A new type of triploid crucian crap?red crucian carp (♀)×allotetraploid (♂)

A new type of triploid hybrids (TC) was produced by crossing red crucian carp (♀) with allotetraploid hybrids (♂). This type of triploids with no barbel was spindle-shaped and gray in color. Compared with their parents, some data of the morphological traits in the triploid hybrids were intermediate to those of their parents, and some were beyond their parents with heterozygous traits. Under the same culture conditions, the growth rate of the new type triploids was faster than their maternal red crucian carp (RC). The gonadal development of triploids was obviously slower than that of diploids, and the germ cells in them were degenerated. Compared with another type of triploids produced by Japanese crucian carp (♀) with allotetraploid hybrids (♂), the new type triploids not only kept faster growth rate and sterility, but also had no barbel that was similar to the common carp. The absence of the barbel in the new type of triploids has the important significance in both the inheritance and fish breeding.     

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.     

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.     

Molecular cloning and polymorphism of major histocompatibility complex class Ⅰ genes from grass carp (Ctenophayngodon idellus)

In order to clarify the molecular sequences, allelic polymorphism and the tertiary structure of grass carp(Ctenophayngodon idellus) MHC class Ⅰ, and to further study their relationship with disease resistances, grass carp MHC class Ⅰ gene (Ctid-MHC I) was cloned from a cDNA library and the allelic polymorphism in the population was investigated. The results showed that most of the variations exist in the peptide-binding domain (PBD) and high polymorphism was identified in the Ctid-MHC I allelic genes from 12 individuals. Based on the genetic distance, Ctid-MHC class Ⅰ can be classified into 6 types (from Ctid-MHC I-UA to Ctid-MHC I-UF) which were subdivided into 9 lineages (from A to I). Comparison of the Ctid-MHC I among animals and humans showed that the key amino acids of the peptide binding sites are conserved. Analysis of the tertiary structure of the PBD between Grass carp and human crystallographic data of HLA-A2, the variation with insertion or deletion was found in eight regions (A-H). The p     

Molecular cloning and polymorphism of major histocompatibility complex class I genes from grass carp (Ctenophayngodon idellus)

In order to clarify the molecular sequences,allelic polymorphism and the tertiary structure of grass carp (Ctenophayngodon idellus) MHC class I,and to further study their relationship with disease resistances,grass carp MHC class I gene (Ctid-MHC I) was cloned from a cDNA library and the allelic polymorphism in the population was investigated.The results showed that most of the variations exist in the peptide-binding domain (PBD) and high polymorphism was identified in the Ctid-MHC I allelic genes from 12 individuals.Based on the genetic distance,Ctid-MHC class I can be classified into 6 types (from Ctid-MHC I-UA to Ctid-MHC I-UF) which were subdivided into 9 lineages (from A to I).Comparison of the Ctid-MHC I among animals and humans showed that the key amino acids of the peptide binding sites are conserved.Analysis of the tertiary structure of the PBD between Grass carp and human crystallographic data of HLA-A2,the variation with insertion or deletion was found in eight regions (A～H).The phylogenetic tree of MHC class I indicates the evolution of MHC class I among grass carp,fish,amphibian,birds,higher vertebrates and humans.     

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.     

Genetic mapping of telomere-associated sequences in soybean ( Glycine max )

Two telomere-associated sequences (TAS), named STAS8 and STAS10, were cloned from soybean genomic DNA using polymerase chain reaction (PCR) amplification. Southern analysis showed that they were sequences with moderate copy number in soybean genome. Sequence analysis demonstrated that STAS10 had tandemly arrayed con sensus sequences of TTTAGGG and TIAGGG . The mapping of these two TAS was performed with a population of F8 re combinant inbred line using restriction fragment length polymorphisms(RFLP). Seven out of nine polymorphic fragments were mapped to the most distal position of five linkage groups, Dla, F, G2, H and Q of soybean, and the other two loci were closely linked and mapped to two interstitial positions within linkage group D1a. The mapping of TAS in soybean is essential for completeness of a molecular genetic map of soybean.     

Genetic diversity, geographic differentiation and evolutionary relationship among ecotypes of Glycine max and G. soja in China

The knowledge of origin and evolution of cultivated soybeans is one of the basic issues in both biology and agronomy of the crop. In order to investigate the nuclear and cytoplasmic genetic diversity, geographic differentiation and genetic relationship among geographic ecotypes of cultivated （Glycine max） and wild （G. soja） soybeans, the allelic profiles at 60 nuclear simple-sequence repeat （nuSSR） loci and 11 chloroplastic SSR （cpSSR） loci evenly distributed on whole genome of 393 landraces and 196 wild accessions from nation-wide growing areas in China were analyzed. （i） The genetic diversity of the wild soybean was obviously larger than that of the cultivated soybean, with their nuSSR and cpSSR alleles as 1067 vs. 980 and 57 vs 44, respectively. Of the 980 nuclear alleles detected in the cultivated soybean, 377 new ones （38.5%） emerged, while of the 44 chloroplastic alleles in the cultivated soybean, seven new ones （15.9%） emerged after domestication. （ii） Among the cultivated geographic ecotypes, those from southern China, including South-Central China, Southwest China and South China possessed relatively great genetic diversity than those from northern China, while among the wild geographic ecotypes, the Middle and Lower Changjiang Valleys wild ecotype showed the highest genetic diversity. （iii） The analysis of molecular variance, association analysis between geographic grouping and molecular marker clustering and analysis of specific-present alleles of ecotypes demonstrated that the geographic differentiation of both cultivated and wild soybeans associated with their genetic differentiation, or in other words, had their relevant genetic bases. （iv） The cluster analysis of all accessions clearly showed that the wild accessions from Middle and Lower Changjiang Valleys and South-Central ＆ Southwest China had relatively small genetic distances with all cultivated accessions. The UPGMA dendrogram among geographic ecotypes further showed that the genetic distances between all cultivated ecotypes and the Middle and Lower Changjiang Valleys wild ecotype were smaller than those with other wild ones, including their local wild counterparts. Therefore, it is inferred that the wild ancestors in southern China, especially those from Middle and Lower Changjiang Valleys might be the common ancestor of all the cultivated soybeans.     

Cloning and expression profiles of 15 genes encoding WRKY transcription factor in wheat （Triticum aestivem L.）

WRKY proteins are involved in various physiological processes, including biotic and abiotic stress responses, hormone responses and development. However, no systematic identification, expression and function analysis of WRKY genes in wheat were reported. In this study, we isolated 15 wheat cDNAs with complete open reading frame （ORF） encoding putative WRKY proteins using in silico cloning. Phylogenetic analysis indicated that the 15 wheat WRKY genes belonged to three major WRKY groups. Expression analysis revealed that most genes expressed drastically in leaf, except Ta WRKYIO which expressed in crown intensively. Four genes were strongly up-regulated with the senescence of leaves. Eight genes were responsive to low temperature, high temperature, NaCl or PEG treatment. Moreover, differential expression patterns were also observed between wheat hybrid and its parents, and some genes were more responsive to PEG treatment in the hybrid. These results demonstrated that wheat WRKY genes are involved in leaf senescing and abiotic stresses. And the changed expression of these WRKY genes in hybrid might contribute to the heterosis by improving the stress tolerance in hybrids.     

Cloning of a down-regulated gene encoding small GTP binding protein in hybrid wheat

Previous studies showed that differential gene expression between wheathybrids and their parents was responsible for the heterosis. To provide an insight into the molecular basis of wheat heterosis, one cDNA, designated TaRab, was identified from the cDNA library of wheat seedling leaves. The sequence comparison in GenBank revealed that TaRab is homologous to a group of genes encoding Rab-GTP binding protein. Semi-quantitative RT-PCR analysis indicated that TaRab was expressed in all plant tissues examined, but at slightly higher level in leaves. Further analysis exhibited that TaRab displayed lower expression in hybrid than in its patents in both roots and leaves, which was in agreement with the original results of suppression subtractive hybridization. TaRab was located on chromosome 7B and C-7DS5-0.36 by in silico mapping. The relationship between differential expression of TaRab and the molecular basis of wheat heterosis was also discussed.     

Construction and initial analysis of five Fosmid libraries of mitochondrial genomes of cotton （Gossypium）

Cotton （Gossypium） is an important crop providing textile fiber and edible oil. To gain the insights into mechanism of the cyto- plasmic male sterility （CMS） inheritance, we constructed five fosmid libraries of mitochondrial genomes from mitotype of G. harknessii Brandegee. （one CMS line and its restorer）, mitotype of G. hirsutum L. （one CMS line and its maintainer）, and G. bar- badense L. The numbers of the clones in these libraries ranged from 1152 to 2016 with an average insert size of 36.2 to 38.4 kb, equivalent to 70-119.3 mitogenomes. The libraries were screened with 28 markers derived from the conservative sequences and yielded 22, 19, 26, 21, and 23 positive clones, respectively. These positive clones were used to construct the physical map of G. harknessii Brandegee. CMS line and G. barbadense L. mitogenomes that shared six syntenis regions. A total of 30 genes in nine clusters showed conservative and had high similarity with those in the mitochondrial genomes of cotton, Carica papaya, Cucur- bita pepo and Nicotiana tabacum. Further investigation indicated that gene rrn26 had two copies in all five cotton mitogenomes, while genes atpl, rrn5 and rrn18 had two copies only in G. barbadense L. The positive clones and physical map are considered being useful resources in cotton genomics research.     

Phylogenetic relationships of host insects of Cordyceps sinensis inferred from mitochondrial Cytochrome b sequences

This study used the sequence of the mitochondrial Cytochrome b(Cytb)to estimate phylogenetic relationships among host Hepialidae insects of Cordyceps sinensis.Genome DNA of host insect was extracted from the dead larva head part of 18 cordyceps populations and 2 species of Hepialus,and the Cytb fragment of host insect was amplified with PCR technique.The nucleotide sequence alignments and their homologous sequences of 24 species host Hepialidae insects of Cordyceps sinensis were obtained from GenBank and were used to construct phylogenetic trees based on neighbor-joining method.The results showed that genus Bipectilus diverged earlier than genus Hepialus and Hepialiscus.Hepialus host insects of Cordyceps sinensis have multitudinous species with different morphological characteristics and geographical distributions.The interspecific genetic differentiations are obvious in Hepialus.Thus,the genus Hepialus might be considered as polyphyletic origin.Cytb sequences have abundant variations among the host insects of Cordyceps sinensis on spe- cific and generic level.The divergence rate of Cytb sequences among the species in Hepialus ranged from 0.23% to 9.24%,except that Hepialus pratensis and Hepialus jinshaensis have the same sequence.Cytb sequence can be used for species identification of host insects of Cordyceps sinensis,but further confirmation in more host insect species is needed.To obtain the Cytb sequence of host insect by ampli- fying DNA extracted from the head part of dead larva in cordyceps turns out to be an effective and accurate approach,which will be useful for studies on phylogeny and genetic structure of host insects of cordyceps populations,especially for analyzing relationships between C. sinensis and its host insects.     

Disease-tolerance of transgenic tobacco plants expressing Ah-AMP gene of Amaranthus hypochondriacus

An antimicrobial peptide gene from Amaranthus hypochondriacus, Ah-AMP, was amplified by PCR and cloned. Sequence analysis results revealed that this gene is 261 bp in length encoding a precursor polypeptide of 87 amino acid residues. Ah-AMP gene was inserted in the binary vector pBin438 to construct a plant expression vector pBinAH916. Leave explants of Nicotiana tabacum var. SR1 were transformed with Agrobacterium tumefaciens LBA4404 harboring the above expression vector. Results from PCR, Southern and Northern blot analyses confirmed that the Ah-AMP gene had been integrated into the tobacco genome and was transcribed at mRNA level. Two bacterial-resistant transgenic plants were selected by inoculating the plants with Pseudomonas solanacearum and statistic analysis of two T1 lines showed that the resistance increased by 2.24 and 1.62 grade and the disease index decreased by 49.6% and 37.3% respectively when compared with the non-transformed control plants SR1. The results from challenging the plants with inoculums of Phytophthora parasitica showed that the symptom development was delayed and disease index was significantly reduced. These results suggest that Ah-AMP gene may be a potentially valuable gene for genetic engineering of plant for disease-resistance.     

Construction and genetic analysis of mutator insertion mutant population in maize

A total of 26718 M1 plants were ob- tained by crossing the active mutator transposon donor parents (Q105, WW51, 115F, V26-2 and 919J) with the recipient parents (Hz85,W328 with Bz gene and S-Mo17Rf3Rf3). The phenotypes of M1 plants were observed in the field. M1 plants were self-pollinated to develop the mutator insertion-mutagenized M2 seeds. The transposition frequency of the mutator in the genome was calculated based on the spotted aleurone phenotype of the M2 seeds. The results showed that: (1) the mutation frequency of M1 phe- notypes in the field was 0.07 in the population of W328×Mu; (2) the mutation frequency of spotted aleurone seeds on the M2 ears was 0.122 in the population of W328×Mu; (3) five S-cytoplasm male-sterile plants were found among 22500 M1 plants of S-Mo17Rf3Rf3×Mu, with the transposition frequency about 2.2×10?4 per locus. 99 flanking se- quences of mutator transposition were amplified by the modified MuTAIL-PCR, and 59 non-redundant sequences with length around 400 bp were obtained. After bioinformatic analysis, 27 sequences of them could be annotated, using non-redundant nucleotide database of maize, rice, and Arabidopsis. 36 se- quences of them were located on the genetic map of maize by comparative genomics, and several flank- ing sequences of mutator insertion were mapped on the single marker locus. Hotspot sequences of mu- tator transposition were revealed by comparing the homologies between the 9-bp target site duplication of the mutator insertion. The putative functions of 8 flanking sequences of mutator transposition had identity with the functions of their correspondingmarker. The constructed mutator insertion mutant population in maize will facilitate the new gene discovery and functional genomics study in maize.     

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.