Genetic Diversity and Azole Fungicide Sensitivity in Pseudocercospora musae Field Populations in Brazil.

Pseudocercospora musae, causal agent of Sigatoka leaf spot, or yellow Sigatoka disease, is considered a major pathogen of banana (Musa spp.). Widely disseminated in Brazil, this study explored the genetic diversity in field populations of the pathogen from production areas in the Distrito Federal and the States of Bahia, Minas Gerais, and Rio Grande do Norte. Resistance to demethylation inhibitor (DMI) fungicides was also examined. For 162 isolates from 10 banana growing regions, analysis of mating type idiomorph frequency was conducted, together with estimation of genetic diversity at 15 microsatellite loci. A total of 149 haplotypes were identified across the examined populations, with an average genetic diversity of 4.06. In general, populations displayed 1:1 proportions of idiomorphs MAT1-1 and MAT1-2, providing evidence for sexual recombination. Multilocus linkage disequilibrium also indicated asexual reproduction contributing to the genetic structure of certain populations. AMOVA revealed that 86.3% of the genetic differentiation of the pathogen occurred among isolates within populations. Discriminant Analysis of Principal Components (DAPC) identified six most probable genetic groups, with no population structure associated with geographic origin or collection site. Although genetic similarity was observed among certain populations from different states, data revealed increasing genetic differentiation with increasing geographic distance, as validated by Mantel's test (r = 0.19, P < 0.001). On the basis of DMI fungicide sensitivity testing and CYP51 gene sequence polymorphism, isolates from the Distrito Federal separated into two main groups, one with generally higher EC50 values against eight DMI fungicides. A clear phenotype-to-genotype relationship was observed for isolates carrying the CYP51 alteration Y461N. Conventionally adopted fungicides for control of Sigatoka leaf spot are likely to be overcome by combined sexual and asexual reproduction mechanisms in P. musae driving genetic variability. Continued analysis of pathogen genetic diversity and monitoring of DMI sensitivity profiles of Brazilian field populations is essential for the development of integrated control strategies based on host resistance breeding and rational design of fungicide regimes.

The genome of cowpea (Vigna unguiculata [L.] Walp.).

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub-Saharan Africa, that is resilient to hot and drought-prone environments. An assembly of the single-haplotype inbred genome of cowpea IT97K-499-35 was developed by exploiting the synergies between single-molecule real-time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination-poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high-recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS-LRR and the SAUR-like auxin superfamilies compared with other warm-season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.

A Novel Root-Knot Nematode Resistance QTL on Chromosome Vu01 in Cowpea.

The root-knot nematode (RKN) species Meloidogyne incognita and M. javanica cause substantial root system damage and suppress yield of susceptible cowpea cultivars. The narrow-based genetic resistance conferred by the Rk gene, present in some commercial cultivars, is not effective against Rk-virulent populations found in several cowpea production areas. The dynamics of virulence within RKN populations require a broadening of the genetic base of resistance in elite cowpea cultivars. As part of this goal, F1 and F2 populations from the cross CB46-Null (susceptible) x FN-2-9-04 (resistant) were phenotyped for M. javanica induced root-galling (RG) and egg-mass production (EM) in controlled growth chamber and greenhouse infection assays. In addition, F[Formula: see text] families of the same cross were phenotyped for RG on field sites infested with Rk-avirulent M. incognita and M. javanica The response of F1 to RG and EM indicated that resistance to RKN in FN-2-9-04 is partially dominant, as supported by the degree of dominance in the F2 and F[Formula: see text] populations. Two QTL associated with both RG and EM resistance were detected on chromosomes Vu01 and Vu04. The QTL on Vu01 was most effective against aggressive M. javanica, whereas both QTL were effective against avirulent M. incognita Allelism tests with CB46 x FN-2-9-04 progeny indicated that these parents share the same RKN resistance locus on Vu04, but the strong, broad-based resistance in FN-2-9-04 is conferred by the additive effect of the novel resistance QTL on Vu01. This novel resistance in FN-2-9-04 is an important resource for broadening RKN resistance in elite cowpea cultivars.

A multi-parent advanced generation inter-cross (MAGIC) population for genetic analysis and improvement of cowpea (Vigna unguiculata L. Walp.).

Multi-parent advanced generation inter-cross (MAGIC) populations are an emerging type of resource for dissecting the genetic structure of traits and improving breeding populations. We developed a MAGIC population for cowpea (Vigna unguiculata L. Walp.) from eight founder parents. These founders were genetically diverse and carried many abiotic and biotic stress resistance, seed quality and agronomic traits relevant to cowpea improvement in the United States and sub-Saharan Africa, where cowpea is vitally important in the human diet and local economies. The eight parents were inter-crossed using structured matings to ensure that the population would have balanced representation from each parent, followed by single-seed descent, resulting in 305 F8 recombinant inbred lines each carrying a mosaic of genome blocks contributed by all founders. This was confirmed by single nucleotide polymorphism genotyping with the Illumina Cowpea Consortium Array. These lines were on average 99.74% homozygous but also diverse in agronomic traits across environments. Quantitative trait loci (QTLs) were identified for several parental traits. Loci with major effects on photoperiod sensitivity and seed size were also verified by biparental genetic mapping. The recombination events were concentrated in telomeric regions. Due to its broad genetic base, this cowpea MAGIC population promises breakthroughs in genetic gain, QTL and gene discovery, enhancement of breeding populations and, for some lines, direct releases as new varieties.

Broad-based root-knot nematode resistance identified in cowpea gene-pool two.

Cowpea (Vigna unguiculata L. Walp) is an affordable source of protein and strategic legume crop for food security in Africa and other developing regions; however, damage from infection by root-knot nematodes (RKN) suppresses cowpea yield. The deployment through breeding of resistance gene Rk in cowpea cultivars has provided protection to cowpea growers worldwide for many years. However, occurrence of more aggressive nematode isolates threatens the effectiveness of this monogenic resistance. A cowpea germplasm collection of 48 genotypes representing the cowpea gene-pool from Eastern and Southern Africa (cowpea has two major pools of genetic resources - Western Africa and Eastern/Southern Africa) was screened in replicated experiments under field, greenhouse and controlled-growth conditions to identify resistance to RKN, to determine the spectrum of resistance to RKN, the relative virulence (VI) among RKN species and isolates, and the relationship between root-galling (RG) and egg-mass production (EM). Analysis of variance of data for RG and EM per root system identified seven genotypes with broad-based resistance to Meloidogyne javanica (Mj), avirulent (Avr-Mi), and virulent (Mi) M. incognita isolates. Two of the 48 genotypes exhibited specific resistance to both Mi isolates. Most of the genotypes were resistant to Avr-Mi indicating predominance of Rk gene in the collection. Based on RG data, both Mj (VI = 50%) and Mi (VI = 42%) were fourfold more virulent than Avr-Mi (VI = 12%). Resistant genotypes had more effective resistance than the Rk-based resistance in cowpea genotype CB46 against Mj and Mi. Root-galling was correlated across isolates (Avr-Mi/Mj: r = 0.72; Mi/Mj: r = 0.98), and RG was correlated with EM (r = 0.60), indicating resistance to RG and EM is under control by the same genetic factors. These new sources of resistance identified in cowpea gene-pool two provide valuable target traits for breeders to improve cowpea production on RKN-infested fields.Cowpea (Vigna unguiculata L. Walp) is an affordable source of protein and strategic legume crop for food security in Africa and other developing regions; however, damage from infection by root-knot nematodes (RKN) suppresses cowpea yield. The deployment through breeding of resistance gene Rk in cowpea cultivars has provided protection to cowpea growers worldwide for many years. However, occurrence of more aggressive nematode isolates threatens the effectiveness of this monogenic resistance. A cowpea germplasm collection of 48 genotypes representing the cowpea gene-pool from Eastern and Southern Africa (cowpea has two major pools of genetic resources ­ Western Africa and Eastern/Southern Africa) was screened in replicated experiments under field, greenhouse and controlled-growth conditions to identify resistance to RKN, to determine the spectrum of resistance to RKN, the relative virulence (VI) among RKN species and isolates, and the relationship between root-galling (RG) and egg-mass production (EM). Analysis of variance of data for RG and EM per root system identified seven genotypes with broad-based resistance to Meloidogyne javanica (Mj), avirulent (Avr-Mi), and virulent (Mi) M. incognita isolates. Two of the 48 genotypes exhibited specific resistance to both Mi isolates. Most of the genotypes were resistant to Avr-Mi indicating predominance of Rk gene in the collection. Based on RG data, both Mj (VI = 50%) and Mi (VI = 42%) were fourfold more virulent than Avr-Mi (VI = 12%). Resistant genotypes had more effective resistance than the Rk-based resistance in cowpea genotype CB46 against Mj and Mi. Root-galling was correlated across isolates (Avr-Mi/Mj: r = 0.72; Mi/Mj: r = 0.98), and RG was correlated with EM (r = 0.60), indicating resistance to RG and EM is under control by the same genetic factors. These new sources of resistance identified in cowpea gene-pool two provide valuable target traits for breeders to improve cowpea production on RKN-infested fields.

A major QTL corresponding to the Rk locus for resistance to root-knot nematodes in cowpea (Vigna unguiculata L. Walp.).

KEY MESSAGE: Genome resolution of a major QTL associated with the Rk locus in cowpea for resistance to root-knot nematodes has significance for plant breeding programs and R gene characterization. Cowpea (Vigna unguiculata L. Walp.) is a susceptible host of root-knot nematodes (Meloidogyne spp.) (RKN), major plant-parasitic pests in global agriculture. To date, breeding for host resistance in cowpea has relied on phenotypic selection which requires time-consuming and expensive controlled infection assays. To facilitate marker-based selection, we aimed to identify and map quantitative trait loci (QTL) conferring the resistance trait. One recombinant inbred line (RIL) and two F2:3 populations, each derived from a cross between a susceptible and a resistant parent, were genotyped with genome-wide single nucleotide polymorphism (SNP) markers. The populations were screened in the field for root-galling symptoms and/or under growth-chamber conditions for nematode reproduction levels using M. incognita and M. javanica biotypes. One major QTL was mapped consistently on linkage group VuLG11 of each population. By genotyping additional cowpea lines and near-isogenic lines derived from conventional backcrossing, we confirmed that the detected QTL co-localized with the genome region associated with the Rk locus for RKN resistance that has been used in conventional breeding for many decades. This chromosomal location defined with flanking markers will be a valuable target in marker-assisted breeding and for positional cloning of genes controlling RKN resistance.

Blastocyst trophectoderm biopsy and preimplantation genetic diagnosis for familial monogenic disorders and chromosomal translocations.

OBJECTIVE: Modern in vitro fertilization practices involve transfer of embryos as blastocysts, when anabolic metabolism is well established and pregnancy rates can be maintained while transferring embryos singly to avoid multiple pregnancies. Embryo biopsy for preimplantation genetic diagnosis (PGD), however, is generally performed on day 3, when the embryo comprises just 6 to 8 cells, one or two of which are removed for testing. Implantation rates and clinical pregnancy rates have remained relatively low and a harmful effect from losing one or more cells from such early embryos has not been excluded. METHODS: We performed a sequential study involving 399 egg retrievals and 1879 embryo biopsies for patients undergoing PGD to avoid a serious monogenic disease or an unbalanced chromosomal translocation. We compared implantation and viable pregnancy rates after biopsies taken on day 3 (cleavage-stage biopsy) with biopsies delayed until day 5 or 6, when the embryo is a blastocyst and 5 or more cells can be sampled from the trophectoderm while the inner cell mass, from which the fetus develops, remains intact. All embryos were transferred as blastocysts. RESULTS: Despite fewer blastocysts than cleavage embryos biopsied and tested (3.6 compared to 6.6), implantation rates per embryo transferred were 43.4% if biopsied at the blastocyst stage and 25.6% if biopsied at the cleavage stage (P < 0.01), with ongoing or live-birth pregnancy rates per egg retrieval of 34.2% (average transfer number 1.1) for blastocyst biopsies and 25.5% (transfer number 1.6) for cleavage stage biopsies (P < 0.05, 1-tailed). The multiple pregnancy rate for monogenic disease exclusion fell from 16.7% to 2% (P = 0.04, 1-tailed). CONCLUSIONS: For exclusion of genetic disease, day 5-6 blastocyst-stage biopsies are more likely to be followed by implantation and singleton births than is the case after PGD performed on day 3.

First recorded pregnancy and normal birth after ICSI using electrophoretically isolated spermatozoa.

BACKGROUND: DNA damage in the male germ line is associated with poor fertilization and cleavage rates, impaired embryo quality and early pregnancy loss. Given these associations, embryologists are keen to develop techniques that will allow the selection of viable spermatozoa exhibiting low levels of DNA damage for assisted conception purposes. METHODS: In this article, we describe a novel electrophoretic approach for the rapid isolation of cells possessing little DNA damage. The limits of the method were examined using cryostored and snap-frozen semen samples as well as testicular biopsy material. In addition, clinical utility was demonstrated in a case study involving treatment of a patient exhibiting persistently high levels of DNA damage in his spermatozoa. RESULTS: From a range of difficult starting materials (biopsies, cryostored semen and snap-frozen sperm suspensions), the electrophoretic system rapidly isolated populations of motile, viable, morphologically normal spermatozoa exhibiting high levels of DNA integrity. Clinical application in a couple suffering from long-term infertility associated with extensive DNA damage in the male germ line led to the first human pregnancy following such electrophoretic sperm isolation. CONCLUSIONS: The electrophoretic procedure holds promise as a convenient method for the rapid preparation of high-quality spermatozoa for assisted conception purposes.

Benefits and challenges brought by improved results from in vitro fertilization.

Abstract In vitro fertilization (IVF), in which preimplantation-stage embryos are produced after ovarian stimulation and retrieval of preovulatory oocytes, now accounts for almost 2% of all births in Australia. For clinics performing in the top quartile of national results, the chance of a live birth for a woman under 35 years from one round of egg retrieval and IVF treatment is greater than 50%, albeit still with a greater than 20% risk of twins or higher order multiple pregnancy. Similar or better live birth rates are now obtainable with the elective transfer of a single embryo at the stage of blastocyst (5-6 days in culture), a policy that if adopted for younger women can reduce the risk of twins in a clinic to less than 15%. Current developments centre around improvements to embryo culture and the testing of embryos for chromosomal normality and other genetic and epigenetic variables before transfer, made possible by licences for embryo research protocols now being issued under the Commonwealth's Research Involving Human Embryos Act 2002.

mRNA localization: message on the move.

Cytoplasmic messenger RNA localization is a key post-transcriptional mechanism of establishing spatially restricted protein synthesis. The characterization of cis-acting signals within localized mRNAs, and the identification of trans-acting factors that recognize these signals, has opened avenues towards identifying the machinery and mechanisms involved in mRNA transport and localization.

De novo recurrent germline mutation of the BRCA2 gene in a patient with early onset breast cancer.

Germline mutations in either of the two major breast cancer predisposition genes, BRCA1 and BRCA2, account for a significant proportion of hereditary breast/ovarian cancer. Identification of breast cancer patients carrying mutations of these genes is primarily based on a positive family history of breast/ovarian cancer or early onset of the disease or both. In the course of mutation screening of the BRCA1 and BRCA2 genes in a hospital based series of patients with risk factors for hereditary breast/ovarian cancer, we identified a germline mutation in the BRCA2 gene (3034del4) in a patient with early onset breast cancer and no strong family history of the disease. Subsequent molecular analysis in her parents showed that neither of them carried the mutation. Paternity was confirmed using a set of highly polymorphic markers, showing that the proband carried a de novo germline mutation in the BRCA2 gene. Interestingly, 3034del4 is a recurrent mutation occurring in a putative mutation prone region of the BRCA2 gene. Our study presents the first case in which a de novo germline mutation in the BRCA2 gene has been identified, and supports previous results of haplotype studies, confirming that the 3034del4 mutation has multiple independent origins.

Origin and persistence of the mitochondrial genome.

The mitochondrial genome comprises a circular, histone-free 'chromosome' of 16.6 kb of DNA, present in one or more copies in every mitochondrion. This chromosome has been tightly conserved for more than half a billion years, coding in every multicellular animal so far investigated, both vertebrate and invertebrate: (i) the same 13 protein subunits required for oxidative phosphorylation; (ii) a component of each of the two mitochondrial ribosome subunits; and (iii) the 22 transport RNAs present within the mitochondrion. Exons on the circle are tightly packed, with no spacing introns. Mitochondrial DNA is histone-free, has limited repair ability, and has a relatively high mutation-fixation rate. Inheritance is cytoplasmic and maternal, with epidemiological evidence (namely the familial distribution of polymorphisms) indicating that recombination with mtDNA of paternal origin is exceedingly rare. Thus the maintenance and evolution of mtDNA (its remarkably successful symbiotic persistence with the nuclear genome) has been essentially asexual. The machinery for homologous recombination is present in mitochondria of at least some species, however, and it might be surprising if it did not occur between circles in some circumstances. By bringing together the fields of mitochondrial biochemistry, evolutionary genetics, reproductive physiology, and neuromuscular medicine in focusing on the inheritance of normal and abnormal human mtDNA, we can hope to better understand the forces behind this genome's inheritance and what might be required of ovarian function to satisfy its accurate persistence over millions of years. Clinically we can hope also for a better understanding of ooplasmic factors in human fertility and in the wide manifestations of mitochondrial genomic disease.

Germline passage of mitochondria: quantitative considerations and possible embryological sequelae.

Using a semi-quantitative review of published electron micrographs, we have explored the passage of mitochondria from one generation to the next through the cytoplasm of the human female germ cell. We propose a testable hypothesis that the mitochondria of the germline are persistently 'haploid' (effectively carrying just one mitochondrial chromosome per organelle). For mitochondria, the passage through germ cell differentiation, oogenesis, follicle formation and loss could constitute a restriction/amplification/constraint event of a type previously demonstrated for asexual purification and refinement of a nonrecombining genome. At the restriction event (or 'bottleneck') in the human primordial germ cell, which differentiates in embryos after gastrulation, there appear to be <10 mitochondria per cell. From approximately 100 or so such cells, a population of > or =7 x 10(6) oogonia and primary oocytes is produced in the fetal ovaries during mid-gestation, with mitochondria numbering up to 10000 per cell, implying a massive amplification of the mitochondrial genome. A further 10-fold or greater increase in mitochondrial numbers per oocyte occurs during adult follicular growth and development, as resting primordial follicles develop to preovulatory maturity. So few are the numbers of oocytes that fertilize and successfully cleave to form an embryo of the new generation, that biologists have long suspected that a competitive constraint lies behind the generational completion of this genetic cycle. I propose that maintaining the integrity of mitochondrial inheritance is such a strong evolutionary imperative that features of ovarian follicular formation, function, and loss could be expected to have been primarily adapted to this special purpose. To extend the hypothesis further, the imperative of maintaining mitochondrial genomic integrity in a population could explain why women normally become sterile a number of years before there is depletion of ovarian follicles and endocrine ovarian failure (i.e. why there is 'an oopause' preceding the menopause). Plausible explanations might also follow for several well-known and puzzling reproductive difficulties, including recurrent miscarriage, unexplained infertility, and persistent failure of IVF embryos to cleave or to implant. Current experimental laboratory manoeuvres that might circumvent mitochondrial shortcomings (such as cytoplasmic transfusion and karyoplast exchange) are examined and possible clinical hazards identified.

She2p, a novel RNA-binding protein tethers ASH1 mRNA to the Myo4p myosin motor via She3p.

RNA localization is a widespread mechanism to achieve localized protein synthesis. In budding yeast, localization of ASH1 mRNA controls daughter cell-specific accumulation of the transcriptional regulator Ash1p, which determines mating type switching. ASH1 mRNA localization depends on four independently acting sequences ('zipcodes') within the mRNA. In addition, the class V myosin Myo4p and a set of She proteins with as yet unknown function are essential for ASH1 localization. Here we show that She2p is a novel RNA-binding protein that binds specifically to ASH1 mRNA in vivo and to ASH1 RNA zip codes in vitro. She2p can interact with She3 protein via She3p's C-terminus and becomes localized to the daughter cell tip upon ASH1 expression. The N-terminal coiled-coil domain of She3p is required to form an RNA-independent complex with the heavy chain of the myosin motor protein Myo4p. She2p and She3p are the first examples of adapters for tethering a localized mRNA to the motor protein and might serve as prototypes for RNA-motor protein adapters.

Association of the class V myosin Myo4p with a localised messenger RNA in budding yeast depends on She proteins.

Asymmetric distribution of messenger RNAs is a widespread mechanism to localize synthesis of specific protein to distinct sites in the cell. Although not proven yet there is considerable evidence that mRNA localisation is an active process that depends on the activity of cytoskeletal motor proteins. To date, the only motor protein with a specific role in mRNA localisation is the budding yeast type V myosin Myo4p. Myo4p is required for the localisation of ASH1 mRNA, encoding a transcriptional repressor that is essential for differential expression of the HO gene and mating type switching in budding yeast. Mutations in Myo4p, in proteins of the actin cytoskeleton, and in four other specific genes, SHE2-SHE5 disrupt the daughter-specific localisation of ASH1 mRNA. In order to understand if Myo4p is directly participating in mRNA transport, we used in situ colocalisation and coprecipitation of Myo4p and ASH1 mRNA to test for their interaction. Our results indicate an association of Myo4p and ASH1 mRNA that depends on the activity of two other genes involved in ASH1 mRNA localisation, SHE2 and SHE3. This strongly suggests a direct role of Myo4p myosin as a transporter of localised mRNAs, convincingly supporting the concept of motor-protein based mRNA localisation.

RNA-cytoskeletal associations.

It has become evident over the past years that a large fraction of messenger RNAs is tightly associated with the cytoskeleton. Whereas microtubules are involved in RNA-cytoskeletal association in large cells like oocytes, neurons, or oligodendrocytes, microfilaments play the major role in smaller somatic cell types. Association of RNA with cytoskeletal filaments clearly is required for mRNA transport, but also appears to be crucial for efficient protein synthesis. Recent data now shed light on how mRNAs attach to the cytoskeleton. Messenger RNA seems to interact with microtubules or microfilaments in the form of large ribonucleoprotein particles, which in some cases also contain components of the protein synthesis apparatus. Recently, a number of RNA binding proteins have been identified in flies, amphibians, and mammals that are essential for the interaction of mRNA with cytoskeletal filaments or with microtubule- or actin-associated proteins. Such proteins include heterologous ribonucleoproteins, which are also involved in nuclear export of RNA.

The bottleneck: mitochondrial imperatives in oogenesis and ovarian follicular fate.

Molecular geneticists and ovarian physiologists today face the challenge of defining and reconciling two major biological imperatives that each center on oogenesis, folliculogenesis and competition between ovarian follicles: (1), defining how the mitochondrial genome--important in both aging and a number of serious mitochondrial diseases--is refreshed and purified as it passes, via the oocyte's cytoplasm, from one generation to the next; and (2), endeavouring to discover what cytoplasmic factor(s) it is that permits some eggs but not others to produce viable embryos and ongoing pregnancies. We review here in detail the passage of mitochondria through the female germ cell line. For mitochondria, the processes of oogenesis, follicle formation and loss constitute a restriction/amplification/constraint event of the kind predicted by L. Chao for purification and refinement of a haploid genome. We argue that maintaining the integrity of mitochondrial inheritance is such a strong evolutionary imperative that we should expect at least some features of ovarian follicular formation, function and loss to be primarily adapted to this specific purpose. We predict, moreover, that to prevent accumulation of mild mitochondrial genomes in the population there is a need for physiological female sterility prior to total depletion of ovarian oocytes, a phenomenon for which there is empirical evidence and which we term the oöpause.