Chimeras in Merlot grapevine revealed by phased assembly.

Chimerism is the phenomenon when several genotypes coexist in a single individual. Used to understand plant ontogenesis they also have been valorised through new cultivar breeding. Viticulture has been taking economic advantage out of chimeras when the variant induced an important modification of wine type such as berry skin colour. Crucial agronomic characters may also be impacted by chimeras that aren't identified yet. Periclinal chimera where the variant has entirely colonised a cell layer is the most stable and can be propagated through cuttings. In grapevine, leaves are derived from both meristem layers, L1 and L2. However, lateral roots are formed from the L2 cell layer only. Thus, comparing DNA sequences of roots and leaves allows chimera detection. In this study we used new generation Hifi long reads sequencing, recent bioinformatics tools and trio-binning with parental sequences to detect periclinal chimeras on 'Merlot' grapevine cultivar. Sequencing of cv. 'Magdeleine Noire des Charentes' and 'Cabernet Franc', the parents of cv. 'Merlot', allowed haplotype resolved assembly. Pseudomolecules were built with a total of 33 to 47 contigs and in few occasions a unique contig for one chromosome. This high resolution allowed haplotype comparison. Annotation was transferred from PN40024 VCost.v3 to all pseudomolecules. After strong selection of variants, 51 and 53 'Merlot' specific periclinal chimeras were found on the Merlot-haplotype-CF and Merlot-haplotype-MG respectively, 9 and 7 been located in a coding region. A subset of positions was analysed using Molecular Inversion Probes (MIPseq) and 69% were unambiguously validated, 25% are doubtful because of technological noise or weak depth and 6% invalidated. These results open new perspectives on chimera detection as an important resource to improve cultivars through clonal selection or breeding.

Novel measures of linkage disequilibrium that correct the bias due to population structure and relatedness.

Among the several linkage disequilibrium measures known to capture different features of the non-independence between alleles at different loci, the most commonly used for diallelic loci is the r(2) measure. In the present study, we tackled the problem of the bias of r(2) estimate, which results from the sample structure and/or the relatedness between genotyped individuals. We derived two novel linkage disequilibrium measures for diallelic loci that are both extensions of the usual r(2) measure. The first one, r(S)(2), uses the population structure matrix, which consists of information about the origins of each individual and the admixture proportions of each individual genome. The second one, r(V)(2), includes the kinship matrix into the calculation. These two corrections can be applied together in order to correct for both biases and are defined either on phased or unphased genotypes.We proved that these novel measures are linked to the power of association tests under the mixed linear model including structure and kinship corrections. We validated them on simulated data and applied them to real data sets collected on Vitis vinifera plants. Our results clearly showed the usefulness of the two corrected r(2) measures, which actually captured 'true' linkage disequilibrium unlike the usual r(2) measure.

High throughput analysis of grape genetic diversity as a tool for germplasm collection management.

Using 20 SSR markers well scattered across the 19 grape chromosomes, we analyzed 4,370 accessions of the INRA grape repository at Vassal, mostly cultivars of Vitis vinifera subsp. sativa (3,727), but also accessions of V. vinifera subsp. sylvestris (80), interspecific hybrids (364), and rootstocks (199). The analysis revealed 2,836 SSR single profiles: 2,323 sativa cultivars, 72 wild individuals (sylvestris), 306 interspecific hybrids, and 135 rootstocks, corresponding to 2,739 different cultivars in all. A total of 524 alleles were detected, with a mean of 26.20 alleles per locus. For the 2,323 cultivars of V. vinifera, 338 alleles were detected with a mean of 16.9 alleles per locus. The mean genetic diversity (GDI) was 0.797 and the level of heterozygosity was 0.76, with broad variation from 0.20 to 1. Interspecific hybrids and rootstocks were more heterozygous and more diverse (GDI = 0.839 and 0.865, respectively) than V. vinifera cultivars (GDI = 0.769), Vitis vinifera subsp. sylvestris being the least divergent with GDI = 0.708. Principal coordinates analysis distinguished the four groups. Slight clonal polymorphism was detected. The limit between clonal variation and cultivar polymorphism was set at four allelic differences out of 40. SSR markers were useful as a complementary tool to traditional ampelography for cultivar identification. Finally, a set of nine SSR markers was defined that was sufficient to distinguish 99.8% of the analyzed accessions. This set is suitable for routine characterization and will be valuable for germplasm management.

Prevalence and associated factors of sexual problems after early-stage breast cancer treatment: results of a French exploratory survey.

OBJECTIVE: The objective of this study was to assess the prevalence and associated factors of sexual activity, sexual problems or sexual satisfaction in French early-stage breast cancer survivors (BCS). METHODS: Eight hundred and fifty eligible, post-treatment (6 months-5 years) female patients, aged 18-70 years, randomly selected from a consultation list, were invited to fill in questionnaires exploring quality of life (EORTC QLQ-C30 and QLQ-BR23), body image scale, and sexuality (Sexual Activity Questionnaire-SAQ; Relationship and Sexuality Scale; French Sexual Behaviour Survey-CSF). RESULTS: Fifty-three percent of BCS agreed to participate. Participating women (n=378) were younger, more often premenopausal at diagnosis and with a more recent diagnosis than non-respondents. The prevalence of sexual problems was significantly higher in BCS compared with adjusted data from a French female representative sample (p<0.0001). In logistic regression, no sexual activity (R(2) =0.37) or sexual dissatisfaction (R(2) =0.28) were associated with the feeling of emotional separation in the couple or of partner's fear of sexual intercourse, lower emotional functioning, poorer body image, or co-morbidities. In sexually active women (71% of respondents), lower frequency of sexual activity (R(2) =0.26), lower sexual pleasure (R(2) =0.22), or higher sexual discomfort (R(2) =0.22) were associated with the feeling of emotional separation in the couple or of partner's fear of sexual intercourse, lower emotional functioning, age (>50 years), nausea, or insomnia (all Hosmer-Lemeshow tests: p=NS). CONCLUSIONS: Psychological factors including the perception of the couple relationship appeared prominent in BCS women's experience of sexual problems.

Linkage disequilibrium in wild French grapevine, Vitis vinifera L. subsp. silvestris.

Association mapping based on linkage disequilibrium (LD) can provide high resolution for whole-genome mapping of genes underlying phenotypic variation. This field has received considerable attention over the last decade. We present here the first characterization of LD in wild French grapevine, Vitis vinifera L. subsp. silvestris. To assess the pattern and extent of LD, we used a sample of 85 plants from southern France and 36 microsatellite markers distributed over 5 linkage groups. LD was evaluated with independence tests and multiallelic r(2), using both unphased genotypic data and reconstructed haplotypic data. LD decayed rapidly, with r(2) values decreasing to 0.1 within 2.7 cM for genotypic data and within 1.4 cM for haplotypic data. Compared to the results of a previous study on cultivated grapevine subsp. sativa, where significant LD was found up to 16.8 cM, LD in subsp. silvestris was no longer significant past 1.4 cM. LD was therefore 12 times further extended in cultivated than wild grapevine, even though LD in wild grapevine seemed to extend slightly further than in wild relatives of other crops. Domestication bottlenecks and vegetative propagation are the primary factors responsible for this difference between cultivated and wild grapevine. The rapid decay of LD observed in this study seems promising for future association mapping studies of functional variation in wild V. vinifera grapevine.

Evolution of the VvMybA gene family, the major determinant of berry colour in cultivated grapevine (Vitis vinifera L.).

Polymorphisms in the grape transcription factor family VvMybA are responsible for variation in anthocyanin content in the berries of cultivated grapevine (Vitis vinifera L. subsp. sativa). Previous study has shown that white grapes arose through the mutation of two adjacent genes: a retroelement insertion in VvMybA1 and a single-nucleotide polymorphism mutation in VvMybA2. The purpose of this study was to understand how these mutations emerged and affected genetic diversity at neighbouring sites and how they structured the genetic diversity of cultivated grapevines. We sequenced a total of 3225 bp of these genes in a core collection of genetic resources, and carried out empirical selection tests, phylogenetic- and coalescence-based demographic analyses. The insertion in the VvMybA1 promoter was shown to have occurred recently, after the mutation of VvMybA2, both mutations followed by a selective sweep. The mutational pattern for these colour genes is consistent with progressively relaxed selection from constrained ancestral coloured haplotypes to light coloured and finally white haplotypes. Dynamics of population size in the VvMybA genes showed an initial exponential growth, followed by population size stabilization. Most ancestral haplotypes are found in cultivars from western region, whereas recent haplotypes are essentially present in table cultivars from eastern regions where intense breeding practices may have replaced the original diversity. Finally, the emergence of the white allele was followed by a recent strong exponential growth, showing a very fast diffusion of the initial white allele.

Phytoestrogens after breast cancer.

The current extension of the indications for adjuvant chemotherapy, which predisposes to early menopause, and the media coverage of the benefits of hormone replacement therapy (HRT) have led patients with a history of breast cancer to seek treatments for estrogen deprivation. In breast cancer survivors, most physicians avoid HRT because of concern regarding the potential promotion of growth of occult malignant cells by estrogens, due to the estrogen dependence of breast cancer. Soy phytoestrogens are being promoted as the 'natural alternative' to HRT and have been available without restrictions for several years as nutritional supplements. In this paper, data on the complex mammary effects of phytoestrogens in epidemiological studies, in in vitro studies, as well as in in vivo studies on animal carcinogenesis are reviewed. The potential benefits and risks of phytoestrogens are analyzed, and the prescription of phytoestrogens to postmenopausal women after breast cancer and the coprescription with the anti-estrogen tamoxifen are discussed. The absence of controlled trials and technical checking of extraction and titration in these preparations on 'free sale' raise a new problem in terms of public health and justify close reasoning and a cautious attitude of physicians, as well as straight information given to women, especially after breast cancer.

Genetic variability and evolution of the Schistosoma genome analysed by using random amplified polymorphic DNA markers.

The usefulness of random amplified polymorphic DNA markers (RAPD) was assayed in an attempt to discriminate among species, strains and individuals within the genus Schistosoma. Depending on the species, 40-50 arbitrary decamer oligonucleotides were used as primers to amplify total DNA by the polymerase chain reaction (PCR). An important polymorphism was observed among 5 species, allowing a phylogenetic tree to be outlined. These differences can be used for rapid and accurate identification. A limited but easily detectable polymorphism was revealed among 3 strains of a single species (Schistosoma mansoni). Minor differences were observed among individuals of a single strain. A RAPD marker allows sexual discrimination between individuals from the terminal spined-egg species group. Although a limited number of strains have been examined, the results already indicate clearly that RAPD markers constitute a powerful tool for the analysis of genetic variability. This new tool will considerably extend the information available from morphology, isozyme and limited restriction fragment length polymorphism data and opens the way to genetic analysis of these species.

Genetic mapping of grapevine ( Vitis vinifera L.) applied to the detection of QTLs for seedlessness and berry weight.

Parental and consensus genetic maps of Vitis vinifera L. (2n = 38) were constructed using a F(1) progeny of 139 individuals from a cross between two partially seedless genotypes. The consensus map contained 301 markers [250 amplification fragment length polymorphisms (AFLPs), 44 simple sequence repeats (SSRs), three isozymes, two random amplified polymorphic DNAs (RAPDs), one sequence-characterized amplified region (SCAR), and one phenotypic marker, berry color] mapped onto 20 linkage groups, and covered 1,002 cM. The maternal map consisted of 157 markers covering 767 cM (22 groups). The paternal map consisted of 144 markers covering 816 cM (23 groups). Differences in recombination rates between these maps and another unpublished map are discussed. The major gene for berry color was mapped on both the paternal and consensus maps. Quantitative trait loci (QTLs) for several quantitative subtraits of seedlessness in 3 successive years were searched for, based on parental maps: berry weight, seed number, seed total fresh and dry weights, seed percent dry matter, and seed mean fresh and dry weights. QTLs with large effects (R(2) up to 51%) were detected for all traits and years at the same location on one linkage group, with some evidence for the existence of a second linked major QTL for some of them. For these major QTLs, differences in relative parental effects were observed between traits. Three QTLs with small effects (R(2) from 6% to 11%) were also found on three other linkage groups, for berry weight and seed number in a single year, and for seed dry matter in 2 different years.

Toward the authentication of varietal wines by the analysis of grape (Vitis vinifera L.) residual DNA in must and wine using microsatellite markers.

In an attempt to develop a technique for the identification of grape cultivars in commercial wines, a method for the extraction of DNA from must and experimental wines was adapted and optimal PCR conditions for the amplification of this DNA were established. DNA was analyzed during the fermentation process for six cultivars (Chardonnay, Clairette blanche, Grenache noir, Merlot, Muscat blanc à petits grains, and Syrah). The extractions were performed on solid parts in suspension as well as on the aqueous fraction. Expected profiles for these cultivars were obtained with DNA extracted from the solid parts during all of the fermentation process and for the wine. The analysis of DNA extracted from aqueous fractions was less reproducible, and microsatellite amplifications were obtained only in the case of Clairette blanche, Merlot, and Syrah wines. Results demonstrate that the purification process is adequate for the analysis but that the DNA concentration represents the main limiting factor. Technical improvements of the method are discussed.

[Raloxifene and breast: from the SERMs concept to its place in clinical practice]. / Raloxifène et tissu mammaire : du concept des SERMs à la place de ce composé en pratique clinique.

Raloxifene is a second generation Selective Estrogen Receptor Modulator. It is indicated in prevention and treatment of post-menopausal osteoporosis. This drug has an estrogen agonistic effect on bone tissue, an estrogen-antagonistic effect in breast and endometrial tissue. It inhibits estrogen induced breast cells proliferation and, in animal studies, it prevents the growth of chemically induced mammary tumors. Raloxifene has a limited effectiveness in women with advanced breast cancers. The effects of raloxifene after treated breast cancer and sequential use of tamoxifene after raloxifene (or the contrary) are not known and should be tested in specific pre-clinical and clinical studies. In osteoporotic women included in the MORE trial, the risk reduction of hormone-dependent breast cancer due to raloxifene raises the issue of some effectiveness of raloxifene in breast cancer prevention. The STAR trial is currently comparing tamoxifene vs raloxifene in breast cancer prevention in women at increased risk of breast cancer. Like tamoxifene, raloxifene increases the risk of venous thrombo-embolic events. On the other hand, in a subgroup of women at increased vascular risk in the MORE trial, the reduction of cardiovascular events raises the issue of an effect of raloxifene in the prevention of coronary events. The ongoing RUTH trial is testing this assumption and it is comparing raloxifene to a placebo in women at increased vascular risk. Today, in clinical practice, before the results of ongoing trail, raloxifene should be used in the setting of osteoporosis prevention and treatment.

[Approach of menopause in women at risk for breast cancer]. / Approche de la ménopause chez la femme à risque de cancer du sein.

The small but significant increase in risk of discovering breast cancer in women with hormone replacement therapy and the recent discussion of coronary benefit of this treatment have led many authors to insist on the necessity to evaluate the benefit/risks ratio before administration. This evaluation is particularly important for women that are already at high risk of breast cancer because of some genetic predisposition, family history or some benign breast diseases. In these cases, it is important to evaluate the absolute risk of breast cancer, to define the patient's needs more precisely, to specify menopausal symptoms; it is also important to evaluate the risk of osteoporosis, to review the various therapeutic possibilities, which are not only estrogen/progestin treatments (there are alternative treatments), and to give the patients honest information. Before obtaining the results of current trials, we are proposing here a pragmatic attitude and a decision algorithm to adopt a therapeutic attitude more easily which will be decided together by both patients and their physicians.

[Explosive growth of uterine leiomyomas and carcinologic ovarian risk in a non-menopausal patient with BRCA1-BRCA2 mutation treated by tamoxifen]. / Croissance explosive de myome utérin et risque carcinologique ovarien sous tamoxifène chez une patiente non ménopausée porteuse d'une mutation BRCA1-BRCA2.

OBJECTIVE: To report the case of significant growth of a myoma in a premenopausal woman with a suspicion of BRCA1 and BRCA2 mutation, treated by tamoxifen for a hormonodependent breast cancer and to point out the carcinologic ovarian risks with a mutation BRCA1-BRCA2 in this context. Case. Four months after surgical treatment, chemotherapy and the beginning of tamoxifen, an explosive growth of the abdomen justified pelvic echography and laparoscopy confirming the diagnosis of uterine myoma. A polymyomectomy by laparotomy was performed. CONCLUSION: Use of tamoxifen in premenopausal woman with subjacent gynecological pathologies, whether BRCA1-BRCA2 mutation is present or not can prove to be delicate. A regular clinical and echographic monitoring is necessary.

[Hormone replacement therapy in menopausal women previously treated for breast cancer]. / Traitement hormonal substitutif de la ménopause chez une femme antérieurement traitée pour un cancer du sein.

Is it possible to give a hormonal replacement therapy to a post-menopausal woman with a past history of breast cancer? As estrogens exert a proliferative effect on mammary cells, it remains a controversial issue. Only few studies have been conducted, generally uncontrolled, and involving only a small number of women given short-duration treatment. The endocrinologic arguments are discussed here. Hormone replacement therapy must be replaced in a general discussion on the potential risks for the breast and uterus, and the beneficial effect on coronary heart disease, osteoporosis, and quality of life. The risks and benefits must be weighted for each woman. The circumstances of prescription of hormone replacement therapy, and of the antiestrogen tamoxifen, which has proven a beneficial effect as adjuvant therapy for the menopausal women with a early breast cancer, must be discussed. Some physicians propose a prospective clinical trial to give an answer. Another way could be improvement of our knowledge about the prognostic factors of breast cancer, and better identification of possible "hormonal independance".

[Contraception's choice: women's opinion, satisfaction and profile. Results of a French national survey of a representative sample of 5963 women]. / Contraception des femmes françaises de 15 à 45ans: enquête nationale sur un échantillon représentatif de 5963 femmes.

OBJECTIVES: To describe French women's use of contraceptive methods and study their satisfaction and expectations about contraception. PATIENTS AND METHODS: A survey carried out by mail questionnaire filled in by a representative sample group of French women aged 15 to 45 years (Panel Postal Metascope TNS Sofres) in June 2007. RESULTS: Of 10,000 questionnaires sent out, 6044 questionnaires were returned, of which 5963 were usable. Seventy-three percent of women surveyed used some method of contraception, of which 46% was an oral contraceptive, 15% an IUD and 7% used condoms. A minority used new contraceptive methods (implant, patch, vaginal ring). The average age at first sexual relationship was 17.8 years. It was 18.5 among 40-45-year-olds and gradually reduced to 16.7 in the under 25-year-olds. Twenty-three percent of pregnancies remain unwanted and unplanned. For 96% of patients, the choice of contraception was considered to be important. Thirty-six percent thought that contraception could be harmful to health. Only 63% of women believed themselves to be well informed the first time they used contraception. Satisfaction for contraception method was evaluated from 7.3/10 (condom) to 9.7/10 (sterilization). DISCUSSION AND CONCLUSION: Twenty-seven percent of women do not use any contraception and 23% of pregnancies are unwanted despite a large offering of contraceptives. Our study enables the drawing of 'patient-profiles' for the main methods of contraception in order to better determine women's expectations and to pass the findings on to health professionals.

[Reducing the frequency of menses: extended contraception, a review]. / Diminuer la fréquence des règles: le point sur la contraception étendue.

The extended use of combined hormonal contraception contributes to a reduction of the frequency of menses by shortening the hormone free interval or the number of withdrawal weeks by year. Numerous regimen have been studied, with cycles from 49 to 365days. Most extended/continuous cycle regimens result in fewer scheduled bleeding episodes but in more unscheduled bleedings or spottings, that decrease with time. An extended contraception may be effective in reducing some menstrual symptoms such as menorrhagia or dysmenorrhea, or some menstrual related symptoms such as migraine headaches. This article reviews existing data about extended combined hormonal contraception, women's and providers' attitude, bleeding pattern, efficacy and tolerance.

[Shared medical decision making in gynaecology]. / La décision médicale partagée en gynécologie.

When two options or more can be chosen in medical care, the final decision implies two steps: facts analysis, and patient evaluation of preferences. Shared Medical Decision-Making is a rational conceptual frame that can be used in such cases. In this paper, we describe the concept, its practical modalities, and the questions raised by its use. In gynaecology, many medical situations involve "sensitive preferences choice": for example, contraceptive choice, menorrhagia treatment, and approach of menopause. Some tools from the "Shared Medical Decision Making" concept are useful to structure medical consultations, to convey information, and to reveal patients preferences. Decision aid are used in clinical research settings, but some of them may also be easily used in usual practice, and help physicians to improve both quality and traceability of the decisional process.