF genotypes (P＜0.05). And individuals with allele E had lower milk fat rate and total solids percentage than those with missing allele E (P＜0.01 or P＜0.05). The double-bases mutation c.691-c.692 AA>GC(K232A) was found in Gannan yak and Tianzhu white yak, the allele K was predominant, but those mutations were not detected in Qinghai yak and Wild-blood yak. The presence of allele K was beneficial to increasing the milk fat rate. The results suggested that 3'-untranslated region of yak DGAT1 gene may have a certainly genetic effect on milk quality traits. These results enrich the molecular genetic data for dairy quality traits of yak.

Abstract Molecular genetics and breeding of maize have been promoted and developed more efficiently, during the further study of maize genomics. The rapid development of the second generation sequencing technology possesses the characteristics of high throughput and low cost. Nowadays, more and more researchers began to conduct the population size of re-sequencing work, which promoted the analysis of maize genotype and the mining information of genetic variation. In this study, we constructed a recombination inbred line (RIL) mapping population crossed from two elite inbred lines of maize, X178 and NX531, consisting of 150 individuals. The library building and high-throughput sequencing of RIL population were conducted by genotype by sequencing method (GBS). Besides, the depth re-sequencing was applied for the two parents of the recombinant inbred lines, and the high throughput SNP markers of the populations were obtained. Using a sliding window method, the genotyping of the RIL population was carried out, and then the genetic linkage map was constructed based on bin markers. In order to verify the reliability of this map, the R/qtl packages and composite interval mapping (CIM) model was used for the mapping of QTLs responsible for nutritional quality traits of maize kernel. 5.0 × sequencing of the parents X178 and NX531 was performed, 881 259 high quality SNPs was obtained and there were polymorphisms between the parents. Based on the SNP between parents, a total of 1 842 109 SNP not only showed genotype homozygosity but also had the same genotype of one parental in the 150 lines of the RIL population. The minimum number of the SNP in one inbred line was 1 673, while the maximum number of the SNPs in one inbred lines was 20 403 and the mean SNP number in every inbred lines was 12, 281. After the screening, 124 RIL strain were chosen for further genotyping and the bin map containing 7 278 recombination bin were obtained. The maximum physical distance between two adjacent bins was 3.28 Mb, and the minimum physical distance was 80 kb, with the average of 277 kb. Among the bin makers, the physical length of 110 bin was more than 1 Mb, three more than 2 Mb. According to the genotyping of 7 278 restructuring bin markers and the information of each strain, a high density genetic linkage map was constructed by using R/qtl software. The total length of the genetic map was 2 569 cM, and the average genetic distance between adjacent bin markers was 0.35 cM. Besides, genetic distance of 35 restructuring bin markers was larger than 10 cM. Totally, 20 QTLs were identified for oil, lysine, protein and starch in the 2 years, locating on 8 chromosomes with exception of the chromosome 2 and 9. Each of these QTLs could explain 0.17% to 20.53% phenotypic variation, and 9 QTLs were more than 10% among them. Most of the QTLs were detected in single environment, indicating the significant interactions between QTL and environments. Therefore, this work could lay the foundation for the QTLs mapping and genes cloning of main agronomic.

Abstract The grass carp (Ctenopharyngodon idellus) growth hormone gene transgenic common carp (Cyprinus carpio) is a new variety of fish which aims to enhance growth. However, transgenisis may alter the response of the transgenic carp to different environmental conditions. To examine the effect of feeding rations on energy allocation and overwinter of fast growth transgenic common carp, a growth and overwinter trial on transgenic and nontransgenic carp was conducted under 2 feeding rations (satiation and half-satiation). At the growth trial in summer and autumn, the growth rate of body weight of satiation transgenic (ST) fish was 1.33 times faster than that of satiation nontransgenic (SN) fish, and half-satiation transgenic (HT) fish was 1.51 times faster than half-satiation nontransgenic (HN) fish. However, no significant difference was found between HT and SN in the growth rate of body weight. The moisture content of HN, SN, HT and ST in turn significantly reduced. There was no significant difference in protein content within genotype, but the protein content of ST was significantly higher than that of SN. The lipid content and energy content of HN, SN, HT and ST were increased significantly by degrees. The transgenic carp did not reduce satiation ration in late autumn while the nontransgenic carp did. There were no significant difference in overwinter survival rate among treatments, and feeding history only affected the energy content after overwinter. Our study indicated that the energy allocation in response to feeding ration and overwinter was consistent between transgenic and nontransgenic carp. The transgenic carp would assign more energy to growth, but less energy to storage under food restriction. There was no significant difference in overwinter ability among transgenic and nontransgenic carp. The present study may provide reference for ecological safety assessment of transgenic carp.

Abstract Multiplex PCR technique of microsatellites has been wildly used in the field of parent assignment, population genetic analysis and family management. Rhinogobio ventralis is endemic and rare fish in the upper Yangtze River, China. From the species vale and threatened degreed evaluation, it has reached three-level desperate protection sate and has been classified as low risk fish. In order to develop a rapid, economically efficient and robust approach for genetic studies of R. ventralis, a technique for the parentage assignment in R. ventralis was established based on 5 multiplex PCR panels using previously reported microsatellites loci with high polymorphism. The annealing temperatures, the primer ratio and the concentrations of reaction system were optimized in this technique. With the multiplex PCR tool, the genotyping and the genetic diversity analysis on 136 individuals of R. ventralis were performed using ABI 3730 genetic analyzer and Cervus v.3.0 software. The results showed that the average number of alleles (Na) was 15.600; the average expected heterozygosity (He) was 0.825; the average observed heterozygosity (Ho) was 0.813; the average polymorphism information content (PIC) was 0.801. The parentage assignment was performed on 40 candidate parents and 96 offspring. The combined exclusion probability of the first parent (CE-1P), the second parent (CE-2P), and a parent pair (CE-PP) were shown to be 0.999 986 07, 0.999 999 97 and 1.000 000 00, respectively. When using multiplex PCR group A, B and C, the CE-1P was 0.998 348 13 and CE-2P was 0.999 960 49. All of offspring could correctly find their real parents. The accuracy rate of parentage assignment was 100% using 5 multiplex PCR panels. These results indicated that 15 polymorphic loci and microsatellite multiplex PCR technique were suitable for paternity test of R. ventralis. The development of microsatellite multiplex PCR system provides a high efficiency technical method for parentage assignment, genetic diversity analysis, and marker-assisted family management in R. ventralis.

Abstract Growth and reproduction are closely related in fish. Herely, the hormone cross-talk between the two systems in the gonad has been analyzed with Common carp (Cyprinus carpio) and growth hormone (gh) transgenic common carp by using real qRT-PCR, immunofluorescence, in situ hybridization, and in vitro incubation. The expression of gonadotropin hormone alpha (gthα), follicle-stimulating hormone beta (fshβ), luteinizing-stimulating hormone beta (lhβ), gonadotropin-releasing hormone 3 (gnrh3), growth hormone receptor (ghr) and 4 GnRH receptor mRNA were detected in testis and ovary of common carp. Immunofluorescence showed that GTHα and LHβ were localized mainly in the somatic cells and primary sperm cells of the gonads. In vitro incubation of ovary fractions with GnRH confirmed the existence of gonadal GnRH-GtH paracrine system, which was independent of the hypothalamus-pituitary-gonad axis. The expression levels of gnrh3, gthα, fshβ and lhβ mRNA were significantly different in the gonad of GH-transgenic carp when compared with non-transgenic carp. Further analysis in vitro showed that GH up-regulated the expression of gnrh3, gthα, fshβ and lhβ in the ovarian GnRH-GtH system at primary growth stages. But the signals were inhibited in the ovarian at the secondary growth stages. Our results confirmed the GnRH-GtH paracrine system in gonad and uncovered its important role in the interaction between growth and reproduction in common carp, which provides scientific data to reveal the regulation mechanism between growth and reproduction in fish.

Abstract In order to study the feasibility of injecting Chinese shrimp (Fenneropenaeus chinensis) total DNA into common carp (Cyprinus carpio) and its effects on muscle nutritional composition of experimental carp, total DNA of Chinese shrimp was fragmented and injected into oosperm of common carp to breed a batch of microinjection offspring. The amplified fragment length polymorphism (AFLP) detection showed that all F2 generation of microinjection contained exogenous gene fragments from the total DNA of Chinese shrimp genome. Furthermore, PCR experiments were conducted using E-AAG and M-CTC primers to detect the Chinese shrimp DNA fragments, which presence in F2 generation of microinjection carp, but absent in control group of carps. Results showed that Chinese shrimp gene fragments had been incorporated into the genome of microinjection carp offspring and can be stably transmitted to the progeny generation. Based on the confirmation of genetic stability of exogenous gene, nutrient content and metal content were tested in the muscle of F2 generation. The results showed that crude protein, fat content and ash content in muscle of microinjection carp showed significant difference in that of control carp. Crude protein content was 18.37% in microinjection carp and 16.49% in control carp, respectively. Fat content was 2.49% in microinjection carp, and it was lower than 3.51% in control carp. Ash content was 1.17% in microinjection carp, and it was higher than 1.05% in control carp. Total amino acid content was 79.92% and 74.16% in microinjection carp and control carp, respectively. Of which, glutamic acid was the highest amino acid in all the 17 tested amino acids, accounting for 16.60% and 16.22% of the total amino acid, with a percentage of 13.27% and 12.03% in microinjection carp and control carp, respectively. The top 5 amino acids in microinjection carp were glutamic acid, aspartic acid, lysine, leucine and alanine, which were consistent with that in control carp. These results suggested that there was no differences in amino acid composition between microinjection carp and common carp. The delicious amino acid was 31.16% in microinjection carp, and it was 28.04% in control carp, accounting for 38.99% and 37.81% of the total amino acid, separately. In addition, tryptophan, isoleucine, leucine, threonine, valine, methionine, phenylalanine and lysine were another 7 essential amino acids in 18 amino acids. The essential amino acid content was 31.5% and 29.54% in microinjection carp and control carp, accounting for 39.41% and 39.83% of total amino acids, respectively. The limited amino acid was valine in both microinjection carp and control carp. Above all, microinjection carp had certain advantages in nutrients when compared to common carp. Amino acid score (AAS), chemical score (CS) and essential amino acid index (EAAI) were computed by converting the amino acid content into amino acid weight per gram of nitrogen following by the standard evaluation of protein made by FAO/WHO and the amino acid pattern of egg protein. The content of heavy metals including Cu, Zn, Cr, Hg, Cd and Pb in both microinjection carp and common carp met the requirements of relevant food safety standards. This study provides a basis for the commercialization of microinjection carp.