- Record: found
- Abstract: found
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Maize Transposable Elements Ac/ Ds as Insertion Mutagenesis Tools in Candida albicans
research-article
Author(s):
Kevin Mielich
* ,
Ella Shtifman-Segal
† ,
Julia C. Golz
* ,
Guisheng Zeng
‡ ,
Yue Wang
‡ ,
Judith Berman
† ,
Reinhard Kunze
*
,
2
Publication date (Electronic):
29 January 2018
Journal:
G3: Genes|Genomes|Genetics
Publisher:
Genetics Society of America
There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
In nonmodel systems, genetic research is often limited by the lack of techniques for the generation and identification of gene mutations. One approach to overcome this bottleneck is the application of transposons for gene tagging. We have established a two-element transposon tagging system, based on the transposable elements Activator ( Ac)/ Dissociation ( Ds) from maize, for in vivo insertion mutagenesis in the fungal human pathogen Candida albicans. A nonautonomous Ds transposon carrying a selectable marker was constructed into the ADE2 promoter on chromosome 3 and a codon usage-adapted Ac transposase gene was inserted into the neutral NEUT5L locus on chromosome 5. In C. albicans cells expressing the transposase, the Ds element efficiently excised and reintegrated elsewhere in the genome, which makes the Ac/ Ds transposons promising tools for saturating insertion mutagenesis in clinical strains of C. albicans.
Most cited references39
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A hyperactive piggyBac transposase for mammalian applications.
Kosuke Yusa, Liqin Zhou, Meng Amy Li … (2011)
DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. Among the transposons active in mammalian cells, the moth-derived transposon piggyBac is most promising with its highly efficient transposition, large cargo capacity, and precise repair of the donor site. Here we report the generation of a hyperactive piggyBac transposase. The active transposition of piggyBac in multiple organisms allowed us to screen a transposase mutant library in yeast for hyperactive mutants and then to test candidates in mouse ES cells. We isolated 18 hyperactive mutants in yeast, among which five were also hyperactive in mammalian cells. By combining all mutations, a total of 7 aa substitutions, into a single reading frame, we generated a unique hyperactive piggyBac transposase with 17-fold and ninefold increases in excision and integration, respectively. We showed its applicability by demonstrating an increased efficiency of generation of transgene-free mouse induced pluripotent stem cells. We also analyzed whether this hyperactive piggyBac transposase affects the genomic integrity of the host cells. The frequency of footprints left by the hyperactive piggyBac transposase was as low as WT transposase (~1%) and we found no evidence that the expression of the transposase affects genomic integrity. This hyperactive piggyBac transposase expands the utility of the piggyBac transposon for applications in mammalian genetics and gene therapy.
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The ‘obligate diploid’ Candida albicans forms mating-competent haploids
Meleah A. Hickman, Guisheng Zeng, Anja Forche … (2013)
Candida albicans, the most prevalent human fungal pathogen, was considered an obligate diploid that carried recessive lethal mutations throughout the genome. Here, we demonstrate that C. albicans has a viable haploid state that can be derived from diploid cells under in vitro and in vivo conditions and appears to arise via a concerted chromosome loss mechanism. Haploids undergo morphogenetic changes like those of diploids including the yeast-hyphal transition, chlamydospore formation, and a white-opaque switch that facilitates mating. Haploid opaque cells of opposite mating type mate efficiently to regenerate the diploid form, restoring heterozygosity and fitness. Homozygous diploids arise spontaneously by auto-diploidization and both haploids and auto-diploids display a similar reduction in fitness, in vitro and in vivo, relative to heterozygous diploids, suggesting that homozygous cell types are transient in mixed populations. Finally, we constructed stable haploid strains with multiple auxotrophies that will facilitate molecular and genetic analyses of this important pathogen. Background The opportunistic fungal pathogen C. albicans has been studied extensively since the 1800’s and has been considered a strictly diploid organism with no haploid state. The diploid nature of the organism has complicated genetic and genomic analyses of C. albicans biology and virulence. Studies in the 1960’s through the early 1980’s debated the ploidy of C. albicans, and proposed that a haploid state existed based on cell size heterogeneity 1 , parasexual genetics 2 and estimates of DNA content by fluorimetry 1,3 . Haploid clinical isolates were reported 2,3 , but were later found to be diploid in genome content or were shown to be non-albicans Candida species (T. Suzuki, personal comm. and Fig. S1). Research supporting the diploid nature of the organism included DNA content measurements that suggested a genome size similar to that of diploid Saccharomyces cerevisiae 4,5 , molecular studies that demonstrated the need to delete two gene copies to produce a null mutant 6,7 , genetic studies that demonstrated heterozygosity of specific alleles 8,9 , and ultimately the complete genome sequence, which revealed heterozygosity throughout much of the genome 10 . This, together with the predominantly clonal nature of C. albicans within an individual host 11,12 , was interpreted as evidence that the organism spent most, if not all, of its life cycle in a diploid state. The ‘obligate diploid’ nature of C. albicans was proposed to be due to recessive lethal mutations dispersed throughout the genome 13,14 . However, studies of C. albicans chromosome monosomy 15,16 , recombination 17 and haplotype mapping 18 demonstrated that homozygosis of certain chromosomes can occur, arguing against this balanced lethal mutation hypothesis. A diploid-tetraploid parasexual cycle was also discovered in C. albicans and shown to involve a switch to the ‘opaque’ physiological state that renders cells mating-competent 19,20 , conjugation between opaque diploids to form tetraploids 21,22 , and subsequent ploidy reduction resulting in diploid progeny that often carry multiple aneuploid chromosomes 18,23 . Importantly, specific aneuploid chromosomes can provide a selective advantage under stressful conditions such as exposure to antifungal drugs 24 . Parasexual ploidy reduction in tetraploids occurs via a non-meiotic process termed ‘concerted chromosome loss’. This process can facilitate the rapid generation of diversity through the production of homozygous and aneuploid progeny, under conditions where outcrossing in the host is unlikely 25 . If a haploid state for C. albicans were to exist, it could facilitate the elimination of lethal alleles from the population. Furthermore, mating between different haploids could promote adaptation to changing conditions within the mammalian host. With the advent of whole genome approaches that can distinguish ploidy states, it is now possible to ask if C. albicans exists, even transiently, in the tetraploid or the haploid state. Detection of haploid C. albicans cells Haploid C. albicans was serendipitously discovered during experiments to follow loss of heterozygosity (LOH) at multiple independent loci. Using a multiply marked derivative of SC5314, the laboratory reference strain 10 , we selected for an initial LOH event at the GAL1 locus by growth on 2-deoxygalactose (2-DOG) and subsequently screened for additional LOH events at four other heterozygous loci. Importantly, growth in 2-DOG does not affect LOH rates (Fig. S2) and as such, the selection for cells with an LOH event does not artificially induce LOH. Amongst the ~2500 Gal− isolates, 42 exhibited additional LOH events and intriguingly one strain, Haploid I, was homozygous for all of the markers tested, as well as for multiple SNPs on every chromosome. Analysis of DNA content in Haploid I by flow cytometry indicated that the genome had half the amount of DNA of that in a diploid control (Fig. 1a). Once we became aware that haploid C. albicans cells could be detected, we used flow cytometry to screen isolates from many sources, including in vitro stresses, 26 from which one haploid isolate (Haploid IV) was identified amongst small colonies growing in the presence of fluconazole (in the halo of an E-test strip), a commonly used antifungal drug. We also screened C. albicans cells isolated from in vivo mouse models of candidemia 27 and candidiasis 28 and discovered several additional isolates with 1N DNA content. The overall population of post-in vivo isolates screened for ploidy included ~300 isolates from YPD plates (Gal+), and ~740 isolates from the 2-DOG selection plates (Gal−) and only the DOG-resistant colonies contained isolates that were haploid. We found that 3.2% of DOG-resistant colonies from the OPC model were haploid (10 out of 312), while 1.2% of DOG-resistant colonies from the systemic model were haploid (5 out of 431). Since LOH frequencies in vivo are ~10−3, 29 , this indicates that detectable haploids appeared at a frequency of 1–3 per 100,000 cells. Loss of GAL1 is often, but not always associated with the haploid state (e.g. Haploid IV), especially in diploid progenitors that are heterozygous for this locus. In total, we characterized eleven strains with haploid (or close to haploid) flow cytometry profiles (Fig. 1a, S3 and Table S1) that were derived from multiple independent and varied sources. Since previous reports of haploid C. albicans 3 included misidentified species (Fig. S1), we determined if these newly identified isolates were bona fide C. albicans by PCR amplifying and sequencing the mating-type-like (MTL) locus. All isolates contained sequences identical to either the MTL a or MTLα alleles of SC5314 (Fig. S4 and data not shown). Next, we analyzed the haploid genomes by hybridization to SNP/CGH arrays 30 which measure allelic ratios and also infer relative chromosome copy numbers. Six isolates were completely euploid (Fig. 1b and S5); in the remaining five haploids, one or two chromosomes (Chr6 and Chr7) were disomic. These disomic chromosomes were heterozygous except in one isolate (Haploid IV) where the disomic chromosome (Chr6) was homozygous. Based upon the C. albicans haplotype map 30 , each haploid chromosome primarily contained alleles from only one parental homolog with few, if any, obvious crossovers detectable (Fig. 1b and S5). This implies that haploids did not arise through conventional meiosis, which usually requires at least one crossover per chromosome 31 . The few crossovers detected likely arose by mitotic recombination prior to selection for LOH events. For example, Haploid I was isolated following selection for loss of GAL1, which is within the crossover region on Chr1. Furthermore, in C. albicans haploids (Fig. 1b and S5), the majority of disomic chromosomes were heterozygous, which would appear following segregation of homologs in meiosis I. Such meiosis I segregation is not seen in Drosophila male meioses, which lack recombination 32 ; it is seen in Candida lusitaniae meiotic progeny, which undergo high levels of recombination 33 . The low level of genetic recombination together with the presence of heterozygous disomic chromosomes remains most consistent with random homolog segregation in C. albicans. Accordingly, we propose that haploids arise via a concerted chromosome loss mechanism akin to that described for the diploid-tetraploid C. albicans parasexual cycle 23 , although a non-conventional meiotic program cannot be completely discounted. The existence of haploids refutes the argument that recessive lethal alleles are present on each C. albicans homolog 13,14 . Nonetheless, the absence of some chromosomal homologs from the haploid progeny suggests that recessive lethal alleles may exist. Indeed, only the ‘b’ homologs for Chr3, Chr4, Chr6, Chr7 and most of Chr1 were detected in the haploids analyzed (Fig. 1b and S5). Interestingly, a similar homolog bias was seen in diploid parasexual progeny derived from SC5314 18 . In contrast, homologs of Chr5, which carries the MTL, appeared in equal numbers (six MTL a and five MTLα) and were entirely of one or the other parental haplotype. Similarly, both homologs of ChrR and Chr2 were observed. As such, homologs from more than half the chromosomes (Chr3, Chr4, Chr6, Chr7 and most of Chr1) potentially carry at least one recessive lethal mutation and may limit the frequency with which viable haploids appear. Accordingly, we propose that haploidization may provide an effective mechanism for eliminating recessive lethal mutations from the predominantly diploid populations of C. albicans. Auto-diploidization of haploids In the course of these studies, prolonged propagation of haploid isolates yielded cultures with a mixed population of haploid and diploid cells (Fig. 1c). Subsequent colony purification yielded distinct haploid and/or diploid populations (Fig. 1d). Surprisingly, some haploid isolates also diploidized during the process of strain shipping, which involved storage, transit, and revival from partial dehydration on a solid surface. For example, a potential haploid isolate identified in Taiwan was sent to Minnesota on sterile filter paper. Once revived and grown in liquid culture, the genome was diploid (Fig. 1e) and homozygous for all SNPs (Fig. 1b, ‘Auto-dip.’), suggesting auto-diploidization from a haploid phase. While homozygous diploids could arise through mitotic defects or by self-mating 34 , mating between cells of the same mating-type was not detectable, as discussed below. Thus, we suggest that auto-diploids arise through mitotic defects that may be analogous to the auto-diploidization events in vertebrate haploid stem cell cultures 35–37 . Haploid Morphogenesis and Mating Consistent with reduced ploidy content in other yeast species 38 , haploid C. albicans cells were smaller, on average, for both cell and nuclear size compared to diploid cells (Fig. 2a and S6). The bud site selection patterns of haploids, like diploid C. albicans were predominantly axial, with a small fraction of cells displaying a bipolar budding pattern (Fig. 2b) 39 . Significantly, under conditions that induce morphogenesis of diploid C. albicans to form true hyphae, pseudohyphae, and chlamydospores 40,41 , similar morphogenetic events occurred in haploids (Fig. 2c). C. albicans diploid cells that are homozygous at the MTL can undergo a transcriptionally induced switch to the opaque state that renders them competent for mating 19,42 . Similarly, haploids readily switched from the white (Wh) to the opaque (Op) state, as detected by colony sectoring (Fig. 2d). Haploid opaque cells had elongated opaque-like cell morphology 43 , yet were considerably smaller than diploid opaque cells (Fig. 2d). Importantly, haploid opaque cells of opposite mating type were mating competent. Mating between cells with complementary genetic markers resulted in the formation of heterozygous diploids (Fig. 2e, Op x Op). In contrast, neither opaque cells of the same mating type, nor white cells of opposite mating type, mated efficiently (Fig. 2e). This is consistent with opposite mating types and the opaque state being prerequisites for conventional mating 19,42 . Thus, we propose that haploids participate in a non-meiotic haploid-diploid parasexual cycle. Reduced growth and virulence of haploids The products from haploid a and α mating were heterozygous for MTL (Fig. S7), near-diploid in genome content (Fig. S7), and likely inherited the extra aneuploid copies of Chr6 and/or Chr7 from their MTLα parent. All the haploid isolates grew significantly slower than SC5314 (Fig. 3a and S8, p < 0.001 for all haploids). However, the mating products grew significantly faster than either of their haploid parents (Fig. 3a), as expected if heterozygosity restores fitness through the complementation of recessive alleles 44 . Consistent with this, auto-diploids exhibited growth rates indistinguishable from their haploid progenitors (Fig. 3a and S8), with the single exception of Haploid VIII (p = 0.02). We propose that the low fitness of haploids, as well as their corresponding auto-diploids, is a consequence of unmasking recessive alleles that reduce growth potential. Furthermore, the variability in fitness between haploids is likely due to the specific combination of alleles inherited by each haploid isolate. Significantly, the diploid mating products from haploid crosses grew more slowly than the highly heterozygous diploid SC5314. The generation of robust and diverse diploid progeny from haploid mating would require outcrossing, which is predicted to occur infrequently 11,12 , as many humans are colonized with a single strain of C. albicans. As it appears that some lethal mutations have accumulated on one of the two homologs for more than half the chromosomes (Fig. 1 and S5), mating between related haploids would not reestablish complete genome heterozygosity. Thus, even though the formation of haploids potentially eliminates lethal mutations, continued inbreeding between related haploids would perpetuate homozygosity of most chromosomes and consequently would reduce overall fitness. Consistent with reduced growth rates in vitro, Haploid II was avirulent in a mouse model of systemic candidiasis (Fig. 3b) and was cleared from the mouse after ten days. However, after 48 hours of infection with a haploid strain, cells could be recovered (Fig. S9a) and the majority of these isolates remained haploid. Subsequent in vivo experiments compared the colony forming units (CFUs) of haploids, auto-diploids and SC5314 isolated from mouse kidneys 48 hours post-inoculation in individual and direct competition experiments (Fig. 3c and S9b). The number of CFUs recovered from mice inoculated with haploids and auto-diploids was several orders of magnitude lower than the number of CFUs recovered from mice inoculated with SC5314 (Fig. 3c). Furthermore, competition experiments indicated that haploids and auto-diploids exhibited similar fitness in vivo, whereas both of these forms were outcompeted by heterozygous diploids (Fig. S9b). Similar to growth rates in vitro, the low fitness of haploids and their corresponding auto-diploids in vivo, indicates that it is not the diploid state, per se, that is beneficial to growth but rather extensive allelic heterozygosity, as evidenced with SC5314. From these data, we hypothesize that haploid formation may not be rare, but that haploids likely represent a very small fraction of the overall population due to their low competitive fitness relative to heterozygous diploids. Haploid strains as genetic tools The haploid state greatly facilitates experimental approaches such as classical genetic screens for recessive alleles and a single-round of gene-knockout phenotyping. To illustrate this potential, a set of auxotrophic C. albicans strains was derived from a relatively stable haploid, GZY792 (Fig. 4a and S10) and genes important for morphogenesis in diploids (HGC1, RVS167, SLA1, SEC3 and ACE2) were deleted in a single step. The resulting mutants exhibited morphogenesis defects closely resembling those of the corresponding diploid null mutants (Fig. 4c) 45–48 . Furthermore, because the deleted genes map to all eight chromosomes (Fig. 4b), the ability to delete each gene in a single step confirms that all chromosomes were monosomic in the parental haploid. Concluding remarks In summary, C. albicans can no longer be considered to be an obligate diploid. Rather, it has the ability to form haploids that subsequently mate to form diploids or undergo auto-diploidization. The lower fitness of haploids and auto-diploids suggests they will not persist in the population and thus should be detected only rarely. Nonetheless, efficient mating between viable haploids can produce heterozygous diploids that have increased fitness, presumably due to the complementation of detrimental recessive alleles. Furthermore, the reduction to a haploid state can serve as a vehicle to eliminate recessive lethal alleles from a heterozygous diploid population. We propose that C. albicans rapidly generates genetic diversity by producing a broad range of different ploidy states, including haploid, diploid, tetraploid, and aneuploid 25 . Each of these ploidy states are mating competent and genetic outcrossing can introduce, albeit infrequently, further genetic diversity into the population. While the ploidy reduction mechanism(s) used by C. albicans remain an enigma, the discovery of a haploid form and the potential for a haploid-diploid parasexual cycle significantly expands our ability to manipulate, and thereby better understand, this opportunistic pathogen. Furthermore, this study reveals how whole-genome analyses can lead to a re-evaluation of common assumptions about genomic structure in microbial organisms. Supplemental Methods Haploid screening from the mouse models of infection Bloodstream infections were performed by injecting 106 cells of parent strain AF7 (gal1Δ::URA3/GAL1), into the tail vein of 13 outbred ICR male mice (22–25 g, Harlan, Indianapolis) 27 . When moribund (at 5–7 days), mice were anesthetized using isofluorane, euthanized, and both kidneys were removed. Kidneys were combined, homogenized with 1 mL of water. 1:1000 dilutions of kidney homogenates were plated for total cell count onto YPD, and 1:10 dilutions of the same homogenate were plated onto 2-DOG medium to obtain Gal− colony counts at 3 days. For the oropharyngeal model of infection, Balb/C mice were immunosuppressed with cortisone on days −1, 1 and 3 of infection. Calcium alginate swabs were saturated with a suspension of 106 cells/ml of YJB9318 (gal1Δ::URA3/GAL1) under their tongues for 75 min. Mice were sacrificed at 1, 2, 3, and 5 days post-infection. 1:1000 dilutions of tongue tissue homogenates were plated for total cell count onto YPD, and 1:10 dilutions of the same homogenate were plated onto 2-DOG medium to obtain Gal− colony counts at 3 days. Importantly, to confirm that 2-DOG resistant cells only arose during in vivo passage and were not selected for by plating on 2-DOG medium, a gal1/gal1 strain and a gal1/GAL1 heterozygote strain were plated onto 2-DOG medium and observed for growth. 2-DOG resistant colonies grew up from the gal1/gal1 mutant but not from the GAL1/gal1 heterozygote by day 3 after plating 27 . Therefore, we used a cut-off of day 3 for picking 2-DOG resistant colonies after plating. Approximately 300 colonies were transferred from YPD plates and ~740 colonies from 2-DOG plates (312 from OPC and 431 BSI isolates) into 96-well plates containing 50% glycerol and stored at −80°. All C. albicans cells isolated following in vivo passaging were analyzed by flow cytometry to determine cell ploidy. Flow cytometry preparation and analysis Mid-log phase cells were harvested, washed and resuspended in 50:50 TE (50mM Tris pH8 : 50mM EDTA) and fixed with 95% ethanol. Cells were washed with 50:50 TE and treated with 1 mg/ml RNAse A and then 5 mg/ml Proteinase K. Cells were washed with 50:50 TE and resuspended in SybrGreenI (1:85 dilution in 50:50 TE) incubated overnight at 4°. Stained cells were collected and resuspended in 50:50 TE and analyzed using a FACScaliber. Whole genome ploidy was estimated by fitting DNA content data with a multi-Gaussian cell cycle model that assumes the G2 peak has twice the fluorescence of the G1 peak and that minimizes S-phase cell contribution to the error function. Ploidy values were calculated by comparing the ratio of peak locations in experimental samples to those of diploid and tetraploid controls. Mating Assays Opaque or white cells were mixed together in equal cell numbers and incubated on Spider media 34 for 18 hours prior to replica-plating onto SDC −Ade + Nat (to select for mating products), as well as YPAD, YPAD + Nat, SDC −Ade and SDC −Ade + Nat to detect parental auxotrophies and then photographed 24 hours later. In vitro growth assays Strains were grown in YPD media supplemented with adenine, uridine and histidine in a 96-well microtiter plate and optical density was measured every 15 minutes with a plate reader (Tecan Sunrise) for 24 hours. Doubling times were calculated as previously described 30 . In vivo assays For virulence assays, eight mice per C. albicans strain were inoculated with ~6.0 × 105 CFUs by tail vein injection 49 . For survival assays, three mice per strain were inoculated by tail vein injection, both kidneys were harvested at 48h and CFUs were determined by plating on YPAD. Ploidy of randomly selected isolates was determined by flow cytometry. Supplementary Material 1 2
- Record: found
- Abstract: found
- Article: not found
Haploinsufficiency-based large-scale forward genetic analysis of filamentous growth in the diploid human fungal pathogen C.albicans.
Nancy Craig, Matthew D. Johnson, M Uhl … (2003)
Candida albicans is the most prevalent human fungal pathogen. Here, we take advantage of haploinsufficiency and transposon mutagenesis to perform large-scale loss-of-function genetic screen in this organism. We identified mutations in 146 genes that affect the switch between its single-cell (yeast) form and filamentous forms of growth; this switch appears central to the virulence of C.albicans. The encoded proteins include those involved in nutrient sensing, signal transduction, transcriptional control, cytoskeletal organization and cell wall construction. Approximately one-third of the genes identified in the screen lack homologs in Saccharomyces cerevisiae and other model organisms and thus constitute candidate antifungal drug targets. These results illustrate the value of performing forward genetic studies in bona fide pathogens.
Author and article information
Journal
Journal ID (nlm-ta): G3 (Bethesda)
Journal ID (iso-abbrev): Genetics
Journal ID (hwp): G3: Genes, Genomes, Genetics
Journal ID (pmc): G3: Genes, Genomes, Genetics
Journal ID (publisher-id): G3: Genes, Genomes, Genetics
Title:
G3: Genes|Genomes|Genetics
Publisher:
Genetics Society of America
ISSN
(Electronic):
2160-1836
Publication date
(Electronic):
29
January
2018
Publication date Collection:
April
2018
Volume: 8
Issue: 4
Pages: 1139-1145
Affiliations
[*
]Institute of Biology, Dahlem Centre of Plant Sciences, Free University pf Berlin,
14195, Germany
Author notes
[2
]Corresponding author: Institute of Biology, Dahlem Centre of Plant Sciences, Free
University of Berlin, Albrecht-Thaer-Weg 6, 14195 Berlin, Germany. E-mail:
reinhard.kunze@
123456fu-berlin.de
Author information
Yue Wang
http://orcid.org/0000-0002-1211-2822
Judith Berman
http://orcid.org/0000-0002-8577-0084
Reinhard Kunze
http://orcid.org/0000-0002-3304-5550
Article
Publisher ID: GGG_300388DOI: 10.1534/g3.117.300388
PMC ID: 5873905
PubMed ID: 29378819
SO-VID: 045ec7bf-4766-4ce0-b097-53910dd6cb93
Copyright © Copyright © 2018 Mielich
et al.
License:
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
History
Date
received
: 25
October
2017
Date
accepted
: 26
January
2018
Page count
Figures: 2,
Tables: 0,
Equations: 0,
References: 45,
Pages: 7
Categories
Subject:
Investigations
ScienceOpen disciplines: Genetics
Data availability:
ScienceOpen disciplines: Genetics