Tracking the adoption of bread wheat varieties in Afghanistan using DNA fingerprinting.

BACKGROUND: Wheat is the most important staple crop in Afghanistan and accounts for the main part of cereal production. However, wheat production has been unstable during the last decades and the country depends on seed imports. Wheat research in Afghanistan has emphasized releases of new, high-yielding and disease resistant varieties but rates of adoption of improved varieties are uncertain. We applied DNA fingerprinting to assess wheat varieties grown in farmers' fields in four Afghan provinces. RESULTS: Of 560 samples collected from farmers' fields during the 2015-16 cropping season, 74% were identified as varieties released after 2000, which was more than the number reported by farmers and indicates the general prevalence of use of improved varieties, albeit unknowingly. At the same time, we found that local varieties and landraces have been replaced and were grown by 4% fewer farmers than previously reported. In 309 cases (58.5%), farmers correctly identified the variety they were growing, while in 219 cases (41.5%) farmers did not. We also established a reference library of released varieties, elite breeding lines, and Afghan landraces, which confirms the greater genetic diversity of the landraces and their potential importance as a genetic resource. CONCLUSIONS: Our study is the first in wheat to apply DNA fingerprinting at scale for an accurate assessment of wheat varietal adoption and our findings point up the importance of DNA fingerprinting for accuracy in varietal adoption studies.

Radiation hybrid QTL mapping of Tdes2 involved in the first meiotic division of wheat.

Since the dawn of wheat cytogenetics, chromosome 3B has been known to harbor a gene(s) that, when removed, causes chromosome desynapsis and gametic sterility. The lack of natural genetic diversity for this gene(s) has prevented any attempt to fine map and further characterize it. Here, gamma radiation treatment was used to create artificial diversity for this locus. A total of 696 radiation hybrid lines were genotyped with a custom mini array of 140 DArT markers, selected to evenly span the whole 3B chromosome. The resulting map spanned 2,852 centi Ray with a calculated resolution of 0.384 Mb. Phenotyping for the occurrence of meiotic desynapsis was conducted by measuring the level of gametic sterility as seeds produced per spikelet and pollen viability at booting. Composite interval mapping revealed a single QTL with LOD of 16.2 and r (2) of 25.6 % between markers wmc326 and wPt-8983 on the long arm of chromosome 3B. By independent analysis, the location of the QTL was confirmed to be within the deletion bin 3BL7-0.63-1.00 and to correspond to a single gene located ~1.4 Mb away from wPt-8983. The meiotic behavior of lines lacking this gene was characterized cytogenetically to reveal striking similarities with mutants for the dy locus, located on the syntenic chromosome 3 of maize. This represents the first example to date of employing radiation hybrids for QTL analysis. The success achieved by this approach provides an ideal starting point for the final cloning of this interesting gene involved in meiosis of cereals.

Development of wild barley (Hordeum chilense)-derived DArT markers and their use into genetic and physical mapping.

Diversity arrays technology (DArT) genomic libraries were developed from H. chilense accessions to support robust genotyping of this species and a novel crop comprising H. chilense genome (e.g., tritordeums). Over 11,000 DArT clones were obtained using two complexity reduction methods. A subset of 2,209 DArT markers was identified on the arrays containing these clones as polymorphic between parents and segregating in a population of 92 recombinant inbred lines (RIL) developed from the cross between H. chilense accessions H1 and H7. Using the segregation data a high-density map of 1,503 cM was constructed with average inter-bin density of 2.33 cM. A subset of DArT markers was also mapped physically using a set of wheat-H. chilense chromosome addition lines. It allowed the unambiguous assignment of linkage groups to chromosomes. Four segregation distortion regions (SDRs) were found on the chromosomes 2H(ch), 3H(ch) and 5H(ch) in agreement with previous findings in barley. The new map improves the genome coverage of previous H. chilense maps. H. chilense-derived DArT markers will enable further genetic studies in ongoing projects on hybrid wheat, seed carotenoid content improvement or tritordeum breeding program. Besides, the genetic map reported here will be very useful as the basis to develop comparative genomics studies with barley and model species.

Development and assessment of DArT markers in triticale.

Triticale (X Triticosecale Wittm.) is a hybrid derived by crossing wheat (Triticum sp.) and rye (Secale sp.). Till date, only a limited number of simple sequence repeat (SSRs) markers have been used in triticale molecular analyses and there is a need to identify dedicated high-throughput molecular markers to better exploit this crop. The objective of this study was to develop and evaluate diversity arrays technology (DArT) markers in triticale. DArT marker technology offers a high level of multiplexing. Development of new markers from triticale accessions was combined with mining the large collection of previously developed markers in rye and wheat. Three genotyping arrays were used to analyze a collection of 144 triticale accessions. The polymorphism level ranged from 8.6 to 23.8% for wheat and rye DArT markers, respectively. Among the polymorphic markers, rye markers were the most abundant (3,109) followed by wheat (2,214) and triticale (719). The mean polymorphism information content values were 0.34 for rye DArT markers and 0.37 for those from triticale and wheat. High correlation was observed between similarity matrices derived from rye, triticale, wheat and combined marker sets, as well as for the cophenetic values matrices. Cluster analysis revealed genetic relationships among the accessions consistent with the agronomic and pedigree information available. The newly developed triticale DArT markers as well as those originated from rye and wheat provide high quality markers that can be used for diversity analyses and might be exploited in a range of molecular breeding and genomics applications in triticale.

Assessment of genotoxic damage in nurses occupationally exposed to antineoplastics by the analysis of chromosomal aberrations.

To estimate the genotoxic risk of occupational exposure to antineoplastic drugs, chromosomal aberration (CAs) frequencies in peripheral lymphocytes were determined for 20 nurses handling antineoplastics and 18 referents matched for age and sex. Urinary cyclophosphamide (CP) excretion rates, which are used as a marker for drug handling, were also measured on these nurses. We have observed significant frequencies of CAs (about 2.5-fold increase) including chromatid breaks, gaps, and acentric fragments for nurses handling antineoplastics as compared to control subjects (p < 0.05, p < 0.01, excluding and including gaps, respectively). The mean value of CP excretion rate for 12 nurses was 1.63 microg/24 h, suggesting that when the nurses handled CP (and other antineoplastic drugs) this particular compound was absorbed. Our study has shown that increased genetic damage was evident in nurses, at population level, due to occupational exposure to antineoplastics. Until the effects of handling antineoplastics from low-level exposure are known, it will be important to keep the exposure to a minimum.

Ribozyme genes protecting transgenic melon plants against potyviruses.

Potyviruses are the most important viral pathogens of crops worldwide. Under a contract with Gene Shears Pty Limited, we are using ribozyme genes to protect melon plants against two potyviruses: WMV2 and ZYMV. Different polyribozyme genes were designed, built and introduced into melons plants. Transgenic melon plants containing a resistance gene were obtained and their progeny was challenged by the appropriate virus. Most of the genes tested conferred some degree of resistance to the viruses in glasshouse trials. Melon plants from one family containing one gene directed against WMV2 were also field-trialed on small plots under natural infection pressure and were found immune to WMV2. Field trial is in progress for plants containing genes against ZYMV. Some of the ribozyme genes used in the plants were also assayed in a transient expression system in tobacco cells. This enabled us to study the sequence discrimination capacity of the ribozyme in the case of one ribozyme target site. We found that a mutated target GUG (non cleavable) was less susceptible to inhibition by the ribozyme gene than the corresponding wild type target GUA (cleavable). Work is now in progress to incorporate multiple resistance genes in melon plants, in constructs designed in compliance with the evolving European regulations concerning transgenic plants. The use of ribozyme genes to protect plants against viruses provides an alternative to the technologies currently used for protecting crops against viruses, based on the concept of Pathogen Derived Resistance (see for example 14). In the light of concerns expressed by some plant virologists (13) about the use of viral genes in transgenic plants, it may be that ribozyme genes will find many uses in this area of agricultural biotechnology.

Frequency of HPRT mutants in humans exposed to vinyl chloride via an environmental accident.

The mutant frequency (MF) in the hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) locus of peripheral blood T-lymphocytes was measured in a population environmentally exposed to vinyl chloride - a toxic and carcinogenic substance through an accidental release into the atmosphere. It was compared to MF in a control group of unexposed individuals. Both groups were re-investigated in a follow-up study, 2 years later. No significant difference could be observed in MF between exposed and controls either at the accident nor in the follow-up study. Approximately the same mean HPRT mutant frequencies were observed for both groups in T-lymphocytes from blood samples obtained shortly after the accident and from the follow-up blood samples. Both groups showed a higher mean MF in the re-investigation samples which is most probably due to the significantly lower average cloning efficiency (CE) under non-selective conditions and because of the inverse relationship between CE and MF. The exposed population showed a higher mean T-cell CE at the initial blood sampling as compared to the control group. The concurrent cytogenetic analyses of peripheral lymphocytes showed a significant increase in cells with aberrations in the exposed population. Clastogenic but not mutagenic activity of vinyl chloride was observed in our study.

Chromosomal aberrations in humans as genetic endpoints to assess the impact of pollution.

The chromosomal aberration assay with peripheral blood lymphocytes has been used routinely during the last three decades to survey exposure of humans to various genotoxic agents. A large number of biomonitoring studies are based on this genetic endpoint. A great deal of data exists on occupational, life-style or medical exposure situations but less evidence of the validity of the assay is available with regards to environmental exposure. In the present paper we report our investigations on the impact of pollution in two different populations using chromosomal aberrations in human peripheral blood lymphocytes as a biomarker of chronic exposure to heavy metals and dioxins/furans for a long period and as a biomarker of acute exposure to accidentally released vinyl chloride in the air. In order to study genotoxic effects (chromosomal aberrations) of heavy metals and dioxins/furans, 52 exposed individuals from a polluted area were compared to 51 matched controls from a distant non-industrialized area. A statistically significant increase was observed in the frequency of chromosomal aberrations in peripheral blood lymphocytes from the exposed population (1.90% aberrant cells vs. 1.11% for the controls). In the case of the vinyl chloride accident, chromosomal aberrations were analyzed in peripheral blood lymphocytes from 29 potentially exposed and 29 non-exposed individuals (matched controls). The exposed group showed a statistically significant increase in the frequency of aberrant cells (1.47% vs. 1.07% for the controls).

A follow-up study on chromosomal aberrations in lymphocytes of alcoholics during early, medium, and long-term abstinence.

The frequencies of structural chromosomal aberrations were analyzed in peripheral blood lymphocytes of 31 chronic alcoholics at the beginning of an intensive outpatient treatment program at a neuropsychiatric clinic and were compared with 31 controls matched for gender, age, smoking habits, and nondrinkers. A statistically significant difference was observed in the level of chromosomal aberrations in somatic cells from alcoholics when compared with controls (3.01% vs. 1.28%, p < or = 0.001). A follow-up study was carried out for a subset of the patients after 3 months (8 subjects) and 12 months (14 subjects) of controlled abstinence. A statistically significant increase in the mean frequency of cells with aberrations was observed in the group of 14 subjects reinvestigated after 12 months of abstinence when compared with the mean value of the first blood samples immediately after hospitalization (4.61% vs. 3.01%; p < or = 0.001). An excessive increase in cigarette consumption during alcohol abstinence, reflected by a dramatic elevation of CO-hemoglobin levels, may, at least in part, account for this finding. In conclusion, chronic alcoholism leads to genotoxic effects that, instead of recovering after 1 year of alcohol abstinence, are even enhanced, most likely due to the "shift in addictive behavior."

Validation of the human T-lymphocyte cloning assay--ring test report from the EU concerted action on HPRT mutation (EUCAHM).

The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P = 0.0004) and lnMF (P = 0.03), but there was no significant laboratory effect on the lnCE (P = 0.38) or lnMF (P = 0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations.

Cytogenetic analysis of peripheral lymphocytes in a population exposed to vinyl chloride through an accidental release into the environment.

On the first of June, 1996 an environmental accident occurred in Schönebeck, Germany in which free vinyl chloride was evaporated into the atmosphere. Thereby, the human population living in this area was exposed to vinyl chloride and its byproducts. Chromosomal aberrations were measured in peripheral blood lymphocytes from 29 potentially exposed and 29 non-exposed (control) individuals. Both groups were matched regarding age, gender and smoking habits. Two hundred metaphases were analysed for chromosomal aberrations per each individual. The exposed group showed a statistically significant increase in the mean frequency of aberrant cells (1.47% versus 1.07% in the control group). Chromosomal aberrations in peripheral lymphocytes have been shown to be a very sensitive biomarker of genotoxic effects not only for occupational exposure to vinyl chloride as reported several times during the last 20 years, but also for an accidental environmental exposure. A follow-up cytogenetic study is recommendable.

Transfer RNA-mediated suppression of amber stop codons in transgenic Arabidopsis thaliana.

An artificial amber suppressor tRNA(Leu) gene (supL) was physically linked to a mutated gus reporter gene, p35S-gus(amL), which was inactivated by an amber stop codon (amL). Upon introduction into Arabidopsis thaliana, the presence of the supL gene was found to be correlated with cytotoxic effects observed during tissue culture and in mature plants. Those primary transformants that displayed cytotoxic symptoms were shown by X-Glu staining to express GUS as a result of amber stop codon suppression in vivo. Phenotypically normal lines were found by RT-PCR to express supL. GUS activity above background level was barely detectable in these plants, indicating a low level expression of supL. However, the remaining suppressor activity was still sufficient to transactivate an amber-mutated male sterility gene, pA9-barnase(amL1) when combined within the same plant by crossing. The suppressor tRNA(Leu) gene may thus be used in transgenic plants for gene transactivation.

HPRT mutant frequencies and detection of large deletions by multiplex-PCR in human lymphocytes of vinyl chloride exposed and non-exposed populations.

The frequency of hypoxanthine guanine phosphoribosyltransferase (HPRT) mutations was determined in human T-lymphocytes isolated from peripheral blood of three populations: (1) 24 employees occupationally exposed to vinyl chloride monomer; (2) 23 healthy non-exposed matched control individuals and (3) 41 regular blood donors. In addition, mutant clones of all studied groups were examined by multiplex-PCR for visible abnormalities of the gene (large deletions). Surprisingly, the mutation frequency of all three investigated populations was approximately the same (7-8 x 10(-6)). However, great differences occurred for the spectrum of mutants. Among the mutant clones of the non-exposed healthy individuals only 6% (blood donors) and 11% (matched control group) showed large deletions. The corresponding percentage of large deletions in the occupationally exposed group was, at 21%, much higher.

Adaptive and synergistic effects of a low-dose ENU pretreatment on the frequency of chromosomal aberrations induced by a challenge dose of ENU in human peripheral blood lymphocytes in vitro.

N-Ethyl-N-nitrosourea (ENU) is an alkylating agent whose mutagenic and carcinogenic potential has been extensively studied but its ability to induce cytogenetic adaptive responses in normal human cells has not been investigated so far. The aim of our present experiments was to study the effect of a pretreatment with a low concentration of ENU (2 x 10(-5) M) on the frequency of chromosomal aberrations induced by a subsequent 50 times higher concentration of ENU (10(-3) M) in human lymphocytes isolated from buffy coats of 4 donors. Two different inter treatment times and three harvesting times were applied to the lymphocytes from each donor. A cytogenetic adaptive response was shown by the lymphocytes of one donor only when the time span between the low adapting and the higher challenging concentration was 4 h. The other three donors did not respond with significant differences in the yield of cells with aberrations. The complex interaction between the ENU-induced multiple primary DNA lesions and various DNA repair mechanisms as well as the influence of cell cycle effects on the induction of clastogenic adaptive response are discussed.

AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth.

To understand better the role of genes in controlling ovule development, a female-sterile mutant, aintegumenta (ant), was isolated from Arabidopsis. In ovules of this mutant, integuments do not develop and megasporogenesis is blocked at the tetrad stage. As a pleiotropic effect, narrower floral organs arise in reduced numbers. More complete loss of floral organs occurs when the ant mutant is combined with the floral homeotic mutant apetala2, suggesting that the two genes share functions in initiating floral organ development. The ANT gene was cloned by transposon tagging, and sequence analysis showed that it is a member of the APETALA2-like family of transcription factor genes. The expression pattern of ANT in floral and vegetative tissues indicates that it is involved not only in the initiation of integuments but also in the initiation and early growth of all primorida except roots.

Frequency of HPRT mutant lymphocytes in a human control population as determined by the T-cell cloning procedure.

The T-cell-cloning assay was established to determine the frequency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutant lymphocytes in the presence of the selective agent 6-thioguanine in peripheral blood from a human control population. We investigated 44 healthy adults (blood donors) and found a mean mutant frequency of 7.2 x 10(-6) (geometric mean 5.6 x 10(-6). An elevated mean mutant frequency occurred in smokers as compared to non-smokers. However, a statistically significant increase was only observed between female smokers and female non-smokers while there was only a slight difference in the male group. A significant difference in mutant frequency could be found between individuals younger than 35 years and those above 35. But the difference of the mutant frequency with age showed up only among smokers. No significant effect of the gender was observed. Mutant frequency was inversely related to the cloning efficiency.

Expression of the tobacco Tnt1 retrotransposon promoter in heterologous species.

The expression of the tobacco (Nicotiana tabacum) retrotransposon Tnt1 has previously been shown to be strongly regulated and driven from the 5' long terminal repeat (LTR). We report here that the Tnt1 LTR can promote activity of the beta-glucuronidase (GUS) reporter gene in two heterologous species of the Brassicaceae family, namely rapeseed (Brassica napus) and Arabidopsis thaliana. The translational LTR-GUS fusion was active in transient expression studies performed with tobacco and rapeseed protoplasts, indicating that the LTR sequences are recognized in heterologous species. Our results also showed that Tnt1 LTR-promoted GUS expression in transgenic Arabidopsis is strongly regulated, and that, in contrast to tobacco, hormonal activation plays a significant role in the expression of the Tnt1 LTR in Arabidopsis. LTR sequences were shown to be more effective than the CaMV 35S enhancer region in transient expression studies performed with tobacco or rapeseed protoplasts, and substitution of the LTR sequences upstream from the major transcriptional start with the CaMV 35S enhancer region gave high levels of expression in transgenic tobacco and Arabidopsis leaves, suggesting that a Tnt1 element with similar substitutions in its 5' LTR might be suited for gene-tagging experiments in heterologous species.

Genotoxicity studies in semiconductor industry. 1. In vitro mutagenicity and genotoxicity studies of waste samples resulting from plasma etching.

Solid waste samples taken from the etching reactor, the turbo pump, and the waste air system of a plasma etching technology line in semiconductor production were studied as to their genotoxic properties in a bacterial repair test, in the Ames/Salmonella microsome assay, in the SOS chromotest, in primary mouse hepatocytes, and in Chinese hamster V79 cell cultures. All three waste samples were found to be active by inducing of unscheduled DNA-synthesis in mouse hepatocytes in vitro. In the bacterial rec-type repair test with Proteus mirabilis, waste samples taken from the turbo pump and the vacuum pipe system were not genotoxic. The waste sample taken from the chlorine-mediated plasma reactor was clearly positive in the bacterial repair assay and in the SOS chromotest wit Escherichia coli. Mutagenic activity was demonstrated for all samples in the presence and absence of S9 mix made from mouse liver homogenate. Again, highest mutagenic activity was recorded for the waste sample taken from the plasma reactor, while samples collected from the turbo pump and from the waste air system before dilution and liberation of the air were less mutagenic. For all samples chromosomal damage in V79 cells was not detected, indicating absence of clastogenic activity in vitro. Altogether, these results indicate generation of genotoxic and mutagenic products as a consequence of chlorine-mediated plasma etching in the microelectronics industry and the presence of genotoxins even in places distant from the plasma reactor. Occupational exposure can be expected both from the precipitated wastes and from chemicals reaching the environment with the air stream.

Specific expression of the tobacco Tnt1 retrotransposon in protoplasts.

The Tnt1 transposable element of tobacco belongs to the retrotransposon family and shares the structural features of viral retroelements including two long terminal repeats (LTRs) which are known to contain promoter regions. We show that two Tnt1 RNAs of 5.2 and 6.5 kb can be found. The 5.2 kb RNA matches with the size of the Tnt1 elements so far isolated (5.3 kb), whilst the evidence suggests that the 6.5 kb RNA could be a chimaeric RNA initiated in a gene in which Tnt1 has inserted. The Tnt1 5.2 kb RNA starts in the LTR, and the LTR can promote the expression of a translational LTR-beta-glucuronidase (GUS) fusion at a high level in transient expression assays. The Tnt1 5.2 kb RNA and the LTR-GUS fusion of transgenic tobacco plants are specifically expressed in leaf-derived protoplasts whereas they are not expressed in leaf tissue. The 5.2 kb RNA is also transcribed at low levels in roots. This RNA is induced after 2 h of maceration in the protoplast isolation medium, and its level declines rapidly after protoplast isolation. The induction requires only the presence of cell wall hydrolases, and is independent of wounding and plasmolysis. The induction of Tnt1 expression is not mediated by typical oligosaccharide elicitors released from the cell wall known to mediate defense gene responses. Tnt1 transcription features provide a first example of tissue culture-induced mutagenesis in plants and a molecular basis for some of the somaclonal variation events.