Assessing the genetic diversity of tobacco germplasm using intersimple sequence repeat and inter-retrotransposon amplification polymorphism markers

The genetic diversity of 118 tobacco accessions, including flue‐cured tobacco, sun‐/air‐cured tobacco, burley tobacco, oriental tobacco and wild tobacco, was characterised using intersimple sequence repeat (ISSR) and inter‐retrotransposon amplification polymorphism (IRAP) markers. ISSR and IRAP banding patterns and genetic distance (GD) values showed the low level of genetic diversity within and among cultivated tobacco types. There was higher GD and average heterozygosity among wild tobacco types than those among cultivated tobacco. Genetic diversity of tobacco germplasm was low, with a high level of genetic identity (>0.77) between the different types. However, neighbour‐joining cluster analysis of marker‐based GDs showed that the accessions from the same tobacco type, as classified by manufacturing quality traits, were nearly clustered into the same group. These results will help in the formulation of appropriate strategies for variety improvement in tobacco, and ISSR and IRAP markers of the genetic diversity will contribute to further study and improvement of tobacco.     

Identification of trait-improving quantitative trait loci alleles from a wild rice relative,Oryza rufipogon.

Wild species are valued as a unique source of genetic variation, but they have rarely been used for the genetic improvement of quantitative traits. To identify trait-improving quantitative trait loci (QTL) alleles from exotic species, an accession of Oryza rufipogon, a relative of cultivated rice, was chosen on the basis of a genetic diversity study. An interspecific BC2 testcross population (V20A/O. rufipogon//V20B///V20B////Ce64) consisting of 300 families was evaluated for 12 agronomically important quantitative traits. The O. rufipogon accession was phenotypically inferior for all 12 traits. However, transgressive segregants that outperformed the original elite hybrid variety, V20A/Ce64, were observed for all traits examined. A set of 122 RFLP and microsatellite markers was used to identify QTL. A total of 68 significant QTL were identified, and of these, 35 (51%) had beneficial alleles derived from the phenotypically inferior O. rufipogon parent. Nineteen (54%) of these beneficial QTL alleles were free of deleterious effects on other characters. O. rufipogon alleles at two QTL on chromosomes 1 and 2 were associated with an 18 and 17% increase in grain yield per plant, respectively, without delaying maturity or increasing plant height. This discovery suggests that the innovative use of molecular maps and markers can alter the way geneticists utilize wild and exotic germplasm.     

Optimized production of high-titer recombinant adeno-associated virus in roller bottles

Adeno-associated viral (AAV) vectors are used for in vivo gene transfer in a number of preclinical models of genetic diseases (including large-animal models) and are currently being tested in clinical trials for treatment of hemophilia B and cystic fibrosis. Protocols for production of AAV vectors in a helper virus-free system are available and are based on transient transfection of HEK-293 cells with multiple plasmids. Scale-up of vector production has been labor intensive and inefficient because of a lack of larger culture vessels suitable for growth of adherent cells, large-scale transfection, and vector production. Here we report efficient production of AAV vector in roller bottles, which represents a 10-fold scale-up from the conventional flask or plate method. Optimized production yielded greater than 10(13) vector genomes per bottle and was as cost effective as published protocols using plates. Successful vector production by this method was dependent on optimization of transfection by calcium phosphate precipitation, of monitoring of cell growth (by measurement of glucose consumption), of cell culture conditions, and CO2/air exchange with the culture vessel.     

Kinetic FP-TDI assay for SNP allele frequency determination

Strategies for identifying genetic risk factors in complex diseases by association studies require the comparison of allele frequencies of numerous SNPs between affected and control populations. Theoretically, hundreds of thousands of SNP markers across the genome will have to be genotyped in these studies. Genotyping SNPs one sample at a time is extremely costly and time consuming. To streamline whole genome association studies, some have proposed to screen SNPs by pooling the DNA samples initially for allele frequency determination and perform individual genotyping only when there is a significant discrepancy in allele frequencies between the affected and control populations. Here we describe a new method for determining the allele frequency of SNPs in pooled DNA samples using a two-color primer extension assay with real-time monitoring of fluorescence polarization (named kinetic FP-TDI assay). By comparing the ratio of the rate of incorporation of the two allele-specific dye-terminators, one can calculate the relative amounts of each allele in the pooled sample. The accuracy of allele frequency determination with pooled samples is within 3.3 +/- 0.8% of that determined by genotyping individual samples that make up the pool.     

New records of Stixis Lour. (Capparaceae) from China

1CHEN Wen-Hong,1SHUI Yu-Min,2YANG Zhi-Guo,1CHENG Xiao 1(Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming 650204, China) 2 (Hekou Forestry Bureau, Forestry Department of Yunnan Province, Hekou 661300, China) 2003,41（1）.—89～90 Abstract：Two taxa of Stixis　Lour., S.ovata(Korth.) Hall.　f. ssp. fasciculata(King) Jacobs and S. scandens　Lour., are reported as new records from China .     

Studies of ice melting using molecular dynamics

In this paper, the melting of ice 1h is studied using molecular dynamics (MD). Common potential functions employed in the MD simulations include SPC/E, TIP4P, TIP5P, TIP4/ice and TIP5P/E. We first conducted melting of ice bulks and then studied the melting speed of the ice/water interface during ice melting. It is found that various potentials result in similar ice-melting phenomena. The result is compared with the analytical solution for phase change problem. We also studied size effects and temperature gradient effects on ice melting.     

Pol32 is required for Pol zeta-dependent translesion synthesis and prevents double-strand breaks at the replication fork

POL32 encodes a non-essential subunit of Polδ and plays a role in Polδ processivity and DNA repair. In order to understand how Pol32 is involved in these processes, we performed extensive genetic analysis and demonstrated that POL32 is required for Polζ-mediated translesion synthesis, but not for Polη-mediated activity. Unlike Polζ, inactivation of Pol32 does not result in decreased spontaneous mutagenesis, nor does it limit genome instability in the absence of the error-free postreplication repair pathway. In contrast, inactivation of Pol32 results in an increased rate of replication slippage and recombination. A genome-wide synthetic lethal screen revealed that in the absence of Pol32, homologous recombination repair and cell cycle checkpoints play crucial roles in maintaining cell survival and growth. These results are consistent with a model in which Pol32 functions as a coupling factor to facilitate a switch from replication to translesion synthesis when Polδ encounters replication-blocking lesions. When Pol32 is absent, the S-phase checkpoint complex Mrc1–Tof1 becomes crucial to stabilize the stalled replication fork and recruit Top3 and Sgs1. Lack of any of the above activities will cause double strand breaks at or near the replication fork that require recombination as well as Rad9 for cell survival.     

Two-component sensor RhpS promotes induction of Pseudomonas syringae type III secretion system by repressing negative regulator RhpR

The Pseudomonas syringae type III secretion system (T3SS) is induced during interaction with the plant or culture in minimal medium (MM). How the bacterium senses these environments to activate the T3SS is poorly understood. Here, we report the identification of a novel two-component system (TCS), RhpRS, that regulates the induction of P. syringae T3SS genes. The rhpR and rhpS genes are organized in an operon with rhpR encoding a putative TCS response regulator and rhpS encoding a putative biphasic sensor kinase. Transposon insertion in rhpS severely reduced the induction of P. syringae T3SS genes in the plant as well as in MM and significantly compromised the pathogenicity on host plants and hypersensitive response-inducing activity on nonhost plants. However, deletion of the rhpRS locus allowed the induction of T3SS genes to the same level as in the wild-type strain and the recovery of pathogenicity upon infiltration into plants. Overexpression of RhpR in the deltarhpRS deletion strain abolished the induction of T3SS genes. However, overexpression of RhpR in the wild-type strain or overexpression of RhpR(D70A), a mutant of the predicted phosphorylation site of RhpR, in the deltarhpRS deletion strain only slightly reduced the induction of T3SS genes. Based on these results, we propose that the phosphorylated RhpR represses the induction of T3SS genes and that RhpS reverses phosphorylation of RhpR under the T3SS-inducing conditions. Epistasis analysis indicated that rhpS and rhpR act upstream of hrpR to regulate T3SS genes.