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The development of Fusarium head blight (FHB)-resistant winter wheat cultivars using the gene Fhb1 has been conducted in northern China. Sumai 3, a Chinese FHB-resistant spring wheat cultivar, carries three FHB resistance genes: Fhb1, Fhb2 and Fhb5. To better use these genes for increasing FHB resistance in northern China, it is necessary to elucidate the pyramiding effects of Fhb1, Fhb2 and Fhb5 in winter wheat backgrounds. Eight gene combinations involving Fhb1, Fhb2 and Fhb5 were identified in a double haploid (DH) population, and the effects on FHB resistance were evaluated in six tests. At the single gene level, Fhb1 was more efficient than the other two genes in single-floret inoculation tests, whereas Fhb5 showed better resistance than Fhb1 and Fhb2 under a natural infection test. Pyramiding Fhb1, Fhb2 and Fhb5 showed better FHB resistance than the other gene combinations. Forty-nine DH lines showing consistently better resistance than the moderately susceptible control Huaimai 20 in multiple tests were evaluated for main agronomic traits, and no difference in grain yield was found between the mean values of DH lines and the recipient parents Lunxuan 136 and Lunxuan 6, which are higher than those of recipient parent Zhoumai 16 and the donor parent Sumai 3 (p < 0.05). Based on the phenotypic and genomic composition analyses, five promising DH lines fully combined the FHB resistance of donor Sumai 3 and the elite agronomic traits from the recipient parents. This study elucidates the pyramiding effects of three FHB resistance genes and that the promising DH lines with resistance to FHB can be directly applied in wheat production or as parents in winter wheat breeding programs.

Breeding studies in Cucurbitaceae species take a long time. It has become necessary to shorten the time and supporttraditional breeding methods with modern biotechnological methods to get qualified domestic cucumber varieties.Cytoplasmic genome prediction within the scope of molecular-based breeding is a very important application. To increaseheterosis in test crosses, reciprocal ‘double way’ crosses can be made as well as single crosses. Cytoplasmic organelles‘plastid and mitochondria’ are considered to be different from each other between individuals and reciprocal crosses aremade based on this idea. However it significantly increases the labor. In this study, 4 plastid genome regions (rbcL, psbtrnS, trnHK, trnSt) located within non-conserved regions therefore expected to be variable of 50 donor genotypes weresequenced, analyzed and their cytoplasmic genome prediction was estimated. A total of 6300 bp including four plastidregions indicated no polyfmorphism and all sequences were identical among the 50 donor genotypes analyzed. This mayimply no cytoplasmic organelle variation. In conclusion, reciprocal crosses were excluded from our breeding studies. Socytoplasmic genome prediction can provide rapidity and savings in breeding by eliminating unnecessary reciprocal testcrosses.

To facilate breeding process of Brassica napus, a microspore culture and molecular marker-assisted screening combined system were proposed in this research. At early flowering stage, F1 offspring of hybridized combination HY15A×HF06 was used as donor for microspore culture to analyze effects of colchicine concentration on embryogenic and diploid rates of microspore. Treatment with 50 mg/L colchicine resulted in embryogenic rate of 3.56 embryos/bud, which was substantially higher than control (0.78 embryos/bud). A total of 1387 embryos were obtained and 862 single plants were obtained after induction culture. Ploidy detection was performed for the regenerated plants by flow cytometry. Diploid rates of microspores treated with 50 mg/L and 70 mg/L colchicine were 17.2% and 21.0% respectively, which was significantly higher than control (10.5%). A total of 108 single plants that doubled successfully were randomly selected and screened using molecular marker BE10. Approximately 54 of 108 plants generated a 305 bp amplification product, whereas the other 54 plants showed a 398 bp band, thereby satisfying 1:1 separation ratio (x20.05=0.0093). These results coincided with field identification results. Findings of this study indicated that homozygous breeding material could be obtained by microspore culture in a short time, thereby remarkably accelerate the breeding.

Double haploids (DHs) production and utilization is an important aspect of wheat breeding programs in worldwide, because, it provides many advantages over the conventional breeding program. Therefore, in vitro and in vivo responses of anther derived two bread wheat cultivars and their F1 crosses were investigated. Results showed significant genetic variation among the tested bread wheat genotypes. The pre-cold treated anther derived embryonic calli and shoot induction (%) were obtained from all genotypes that ranged 44 - 76% and 32 - 58% respectively. The highest shoot induction from embryonic calli was achieved from F1 cross of Zubkov x Atay-85, whereas, lowest induction was obtained from Atay-85 cultivar. The F1 cross (Zubkov x Atay-85) showed a better response in anther culture in term of shoots per plant and root induction than their respective parental genotypes.The present results indicated positive and significant heterotic effects of F1 crosses for calli and shoot induction that also showed less albino plant regeneration after colchicine treatments. Total number of 114 anthers driven DHs lines from two genotypes and their F1 crosses were regenerated, confirmed by flow-cytometry and evaluated under field condition. Some DHs lines were found to be significantly superior for agronomical traits including seed yield than the parental genotypes as well as local bread wheat (control) cultivars.

Background Farmed Atlantic salmon are one of the most economically significant global aquaculture products. Early sexual maturation of farmed males represents a significant challenge to this industry and has been linked with the vgll3 genotype. However, tools to aid research of this topic, such as all-male and clonal fish, are still lacking. The present 6-year study examined if all-male production is possible in Atlantic salmon, a species with heteromorphic sex chromosomes (males being XY, females XX), and if all-male fish can be applied to further explore the vgll3 contribution on the likelihood of early maturation. Results Estrogen treatment of mixed sex yolk sac larvae gave rise to one sexually mature hermaphrodite with a male genotype (XY) that was used to produce both self-fertilized offspring and androgenetic double haploid (dh) offspring following egg activation with UV treated sperm and pressure shock to block the first mitotic division. There were YY supermales among both offspring types, which were crossed with dh females. Between 1 and 8% of the putative all-male offspring from the eight crosses with self-fertilized supermales were found to have ovaries, and 95% of these phenotypic females were also genetically female. None of the offspring from the one dh supermale cross had ovaries. When assessing the general contribution of the vgll3 locus on the likelihood of early post-smolt sexual maturation (jacking) in the all-male populations we found individuals that were homozygous for the early maturing genotype (97%) were more likely to enter puberty than individuals that were homozygous for the late maturing genotype (26%). However, the likelihood of jacking within individuals with an early/late heterozygous genotype was higher when the early allele came from the dam (94%) compared to the sire (45%). Conclusions The present results show that supermale Atlantic salmon are viable and fertile and can be used as a research tool to study important aspects of sexual maturation, such as to further explore the sex dependent parental genetic contribution to age at puberty in Atlantic salmon. In addition, we report the production of viable double haploid supermale fish.

[ES] El aumento de la población humana previsto en los próximos años, irá acompañado por una demanda mayor de productos vegetales. Esta necesidad de un aumento de producción se verá acompañada por el impacto del cambio climático, que, entre otras cosas, agravará los estreses abióticos (sequía, altas temperaturas, salinidad). La exigencia de una mayor producción y de disponer de variedades resistentes a distintos estreses abióticos hace necesario el abordaje de distintas estrategias de mejora genética vegetal que nos permitan disponer de nuevas variedades en un corto espacio de tiempo que permita reaccionar a las nuevas necesidades que puedan ir surgiendo. Es bien conocido que la agricultura actual se basa en gran medida en la productividad que permite el uso de híbridos entre líneas homocigotas (puras) con caracteres definidos. En los últimos años se vienen utilizando en ciertas especies técnicas alternativas a las clásicas. Estas técnicas, basadas fundamentalmente en la embriogénesis de microsporas, permiten obtener líneas puras, doble haploides (DHs), mediante la regeneración in vitro de plantas partiendo de microsporas o polen recién dividido en una sola generación, siendo pues mucho más rápida y económica. Por un lado, un objetivo del trabajo es la aplicación de nuevas iHDACs para favorecer la inducción de embriogénesis de microsporas. La aplicación del inhibidor de histona desacetilasas (HDACs) Tricostatina A (TSA), provoca un aumento de la respuesta embriogénica en genotipos de baja respuesta de colza y otras especies de interés agronómico. En este trabajo se van a probar distintos tipos de inhibidores de HDACs, selectivos y no selectivos, y a analizar su respuesta embriogénica en términos de número de embriones producidos. Por otro lado, está estudiado el papel del estrés del retículo endoplásmico en la respuesta a estreses bióticos y abióticos. Bajo condiciones de estrés, la capacidad de plegado del RE no puede cubrir la demanda y las proteínas se acumulan sin plegar, causando un estrés en el RE. Existe un mecanismo de control de calidad, la respuesta a proteínas mal plegadas o UPR para tratar que el plegado funcione correctamente con la demanda que aparece tras el estrés y disminuir la acumulación de proteínas mal plegadas en el RE. Por lo que en el segundo objetivo se van a probar compuestos que reducen el estrés del RE. Los compuestos serán aplicados a distintas concentraciones y en distintos momentos del cultivo para determinar la concentración y el momento de aplicación óptimo., [EN] Today¿s agriculture relies heavily on the productivity provided by the use of hybrids between homozygous (pure) lines with defined traits. Traditionally, these pure lines were obtained through successive cycles of self-fertilization-selection, requiring a large amount of economic resources and time. However, current plant breeding programs rely on double haploid (DH) technology, based mainly on the reprogramming of microspores towards embryogenesis, which allows obtaining pure lines, double haploid, through the in vitro regeneration of plants from microspores or newly divided pollen. This technology is of high agronomic interest since it reduces the time needed to obtain hybrids to only one generation, which means a great temporal and economic saving. One of the factors that reduce the efficiency of microspore cultivation for the obtention of DHs is the stress of the endoplasmic reticulum (ER). Under stress conditions, the folding capacity of ER cannot cover the demand, so proteins accumulate without folding, causing ER stress. There is a quality control mechanism, the UPR or Unfolded Protein Response, to try to match the protein folding capacity with the demand appeared after the stress and to reduce the accumulation of badly folded or unfolded proteins in the ER. Another factor affecting the efficiency of microspore culture is the low rate of cell reprogramming towards embryogenesis. This rate increases when DNA histones are acetylated, providing an uncondensed structure to the chromatin and favoring the change of developmental program of the microspore to embryogenic. Consequently, the application of compounds with the ability to inhibit the stress on ER and histone deacetylases (HDACs) increases the efficiency of microspore culture. In the present work we study the effect of the application of the ER stress inhibitor compounds: TUDCA and DHA, and the HDACs inhibitors: Panobinostat and iHDAC Cocktail, on the efficiency of rapeseed and eggplant microspore cultures. The compounds were applied at different concentrations and times to determine the optimal concentration and application time.

Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

For Russia countries with a temperate climate, with direct sowing of rice increase of cold tolerant varieties in seed germination and shoot formation are important. This trait allows to begin rice sowing earlier using for plant growing the favorable period by temperature to obtain optimum shoots by density that gives the chance to realize potential efficiency of varieties. A research objective is to create and estimate the initial stock resistant to low positive temperatures for use it for breeding of new cold tolerant rice varieties.Display of molecular markers - is neutral in relation to a phenotype and they can be found out at any stage of development of plants in ontogenesis. Methods of DNA-genotipirovanija and PCR analysis by means of molecular markers allow accelerating carrying over of the necessary genes in the course of breeding and to provide creation of new varieties with a complex of the set properties Therefore the role of biotechnological methods is important at creation new cold tolerant varieties.According to results of PCR analysis by use of SSR - marker lines of rice with dominant alleles of cold resistance genes were screened. Double haploids were obtained by anther culture method in vitro and resistant forms were found. Plants with economically valuable traits were screened. All-round evaluation of obtained material gave the possibility to find initial stock for use breeding process in future for release of new cold resistant rice varieties.

Maize (Zea mays) hybrid breeding programs are increasingly using Double Haploid (DH) technology; however, in developing countries like in the subtropics, its use is still limited. DH is considered to provide 100% homozygous lines faster than the pedigree selfing method, and since production costs are presumably decreasing, it would be necessary to consider incorporating this technology into new and established breeding programs in these countries. This investigation starts with an S1 population from the same heterotic group the belongs to the CIMMYT Genebank, and it was divided into three parts. First, it describes the DH process, then the pedigree selfing method is evaluated for early generation testing, and finally, there was a comparison of both methodologies through a line by tester evaluation. The testcross performance of 15 DH lines and 14 lines from each of the S2, S4, and S6 selfing generation were tested in multilocation yield trials in the subtropics region of Mexico. It would take three years from the S1 population to identify the DH 100% homozygous lines with highest GCA, one year before the S6 lines (98.4% homozygous). For this data set, early generation testing was not found appropriate due to low correlation values. The DH lines reviled to have more GxE interactions than S2 testcrosses and the same heritability than the S6. There was no difference between the S2 evaluations; however, there were statistical differences found with the S4 and the S6 testcrosses. Thus, the DH technology could be implemented gradually in hybrid breeding programs, but the pedigree method should not be abandoned just yet, and testing in selfing generations should be done in S6. Doctorado

Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

Obtaining double haploid organisms is one of the main objectives of many agricultural companies as well as companies of genetic improvement. The cross between two pure lines allows getting hybrid, homozygous for all its characters. This type of organism presents some agronomic traits of great value, as for example hybrid vigor, greater resistance to different biotic and abiotic stress, homogeneity in crops... as well as some genetic features of great interest at the time of getting genetic maps and other studies. Ginogenesis is an alternative method to androgenesis and interspecific hybridization in those species in which these methods do not work to obtain doubled haploids. It presents obvious advantages over the traditional methods, enabling you to get these lines in less time and with fewer economic resources. This work evaluates the gynogenic competence of three commercial onion genotypes (Allium cepa) by adapting the induction protocol of Michalik et al. (2000) for the agroclimatic conditions of the Vera orchard. The aim is verify if is possible to obtain gynogenic embryos and, if it is successful, try to obtain double haploid lines of these genotypes., La obtención de organismos dobles haploides es uno de los objetivos principales de muchas empresas agroalimentarias así como en empresas de mejora genética. El cruce de dos líneas puras permite obtener descendientes híbridos, homocigotos para todos sus caracteres. Este tipo de organismos presenta unas características agronómicas de gran valor, como por ejemplo el vigor hibrido, mayor resistencia a diferentes estreses bióticos y abióticos, homogeneidad en las cosechas¿ así como unas características genéticas de gran interés a la hora de realizar mapas genéticos y otro tipo de estudios. La ginogénesis es un método alternativo a la androgénesis y la hibridación interespecífica en aquellas especies en las que estos métodos no funcionan para la obtención de dobles haploides. Presenta ventajas evidentes para obtener líneas puras respecto a los métodos tradicionales, ya que permite obtener estas líneas en menor tiempo y con menos recursos económicos. En este trabajo se evaluará la competencia ginogénica de tres genotipos comerciales de cebolla (Allium cepa) adaptando el protocolo de inducción de Michalik et al. (2000) para las condiciones agroclimáticas de la huerta de Vera. Con ello se pretende comprobar si es posible o no la obtención de embriones ginogénicos y en caso de que se consiga, intentar obtener líneas dobles haploides de estos genotipos.

This review provides a summary of haploid induction methods and factors affecting the efficacy of specific methodologies as applied to cucumber (Cucumis sativus L.), melon (Cucumis melo L.), watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), winter squash (Cucurbita maxima Duch. ex Lam.), summer squash (Cucurbita pepo L.) and other cucurbits. This report is focused on studies that were carried out during the last 20 years. The main objective of the research on the production of haploid cucurbit plants is to accelerate breeding programs through the use of homozygous double haploid lines (DHL) and to facilitate the selection of desired (e.g. disease-resistant) genotypes for breeding. Unfortunately, currently used protocols result in a low number of double haploids (DH). The most common and best-known method of obtaining haploid cucurbit plants is via pollination with irradiated pollen, which induces parthenogenetic development of haploid embryos in planta. The embryos are extracted from immature seeds and cultured in vitro to facilitate the maturation and development of plants. The studies described below were primarily aimed at the determination of an appropriate dose of radiation and the evaluation of the impact of the genotype and the time of year on the number of haploid embryos and plants obtained. A less popular method of haploid production - ovule and ovary culture - is based on in vitro gynogenesis. The studies related to this method concentrated on optimising the composition of the medium and pre-treatment conditions (primarily temperature) to which the flower buds were subjected. Recently, increasing attention has been paid to anther and microspore culture. As in the case of in vitro ovule and ovary culture, the medium composition and flower bud pre-treatment conditions were optimised. The most recent studies suggest that anther culture is comparable in effectiveness to the irradiated pollen technique.

Çalışmada farklı dönemlerde ıslah edilmiş ekmeklik buğday çeşitleri ve Namık Kemal Üniversitesi Ziraat Fakültesi Tarla Bitkileri Bölümü Tarafından kombinasyon ıslahı ile elde edilen F2 döllerinden anter kültürü ile geliştirilen double haploid hatlar ve bölgede yetiştirilen çeşitler materyal olarak kullanılmştır. Çalışmada fiziksel özellikler olarak; bitki boyu, başak uzunluğu, başaktaki dane sayısı, başak dane ağırlığı, başak ağırlığı, dane verimi ve başaklanma gün sayısı incelenmiş ve bunların hepsi de istatistiki olarak önemli bulunmuştur. Araştırılan özellikler bakımından kalite analizlerinde ise nem, süne zararı, embriyo kararması, protein oranı, gluten ve gluten indeks, sedimantasyon ve bekletilmiş sedimantasyon incelenmiş ve bu özelliklerin tamamı istatistiki olarak önemli bulunmuştur. Yapılan çalışmada gerek kalite gerekse fiziksel özellikler bakımından istenilen değerler veren double haploid melez Flamura85/Golia hattı tespit edilmiştir. In the study different periods have been breeding bread wheat varieties and Namık Kemal University established using the Faculty of Agronomy Department developed by anther culture of the resulting F2 progeny with a combination of breeding by double haploid as materials and their physical and quality characteristics of the double haploid lines derived from hybrids were examined. In the study the physical properties; plant height, spike length, grain piece in spike, spike grain weight, spike weight, grain yield and heading examined the number of days and all of them were statistically significant as well. In terms of quality of analysis investigated the characteristics of moisture, stink bug damage, embryo darkening, protein content, gluten and gluten index, sedimentation and suspended sedimentation was examined and found to be statistically significant all of these characters. In this study, desired values of the terms of quality and psysical properties that are resulted; hybrid double haploid line Flamura85/Golia was determined.

A doubled-haploid (DH) population, obtained by anther culture of F1 plants from a cross between a highlysusceptible rice cultivar Lijiangxintuanheigu and the resistant somaclone (SC09), of the cultivar Araguaia, was used to identifyRAPD markers linked to the blast resistance gene Pi-ar. The 86 DH plants, inoculated with the race IB-9 of Magnaportheoryzae, segregated in 1:1 ratio of resistant and susceptible plants. Of the 67 primers used 31 produced DNA profiles thatdifferentiated resistant and susceptible bulks as well as the parental cultivars. The resistance gene was found linked to theprimer OPS162072 (‘AGGGGGTTCC’) at a distance of 3.6 cM. The selection efficiency of this primer was assessed in a BC3 F1population derived from another cross between a susceptible cultivar IAC 201 and SC09. The marker OPS16 showedefficiency of 86.9%, when six resistant and two susceptible plants were considered as negatives in RAPD analysis.

The cassava crop Manihot esculenta crantz is the third resource more important to provide calories to the population in the marginal tropical countries of Africa, Asia and South America. However, socioeconomic factors, market conditions, pest, diseases, biotic and abiotic constraints affect cassava production of smallholders. Multiple efforts in cassava breeding program have been made, but his high heterozygous and strong inbreeding depression difficult the production of homozygous parental, causing that conventional crossings lead to a time large. For this reason the doubled haploid technology by isolated microspore culture for production of homozygous plant is an interesting alternative, reducing time and cost in hybrid production. This study was conducted in the International Center of Tropical Agriculture, we assessed cytology and cytogenetic features of microspores tetrads in TMS60444 and SM1219-9 cassava genotypes for optimize efficient protocols of doubled haploid production through evaluation of meiotic abnormalities, meiotic index and viability in microspore tetrads of donor plant used for doubled haploid production. Abnormalities during microsporogenesis like multiple nucleoli, sticky chromosomes, micronuclei, irregular spindles, dyads, triads and polyads were evidenced although in a low frequency. However, the great level of meiotic index and high microspore viability lead to conclude that these cassava genotypes are meiotically stable and can be used in breeding programs or conservation strategies. Maestría

Chrysanthemum is one of important ornamental species in the world. Its highly heterozygous state complicates molecular analysis, so it is of interest to derive haploid forms. A total of 2,579 non-fertilized chrysanthemum ovules pollinated by Argyranthemum frutescens were cultured in vitro to isolate haploid progeny. One single regenerant emerged from each of three of the 105 calli produced. Chromosome counts and microsatellite fingerprinting showed that only one of the regenerants was a true haploid. Nine doubled haploid derivatives were subsequently generated by colchicine treatment of 80 in vitro cultured haploid nodal segments. Morphological screening showed that the haploid plant was shorter than the doubled haploids, and developed smaller leaves, flowers and stomata. An in vitro pollen germination test showed that few of the haploid's pollen were able to germinate and those which did so were abnormal. Both the haploid and the doubled haploids produced yellow flowers, whereas those of the maternal parental cultivar were mauve. Methylation-sensitive amplification polymorphism (MSAP) profiling was further used to detect alterations in cytosine methylation caused by the haploidization and/or the chromosome doubling processes. While 52.2% of the resulting amplified fragments were cytosine methylated in the maternal parent's genome, the corresponding proportions for the haploid's and doubled haploids' genomes were, respectively, 47.0% and 51.7%, demonstrating a reduction in global cytosine methylation caused by haploidization and a partial recovery following chromosome doubling.

Homozygosity was induced in transgenic barley by microspore culture. Spikes of transgenic barley plants carrying microspores in the late uni-nucleate stage were cold pretreated. Teflon rod maceration and a density of 100 000 viable micropores per plate were used. The developed calli were regenerated and plantlets were treated with colchicine. The microspore culture of 16 mother plants (three transgenic lines) resulted in 927 green regenerants. Of these plants, 476 were transferred to soil, 380 were transgenic, 358 reached maturity and 350 were fertile with a normal seed-set carrying a yield of 6.9 kg. A production efficiency of 0.8 fertile transgenic doubled haploid barley plants per spike used for microspore isolation was recorded. The produced transgenic seeds were used in malting experiments.

En el último siglo, el desarrollo de híbridos de maíz estuvo basado en la autofecundación de cultivares mejorados para desarrollar líneas con diferente grado de endogamia y luego recombinar aquellas líneas seleccionadas para formar híbridos y sintéticos comerciales. En los últimos años, se viene utilizando la tecnología de doble haploides (TDH) para obtener líneas doble haploides (LDH) en un tiempo menor que el utilizado mediante el método de autofecundación. Los objetivos de este estudio fueron evaluar la posibilidad de utilizar TDH en Colombia y desarrollar híbridos y sintéticos utilizando LDH en comparación con líneas desarrolladas por autofecundación (LS1). Inductores de haploidía (IH) de clima templado e IH tropicalizados fueron evaluados y se seleccionó los de mejor comportamiento para desarrollar LDH. LDH y LS1 fueron utilizados para formar híbridos con probadores (2 líneas y 2 híbridos simples) con la finalidad de identificar los mejores híbridos y para seleccionar las LDH y LS1 que se utilizarán para desarrollar sintéticos. Los resultados muestran que los IH tropicalizados se comportan mejor que los IH de clima templado y permitieron escoger al híbrido RWS x UH400 como IH en este estudio. Los resultados muestran también que no hubo diferencia para rendimiento de grano y otras características agronómicas entre híbridos formados por cruzamiento entre las LDH o las LS1 y los probadores, lo que significa que el comportamiento de los híbridos depende de la población de la cual las líneas fueron derivadas. La mayor ventaja de TDH es el menor tiempo utilizado para la obtención de líneas homocigotas (LDH). Los híbridos formados por el cruzamiento entre LDH con los probadores muestran que el mayor potencial de rendimiento de grano se obtiene cuando el probador es una línea antes que un híbrido simple aunque el comportamiento de los híbridos formados depende de los ambientes de evaluación; sin embargo, el 10% de los híbridos tuvo un buen comportamiento en ambientes contrastantes. Para la formación de sintéticos se seleccionó las 8 LS1 y las 8 LDH con mayor habilidad combinatoria general (HCG). //Abstract: In the last century, maize hybrid development has been done based on selfing improved cultivars to develop inbreeds and by crossing the selected ones to form commercial hybrids and synthetics. In the last few years, double haploid technology (DHT) is being used to obtain double haploid lines (DHL) in a shorter time than by selfing. Objectives for this study were to evaluate the feasibility of using the DHT in Colombia and develop hybrids and synthetics by using DHL as compared with inbreeds (LS1) developed by selfing. Temperate and tropicalized haploid inducers (HI) were tested and the selected ones used to develop DHL. DHL and LS1 were crossed with suitable testers (2 inbreeds and 2 single crosses) to identify the best hybrids and to select parent DHL and LS1 to develop synthetics. The results show that tropicalized HI performs better than temperate HI and allow choosing the hybrid RWS x UH400 as the HI for this study. Results also have shown no difference for grain yield and other agronomic traits between hybrids formed by crossing DHL or LS1 by testers, meaning that yield potential of the line is function of the population from which lines were developed. The main advantage of DHT is the short time used for obtaining homozygous lines (DHL). Hybrids formed by crossing DHL with testers showed that the highest grain yield hybrids were formed by crossing a DHL with an inbred rather than a single cross as tester, although the hybrid performance depends on the environment of evaluation; however, 10% of these hybrids had good performance in contrasting environments. For synthetics development, 8 LS1 and 8 DHL were selected based on their highest general combining ability. Doctorado

Chantier qualité GA; Salmonid genomes are considered to be in a pseudo-tetraploid state as a result of a genome duplication event that occurred between 25 and 100 Ma. This situation complicates single-nucleotide polymorphism (SNP) discovery in rainbow trout as many putative SNPs are actually paralogous sequence variants (PSVs) and not simple allelic variants. To differentiate PSVs from simple allelic variants, we used 19 homozygous doubled haploid (DH) lines that represent a wide geographical range of rainbow trout populations. In the first phase of the study, we analysed SbfI restriction-site associated DNA (RAD) sequence data from all the 19 lines and selected 11 lines for an extended SNP discovery. In the second phase, we conducted the extended SNP discovery using PstI RAD sequence data from the selected 11 lines. The complete data set is composed of 145 168 high-quality putative SNPs that were genotyped in at least nine of the 11 lines, of which 71 446 (49%) had minor allele frequencies (MAF) of at least 18% (i.e. at least two of the 11 lines). Approximately 14% of the RAD SNPs in this data set are from expressed or coding rainbow trout sequences. Our comparison of the current data set with previous SNP discovery data sets revealed that 99% of our SNPs are novel. In the support files for this resource, we provide annotation to the positions of the SNPs in the working draft of the rainbow trout reference genome, provide the genotypes of each sample in the discovery panel and identify SNPs that are likely to be in coding sequences.

An effective method for producing doubled haploid plants of the Asiatic hybrid lily 'Connecticut King' was established with an in vitro colchicine treatment of haploid calli. With an increase in the concentration and duration of colchicine treatment for the haploid calli, both the survival rate and the shoot regeneration from the calli decreased, but the frequency of diploid cells in the callus increased. Treatments of 0.25 and 0.5 mM colchicine for 48 or 72 hrs induced doubled haploid cells and shoot differentiation from calli. Haploid and diploid plantlets were regenerated from colchicine-treated calli, and only haploid plantlets were formed in colchicine-free treatments. This result suggests that these diploid plantlets originated from doubled haploid cells. Double haploids developed bulblets with more scaly leaves with longer stomatal guard cells than did the haploid plantlets., 本研究ではコルヒチン処理によるアジアティックハイブリッドユリ‘コネチカットキング’の倍加半数体植物の獲得方法について検討した．葯由来の半数性カルスの生存率とシュート再生率は，コルヒチン濃度の上昇および処理時間の延長にともなって低下したが，2倍性カルス細胞の割合は増加した．半数性カルス細胞の倍加とシュート再生率には，0.25mMと0.5mMのコルヒチンを用いた48時間または72時間処理が良好であることが明らかとなった．コルヒチン処理された半数性カルスは半数体と2倍体植物を再生したが，対照区(コルヒチン無処理)のカルスは半数性植物のみを再生したことから，これらの2倍体植物は半数性細胞が倍加されたことにより得られた倍加半数体植物であることが推察された．倍加半数体植物では，葉の気孔サイズは半数体植物のものと比べ大きく，子球もよく発達した．

The selection of desirable genotypes with recessive characteristics, such as self-incompatible plants, is often difficult or even impossible and represents a crucial barrier in accelerating the breeding process. Molecular approaches and selection based on molecular markers can allow breeders to overcome this limitation. The use of self-incompatibility is an alternative in hybrid breeding of oilseed rape. Unfortunately, stable self-incompatibility is recessive and phenotype-based selection is very difficult and time-consuming. The development of reliable molecular markers for detecting desirable plants with functional self-incompatible genes is of great importance for breeders and allows selection at early stages of plant growth. Because most of these reliable molecular markers are based on discrimination of class I S-locus genes that are present in self-compatible plants, there is a need to use an internal control in order to detect possible PCR inhibition that gives false results during genotyping. In this study, 269 double haploid F2 oilseed rape plants obtained by microspore embryogenesis were used to verify the applicability of an improved PCR assay based on the detection of the class I SLG gene along with an internal control. Comparative analysis of the PCR genotyping results vs. S phenotype analysis confirmed the applicability of this molecular approach in hybrid breeding programs. This approach allows accurate detection of self-incompatible plants via a different amplification profile.

10 Págs., 3 figs. The definitive version is available at: http://link.springer.com/journal/497, Microspore embryogenesis involves reprogramming of the pollen immature cell towards embryogenesis. We have identified and characterized a collection of 14 genes induced along different morphological phases of microspore-derived embryo development in wheat (Triticum aestivum L.) anther culture. SERKs and FLAs genes previously associated with somatic embryogenesis and reproductive tissues, respectively, were also included in this analysis. Genes involved in signalling mechanisms such as TaTPD1-like and TAA1b, and two glutathione S-transferase (GSTF2 and GSTA2) were induced when microspores had acquired a 'star-like' morphology or had undergone the first divisions. Genes associated with control of plant development and stress response (TaNF-YA, TaAGL14, TaFLA26, CHI3, XIP-R; Tad1 and WALI6) were activated before exine rupture. When the multicellular structures have been released from the exine, TaEXPB4, TaAGP31-like and an unknown embryo-specific gene TaME1 were induced. Comparison of gene expression, between two wheat cultivars with different response to anther culture, showed that the profile of genes activated before exine rupture was shifted to earlier stages in the low responding cultivar. This collection of genes constitutes a value resource for study mechanism of intra-embryo communication, early pattern formation, cell wall modification and embryo differentiation. © 2013 Springer-Verlag Berlin Heidelberg., RA Sánchez-Díaz was recipient of a predoctoral fellowship, from Junta Ampliación de Estudios, Consejo Superior de Investigaciones Científicas (JAE-CSIC) of Spain. This work was supported by Project AGL2010-17509, from ‘Plan Nacional de Recursos y Tecnologías Agroalimentarias’ of Spain and by COST Action FAO0903 ‘Harnessing of Reproduction for Plant Improvement’ (HAPRECI)

Es va analitzar una població doble haploide procedent de l'encreuament entre las varietats d'ordi Albacete i Plaisant per dur a terme la caracterització de la translocació cromosòmica recíproca present en Albacete i valorar la seva possible relació amb la resistència d'Albacete a ambients extrems de fred i sequera. El genotipatge amb marcadors SSR i DART y la hibridació in situ fluorescent (FISH) en preparacions cromosòmiques d'Albacete va confirmar la presència de la translocació recíproca entre els cromosomes 1H i 3H. Per valorar la relació de la translocació recíproca amb caràcters d'interès agronòmic es van realitzar assaigs en dues localitats durant un període de dos anys. El caràcter pes de mil grans va ser superior, de forma significativa, en les línies doble haploides portadores de la translocació recíproca. Es van detectar també QTLs significatius per als caràcters vigor inicial, data de canoneig, data d’espigat, fase d'elongació de la tija i fase d'ompliment de gra., Se analizó una población doble haploide de cebada procedente del cruzamiento de Albacete y Plaisant para llevar a cabo la caracterización de la translocación cromosómica recíproca presente en Albacete y valorar su posible relación con la resistencia de esta variedad a ambientes extremos de frío y sequía. La construcción de un mapa saturado, con marcadores SSR y DArT, y la hibridación in situ fluorescente (FISH) en preparaciones cromosómicas de Albacete determinaron que los cromosomas implicados en la translocación recíproca eran el 1H y el 3H. Para valorar la relación de la translocación recíproca con caracteres de interés agronómico se realizaron ensayos en dos localidades durante un periodo de dos años. El carácter peso de mil granos mostró ser significativamente superior en las líneas doble haploides portadoras de la translocación recíproca. Se detectaron también importantes QTLs para los caracteres vigor inicial, fecha de encañado, fecha de espigado, fase de elongación del tallo y fase de llenado de grano, A double haploid population derived from a cross between two barley cultivars widely grown in Spain, Albacete and Plaisant, was used to characterized the reciprocal chromosomal translocation present in Albacete. The possible relation between this translocation and the drought tolerance present in Albacete was analyzed. A genotyping process, with SSR and DArT markers, and a fluorescence in situ hybridization (FISH), in chromosome preparations of Albacete cultivars, confirmed the presence of a reciprocal translocation between the chromosomes 1H and 3H. In order to assess the relation of the reciprocal translocation with important agronomical traits, different trials were carried out in two locations in two growing seasons. The 1000-kernel weight was significantly higher in DH lines carrying the reciprocal translocation. Moreover, significant QTLs were detected for early vigour, jointing, heading date, stem elongation and grain filling.

The objective of the study was to investigate efficiency of anther culture in the production of spontaneous double haploids from randomly selected heterozygous genotypes of wheat (Triticum aestivum L.). Anthers of 20 F1 wheat combinations were grown in vitro on a modified Potato-2 medium. All of the examined genotypes have shown the ability to produce pollen calluses as well as to regenerate green plants. On average for the whole experiment material, 47.2 calluses were produced per 100 cultured anthers. The green plant regeneration ranged from 0.8 to 13.4 green plants per spike, with an overall mean of 5.8. From the total of 582 regenerated green plants, 47.9% (279) were spontaneous double haploids. The final average yield from the study was 2.8 double haploids per spike. Cilj rada bio je da se ispita efikasnost kulture antera u proizvodnji spontanih dvostrukih haploida iz slučajno odabranih heterozigotnih genotipova pšenice (Triticum aestivum L.). Antere iz 20 F1 kombinacija pšenice gajene su in vitro na modifikovanoj Potato-2 podlozi. Svi ispitivani genotipovi pokazali su sposobnost da proizvedu kaluse, kao i da regenerišu zelene biljke. U proseku za ceo eksperimentalni materijal, proizvedena su 47.2 kalusa na 100 izolovanih antera. Regeneracija zelenih biljaka kretala se od 0.8 do 13.4 zelene biljke po klasu, sa ukupnim prosekom od 5.8. Od ukupno 582 regenerisane zelene biljke, 47.9% (279) bile su spontani dvostruki haploidi. U proseku, u ovom istraživanju, proizvedeno je 2.8 DH biljaka po klasu.

International audience; Blackleg, caused by Leptosphaeria maculans, is a major disease of oilseed rape (Brassica napus), worldwide, including Australia and France. The aims of these studies were first, to determine if higher levels of resistance to L. maculans could be generated in double haploid (DH) lines derived from spring-type B. napus cv. Grouse, which has a good level of field resistance to blackleg; and second, to determine whether the resistance to blackleg disease of individual DH lines responds differentially to different L. maculans field populations within and between the two countries. DH lines were extracted from cv. Grouse and tested in field experiments carried out in both France and Australia against natural L. maculans populations. Extracting and screening DH lines were an effective means to select individual lines with greatly improved expression of resistance to blackleg crown canker disease in comparison with the original parental population. However, relative disease resistance rankings for DH lines were not always consistent between sites. The higher level of resistance in France was shown to be because of a high expression level of quantitative resistance in the French growing conditions. Big differences were observed for some DH lines between the 2004 and the 2005 field sites in Australia where the L. maculans populations differed by their virulence on single dominant gene-based resistant lines derived from Brassica rapa ssp. sylvestris. This differential behaviour could not be clearly explained by the specific resistance genes until now identified in these DH lines. This investigation highlights the potential to derive DH lines with superior levels of resistance to L. maculans compared with parental populations. However, in locations with particularly high pathogen diversity, such as in southern Australia, multiyear and multisite evaluations should be performed to screen for the most efficient material in different situations

International audience

The objective of the study was to investigate the production of homozygous doubled haploid (DH) lines from heterozygous genotypes of wheat (Triticum aestivum L.) using an anther culture. Anthers were isolated from 20 randomly selected F1 wheat combinations and grown in vitro on a modified Potato-2 inductive medium. Plant regeneration was performed on the modified 190-2 medium. The results have shown that all genotypes had the ability to produce pollen calli, as well as, to regenerate green plants. The hybrid Mex.3 x MV 18 had the highest value for androgenous capacity (40.6%), while the combination Kutječanka x Slavija had the lowest androgenous capacity (6.3%). The average androgenous capacity of all genotypes was 19.4%. The regeneration frequency ranged from 1.3 to 21.6 green plants per 100 isolated anthers, with an overall mean of 9.6%. Out of a total of 582 regenerated green plants 279 (47.9%) were spontaneous double haploids. The average production of double haploids of wheat in this study amounted to 4.6 per 100 cultured anthers. Cilj rada bio je da se ispita mogućnost proizvodnje homozigotnih linija dvostrukih haploida (DH) iz heterozigotnih genotipova pšenice (Triticum aestivum L.) korišćenjem kulture antera. Antere su izolovane iz 20 slučajno odabranih F1 kombinacija pšenice i gajene u kulturi in vitro na modifikovanoj potato-2 indukcionoj podlozi. Regeneracija biljaka vršena je na modifikovanoj 190-2 podlozi. Rezultati su pokazali da su svi genotipovi posedovali sposobnost da proizvedu kaluse iz polena, kao i da regenerišu zelene biljke. Hibrid Mex.3 x MV 18 je imao najvišu vrednost za androgeni kapacitet (40,6%), dok je kombinacija Kutječanka x Slavija imala najniži androgeni kapacitet (6,3%). Prosečan androgeni kapacitet za sve genotipove bio je 19,4%. Frekvencija za regeneraciju zelenih biljaka kretala se od 1,3 do 21,6 zelenih biljaka na 100 izolovanih antera, sa prosekom od 9,6%. Od ukupno 582 regenerisane zelene biljke, 279 (47,9%) su bile spontani dvostruki haploidi. U ovom istraživanju prosečno je proizvedeno 4,6 dvostrukih haploida pšenice na 100 izolovanih antera.

Not available, La culture d'ovaires non fécondés, gynogenèse, des 6 génotypes de blé dur (Triticum durum) : Cham 1, Cocorit, Isly, Jori, Oued Zenati et Sarif a été élaborée, et des plantes haploïdes ont été obtenues, tant sur cette espèce (Pl/Ov = 21,8%) que sur l'orge (Hordeum vulgare) (Pl/Ov = 18,3%). Dans chacun des cas des souches régénérantes ont été établies pour plusieurs cultivars (plus de 10 repiquages chez Triticum durum). Des plantes gynogénétiques ont été régénérées, d'une part, à partir de souches non sélectionnées (plus de 700 plantes chez le blé dur et 150 plantes chez l'orge) et aussi, de souches ayant subi des stress salins (NaCl), par application, soit directe, soit progressive. La seconde procédure a permis de garder la viabilité et la régénération des souches malgré l'introduction dans le milieu de quantités de sel allant jusqu'a 9 g/L chez Triticum durum et jusqu'a 15 g/L chez Hordeum vulgare. Après doublement des chromosomes, les descendances d'autofécondation ont été récoltées principalement chez l'orge.

Not available, La culture d'ovaires non fécondés, gynogenèse, des 6 génotypes de blé dur (Triticum durum) : Cham 1, Cocorit, Isly, Jori, Oued Zenati et Sarif a été élaborée, et des plantes haploïdes ont été obtenues, tant sur cette espèce (Pl/Ov = 21,8%) que sur l'orge (Hordeum vulgare) (Pl/Ov = 18,3%). Dans chacun des cas des souches régénérantes ont été établies pour plusieurs cultivars (plus de 10 repiquages chez Triticum durum). Des plantes gynogénétiques ont été régénérées, d'une part, à partir de souches non sélectionnées (plus de 700 plantes chez le blé dur et 150 plantes chez l'orge) et aussi, de souches ayant subi des stress salins (NaCl), par application, soit directe, soit progressive. La seconde procédure a permis de garder la viabilité et la régénération des souches malgré l'introduction dans le milieu de quantités de sel allant jusqu'a 9 g/L chez Triticum durum et jusqu'a 15 g/L chez Hordeum vulgare. Après doublement des chromosomes, les descendances d'autofécondation ont été récoltées principalement chez l'orge.

WOS: 000483480000001, Advanced marker technologies are widely used for evaluation of genetic diversity in cultivated crops, wild ancestors, landraces or any special plant genotypes. Developing agricultural cultivars requires the following steps: (i) determining desired characteristics to be improved, (ii) screening genetic resources to help find a superior cultivar, (iii) intercrossing selected individuals, (iv) generating genetically hybrid populations and screening them for agro-morphological or molecular traits, (v) evaluating the superior cultivar candidates, (vi) testing field performance at different locations, and (vii) certifying. In the cultivar development process valuable genes can be identified by creating special biparental or multiparental populations and analysing their association using suitable markers in given populations. These special populations and advanced marker technologies give us a deeper knowledge about the inherited agronomic characteristics. Unaffected by the changing environmental conditions, these provide a higher understanding of genome dynamics in plants. The last decade witnessed new applications for advanced molecular techniques in the area of breeding, with low costs per sample. These, especially, include next-generation sequencing technologies like reduced representation genome sequencing (genotyping by sequencing, restriction site-associated DNA). These enabled researchers to develop new markers, such as simple sequence repeat and single- nucleotide polymorphism, for expanding the qualitative and quantitative information on population dynamics. Thus, the knowledge acquired from novel technologies is a valuable asset for the breeding process and to better understand the population dynamics, their properties, and analysis methods.