Vegetative compatibility and genetic analysis of Colletotrichum lindemuthianum isolates from Brazil

The causal agent of common bean anthracnose, Colletotrichum lindemuthianum, has considerable genetic and pathogenic variability, which makes the development of resistant cultivars difficult. We examined variability within and between Brazilian pathotypes of C. lindemuthianum through the identification of vegetative compatibility groups (VCGs) and by RAPD analysis. Two hundred and ninety-five nit mutants were obtained from 47 isolates of various pathotypes of the fungus collected from different regions, host cultivars and years. In complementation tests, 45 VCGs were identified. Eighteen RAPD primers were employed in the molecular analyses, producing 111 polymorphic bands. Estimates of genetic similarities, determined from the Sorence-Dice coefficient, ranged from 0.42 to 0.97; the dendrogram obtained by cluster analysis revealed 18 groups of isolates. RAPD and VCG markers presented high genotypic diversity. The number of significant associations (P=0.05) between RAPD, VCG and pathogenicity markers ranged from 0 (VCG) to 80% (pathogenicity). The test of multilocus association (rd) for RAPD markers was significantly different from zero (P<0.001), suggesting linkage disequilibrium. However, the results for VCG markers show the presence of recombination mechanisms. In conclusion, RAPD markers and VCGs were useful for detecting genetic variability among isolates of C. lindemuthianum. We found considerable diversity among isolates from the same geographic origin within a short interval; this suggests rapid evolution. There is a need for further studies to elucidate the population structure of this pathogen in agro-ecosystems.     

Genetic and molecular characterization of pathogenic isolates of Pyricularia grisea from wheat (Triticum aestivum Lam.) and triticale (x Triticosecale Wittmack) in the state of Paraná, Brazil

Isolates of Pyricularia grisea from wheat (Triticum aestivum Lam.) and triticale (x Triticosecale Wittmack) spikes with blast symptoms were analyzed by classical (VCG) and molecular (RAPD) techniques. P. grisea mutants, unable to use sodium nitrate (nit) as nitrogen source, were obtained with potassium chlorate. For vegetative compatibility (VCG) tests, genetically complementary nit mutant pairs were inoculated in a medium with sodium nitrate as a single nitrogen source. P. grisea isolates were divided into two vegetative compatibility groups and two RAPD groups. Since vegetative compatible strains may mutually exchange genetic and cytoplasmatic material, the contribution of the parasexual cycle in the genetic variability of Brazilian P. grisea isolates is discussed.     

Vegetative Compatibility Grouping of Colletotrichum kahawae in Kenya

Vegetative compatibility using nitrate nonutilizing ( nit ) mutants was analysed between 44 isolates of Colletotrichum kahawae from Kenya, one each from Ethiopia and Malawi, one of Colletotrichum gloeosporioides and one of Colletotrichum acutatum . Another isolate of C. kahawae did not generate mutants and thus could not be utilized. The results showed that all the C. kahawae isolates, except a white sector mutant (VCG2), belonged to one vegetative compatibility group (VCG4). The other species belonged to their own unique groups (VCGs 1 and 3). Implications of the results and future research needs on the subject are discussed.     

新月弯孢菌Curvularia lunata营养体亲和性的进一步研究

来自全国39个地区的70株新月弯孢菌Curvularialunata在WAC培养基上诱导培养后,随机挑取10197个抗KClO3突变体,经CDA鉴定获得2207株nit突变体,nit突变体频率为21.64%。在这些nit突变体中,1397个为nit1,占63.30%;734个为NitM,占33.26%;76个为nit3,占3.44%。70个菌株全部获得了稳定的nit突变体,其中52个菌株获得了NitM突变体。结果表明WAC比先前报道的KPS更适合用于C.lunatanit突变体的筛选。通过不同菌株间互补nit突变体配对测试,将其中的65个C.lunata菌株划分为22个营养体亲和群(VCGS),而另外5个菌株因未获得NitM突变体暂时无法确定其VCG。划分出的22个VCGs中,有11个VCGs是由多菌株组成的,VCG3为优势类群,含18个菌株,其地理来源最复杂,主要为致病性中等以上的菌株;其余11个VCGs内均仅有1个自身亲和的菌株。以上结果初步表明,在C.lunata群体内存在丰富的VCG多样性,VCG3可能是与致病性相关的优势VCG,但营养体亲和性与菌株地理来源没有明显的直接关系。     

炭疽菌不利用硝酸盐突变体的筛选及鉴定

将从杉木和大叶黄杨上分离获得的８个胶孢炭疽菌（Colletoctrichum gloeosporioides）分离物培养在含氯酸钾的平板上,得到快速生长抗氯酸钾的不利用硝酸盐的突变体(Nit)。所有的突变体经鉴定分属于３种表现型,即硝酸还原酶结构位点（nit１）,硝酸盐同化途径的专化调节位点（nit３）,和钼辅因位点（nitM）。分离物发生突变的频率随氯酸钾浓度的增加而提高,并且不同的氮源在一定程度上会影响突变表型种类。除ＣＣ３外,所有的分离物都是自身亲和的,即不同表型的突变体能遗传互补,其     

Vegetative compatibility groups in indigenous and mass-released strains of the entomopathogenic fungus Beauveria bassiana: likelihood of recombination in the field

Using nitrate non-utilizing (nit) mutants, we determined vegetative compatibility groups (VCG) among strains of Beauveria bassiana representing strains indigenous to North America, isolated from diverse insect hosts, and strains that have been mass released for insect control. Genetic similarity among these strains was analyzed using random amplified polymorphic DNA (RAPD) markers. Our data revealed 23 VCGs among the 34 strains tested, with most of these groups comprised of only a single strain. We also observed a VCG comprised of eight genetically similar strains isolated from Colorado potato beetles (CPB). Co-inoculation studies of CPB larvae with complementary nit mutants from the same or from different VCGs revealed heterokaryosis in four out of five same-VCG pairs, with only 5-17% of the sporulating cadavers generating few parasexual recombinants. In contrast, none of the infected beetles treated with non-compatible pairs generated recombinants. The large number of VCGs observed and the low frequency of in vivo recombination limited to vegetatively compatible strains indicate that this self/non-self recognition system may be an effective barrier preventing genetic exchange between dissimilar strains in the field.     

Characterization of Fusarium oxysporum f.sp. cepae from Onion in Turkey Based on Vegetative Compatibility and rDNA RFLP Analysis

Seventy‐five isolates of Fusarium oxysporum f.sp. cepae, the causal agent of basal plate rot on onion, were obtained from seven provinces of Turkey. The isolates were characterized by vegetative compatibility grouping (VCGs) and restriction fragment length polymorphism (RFLP) analysis of the nuclear ribosomal DNA intergenic spacer region (IGS). Forty‐eight vegetative compatibility groups were found, each containing a single isolate. Only one isolate formed strong heterokaryons with the reference isolates of VCG 0423. Five isolates were heterokaryon self‐incompatible. Restriction fragment analysis with six different enzymes revealed 13 IGS types among 75 F. oxysporum isolates from Turkey as well as 16 reference isolates from Colorado, USA. The majority of single‐member VCGs produced identical RFLP banding patterns with minor deviations, considerably different from those of the reference VCG isolates. These results suggested that isolates of F. oxysporum f.sp. cepae in Turkey derived from distinct clonal lineages and mutations at one or more vegetative compatibility loci restrict heterokaryon formation.     

Vegetative compatibility and pathogenecity of Verticillium dahliae kleb. isolates from olive in Iran

Verticillium wilt caused by Verticillium dahliae is a serious problem of olive trees leading to significant reduction in yield. Verticillium wilt of olive trees was first recorded in Iran 1996 and confirm as due to Verticillium dahliae Kleb. 101 isolates of V. dahliae from olive trees at deferent locations in north provinces of Iran were assigned to vegetative compatibility groups (VCGS), using nitrate non-utilizing (Nit) mutants. A higher frequency of nit 1/nit 3 mutants (93%) was obtained compared with NitM (7%) with 10% of the isolates being assigned to VCG1 and 51% VCG4B and 19% VCG2A. 20% of isolates could not be classified in standard isolates. The pathogenecity of 15 randomly selected isolates (5 of each VCG) was tested on olive seedling (cv. Zard) and eggplant. The VCGs isolates were similarly aggressive on olive. However, VCG1 isolates were more aggressive on eggplant cv. Local than the VCG2A and VCG4B isolates as indicated by a higher colonization index. The pathogenecity tests of the pathogen on test plants (cotton cv. 'sahel', eggplant cv. 'local' and tomato cv. 'ps') show all isolates category in 2 pathogenecity groups defoliate and non-defoliate (with severe and mild subgroups). The morphology of V. dahliae isolates on C'zapeck's agar and water agar medium were different especially for microsclerotia appearance time in culture and their morphology.     

Vegetative Compatibility Among Isolates of Colletotrichum gloeosporioides from Yam (Dioscorea spp.) in Nigeria

Isolates of Colletotrichum gloeosporioides obtained from yam‐based cropping systems in Nigeria, previously characterized on the basis of morphology, virulence and rDNA internal transcribed spacer (ITS) sequence variation were further compared for vegetative compatibility (VC). Chlorate‐resistant nitrate non‐utilizing (nit) mutants were generated from the isolates and used in complementation (heterokaryon) tests. Tests of VC between complementary mutants from different isolates indicated the presence of several genotypes within a single field, suggesting limited clonal spread. In some cases, isolates obtained from the same lesion were observed to belong to different vegetative compatibility groups (VCGs). No compatibility was observed between isolates of the highly virulent slow‐growing grey (SGG), the moderately virulent fast‐growing salmon (FGS) and the avirulent/weakly virulent fast‐growing grey (FGG) strains. Forty‐one C. gloeosporioides isolates belonged to 28 VCGs, giving a genotype diversity estimate of 0.68. This diversity confirmed the high variability of the pathogen population as revealed by previous characterization studies, however, a correlation between VCGs and isolate groupings based on morphology and virulence was not found. The finding that an isolate from weed was compatible with yam isolates indicated that transfer of important traits, such as virulence, may take place between isolates from yam and non‐yam hosts. The VCG diversity revealed by this study suggests that in addition to asexual reproduction, sexual reproduction may play an important role in the epidemiology of anthracnose on yam.     

尖孢镰孢一新硝酸盐营养突变类型与营养体亲和性

从73个尖孢镰孢(Fusarium oxysporum)不同专化型菌株上获得684个硝酸盐营养突变株(nit mutant)。作相关氮源利用试验及亚硝酸反应后,鉴定出一新硝酸盐营养突变类型:亚硝酸盐还原酶结构基因类型,命名为nit8,占总突变株的6.7%。同时被鉴别的还有nit1、nit3和Nit M三种突变类型,它们分别占突变株总数的81.0%,3.8%和8.5%。此外,首次引入一种亚硝酸反应在这类研究中的应用,还提出了互补指数概念与公式来表示nit突变株营养体之间亲和的能力。     

Characterization of Colletotrichum acutatum causing anthracnose of anemone (Anemone coronaria L.)

Anthracnose, or leaf-curl disease of anemone, caused by Colletotrichum sp., has been reported to occur in Australia, western Europe, and Japan. Symptoms include tissue necrosis, corm rot, leaf crinkles, and characteristic spiral twisting of floral peduncles. Three epidemics of the disease have been recorded in Israel: in 1978, in 1990 to 1993, and in 1996 to 1998. We characterized 92 Colletotrichum isolates associated with anthracnose of anemone (Anemone coronaria L.) for vegetative compatibility (72 isolates) and for molecular genotype (92 isolates) and virulence (4 isolates). Eighty-six of the isolates represented the three epidemics in Israel, one isolate was from Australia, and five isolates originated from western Europe. We divided these isolates into three vegetative-compatibility groups (VCGs). One VCG (ANE-A) included all 10 isolates from the first and second epidemics, and 13 of 62 examined isolates from the third epidemic in Israel, along with the isolate from Australia and 4 of 5 isolates from Europe. Another VCG (ANE-F) included most of the examined isolates (49 of the 62) from the third epidemic, as well as Colletotrichum acutatum from strawberry, in Israel. Based on PCR amplification with species-specific primers, all of the anemone isolates were identified as C. acutatum. Anemone and strawberry isolates of the two VCGs were genotypically similar and indistinguishable when compared by arbitrarily primed PCR of genomic DNA. Only isolate NL-12 from The Netherlands, confirmed as C. acutatum but not compatible with either VCG, had a distinct genotype; this isolate represents a third VCG of C. acutatum. Isolates from anemone and strawberry could infect both plant species in artificial inoculations. VCG ANE-F was recovered from natural infections of both anemone and strawberry, but VCG ANE-A was recovered only from anemone. This study of C. acutatum from anemone illustrates the potential of VCG analysis to reveal distinct subspecific groups within a pathogen population which appears to be genotypically homogeneous by molecular assays.     

Analysis of single nucleotide polymorphisms in three genes shows evidence for genetic isolation of certain Aspergillus flavus vegetative compatibility groups

Genetic exchange by asexual filamentous fungi is presumed to be limited to isolates in the same vegetative compatibility group (VCG). To evaluate genetic isolation of Aspergillus flavus due to vegetative incompatibility, three gene regions were chosen that contained closely spaced nucleotides that were polymorphic among some of the six VCGs examined. A member of each VCG was collected from five regions across the southern United States. Isolates belonging to the same VCG had similar sets of single nucleotide polymorphisms regardless of isolate origin. The six VCGs formed four genetically distinct groups. Although recombination between certain pairs of VCGs could not be excluded, none was found for YV36, the VCG that includes the atoxigenic A. flavus isolate currently used to mitigate aflatoxin contamination in cotton in Arizona.     

Comparisons of Isolates of Fusarium avenaceum from White Lupin and Other Crops by Pathogenicity Tests,DNA Analyses and Vegetative Compatibility Tests

Isolates of Fusarium avenaceum, mostly from crops of white lupin or wheat, were tested for pathogenicity on white lupin and wheat plants and compared by DNA tests and, in a limited study, vegetative compatibility. Most of the 80 isolates were pathogenic on both plant species after inoculation on shoot bases. Disease severity was greater at higher incubation temperatures that ranged from 15/10°C to 25/20°C (day/night temperatures). Isolates from lupin crops tended to be more pathogenic, on average, on lupins than on cereals. Polymerase chain reaction (PCR)‐restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer region of the rDNA distinguished two groups of isolates that occurred in different proportions among isolates from lupins and cereal crops. Random amplified polymorphic DNA (RAPD)‐PCR analyses indicated considerable genetic variation among isolates, but there was some similarity among groups of isolates from populations in the same field. Genetic diversity was confirmed by a high degree of vegetative incompatibility among 20 isolates using nitrate nonutilizing mutants. There were no relationships among pathogenicity, RFLP group, RAPD group and vegetative compatibility group.     

Application of RAPD-PCR for Virulence Type Analysis within Verticillium dahliae and V. longisporum

A collection of 24 isolates of Verticillium dahliae, 11 isolates of V. longisporum and one isolate of V. albo‐atrum originating from different host plants and geographical regions was tested for genetic variability by random amplified polymorphic DNA‐polymerase chain reaction (RAPD–PCR). Based on nine primers, the three Verticillium species could be clearly differentiated. Likewise, this analysis provided a distinct separation of vegetative compatibility groups (VCG) 2B, 4A and 4B of V. dahliae by specific DNA banding patterns. Additionally, V. longisporum was found to segregate into two subgroups with only 88% similarity. This molecular‐genetic approach was used for the analysis of randomly selected Verticillium isolates from a field with high intensity of oilseed rape cultivation (33% in crop rotation). RAPD‐PCR analysis revealed that 95 of 100 isolates tested belonged to V. longisporum and 5 to VCG 2B of V. dahliae. This study demonstrates an adaptation of Verticillium soil populations to a specific cropping history.     

Molecular differences distinguish clonal lineages within East African populations of Fusarium oxysporum f.sp. cubense

Molecular approaches for the assessment of intraspecific diversity within an economically important plant pathogen were compared with traditional physiological methods (vegetative compatibility testing). The vegetative compatibility groups (VCGs) of 14 isolates of Fusarium oxysporum f.sp. cubense (FOC) from Kenya were first assessed using nitrate non-utilizing mutants. Nine of these isolates, from different areas of the country, were compatible with one or more of VCGs 0124, 0125, 0128 and 01220, i.e. they formed a single clonal lineage. Three isolates, all originating from the banana growing district of Kisii, were compatible with the VCG 01212 and formed a second distinct clonal lineage. Mutants could not be recovered from one isolate (62) and two isolates (27 and 30) were not vegetatively compatible with any of the VCG testers and may represent two novel VCGs. Polymerase chain reaction (PCR) fingerprinting, especially when using the M13 derived primer, was found to produce banding patterns that correlated with clonal lineage and also distinguished isolates 27 and 30 when analysed by unweighted pair group method analysis and principle co-ordinate analysis. This approach also distinguished FOC from F. oxysporum IMI350438 isolated from Triticum sp. and from isolates of Colletotrichum gloeosporioides . Total protein profiles were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and although clonal lineages were not separated, isolates 27 and 30 were again distinguishable and FOC produced a different profile to F. oxysporum (IMI 350438) and C. gloeosporioides.     

Characterization of Colletotrichum acutatum Causing Anthracnose of Anemone (Anemone coronaria L.)

Anthracnose, or leaf-curl disease of anemone, caused by Colletotrichum sp., has been reported to occur in Australia, western Europe, and Japan. Symptoms include tissue necrosis, corm rot, leaf crinkles, and characteristic spiral twisting of floral peduncles. Three epidemics of the disease have been recorded in Israel: in 1978, in 1990 to 1993, and in 1996 to 1998. We characterized 92 Colletotrichum isolates associated with anthracnose of anemone (Anemone coronaria L.) for vegetative compatibility (72 isolates) and for molecular genotype (92 isolates) and virulence (4 isolates). Eighty-six of the isolates represented the three epidemics in Israel, one isolate was from Australia, and five isolates originated from western Europe. We divided these isolates into three vegetative-compatibility groups (VCGs). One VCG (ANE-A) included all 10 isolates from the first and second epidemics, and 13 of 62 examined isolates from the third epidemic in Israel, along with the isolate from Australia and 4 of 5 isolates from Europe. Another VCG (ANE-F) included most of the examined isolates (49 of the 62) from the third epidemic, as well as Colletotrichum acutatum from strawberry, in Israel. Based on PCR amplification with species-specific primers, all of the anemone isolates were identified as C. acutatum. Anemone and strawberry isolates of the two VCGs were genotypically similar and indistinguishable when compared by arbitrarily primed PCR of genomic DNA. Only isolate NL-12 from The Netherlands, confirmed as C. acutatum but not compatible with either VCG, had a distinct genotype; this isolate represents a third VCG of C. acutatum. Isolates from anemone and strawberry could infect both plant species in artificial inoculations. VCG ANE-F was recovered from natural infections of both anemone and strawberry, but VCG ANE-A was recovered only from anemone. This study of C. acutatum from anemone illustrates the potential of VCG analysis to reveal distinct subspecific groups within a pathogen population which appears to be genotypically homogeneous by molecular assays.     

Vegetative compatibility of an isolate ofVerticillium dahliae pathogenic to both tomato and pepper

An isolate ofVerticillum dahliae Vdp-4, pathogenic to both tomato and pepper (tomato-pepper pathotype), was examined for its vegetative compatibility with testers of the Japanese vegetative compatibility group (subgroups J1, J2, and J3). Seven isolates ofV. dahliae from the same field as Vdp-4 in Misato, Nagano Pref. and two isolates from Hokkaido were separately determined as either tomato pathotype (B) or pepper pathotype (C). Isolate 5922 previously reported as tomato-pepper pathotype was also examined. Compatiblenit1 and NitM mutants were obtained from all isolates except for isolates Vdp-3 and Vdt-10. The isolate of tomato-pepper pathotype Vdp-4 showed a strong reaction with VCGJ1 and J3 and was thus assigned to J3. Seven of these isolates showed compatibility and were assigned into three provisional subgroups. The isolate 5922 was self-incompatible.     

Intraspecific Variation within Populations of Fusarium oxysporum Based on RFLP Analysis of the Intergenic Spacer Region of the rDNA

Appel, D. J., and Gordon, T. R. 1995. Intraspecific variation within populations of Fusarium oxysporum based on RFLP analysis of the intergenic spacer region of the rDNA. Experimental Mycology 19, 120-128. Fifty-six isolates of Fusarium oxysporum, including F. oxysporum f. sp. melonis and nonpathogenic strains, were chosen from a larger collection to represent diversity in vegetative compatibility groups (VCGs), mitochondrial DNA (mtDNA) haplotype, geographic distribution, and virulence. Using PCR, a 2.6-kb fragment including the intergenic spacer (IGS) region of the ribosomal DNA was amplified from each isolate. The enzymes EcoRI, Sau 3A, Cfo1, and Ava1I, cut this fragment differentially, revealing 5, 6, 6, and 7 patterns, respectively. Among the 56 isolates, a total of 13 unique IGS haplotypes was identified. Among most F. o. melonis isolates. IGS haplotype correlated with VCG and mtDNA haplotype, but did not differentiate among races. However, a race 1 isolate found in VCG 0131 shared virulence, mtDNA, and IGS haplotypes characteristic of VCG 0134; this isolate may represent a conversion in VCG from 0134 to 0131. Four nonpathogens shared the pathogen vegetative compatibility phenotypes. One race 1,2 isolate associated with VCG 0134 shared both IGS haplotype and VCG with a nonpathogen, but these isolates did not share the same mtDNA haplotype. Another nonpathogenic isolate shared mtDNA and IGS haplotypes with pathogen group 0131 and may simply be an avirulent mutant of a pathogenic strain. For the other two nonpathogenic isolates, vegetative compatibility indicated a close relationship to the pathogen, but differences in both mtDNA and IGS haplotype suggest otherwise. Overall, the IGS haplotype was more variable among the nonpathogenic F. oxysporum VCGs among which 12 of the 13 IGS haplotypes were found. Nonpathogenic isolates that shared a common mtDNA haplotype, but were associated with different VCGs, often had different IGS haplotypes.     

Vegetative Compatibility,Pathogenicity and Virulence Diversity of Fusarium oxysporum f. sp. melongenae Recovered from Eggplant

Fusarium oxysporum (Schlechtend.: Fr.) f. sp. melongenae (Fomg) recovered from symptomatic eggplants from five eggplant‐growing areas in Turkey, including the south, west, north‐west, north and south‐east regions. The objective of this study was to investigate the genetic diversity of the Fomg isolates from different geographical location by pathogenicity and VCG tests. Three hundred and seventy‐four Fomg isolates were classified as highly virulent, virulent, moderately virulent and low virulent through pathogenicity assays. No correlation was observed between virulence of Fomg isolates and their locations. The nitrate non‐utilizing mutants (nit) were generated as nit1, nit3 and NitM, based on phenotyping of Fomg growth characteristics of the Fomg isolates on diagnostic media with various sources of nitrogen. The majority of nit mutants (39.4%) recovered were nit1 from minimal medium (MM) containing of 2.0% potassium chlorate (MMC). The most of Fomg isolates were identified as heterokaryon self‐compatible (HSC) based on their ability to form a stable heterokaryon, while four isolates were classified as heterokaryon self‐incompatible (HSI). A large amount of Fomg isolates were vegetatively compatible and assigned as members of the same VCG, whereas nit mutants of 10 Fomg isolates that did not complement with tester strains only paired by themselves (HSC), these isolates were termed vegetative incompatible (vic). The complementation of 33 isolates with tester strains was slow and quite weak, but not paired with themselves even though they are HSC. About 96.3% of the Fomg isolates were assigned to VCG 0320, while the remaining 3.7% were classified as vegetative incompatible group.     

Widespread vegetative compatibility groups in the dry-rot fungus Serpula lacrymans

Serpula lacrymans is the most notorious decayer of wooden buildings in temperate regions. The occurrence of geographically widespread vegetative compatibility groups (VCG) in S. lacrymans in Europe is demonstrated in this study. Among 22 heterokaryotic isolates of S. lacrymans, five VCG were found. The most widespread VCG included isolates from Belgium, south and central Norway, separated by more than 1500 km. No other genetic variation, measured as DNA sequence variation or ISSR polymorphisms, was detected between the investigated S. lacrymans isolates, whereas a considerable level of genetic variation was found among five European isolates of the sister taxon, S. himantioides. It is hypothesised that isolates of S. lacrymans have lost their ability to recognize self from nonself due to sharing of similar VC alleles, caused by a recent genetic bottleneck during the establishment in northern Europe. Isolates re-isolated from overlapping mycelial zones between different compatible isolates had significantly slower growth than that of the original isolates and the different isolates within a VCG had different growth morphology, indicating that isolates within a single VCG may belong to different genets.