Evolutionary analysis of allotetraploid hybrids of red crucian carp × common carp, based on ISSR, AFLP molecular markers and cloning of cyclins genes

The allotetraploid hybrids of red crucian carp x common carp are the first reported artificially cultured polyploid fish with bisexual fertility and stable inheritance in vertebrate. Using ISSR and AFLP markers and the cyclins genes, the genomes and cyclin gene sequence changes were analyzed between the allotetraploid hybrids and their parents. The results indicated that the allotetraploids inherited many genetic characteristics from their parents and the genetic characteristics were stable after 15 generations. However, the allotetraploids had a closer genetic relationship with their original female parents and represented a bias toward the maternal progenitor. DNA fingerprinting analysis showed that the allotetraploids had undergone sequences deletion from their original parents and that the deleted sequences were mostly from the male parent＇s genome. Some non-parental bands were found in the allotetraploid hybrids. Sequences analysis of the cyclin A1 and B1 genes showed nonsynonymous substitutions of single nucleotides in codons that were different from their original parents, leading to non-parental amino acid loci. We speculate that the non-additivity in the allotetraploids, compared with their progenitors, could be an adjustment to the genomic shock from heterozygosity and polyploidy, allowing maintenance of genetic stability.     

Induction of gynogenesis in red crucian carp using spermatozoa of blunt snout bream

Gynogenetic diploid was induced in red crucian carp (RCC) (Carassius auratus Red Variety) eggs using UV-irradiated spermatozoa from blunt snout bream (B) (Megalobrama amblycephala) or from mirror carp (C) (Cyprinus carpio. L). Spermatozoa were genetically inactivated by an appropriate UV dosage, and then the maternal DNA was duplicated with cold shock at 0-4℃. When using the spermatozoa of B, the fertilization rate, hatching rate and survival at first feeding were 52.6±3.0 %, 23.6±4.1 % and 15.7±3.4 %, respectively, and the survival at first feeding was significantly higher than that (11.3±2.2%) when using the spermatozoa of C (Cyprinus carpio. L). According to the morphological characteristics, the chromosome number and the degree of gonadal development, gynogenetic RCC could be distinguished from the control hybrids of RCC♀× B♂. The individuals with red body color, 100 chromosomes and normal gonadal development were successful gynogenetic RCC, while the individuals with 124 or 148 chromosomes and delayed gonadal development were hybrids of (RCC× B). The triploid hybrids (RCC× B) (2 years old) were sterile, but the tetraploid hybrids (RCC× B) were sexually mature age of two. In the present study, compared to the spermatozoa of C, the advantages of spermatozoa of B as the activation source were that could increase the survival at first feeding of gynogenetic individuals and simplify the confirmation of gynogenetic status, which suggested that the sperm of B was an effective activation source for inducing gynogenesis in crucian carp.     

Induction of gynogenesis in red crucian carp using spermatozoa of blunt snout bream

Gynogenetic diploid was induced in red crucian carp (RCC) ( Carassius auratus Red Variety) eggs using UV-irradiated spermatozoa from blunt snout bream (B) (Megalobrama amblycephaia) or from mirror carp (C) (Cyprinus carpio. L) . Spermatozoa were genetically inactivated by an appropriate UV dosage, and then the maternal DNA was duplicated with cold shock at 0-4℃. When using the spermatozoa of B, the fertilization rate, hatching rate and survival at first feeding were 52. 6±3. 0 %, 23.6±4.1 % and15.7±3.4 % , respectively, and the survival at first feeding was significantly higher than that (11.3±2.2%) when using the spermatozoa of C (Cyprinus carpio. L). According to the morphological characteristics, the chromosome number and the degree of gonadal development, gynogenetic RCC could be distinguished from the control hybrids of RCC♀×B♂. The individuals with red body color, 100 chromosomes and normal gonadal development were successful gynogenetic RCC, while the individuals with 124 or 148 chromosomes and delayed gonadal development were hybrids of (RCC×B). The triploid hybrids (RCC×B) (2 years old) were sterile, but the tetraploid hybrids (RCC×B) were sexually mature age of two. In the present study, compared to the spermatozoa of C, the advantages of spermatozoa of B as the activation source were that could increase the survival at first feeding of gynogenetic individuals and simplify the confirmation of gynogenetic status, which suggested that the sperm of B was an effective activation source for inducing gynogenesis in crucian carp.     

Maternal inheritance in polyploid fish inferred from mitochondrial ATPase genes analysis

The sequences of the ATPase8/6 genes for the triploid, tetraploid and pentaploid hybrids as well as for their male parent blunt snout bream were determined. In order to examine mitochondrial maternal inheritance, the sequences were subjected to a comparative sequence analysis with the homologous sequences of red crucian carp, their female parent, and zebrafish as the outgroup. Base composition and variation as well as the divergences based on nucleotide sequences and deduced amino acid sequences were calculated. Phylogenetic trees were also constructed with maximum parsimony (MP), minimum evolution (ME), neighbor joining (NJ) and the unweighted pair group method with arithmetic mean (UPGMA) algorithms in MEGA 3.1. The results showed that most nucleotide substitutions occurred at the third codon position of the two genes and thus represented synonymous mutations. The nucleotide sequence divergences of the ATPase8/6 genes ranged from 0.0% to 21.6% among ingroup samples (three types of polyploids and their parents), and 27.0–28.2% between their ingroup and the outgroup samples. All the polyploids were considerably closer in sequence relationship to the female parent red crucian carp (0.0–3.3%) compared to their male parent blunt snout bream (21.0–21.6%). The phylogenetic trees also showed a similar result. In conclusion, the mitochondrial ATPase8/6 genes of artificial polyploid fish stringently indicated maternal inheritance. Our results also suggested that the ATPase8/6 genes are valuable genetic markers to track genealogies and variations in the progenies of the hybrids.     

Analysis of intron sequence variability of the conservative HMG-box of Sox9 genes in allotetraploids and their original parents

The Sox genes of allotetraploids and their original maternal red crucian carp (Carassius caassius red var.) and original paternal common carp (Cyprinus carpio L.) were detected by PCR with the designed primers based on the conserved HMG-box sequence in different species. Sequencing of Sox genes indicated that two Sox9 genes (Atsox9a and Atsox9b) existed in allotetraploids, while only one Sox9 gene existed in red crucian carp (Rcsox9a) and common carp (Ccsox9b) . All of the four Sox9 genes contained an intron in the HMG-box. with the sizes of 413 bp, 703 bp, 401 bp and 714 bp, respectively. Moreover, the introns obeyed the rule of "GT-AG" . A high similarity was observed between introns of Atsox9a and Rcsox9a (94.4%), Atsox9b and Ccsox9b (97.8%). Interestingly, the deduced amino acid sequences of their corresponding exons all shared 100% identity. Thus, introns of the HMG-domain of Sox9s in allotetraploids and their original parents have not only the length polymorphism but also intron variability. Our results provide significant molecular evidence for the origin and evolution of allotetraploids.     

Analysis of intron sequence variability of the conservative HMG-box of Sox9 genes in allotetraploids and their original parents

The Sox genes of allotetraploids and their original maternal red crucian carp （Carassius caassius red var.） and original paternal common carp (Cyprinus carpio L.) were detected by PCR with the designed primers based on the conserved HMG-box sequence in different species. Sequencing of Sox genes indicated that two Sox9 genes (Atsox9a and Atsox9b) existed in allotetraploids, while only one Sox9 gene existed in red crucian carp (Rcsox9a) and common carp (Ccsox9b). All of the four Sox9 genes contained an intron in the HMG-box, with the sizes of 413 bp, 703 bp, 401 bp and 714 bp, respectively. Moreover, the introns obeyed the rule of “GT-AG”. A high similarity was observed between introns of Atsox9a and Rcsox9a (94.4%), Atsox9b and Ccsox9b (97.8%). Interestingly, the deduced amino acid sequences of their corresponding exons all shared 100% identity. Thus, introns of the HMG-domain of Sox9s in allotetraploids and their original parents have not only the length polymorphism but also intron variability. Our results provide significant molecular evidence for the origin and evolution of allotetraploids.     

Relationship between differential gene expression patterns in functional leaves of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) at milk filling stage and heterosis using cDNA-AFLP

To understand the molecular mechanism of maize heterosis ,differential gene expression patterns in the functional leaves of 35 maize hybrids relative to their parents involving 10 elite inbreds at milk filling stage were analyzed hy using cDNA-AFLP,the correlation analyses of various differential expression patterns with the performance and heterosis of main maize agronomic traits were evaluated.The main results were as follows:For uniparental specific expression ,significant positive correlations were detected with the performance of seed weight per ear and 100-seed weight at 0.01 and 0.05 probability levels respectively.For biparental specific expression ,significant negative correlations were detected with the performance of ear diameter and seed weight per ear at 0.01 probability level .For uniparental specific expression ,significant positive correlations were detected with the heterosis of ear diameter and seed weight per ear at 0.01 and 0.05 probability levls rspectively,For biparental specific expression .significant negtive correlation was detected with theheterosis of ear diameter at 0.05 probability level ,However,for F1-spectific expression,for fragments detected only in one parent and F1,and for fragments detected only in two parents or only in F1,no significant correlation was detected with the performance or heterosis of all agronomic traits surveyed.     

Pedigree transmission disequilibrium test for quantitative traits in farm animals

General pedigrees are very common in farm animals,and the recent availability of large panels of SNPs in domestic species has given new momentum to the search for the mutations underlying variation in quantitative traits.In this paper,we proposed a new transmission disequilibrium test approach,called the pedigree transmission disequilibrium test,which deals with general pedigrees and quantitative traits in farm animals.Compared with the existing pedigree disequilibrium test (PDT) and general linear model-based method QTDT,our approach performed better with higher power and lower type I error,especially in scenarios where the quantitative trait locus (QTL) effect was small.We also investigated the application of our approach in selective genotyping design.Our simulation studies indicated that it was plausible to implement a selective genotyping strategy in the proposed pedigree transmission disequilibrium test.We found that our approach performed equally well or better when only some proportion of the individuals in the two tails were genotyped compared with its performance when all the individuals in the pedigree were genotyped.     

Regeneration and identification of interspecific asymmetric somatic hybrids obtained by donor-recipient fusion in cotton

Asymmetric hybrids between Gossypium hirsutum （YZ-1） and G. davidsonii were obtained by donor-recipient fusion. YZ-1 was considered the recipient and was pretreated with iodoacetamide （IOA）, while G. davidsonii was considered the donor and was irradiated with ultraviolet （UV） before fusion. YZ-1 protoplasts stopped growth when treated with 0.5 mmol/L IOA for 20 min, and G. davidsonii protoplasts stopped growth when irradiated with 38.7 J/cm2 UV for 30 s. Asymmetric somatic hybrids were obtained by electrofusion between the separately treated protoplasts of the 2 species. The regenerated plants were identified by morphological, cytological, and molecular analysis. Most regenerated plants derived from fused protoplasts displayed new morphology; some were intermediate between the two parents and a few displayed recipient-like morphology. Chromosome numbers in these somatic hybrids mostly ranged from 40 to 73. The hybridity was confirmed by random amplified polymorphic DNA and simple sequence repeat analysis. Organelle DNA inheritance of the YZ-1 and G. davidsonii somatic hybrid was investigated by cleaved amplified polymorphism sequence and chloroplast simple sequence repeat analysis, which indicated that recombination and rearrangements might have occurred in some regions of mitochondrial and chloroplastic DNA. This is the first report of completely asymmetric hybrid production via donor–recipient fusion between G. hirsutum and G. davidsonii, which is a novel case in hybrid production following the symmetric fusion and asymmetric fusion based on UV irradiation in cotton.     

Effects of 2,3,7,8-TCDD and benzo[a]pyrene on modulating vitellogenin expression in primary culture of crucian carp （Carassius auratus） hepatocytes

Vitellogenin (Vtg) is the precursor of yolk protein. Its expression and secretion are estrogen-regulated and are crucial for oocyte maturation. An in ritro xenoestrogen screening model was established by measuring Vtg induction in cultured primary hepatocytes from crucian carp. Vtg production was detected by biotin-avidin sandwich ELISA method while Vtg and cytochrome P4501A1 (CYP1A1) mRNA induction were measured by semi- quantitative PCR-primer dropping technique. Vtg and Vtg mRNA were dose-dependently induced by diethylstilbestrol (DES, 0.2-200 ng/mL) in hepatocytes of crucian carp. Co-treatment of the DES-induced hepatocytes with either 2,3,7,8-TCDD (TCDD, 0.1-4 pg/mL) or benzo[a]pyrene (B[a]P, 5-1000 ng/mL) resulted in a reduction of Vtg production and an increment of CYP1A1 mRNA expression both in a dose dependent manner, indicating the anti-estrogenic effects of the compounds. However, at lower tested concentrations, TCDD (0.1, 0.2 pg/mL), B[a]P (5 ng/mL)seemed to have a potentiating effect on Vtg expression and secretion, although by their own these compounds had no observable estrogenic effect on Vtg induction. Tamoxifen (a selective estrogen receptor modulators, 1 nmol/L-1μmol/L),and β-naphtho-flavone (β-NF, an aryl hydrocarbon receptor inducing compounds, 2.5-1000 ng/mL) also were employed to study the possible interactions in DES-induced Vtg expression. In co-treatment of the DES-induced hepatocytes with β-NF or tamoxifen, the decrease in Vtg production did parallel induction of CYP1A1 for β-NF, but tamoxifen inhibited Vtg induction did not parallel induced CYP1A1 expression in all test concentrations. On the contrary, it was found that in co-treatment of the TCDD-induced hepatocytes with DES, TCDD induced CYP1A1 mRNA production was inhibited by DES also. These results implicated a possible cross talk between estrogen receptor- and aryl hydrocarbon receptor-mediated pathways in the hepatocytes.     

A new statistical method for mapping QTLs underlying endosperm traits

Genetic expression for an endosperm trait in seeds of cereal crops may be controlled simultaneously by the triploid endosperm genotypes and the diploid maternal genotypes. However, current statistical methods for mapping quantitative trait loci （QTLs） underlying endosperm traits have not been effective in dealing with the putative maternal genetic effects. Combining the quantitative genetic model for diploid maternal traits with triploid endosperm traits, here we propose a new statistical method for mapping QTLs controlling endosperm traits with maternal genetic effects. This method applies the data set of both DNA molecular marker genotypes of each plant in segregation population and the quantitative observations of single endosperms in each plant to map QTL. The maximum likelihood method implemented via the expectation-maximization algorithm was used to the estimate parameters of a putative QTL. Since this method involves the maternal effect that may contribute to endosperm traits, it might be more congruent with the genetics of endosperm traits and more helpful to increasing the precision of QTL mapping. The simulation results show the proposed method provides accurate estimates of the QTL effects and locations with high statistical power.     

Cytogenetics of intergeneric hybrids between Brassica species and Orychophragmus violaceus

In the sexual intergeneric hybrids between the cultivated Brassica species and Orychophragmus violaceus, both complete separation and partial separation of the parental genomes were found to occur during mitosis and meiosis under genetic control. The cytogenetics of these hybrids was species-specific for Brassica parents. The different chromosome behavior of hybrids with three Brassica diploids ( B. campestris , B. nigra and B. oleracea ) might contribute to the different cytogenetics of hybrids with three tetraploids ( B. napus, B. juncea and B. carinata). Owing to the parental genome separation, Brassica homozygous plants and aneuploids with various chromosome constitutions were identifiable in the progenies of these hybrids, which were valuable for the study of the structure and evolution of Brassica genome and for the breeding of Brassica crops.     

Pedigree transmission disequilibrium test for QTL mapping of threshold trait

With biological and economic importance,threshold traits are discrete in phenotype but have the same polygenic genetic basis as quantitative traits. The traditional linkage analysis for quantitative traits is invalid for threshold traits due to their special characters. The transmission disequilibrium test (TDT) has received great attention recently in localizing human disease genes due to its simplicity and powerfulness. But TDT only deals with data from independent nuclear families and it will lose information about extended pedigree that incorporates information not only from parents and siblings but also from other relatives. The pedigree disequilibrium test (PDT) proposed by Martin in 2001 can be used to analyze the extended pedigree in human.In this study, PDT was introduced into the QTL mapping of threshold traits for farm animals, and was modified in order to accommodate the pedigree structures of farm animals.The modified PDT was renamed pedigree transmission disequilibrium test (PTDT) and its power and type I error were investigated and compared with that of PDT by Monte Carlo simulation. It was shown that PTDT is a robust and valid approach to mapping QTL of threshold trait. When the parental information is complete, PTDT and PDT are almost the same in terms of power and type I error. However, if the parental information is missing to a certain extent, PTDT is higher in power and lower in type I error than PDT. These results imply that PTDT can be a novel approach to QTL fine mapping of threshold traits based on the existing coarse mapping information.     

Transgene for growth hormone in common carp（Cyprinus carpio L.） promotes thymus development

The transgenic carp were produced by micro-injection of CAgcGHc into the fertilized eggs. Observation of the thymus development between the transgenics and non- transgenic controls was carried out. The thymus of one-year- old transgenics F1 showed a great increase in both size and weight. The unilateral thymus of the transgenics weighed from 190 to 295 mg with average 218.6 mg, whereas the uni-lateral thymus of the controls weighed 20—81 mg with av-erage 42.5 mg; i.e. the thymus weight in the transgenics was 5.14 fold over that in the controls. The index of thymus/body weight in the transgenics was 2.97 fold over the controls. Light microscopy observation indicated that the thymus of the transgenics well developed with the thickened outer re-gion and compactly arranged thymocytes, while the thymus in the controls were degenerating with the thinned outer region, scattered thymocytes and groups of fatty cells. Fur-ther analysis with the electron microscopy revealed that pro-liferous cells in the transgenics were mainly small lympho-cytes and no pathological changes were found. The results confirmed that the 揂ll-fish?GH-transgene promotes thy-mus development and thymocyte proliferation, and retards thymus degeneration. The study has laid a foundation for further analysis of the immunobiological function in GH- transgenic carp.     

Production of bacterial blight resistant lines from somatic hybridization between Oryza sativa L. and Oryza meyeriana L

Novel bacterial blight (BB) resistance gene(s) for rice was (were) introduced into a cultivated japonica rice variety Oryza sativa (cv. 8411), via somatic hybridization using the wild rice Oryza meyeriana as the donor of the resistance gene(s). Twenty-nine progenies of somatically hybridized plants were obtained. Seven somatically hybridized plants and their parents were used for AFLP (amplified fragment length polymorphism) analysis using 8 primer pairs. Results confirmed that these plants were somatic hybrids containing the characteristic bands of both parents. The morphology of the regenerated rice showed characters of both O.sativa and O.meyeriana. Two somatic hybrids showed highest BB resistance and the other 8 plants showed moderate resistance. The new germplasms with highest resistance have been used in the rice breeding program for the improvement of bacterial blight resistance.     

The relationship of differential expression of genes in GA biosynthesis and response pathways with heterosis of plant height in a wheat diallel cross

Heterosis in internode elongation and plant height is commonly observed in hybrid plants, but the molecular basis for the increased internode elongation in hybrids is unknown. In this study, midparent heterosis in plant height was determined in a wheat diallel cross involving 16 hybrids and 8 parents, and real-time PCR was used to analyze alterations in gene expression between hybrids and parents. Significant heterosis of plant height and the first internode in length were observed for all 16 hybrids, but the magnitude of heterosis was variable for different cross combinations. Analysis revealed that the heterosis of the first internode was significantly correlated to that of plant height （r = 0.56, P 〈 0.05）, suggesting that the increased elongation of the first internode is the major contributor to the heterosis in plant height. Real-time PCR analysis exhibited that significant difference in heterosis of gene ex- pression was observed among all cross combinations. Moreover, heterosis of the first internode in length was correlated significantly and positively with expression heterosis of KS, GA3ox2-1, GA20ox2, GA20ox1D, GA-MYB and GID1-1, but significantly and negatively with expression heterosis of GAI and GA2ox-1, which is consistent with our recently proposed model of GAs and heterosis in wheat plant height, suggesting the alteration of GA biosynthesis and response pathways might be responsible for the observed heterosis in plant height.     

Restorable variation of cytoplasmic type of male sterile line in rice byin vitro culture

Rice male sterile (MS) lines, IR66707A and IR69700A, which possess the cytoplasm of Oryza perennis and O. glumaepatula respectively, belong to the cytoplasmic type. Their sterility could be maintained but not be restored. By somatic cell culture of these two MS lines, 47 somaclones with 465 R₁ plants were obtained. All of the 465 R₁ plants were sterile in the spring season in Guangzhou. According to the expression of the R₁ plants and the level of similarity to their donor parents, they could be divided into three types. The plants of type Ⅰ were male sterile. The sterility of some somaclones of this type could be restored by the test crossing varieties or alternated to fertile by changes of some environmental conditions. The hybrid F₁ of test cross from the MS somaclones in type Ⅰ was fertile while the hybrid F₁ from the donor MS lines was still sterile. The R₁ plants of type Ⅱ were similar to the donor parents and also male sterile. The hybrid F₁ from all of the plants of type Ⅱ crossed to test variety were still sterile, so they did not possess restorability. For the somaclone of type Ⅲ, all of R₁ plants were sterile in both male and female organs. No seed was set in both conditions of self and cross pollination. The fact that the restorable variants obtained in the cytoplasmic type of MS lines of rice by in vitro culture reported here should be the first sample in somaclonal variation in plant kingdom.     

Heterotic patterns in rapeseed (Brassica napus L.) using exotic germplasm for Chinese semi-winter hybrid breeding

Chinese semi-winter rapeseed, genetically differing from winter and spring rapeseed, has been considered to possess strong potential as parent in winter and spring rapeseed hybrid breeding programs. However, no detailed researches have been documented whether winter and spring rapeseed lines have potential for Chinese semi-winter rapeseed hybrid breeding. The objectives of this study are to estimate the potential of winter and spring rapeseed for semi-winter rapeseed hybrid breeding, and to investigate the association of general combining ability (GCA) with adaptation of parental lines by combining with the data in our previous studies (Qian et al. 2007, 2009). Four winter and four spring male sterile lines were crossed with 14 Chinese semi-winter rapeseed lines to develop 112 hybrids, which were evaluated together with their parents for seed yield under three environments in China. The exotic parental lines were not adapted to local environments as demonstrated by late flowering, low seed weight and poor seed yield per se. However, the hybrids, especially derived from winter rapeseed exhibited strong heterosis for seed yield, indicating that winter rapeseed germplasm has a great potential for rapeseed hybrid breeding in China. Our data suggested a strong association of GCA with their adaptation ability of parental lines, since high to middle correlations were found for local parental lines and low correlations for exotic parental lines under spring, winter and semi-winter eco-growth environments. The hybrid breeding program using exotic germplasm in rapeseed was discussed.     

Effects of heat treatment on the microstructures and mechanical properties of a new type of nitrogen-containing die steel

Nitrogen can increase the strength of steels without weakening the toughness and improve the corrosion resistance at the same time. Compared with conventional nitrogen-free die steels, a new type of nitrogen-containing die steel was developed with many superior properties, such as high strength, high hardness, and good toughness. This paper focused on the effects of heat treatment on the microstructures and mechanical properties of the new type of nitrogen-containing die steel, which were investigated by the optimized deformation process and heat treatment. Isothermal spheroidal annealing and high-temperature quenching as well as high-temperature tempering were applied in the experiment by means of an orthogonal method after the steel was multiply forged. The mechanical properties of nitrogen-containing die steel forgings are better than the standard of NADCA #207-2003.     

海滨钛磁铁矿精矿直接还原制备钛酸镁纯度的因素分析

Magnesium titanate was prepared directly through external coal reduction of seashore titanomagnetite concentrate and magnesium oxide (MgO). The effects of roasting temperature and the type and dosage of reductants on the purity of generated magnesium titanate particles were systematically investigated. Scanning electron microscopy and energy-dispersive spectroscopy analyses were performed to characterize the magnesium titanate particles and observe their purity under different conditions. Results showed that the roasting temperature remarkably influenced the purity of magnesium titanate. At 1200, 1300, and 1400°C, some magnesium ferrite and magnesium aluminate spinel were dissolved in magnesium titanate. However, as the roasting temperature increased to 1500°C, relatively pure magnesium titanate particles were generated because no magnesium ferrite was dissolved in them. The type and dosage of the reductants also remarkably affected the purity of magnesium titanate. The amount of fine metallic iron disseminated in the magnesium titanate particles obviously decreased when lignite was used as a reductant at a dosage of 70wt%. Thus, high-purity magnesium titanate particles formed. At a roasting temperature of 1500°C and with 70wt% lignite, the magnesium titanate product with a yield of 30.63% and an iron content of 3.01wt% was obtained through magnetic separation.