Introgression and targeting of the Pl37 and Pl38 genes for downy mildew resistance from wild Helianthus annuus and H. praecox into cultivated sunflower (Helianthus annuus L.).

KEY MESSAGE: Two new downy mildew resistance genes, Pl37 and Pl38, were introgressed from wild sunflower species into cultivated sunflower and mapped to sunflower chromosomes 4 and 2, respectively Downy mildew (DM), caused by the oomycete pathogen Plasmopara halstedii (Farl.) Berl. & de Toni, is known as the most prevalent disease occurring in global sunflower production areas, especially in North America and Europe. In this study, we report the introgression and molecular mapping of two new DM resistance genes from wild sunflower species, Helianthus annuus and H. praecox, into cultivated sunflower. Two mapping populations were developed from the crosses of HA 89/H. annuus PI 435417 (Pop1) and CMS HA 89/H. praecox PRA-417 (Pop2). The phenotypic evaluation of DM resistance/susceptibility was conducted in the BC1F2-derived BC1F3 populations using P. halstedii race 734. The BC1F2 segregating Pop1 was genotyped using an Optimal GBS AgriSeq™ Panel consisting of 768 mapped SNP markers, while the BC1F2 segregating Pop2 was genotyped using a genotyping-by-sequencing approach. Linkage analysis and subsequent saturation mapping placed the DM resistance gene, designated Pl37, derived from H. annuus PI 435417 in a 1.6 cM genetic interval on sunflower chromosome 4. Pl37 co-segregated with SNP markers SPB0003 and C4_5738736. Similarly, linkage analysis and subsequent saturation mapping placed the DM resistance gene, designated Pl38, derived from H. praecox PRA-417 in a 0.8 cM genetic interval on sunflower chromosome 2. Pl38 co-segregated with seven SNP markers. Multi-pathotype tests revealed that lines with Pl37 or Pl38 are immune to the most prevalent and virulent P. halstedii races tested. Two germplasm lines, HA-DM15 with Pl37 and HA-DM16 with Pl38, were developed for use in sunflower DM-resistance breeding.

Whole-genome sequencing enables molecular dissection and candidate gene identification of the rust resistance gene R12 in sunflower (Helianthus annuus L.).

KEY MESSAGE: We finely mapped the rust resistance gene R12 to a 0.1248-cM region, identified a potential R12 candidate gene in the XRQ reference genome, and developed three diagnostic SNP markers for R12. Rust is a devastating disease in sunflower that is damaging to the sunflower production globally. Identification and utilization of host-plant resistance are proven to be preferable means for disease control. The rust resistance gene R12 with broad-spectrum specificity to rust was previously localized to a 2.4 Mb region on sunflower chromosome 11. To understand the molecular mechanism of resistance, we conducted whole-genome sequencing of RHA 464 (R12 donor line) and reference genome-based fine mapping of the gene R12. Overall, the 213 markers including 186 SNPs and 27 SSRs' were identified from RHA 464 sequences and used to survey polymorphisms between the parents HA 89 and RHA 464. Saturation mapping identified 26 new markers positioned in the R12 region, and fine mapping with a large population of 2004 individuals positioned R12 at a genetic distance of 0.1248 cM flanked by SNP markers C11_150451336 and S11_189205190. One gene, HanXRQChr11g0348661, with a defense-related NB-ARC-LRR domain, was identified in the XRQr1.0 genome assembly in the R12 region; it is predicted to be a potential R12 candidate gene. Comparative analysis clearly distinguished R12 from the rust R14 gene located in the vicinity of the R12 gene on chromosome 11. Three diagnostic SNP markers, C11_147181749, C11_147312085, and C11_149085167, specific for R12 were developed in the current study, facilitating more accurate and efficient selection in sunflower rust resistance breeding. The current study provides a new genetic resource and starting point for cloning R12 in the future.

Discovery and mapping of two new rust resistance genes, R17 and R18, in sunflower using genotyping by sequencing.

KEY MESSAGE: Discovery of two rust resistance genes, R17 and R18, from the sunflower lines introduced from South Africa and genetic mapping of them to sunflower chromosome 13. Rust, caused by the fungus Puccinia helianthi Schw., is one of the most serious diseases of sunflower in the world. The rapid changes that occur in the virulence characteristics of pathogen populations present a continuous threat to the effectiveness of existing rust-resistant hybrids. Thus, there is a continued need for the characterization of genetically diverse sources of rust resistance. In this study, we report to identify two new rust resistance genes, R17 and R18, from the sunflower lines, KP193 and KP199, introduced from South Africa. The inheritance of rust resistance was investigated in both lines using two mapping populations developed by crossing the resistant plants selected from KP193 and KP199 with a common susceptible parent HA 89. The F2 populations were first genotyped using genotyping by sequencing for mapping of the rust genes and further saturated with markers in the target region. Molecular mapping positioned the two genes at the lower end of sunflower chromosome 13 within a large gene cluster. Two co-segregating SNP markers, SFW01497 and SFW08875, were distal to R17 at a 1.9 cM genetic distance, and a cluster of five co-segregating SNPs was proximal to R17 at 0.7 cM. R18 co-segregated with the SNP marker SFW04317 and was proximal to two cosegregating SNPs, SFW01497 and SFW05453, at 1.9 cM. These maps provide markers for stacking R17 or R18 with other broadly effective rust resistance genes to extend the durability of rust resistance. The relationship of the six rust resistance genes in the cluster was discussed.

Calculation of the Probability Distribution of CIBS Score in Different Relationships and Its Application.

ABSTRACT: Objective To derive the probability distribution formula of combined identity by state （CIBS） score among individuals with different relationships based on population data of autosomal multiallelic genetic markers. Methods The probabilities of different identity by state （IBS） scores occurring at a single locus between two individuals with different relationships were derived based on the principle of ITO method. Then the distribution probability formula of CIBS score between two individuals with different relationships when a certain number of genetic markers were used for relationship identification was derived based on the multinomial distribution theory. The formula was compared with the CIBS probability distribution formula based on binomial distribution theory. Results Between individuals with a certain relationship, labelled as RS, the probabilities of IBS=2, 1 and 0 occurring at a certain autosomal genetic marker x （that is, p2（RSx）, p1（RSx） and p0（RSx））, can be calculated based on the allele frequency data of that genetic marker and the probability of two individuals with the corresponding RS relationship sharing 0, 1 or 2 identity by descent （IBD） alleles （that is, φ0, φ1 and φ2）. For a genotyping system with multiple independent genetic markers, the distribution of CIBS score between pairs of individuals with relationships other than parent-child can be deducted using the averages of the 3 probabilities of all genetic markers （that is, p2（RS）, p1（RS） and p0（RS））, based on multinomial distribution theory. Conclusion The calculation of CIBS score distribution formula can be extended to all kinships and has great application value in case interpretation and system effectiveness evaluation. In most situations, the results based on binomial distribution formula are similar to those based on the formula derived in this study, thus, there is little difference between the two methods in actual work.

Diversification of the downy mildew resistance gene pool by introgression of a new gene, Pl35, from wild Helianthus argophyllus into oilseed and confection sunflowers (Helianthus annuus L.).

KEY MESSAGE: We have mapped a new downy mildew resistance gene, Pl35, derived from wild Helianthus argophyllus to sunflower linkage group 1. New germplasms incorporating the Pl35 gene were developed for both oilseed and confection sunflower Sunflower downy mildew (DM), caused by the oomycete pathogen Plasmopara halstedii, is an economically important and widespread sunflower disease worldwide. Non-race-specific resistance is not available in sunflower, and breeding for DM resistance relies on race-specific resistance to control this disease. The discovery of the novel DM resistance genes is a long-term task due to the highly virulent and aggressive nature of the P. halstedii pathogen, which reduces the effectiveness of resistance genes. The objectives of this study were to: (1) transfer DM resistance from a wild sunflower species Helianthus argophyllus (PI 494576) into cultivated sunflowers; (2) map the resistance gene; and (3) develop diagnostic single-nucleotide polymorphism (SNP) markers for efficient targeting of the gene in breeding programs. The H. argophyllus accession PI 494576 previously identified with resistance to the most virulent P. halstedii race 777 was crossed with oilseed and confection sunflower in 2012. Molecular mapping using the BC2F2 and BC2F3 populations derived from the cross CONFSCLB1/PI 494576 located a new resistance gene Pl35 on linkage group 1 of the sunflower genome. The new gene Pl35 was successfully transferred from PI 494576 into cultivated sunflowers. SNP markers flanking Pl35 were surveyed in a validation panel of 548 diversified sunflower lines collected globally. Eleven SNP markers were found to be diagnostic for Pl35 SNP alleles, with four co-segregating with Pl35. The developed oilseed and confection germplasms with diagnostic SNP markers for Pl35 will be very useful resources for breeding of DM resistance in sunflower.

High-throughput genotyping-by-sequencing facilitates molecular tagging of a novel rust resistance gene, R 15 , in sunflower (Helianthus annuus L.).

KEY MESSAGE: A novel rust resistance gene, R 15 , derived from the cultivated sunflower HA-R8 was assigned to linkage group 8 of the sunflower genome using a genotyping-by-sequencing approach. SNP markers closely linked to R 15 were identified, facilitating marker-assisted selection of resistance genes. The rust virulence gene is co-evolving with the resistance gene in sunflower, leading to the emergence of new physiologic pathotypes. This presents a continuous threat to the sunflower crop necessitating the development of resistant sunflower hybrids providing a more efficient, durable, and environmentally friendly host plant resistance. The inbred line HA-R8 carries a gene conferring resistance to all known races of the rust pathogen in North America and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments of 140 F2 individuals derived from a cross of HA 89 with HA-R8, rust resistance in the population was found to be conferred by a single dominant gene (R 15 ) originating from HA-R8. Genotypic analysis with the currently available SSR markers failed to find any association between rust resistance and any markers. Therefore, we used genotyping-by-sequencing (GBS) analysis to achieve better genomic coverage. The GBS data showed that R 15 was located at the top end of linkage group (LG) 8. Saturation with 71 previously mapped SNP markers selected within this region further showed that it was located in a resistance gene cluster on LG8, and mapped to a 1.0-cM region between three co-segregating SNP makers SFW01920, SFW00128, and SFW05824 as well as the NSA_008457 SNP marker. These closely linked markers will facilitate marker-assisted selection and breeding in sunflower.

Genotyping-by-sequencing targeting of a novel downy mildew resistance gene Pl 20 from wild Helianthus argophyllus for sunflower (Helianthus annuus L.).

KEY MESSAGE: Genotyping-by-sequencing revealed a new downy mildew resistance gene, Pl 20 , from wild Helianthus argophyllus located on linkage group 8 of the sunflower genome and closely linked to SNP markers that facilitate the marker-assisted selection of resistance genes. Downy mildew (DM), caused by Plasmopara halstedii, is one of the most devastating and yield-limiting diseases of sunflower. Downy mildew resistance identified in wild Helianthus argophyllus accession PI 494578 was determined to be effective against the predominant and virulent races of P. halstedii occurring in the United States. The evaluation of 114 BC1F2:3 families derived from the cross between HA 89 and PI 494578 against P. halstedii race 734 revealed that single dominant gene controls downy mildew resistance in the population. Genotyping-by-sequencing analysis conducted in the BC1F2 population indicated that the DM resistance gene derived from wild H. argophyllus PI 494578 is located on the upper end of the linkage group (LG) 8 of the sunflower genome, as was determined single nucleotide polymorphism (SNP) markers associated with DM resistance. Analysis of 11 additional SNP markers previously mapped to this region revealed that the resistance gene, named Pl 20 , co-segregated with four markers, SFW02745, SFW09076, S8_11272025, and S8_11272046, and is flanked by SFW04358 and S8_100385559 at an interval of 1.8 cM. The newly discovered P. halstedii resistance gene has been introgressed from wild species into cultivated sunflower to provide a novel gene with DM resistance. The homozygous resistant individuals were selected from BC2F2 progenies with the use of markers linked to the Pl 20 gene, and these lines should benefit the sunflower community for Helianthus improvement.

Discovery and introgression of the wild sunflower-derived novel downy mildew resistance gene Pl 19 in confection sunflower (Helianthus annuus L.).

KEY MESSAGE: A new downy mildew resistance gene, Pl 19 , was identified from wild Helianthus annuus accession PI 435414, introduced to confection sunflower, and genetically mapped to linkage group 4 of the sunflower genome. Wild Helianthus annuus accession PI 435414 exhibited resistance to downy mildew, which is one of the most destructive diseases to sunflower production globally. Evaluation of the 140 BC1F2:3 families derived from the cross of CMS CONFSCLB1 and PI 435414 against Plasmopara halstedii race 734 revealed that a single dominant gene controls downy mildew resistance in the population. Bulked segregant analysis conducted in the BC1F2 population with 860 simple sequence repeat (SSR) markers indicated that the resistance derived from wild H. annuus was associated with SSR markers located on linkage group (LG) 4 of the sunflower genome. To map and tag this resistance locus, designated Pl 19 , 140 BC1F2 individuals were used to construct a linkage map of the gene region. Two SSR markers, ORS963 and HT298, were linked to Pl 19 within a distance of 4.7 cM. After screening 27 additional single nucleotide polymorphism (SNP) markers previously mapped to this region, two flanking SNP markers, NSA_003564 and NSA_006089, were identified as surrounding the Pl 19 gene at a distance of 0.6 cM from each side. Genetic analysis indicated that Pl 19 is different from Pl 17 , which had previously been mapped to LG4, but is closely linked to Pl 17 . This new gene is highly effective against the most predominant and virulent races of P. halstedii currently identified in North America and is the first downy mildew resistance gene that has been transferred to confection sunflower. The selected resistant germplasm derived from homozygous BC2F3 progeny provides a novel gene for use in confection sunflower breeding programs.

An innovative SNP genotyping method adapting to multiple platforms and throughputs.

KEY MESSAGE: An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility. Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3' regions to significantly increase the amplification specificity of the two alleles and tailed at 5' ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion-deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

Relocation of a rust resistance gene R 2 and its marker-assisted gene pyramiding in confection sunflower (Helianthus annuus L.).

KEY MESSAGE: The rust resistance gene R 2 was reassigned to linkage group 14 of the sunflower genome. DNA markers linked to R 2 were identified and used for marker-assisted gene pyramiding in a confection type genetic background. Due to the frequent evolution of new pathogen races, sunflower rust is a recurring threat to sunflower production worldwide. The inbred line Morden Cross 29 (MC29) carries the rust resistance gene, R 2 , conferring resistance to numerous races of rust fungus in the US, Canada, and Australia, and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments and SSR marker analyses on the 117 F2 individuals derived from a cross of HA 89 with MC29 (USDA), R 2 was mapped to linkage group (LG) 14 of the sunflower, and not to the previously reported location on LG9. The closest SSR marker HT567 was located at 4.3 cM distal to R 2 . Furthermore, 36 selected SNP markers from LG14 were used to saturate the R 2 region. Two SNP markers, NSA_002316 and SFW01272, flanked R 2 at a genetic distance of 2.8 and 1.8 cM, respectively. Of the three closely linked markers, SFW00211 amplified an allele specific for the presence of R 2 in a marker validation set of 46 breeding lines, and SFW01272 was also shown to be diagnostic for R 2 . These newly developed markers, together with the previously identified markers linked to the gene R 13a , were used to screen 524 F2 individuals from a cross of a confection R 2 line and HA-R6 carrying R 13a . Eleven homozygous double-resistant F2 plants with the gene combination of R 2 and R 13a were obtained. This double-resistant line will be extremely useful in confection sunflower, where few rust R genes are available, risking evolution of new virulence phenotypes and further disease epidemics.

Pl(17) is a novel gene independent of known downy mildew resistance genes in the cultivated sunflower (Helianthus annuus L.).

KEY MESSAGE: Pl 17, a novel downy mildew resistance gene independent of known downy mildew resistance genes in sunflowers, was genetically mapped to linkage group 4 of the sunflower genome. Downy mildew (DM), caused by Plasmopara halstedii (Farl.). Berl. et de Toni, is one of the serious sunflower diseases in the world due to its high virulence and the variability of the pathogen. DM resistance in the USDA inbred line, HA 458, has been shown to be effective against all virulent races of P. halstedii currently identified in the USA. To determine the chromosomal location of this resistance, 186 F 2:3 families derived from a cross of HA 458 with HA 234 were phenotyped for their resistance to race 734 of P. halstedii. The segregation ratio of the population supported that the resistance was controlled by a single dominant gene, Pl 17. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) primers were used to identify molecular markers linked to Pl 17. Bulked segregant analysis using 849 SSR markers located Pl 17 to linkage group (LG) 4, which is the first DM gene discovered in this linkage group. An F2 population of 186 individuals was screened with polymorphic SSR and SNP primers from LG4. Two flanking markers, SNP SFW04052 and SSR ORS963, delineated Pl 17 in an interval of 3.0 cM. The markers linked to Pl 17 were validated in a BC3 population. A search for the physical location of flanking markers in sunflower genome sequences revealed that the Pl 17 region had a recombination frequency of 0.59 Mb/cM, which was a fourfold higher recombination rate relative to the genomic average. This region can be considered amenable to molecular manipulation for further map-based cloning of Pl 17.

BCL10 as a new candidate gene for immune response in pigs: cloning, expression and association analysis.

BCL10 is an apoptotic regulatory molecule identified through its direct involvement in t(1; 14)(p22; q32) of mucosa-associated lymphoid tissue lymphoma, and was implicated in the pathogenesis of this and several other tumour types. BCL10 was recognized as an antigen receptor-specific regulator of NF-kappaB, which showed close association with immune responses. In this study, we cloned and characterized BCL10 from the porcine spleen and analysed its genomic structure. BCL10 was mapped to SSC4q21-q23 by the IMpRH panels, it is closely linked to the marker S0161 and SW1461. This gene has three exons and two introns. Reverse transcriptase-polymerase chain reaction analyses showed that BCL10 was widely expressed in all the examined tissues. Transient transfection indicated that porcine BCL10 was located in cytoplasm in Pig Kidney Epithelial cells. BCL10 gene displays the opposite expression trend between the two treatments mimic virus and bacteria of polyriboinosinic-polyribocytidylic acid (Poly I:C) and lipopolysaccharide (LPS). The level of the BCL10 mRNA was up-regulated during 12-24 h and peaking at 48 h when treated with LPS, whereas it was down-regulated during 0-48 h and highest at 0 h (cells without treating with Poly I:C) when treated with Poly I:C. One single nucleotide polymorphism (SNP) site was identified in the 3'-untranslated region of porcine BCL10. Association analysis revealed that this SNP was significantly associated with intermediate cell mass (eosinophile granulocyte, basophile granulocyte and histoleucocyte) percentage, absolute intermediate cell mass count and mean red blood cell volume of 0-day-old pigs, and red blood cell count of 17-day-old pigs (P < 0.05), and also had significant associations with red blood cell count and haemoglobin concentration of 32-day-old pigs (P < 0.01).

The in vivo micronucleus test in human capillary blood lymphocytes: methodological studies and effect of ageing.

The in vivo micronucleus test in lymphocytes of human capillary blood collected by skin puncture is described. This method needs only 1-2 drops of finger blood. The normal value of micronucleus frequencies in lymphocytes from 250 healthy persons aged 6-88 years was determined. The upper limits of normal values were estimated by means of percentile. The 95th percentiles were 1/1000 and 1.5/1000 for the 6-45-year age group and the 46-88-year age group, respectively. There was no significant difference in micronucleus frequency between men and women. On the basis of micronucleus assays in more than 3000 cases, we consider that the micronucleus test in human capillary blood lymphocytes is a rapid, convenient and sensitive procedure for monitoring a human population exposed to environmental and occupational mutagens and carcinogens.

Comparative studies on genotoxicity and antigenotoxicity of natural and synthetic beta-carotene stereoisomers.

To evaluate the practical value of natural beta-carotene (NbetaC) and to elucidate the apparent discrepancy between epidemiological observations and intervention trials on the role of beta-carotene (betaC) in tumor prevention, the genotoxicity and the antigenotoxicity of NbetaC and synthetic betaC crystal (SbetaCC) stereoisomers were studied comparatively using chromosome aberration analysis and the micronucleus test in human lymphocytes in vitro. NbetaC was extracted from the halotolerant algae Dunaliella salina. The NbetaC crystal (NbetaCC) preparation is about 70% all-trans (TbetaC) and 8% 9-cis (CbetaC). The NbetaC oil (NbetaCO) preparation is about 40% all-trans and 38% 9-cis. SbetaCC is more than 97% all-trans, and the 9-cis can not be detected. The mixture of betaC (betaCM) preparation is 74% SbetaCC and 26% NbetaC. Our results show no genotoxicity of 1-30 microg/ml NbetaCC, but this concentration of NbetaCC inhibited significantly gamma-ray-induced micronucleus formation in human lymphocytes in vitro. One to thirty microg/ml NbetaCO was most effective against both gamma-ray-induced and spontaneous micronucleus formation. However, no influence of NbetaCO on spontaneous chromosome aberrations in human lymphocytes in vitro was observed. NbetaCO suppressed significantly mitomycin C (MMC)-induced chromosome aberrations. One to thirty microg/ml SbetaCC induced a dose-dependent increase in micronucleus frequency, and also inhibited gamma-ray-induced micronucleus formation. No effect of betaCM on spontaneous chromosome aberrations was found. One to thirty microg/ml betaCM is more effective against MMC-induced chromosome aberrations than NbetaCO. These results suggest that CbetaC might play a critical role in the genotoxicity and antigenotoxicity of SbetaCC and NbetaC. The genotoxic activity of SbetaCC might be involved in carcinogenesis. NbetaC or betaCM could be of practical value in tumor prevention and supplementary treatment.

Suppressing effects of human fetal cell extract on micronuclei induced by cyclophosphamide in mice.

The genotoxicity of human fetal cell extract (HFCE) and its effect on the frequency of micronucleated polychromatic erythrocytes (PCE-MNF) in mice induced by cyclophosphamide (CP) were studied. Statistically significant differences were not found between the control group and each group treated with HFCE (0.3, 3, 30 mg/kg bw). CP (200 mg/kg bw) induced a marked increase in MNF (P < 0.01). Administered together with CP, HFCE suppressed the increase of MNF induced by CP. The reduction effect is dependent on the dose of HFCE. At doses of 3 and 30 mg/kg bw HFCE, MNF decreased markedly (P < 0.05 and < 0.01, respectively). It showed that HFCE did not induce micronucleus formation, while it could suppress the micronucleus formation induced by CP in mice. The results suggested that HFCE might be antimutagenic and have potential value in clinical application.

[Comparative studies of cytokinesis-block and conventional micronucleus test in human lymphocytes cultured in vitro. II. Genotoxicological effects of mitomycin C].

The authors have studied genotoxicological effects of MMC by means of CB- and C-MNT to evaluate practical value of CB-MNT. The main results are as follows: (1) In 0.025-0.4 microgram/ml of MMC MNFs in lymphocytes detected by CB- and C-MNT presented dose-dependent increase, but the MNT detected by C-MNT was higher than that by CB-MNT, this difference became greater as MMC concentrations were increased. The minimum concentrations were 0.1 microgram/ml for CB-MNT and 0.025 microgram/ml for C-MNT respectively. Therefore C-MNT is more sensitive to MMC than to CB-MNT; (2) The mean volume of MN detected by CB- and C-MNT was estimated separately. The volume of MN detected by CB-MNT was about 3 times as large as that by C-MNT. The authors suggested that the fusion between micronuclei in cytoplasm of CB- cells could lead to increase of MN volume and decrease of MN numbers; (3) In healthy donors the background MNF in lymphocytes detected by CB-MNT was significantly higher than that by C-MNT, it could be one of the reasons that led to the decrease of the sensitivity of CB-MNT to low doses of mutagens. Finally, the practical value of CB- and C-MNT was discussed.

[Comparative studies on several indices of nuclear damage in human peripheral lymphocytes].

In order to use appropriately NAT and explore possibility of use of the test in human cells, the authors used gamma-rays as mutagenic and carcinogenic irradiated whole blood in vitro, prepared directly smears of isolated lymphocytes, and studied comparatively dose-response relationship of 4 types of nuclear damage. Main results of the study are given as follows: 1. In doses from 0 to 3 Grays MNF (Frequency of micronucleus), INF (Frequency of irregular nucleus), KNF (Frequency of karyorrhetic nucleus), and PNF (Frequency of pyknotic nucleus) increase along with increase of irradiated doses. At 5 grays INF still increases, but MNF, KNF and PNF decrease. 2. In doses from 0 to 3 grays regression equation of 4 indices of nuclear damage are as follows (D means dose, r means correlative coefficient): MNF: y = 0.3433 + 0.1052D, r = 0.9128, p less than 0.05; INF: y = 7.2178 + 2.1817D, r = 0.8846, p less than 0.05; KNF: y = 0.6462 + 0.2014D, r = 0.7286, p greater than 0.05; PNF: y = 0.2774 + 0.0728D, r = 0.5484, p greater than 0.05. 3. In doses from 0 to 3 grays MNF is linear with INF and KNF. Regression equation are as follows (x means MNF): MNF-INF: y = 2.0598 + 16.6545x, r = 0.778 p greater than 0.05; MNF-KNF: y = 0.2235 + 1.426x, r = 0.5945, p greater than 0.05. Finally according to synthetic analysis of correlative coefficient, intercept, regression coefficient and feasibility of indices of nuclear damage, we could consider it suitable that nuclear anomalies in human peripheral lymphocytes include micronucleus, irregular nucleus and karyorrhexis.

[Preliminary studies of the relationship between micronucleus formation and cell cycle].

For the study of the relationship between micronucleus formation and chromosome aberration. Lymphocytes of human peripheral blood were treated with MMC and chromosome aberrations and micronuclei which produced at various phases of cell cycle were observed. For the study of the main results are as follows: (1) the MMC induced increase in the frequency of aberration cells at metaphase (ACF), that of the frequency of micronucleated cells at G1, S and G2 phases (cultured in vitro, C-MNCF) and that of the frequency of micronucleated cells at G0 phase (Non-cultured, NC-MNCF) are all dose-dependent and their dose-response relation can be described by power regression; (2) the MMC-induced micronucleus formation at G0 phase is not related to chromosome aberration, but the change of NC-MNCF is positively related to the change of CAF as same as C-MNCF; (3) the MMC-induced MNCFs detected in binucleated cells by the cytokinesis block method increase slightly as compared with C-MNCFs, and the ratio of MNCF/ACF is less (about 0.1) and decreases along with the increase of MMC concentrations. The results suggest that the relationship between micronucleus formation and chromosome aberration may be quite complex, that chromosome aberration could be one of the cause of micronucleus formation.

[Effects of rhubarb on electrical and contractive activities of the isolated intestine in rats].

The results indicate that electrical activity of colon is obviously excited by rhubarb (Rheum tanguticum). The characteristics of excitement is cybotaxis discharge. It has been found that the frequency and amplitude of spike potential increase obviously, and the contraction enhances. Spike potential frequency of duodenum excited by rhubarb is increased but that of jejunum is hardly excited. It has also been found that the exciting effect of rhubarb on colon can be blocked by atropine.

[Studies on technology of supercritical-CO2 fluid extraction for volatile oils and saikosaponins in Bupleurum chinense DC].

OBJECTIVE: To study the technology of supercritical-CO2 fluid extraction (SFE-CO2) for the volatile oils and saikosaponins in Bupleurum chinense. METHOD: Exploring the effects of pressure, temperature, extraction time, flow rate of CO2 and entrainers on the yield of the oils and saikosaponin-contained extracts; determining the optimum conditions for SFE-CO2; analyzing the oils by GC/MS and comparing the technology of SFE-CO2 with that of traditional steam distillation. RESULT: The optimum extraction conditions turned out to be--for volatile oils: pressure (EP) = 20 MPa, temperature (ET) = 30 degrees C, isolator I pressure (1P-I) = 12 MPa, temperature(1T-I) = 65 degrees C, isolator II pressure (1P-II) = 6 MPa, temperature (1T-II) = 40 degrees C, extraction time = 4 hours, and CO2 flow rate = 10-20 kg.(h.kg)-1 crude drug; for saikosaponins: EP = 30 MPa, ET = 65 degrees C, 1P I = 12 MPa, 1T I = 55 degrees C, 1P II = 6 MPa, 1T II = 43 degrees C, extraction time = 3 hours, entrainer = 60% ethanol, and CO2 flow rate = 20-25 kg.(h.kg)-1 crude drug. CONCLUSION: SFE-CO2 excels the traditional steam distillation in raising yield and reducing extraction time. The oils are composed of 22 constituents including caproaldehyde, and the saikosaponins can only be extracted with the help of entrainers under higher pressure and temperature.