A study on natural recovery of tassel fertilization and doubling method in maize haploids.

Doubling method is the technical barriers in maize haploid breeding. It was very important to establish the independent intellectual property rights for doubling method. In this experiment, the maize haploid inducer, TG15, was used for producing maternal haploids. Also, haploids were obtained from two kinds of maternal genotypes involved in the experiment, including high-oil type and common type. Significant differences were observed among offspring of various genotypes in the recovery of haploid fertilization. In 21 hybrid offspring haploids, the average powder rate was 8.28%, and the seed setting rate was 4.98%. The experimental results showed that when the hybrids were treated with 0.08% colchicine, the average powder rate and seed setting rate of offspring haploids were 35.53 and 20.30%, respectively, which were significantly higher than the hybrids with natural recovery ability. This study primarily established the doubling method of haploids called "bud seedling method" in China which was very practicably in maize doubled haploid breeding.

Cloning and bioinformatic analysis of transcription factor MYB10 from the red-leaf peach.

In higher plants, the transcription factor MYB10 is an important regulator of anthocyanin biosynthesis. In order to study its role in the development of red coloration in peach leaves, the full-length MYB10 complementary DNA sequence of the red-leaf peach cultivar 'Tsukuba No. 5' (Prunus persica f. atropurpurea) was successfully cloned using reverse transcription-polymerase chain reaction. The sequence was assigned the GenBank accession No. KP315904. Bioinformatic analysis identified the complete MYB10 open reading frame, consisting of 678 bp encoding 225 amino acids. The predicted protein has a molecular weight of 26.56 kDa and a theoretical isoelectric point of 8.97. The secondary structure was found to comprise approximately 34.22% alpha helix, 15.11% extended strand, 10.67% beta turn, and 40% random coil. Subcellular analysis indicated that MYB10 may function in the cytoplasm. Assessment of the amino acid sequence suggested the presence of one serine and two threonine phosphorylation sites. Quantitative real-time polymerase chain reaction revealed that MYB10 expression positively correlated with anthocyanin content in red-leaf peach, indicating that this transcription factor plays a role in the biosynthesis of this pigment in peach trees.

Molecular characterization of the myosatin gene and the effect of fasting on its expression in Chinese perch (Siniperca chuatsi).

Myostatin (MSTN) is an important member of the transforming growth factor-ß (TGF-ß) superfamily and is a muscle growth inhibitor. In the present study, we cloned the Chinese perch MSTN cDNA sequence and analyzed its expression patterns under various conditions. The MSTN full cDNA sequence was 3347 bp long, including an open-reading frame of 1131 bp, which encoded 376 amino acids. Sequence analysis demonstrated that the MSTN shared a highly conserved signal peptide, a TGF-ß functional peptide, a hydrolytic site (RARR), and nine conservative cysteine residues with other members of the TGF-ß superfamily. Sequence alignment and phylogenetic tree analyses indicated that the MSTN had a close relationship with teleostean fish, but they are far separated from mammals. Real-time polymerase chain reaction analysis revealed that the MSTN was strongly expressed in the skeletal muscle and heart tissues. Temporal expression analysis demonstrated that the MSTN gene was expressed in very low levels, from 20 to 90 dph (post-hatching development), and was at its highest level at 150 dph (P < 0.05). The fasting-re-feeding experiment showed that the expression of the MSTN gene was initially decreased in response to a single meal, after seven days of fasting, and subsequently increased significantly, and finally decreased back to its original level. Together, our results provided valuable knowledge regarding the regulation of MSTN gene expression in Chinese perch.

Tissue culture characteristics of maize (Zea mays L.) haploid coleoptile sections.

Doubled haploid (DH) technology, which is used for rapidly purifying genetic resources, is a key technology in modern maize breeding. The present study evaluated the tissue culture characteristics of maize haploid coleoptile sections, in order to provide a new way of haploid doubling. With 20 combinations of haploid coleoptile sections, obtained by hybridization within Reid, Tangsipingtou, and Term-tropical groups, as explants, we analyzed the induction and differentiation rate of callus, observed the number of root tip chromosomes in regenerated plants, and analyzed the pollen fertility. In addition, we used 47 SSR markers to analyze the genotypes of regenerated plants. The Reid and Tangsipingtou groups had significantly higher induction rates of haploid coleoptile callus compared to the Term-tropical group. Fifteen haploid plants were obtained which had 10 chromosomes in the root tips as assessed by I-KI staining. It was also noticed that the pollen of pollinated anthers were partially fertile. The haploid plants had genetic stability and showed no variation. The Reid and Tangsipingtou groups had good culture characteristics of haploid coleoptile sections, while the Term-tropical group had poor culture characteristics. Genotypes of haploid plants generated by tissue culture were evidenced to come from recombinant types of parents. Thus, this study established a tissue culture system of maize haploid coleoptile.

Isolation and characterization of novel polymorphic microsatellite loci in large yellow croaker (Larimichthys crocea).

The large yellow croaker (Larimichthys crocea) is one of the largest marine net-cage cultured species in the oceans around China. In the present study, we isolated and characterized 13 polymorphic microsatellite markers from genomic libraries of L. crocea. Loci were screened for 10 wild specimens from 2 sites in southeast of China. All loci were polymorphic. The number of alleles per locus ranged from 2 to 21. The expected heterozygosity ranged from 0.233 to 0.838 and observed heterozygosity ranged from 0.527 to 0.935. Eleven loci were highly informative (polymorphic information content >0.5). Significant deviation from Hardy-Weinberg equilibrium was observed at 3 loci after Bonferroni's correction. The microsatellite loci may be valuable tools for studying the genetic diversity and genetic structure for conservation planning of the fish.

A novel mutation links to von Hippel-Lindau syndrome in a Chinese family with hemangioblastoma.

Hemangioblastoma of the central nervous system occurs as sporadic tumors or as a part of von Hippel-Lindau (VHL) disease, an autosomal dominant hereditary tumor syndrome caused by a germline mutation in the VHL tumor suppressor gene. We screened a Chinese family with VHL for mutations in the VHL gene and evaluated a genetic test for diagnosing VHL disease and clinical screening of family members. DNA extracted from the peripheral blood of all live members and from tissue of deceased family members with VHL disease was amplified by polymerase chain reaction to 3 VHL gene exons. Mutations in the amplification products were compared against the Human Gene Mutation Database. The involvement of multiple organs among the kindred with VHL disease was confirmed by medical history and radiography. Of the 12 members of the 4-generation family, 5 were diagnosed with VHL disease. Patient age at the initial diagnosis was 26-36 years (mean = 31 years). The mean time was 15 (11-19 months) from symptom appearance to the first patient visit to the hospital. Sequence analysis revealed that the frameshift mutation 327del C (p.Gly39Alafs*26) in exon 1 affected all family members, but not the healthy individuals or 16 unrelated controls. Members without gene mutation showed no clinical manifestation of VHL disease. We detected a conserved novel frameshift mutation in the VHL gene of the family members that contributes to VHL. DNA analysis of VHL is advantageous for VHL diagnosis. We developed a quick and reliable method for VHL diagnosis.

IL-8 -251T/A polymorphism is associated with susceptibility to acute pancreatitis.

We conducted a case-control study to clarify the asso-ciations between inflammatory cytokine, including interleukin (IL)-1b, IL-6, IL-8, and IL-10, polymorphisms and risk of acute pancreatitis. Genotyping analyses of IL-1ß+3954 C/T (rs1143634), IL-1ß-511 C/T (rs16944), IL-6 -174 G/C (rs1800795), IL-6 -634 C/G (rs1800796), IL-8 -251T/A (rs4073), IL-10 -1082A/G (rs1800896), and IL-10 -819C/T (rs1800871) were conducted using polymerase chain reaction-restriction fragment length of polymorphism. Unconditional logistic regression analysis was utilized to assess the potential association be-tween genotype frequencies and risk of acute pancreatitis. Multivari-ate regression analyses showed that subjects carrying the IL-8 -251 AA genotype had a significantly increased risk of acute pancreatitis, with an adjusted odds ratio (95% confidence interval) of 1.55 (1.02-2.36). However, we found no significant association between IL-1ß +3954 C/T, IL-1ß -511 C/T, IL-6 -174 G/C, IL-6 -174 G/C, IL-6 -634 C/G, IL-10 -1082A/G, or IL-10 -819C/T polymorphisms and risk of acute pancreatitis. We found that the IL-8 -251T/A polymorphism was associated with a higher susceptibility to acute pancreatitis in a Chinese population.

Genetic dissection of upland cotton (Gossypium hirsutum) cultivars developed in Hubei Province by mapped SSRs.

The genetic diversity of 51 upland cotton cultivars with different parental origins and breeding periods that were developed in Hubei Province was studied on the basis of 237 mapped simple sequence repeat markers covering the cotton genome. A total of 108 polymorphic primer pairs amplified 196 loci; the polymorphism information content range was 0.04 to 0.83, with an average of 0.46. A model-based clustering analysis (STRUCTURE) of the genomic data identified 3 clear subpopulations, and the result was confirmed by principal components analysis. The genetic similarity coefficient among 51 upland cotton cultivars was 0.598 on average, ranging from 0.378 to 0.817. The unweighted pair group method with arithmetic average cluster analysis revealed inconsistencies in other clustering patterns: "Tianmian1" was distinct from the rest of the materials and formed a separate cluster. This study will provide a guide for breeders to develop new cultivars efficiently and to choose parents, and it supports the need to introduce new alleles into the gene pool of the upland cotton breeding program in Hubei Province.

Association analysis of yield and fiber quality traits in Gossypium barbadense with SSRs and SRAPs.

Cotton is an important cash crop. Mining for quantitative trait loci related to yield and fiber quality traits using association analysis has many advantages for cotton research. In this study, 170 simple sequence repeats (SSRs) and 258 sequence-related amplified polymorphisms (SRAPs) were used to analyze the association of 3 yield component traits and 5 fiber quality traits of 55 Gossypium barbadense accessions in 2009 and 2010. Principal component analysis of SSRs and SRAPs showed 3 and 2 subgroups, respectively. The boundaries between the SRAP groups were much more defined than those of the SSRs. A mixed linear model was used to analyze association of yield and fiber quality traits with SSRs and SRAPs. A total of 72 loci were detected, including 28 loci of SSRs and 44 loci of SRAPs; 26 of these loci were related to yield component traits, and 46 of these loci were related to fiber quality traits. The mean phenotypic variations explained in the SSR and SRAP analysis were 8.89 and 8.61%, respectively. The locus with the highest phenotypic variation explained was NAU1164 (23.33%), which was related to fiber uniformity. The comparison of association results between the two datasets showed that mining quantitative trait loci using association analysis was more efficient with SRAPs than with SSRs.

Target replacement strategy for selection of DNA aptamers against the Fc region of mouse IgG.

Aptamers that recognize the IgG Fc region are of great interest because of their wide application as an immunology probing tool, for diagnostics, and as affinity agents for antibody purification. We developed a target replacement strategy as a modification of conventional Systematic Evolution of Ligands by EXponential enrichment (SELEX) in order to efficiently select and identify novel DNA aptamers against the Fc region of mouse IgG. In this new approach, multiple IgG subclasses (IgG1, IgG2a, mouse IgG Fc, and anti-HBs IgG) were sequentially used to select aptamers in one continuous SELEX. After 8 rounds of selection, the aptamers were analyzed using dot blot and an electrophoretic mobility shift assay, which showed universal binding capability to different IgG subclasses. Secondary structure analysis of the aptamers indicated that the stem-loop structure of the aptamers play an important role in binding to the common site in different mouse IgG subclasses. This demonstrated the feasibility of using multiple target replacement SELEX for the selection of aptamers. This target replacement strategy is also expected to be useful for selecting aptamers that bind common regions of molecules other than antibodies.

Genetic diversity of sea-island cotton (Gossypium barbadense) revealed by mapped SSRs.

In order to evaluate the genetic diversity of sea-island cotton (Gossypium barbadense), 237 commonly mapped SSR markers covering the cotton genome were used to genotype 56 sea-island cotton accessions. A total of 218 polymorphic primer pairs (91.98%) amplified 361 loci, with a mean of 1.66 loci. Polymorphism information content values of the SSR primers ranged from 0.035 to 0.862, with a mean of 0.320. The highest mean polymorphism information content value for the SSR motifs was from a compound motif (0.402), and for the chromosomes it was Chr10 (0.589); the highest ratio of polymorphic primers in Xinjiang accessions was from Chr21 (83.33%). Genetic diversity was high in Xinjiang accessions. AMOVA showed that variation was 8 and 92% among populations and within populations, respectively. The 56 sea-island accessions were divided into three groups in the UPGMA dendrogram: Xinhai5 was in the first group; accessions from Xinjiang, except the five main ones, were in the second group, and the other 34 accessions were in the third group. Accessions from the former Soviet Union and Xinjiang main accessions were closely related. Both PCA and UPGMA confirmed that Xinhai5 was distinct from the other accessions, and accessions from Xinjiang were in an independent group. Given the differences between principal components analysis and UPGMA results, it is necessary to combine molecular markers and pedigree information so that genetic diversity can be objectively analyzed.