Gain from marker‐based recurrent selection for increased heterozygosity in barley

Barley (Hordeum vulgare L.) is a highly adaptable cereal crop grown under a wide range of agro‐ecological conditions. Various literature reports demonstrate that heterozygosity may enhance the yielding level and stability of barley particularly in stress‐prone environments. The major aim of this study was to investigate whether recurrent selection (RS) for heterozygosity at highly polymorphic codominant molecular marker loci increases outcrossing in a genetically broad‐based population. Four to six SSR marker loci were used for this purpose. Selection was conducted over four cycles. Progress from selection was evaluated in a terminal experiment comprised of population samples from all four RS cycles. All experiments were conducted under greenhouse conditions. The starting population was composed of 201 gene bank accessions (178 lines, 12 landraces and 11 wild barley populations) sampled in West Asia and North Africa. Selection led to a stepwise increase in heterozygosity from 0.23% to 1.50%. Correspondingly, the estimated multilocus outbreeding rate rose from 1.4% to 3.5%. This positive selection response offers new perspectives for improving the productivity of barley in stress‐prone areas.     

Breeding systems in Coix lacryma-jobi populations

Summary Six populations of Coix lacryma-jobi with distinct characters were studied to evaluate the presence of autonomous apomixis and pseudogamy, and to determine its implications and effects in the calculations of the rate of breeding. For this reason, two types of genetic markers were used-morphological markers and isozymatic markers.The results show that autonomous apomixis does not seem to occur in these populations. Further experiments are necessary to prove the presence of pseudogamy in one of the populations. The rate of natural outcrossing in the grain types Rosario, Rosadlay and I-47 fluctuated around 35.9% using estimates based on isozymatic markers. The rate of natural outcrossing was 37.4% in the Adlay population using morphological markers. A population composed of tall individuals, named Tailândia, which has a greater vegetative mass and fewer grains, presented a high level of natural outcrossing at 72.8%. This information is important for many reasons, such as: 1) guiding sampling strategies of genetic material, 2) in the organization and upkeep of germplasm banks, and 3) for plant breeding programs.     

Genetic diversity of natural populations of Atriplex halimus L. in Morocco: An isoenzyme-based overview

Based on polyacrylamide gel electrophoresis, nine natural populations of Atriplex halimus L., a perennial shrub, collected in different regions of Morocco, were studied for their genetic variation using isoenzyme polymorphism of the highly active enzyme systems: esterases (EST), acid phosphatases (ACP) and glutamate oxaloacetate transaminase (GOT). Different allozyme frequencies from 7 different loci were obtained for all populations of this halophyte species. High levels of genetic diversity were revealed. The mean number of alleles per locus (A = 1.9–2.0), the percentage of polymorphic loci (p = 71.4–85.7) and the mean expected heterozygosity (H_e = 0.339–0.385) showed an important variability in all populations. Gene diversity was essentially explained by the within population component. The between populations differentiation accounted for 8% of the whole diversity (F_ST, averaged over all loci, is 0.08). The relationships among the 9 populations were inferred from the Nei’s genetic distances. Four major groups were formed. The northern population ‘Tanger’, forming a unique group, was highly divergent from the other groups. It appeared that the genetic distance between all groups was related to the geographic distance that separates them. This revised version was published online in August 2006 with corrections to the Cover Date.     

Mating systems in tropical forages: <Emphasis Type="Italic">Stylosanthes capitata</Emphasis> Vog. and <Emphasis Type="Italic">Stylosanthes guianensis</Emphasis> (Aubl.) Sw.

Stylosanthes capitata and S. guianensis are important forage legumes for tropical areas. The only available estimates of S. capitata and S. guianensis outcrossing rates were based on morphological markers, and the genus is considered as being mainly self-pollinated. Here we describe an estimation of the outcrossing rate in S. capitata and S. guianensis using microsatellite markers. The outcrossing rates were estimated in S. capitata and S. guianensis open-pollinated populations of 20 progenies consisting of ten individuals each. The multi locus outcrossing rate for S. capitata was estimated using 10 polymorphic loci, whereas five microsatellites were used for S. guianensis. The multi locus outcrossing rates for S. capitata and S. guianensis were 31 and 26%, respectively, suggesting a mixed mating system with predominance of autogamy. Comparison of single locus and multi locus estimates of outcrossing rates indicated that little inbreeding other than selfing occurred. The estimated Wright’s fixation index of the parental generation was lower than expected based on the multi locus outcrossing rate, possibly resulting from the use of some heterozygous breeding genotypes for the study. The data on the outcrossing rate described here are potentially useful for breeding programs and for maintenance of germplasm collections of these Stylosanthes species.     

Genetic studies in <Emphasis Type="Italic">Centrosema pubescens</Emphasis> benth,a tropical forage legume: the mating system,genetic variability and genetic relationships between <Emphasis Type="Italic">Centrosema</Emphasis> species

In this study, we used microsatellite loci to estimate the outcrossing rate of Centrosema pubescens in open-pollinated populations of 10 progenies that each contained 20 genotypes. The multilocus outcrossing rate was 27%, which suggested a mixed mating system with a predominance of autogamy. The single locus outcrossing rate was 13%. The difference was 0.040, which indicated that only 4% of outcrossing occurs in related individuals. A paternity correlation of 14% suggests that there is a low probability of finding full sibs in the progeny. Cross-amplification of the 26 microsatellite loci available for C. pubescens was evaluated across 11 different Centrosema species. Nineteen of the 26 tested microsatellites were successfully transferable across the Centrosema species. The polymorphism information content and discriminating power evaluated had averages of 0.64 and 0.77, respectively. A total of three clusters were assembled to demonstrate the genetic relationships between Centrosema species. The transferable microsatellite loci should be useful for exploiting the genetic resources of the Centrosema species and determining the outcrossing rate, which are essential for proposing effective approaches for conservation and for establishing strategies for the selection and improvement of Centrosema spp.     

Outcrossing Rates of Experimental Populations of Cuphea lanceolate

Outcrossing rates of Cuphea lanceolata Ait., a new Dilseed crop, have not been estimated; however, this species is known to be insect-pollinated, self-compatible, and autosterile. We used five allozyme loci and the mixed mating model to estimate the outcrossing rates of experimental populations of Cuphea lanceolata Ait. grown at Corvallis, Oregon in 1987 and 1988. Populations were grown using densities of one and 27 plants m^?2. Multilocus out-crossing rate estimates of populations grown at low and high density ranged from 0.61 to 0.85 and 0.93 to 0.94, respectively. Density caused differences may be caused by differences in pollinator movement. In open plantings, e.g., space-plant nurseries, selfing rates are increased by increased within plant pollination by bumblebees, the primary pollinators of C. lanceolata in temperate zones. The movement of pollinators between plants is enforced in dense plantings. We found C. lanceolata is predominantly al-logamous, but selfing rates as great as 39.0 % were observed among space-plants; thus, the progeny of an open-pollinated individual plant grown in this type of nursery is a mixed half-sib and S₁ family. Maximum outcrossing can be achieved by using dense plantings.     

Isozyme polymorphism in Festuca rubraL.

Summary Six Festuca rubra populations from Europe and Scandinavia were studied for variation at three isozyme loci; phosphoglucoisomerase (PGI-2), glutamate oxaloacetate transaminase (GOT-3) and superoxide dismutase (SOD-1). Seven alleles were found at the Pgi-2 locus, four at the Got-3 locus and five at the Sod-1 locus. Most plants were heterozygous and up to five alleles were found in the same plant at the Pgi-2 locus. Each population could be distinguished by the presence or absence of certain alleles or by differences in the frequencies of the alleles present. Values for the Shannon diversity index were calculated which showed that there was considerable heterogeneity both within and between loci. In general, 53% of this diversity could be attributed to within population variation.     

Outcrossing rates of barley landraces from Syria

Diversity levels in populations of barley landraces may be influenced by varying levels of natural outcrossing caused by environmental conditions. Outcrossing was studied in 10 accessions of the barley landrace Arabi Aswad from different environments in Syria. Electrophoretic variation at two codominant isoenzymes (Est1 and Est2) in six seeds of 50 families per population were analysed and multilocus outcrossing rates calculated. Results were correlated with interpolated environmental conditions. Outcrossing was, on average, 1.7% and not significantly different from the outcrossing rate of wild barley. A significant increase of outcrossing was observed with increasing inter‐annual variation in rainfall and decreasing minimum temperatures of the coldest month.     

The use of allozymes at three life stages influences genetic variation analysis and clustering in Cicer arietinum germplasm collections

Levels of genetic variation using 6 enzyme systems for a total of 11 interpretable loci were examined in chickpea (Cicer arietinum) originating from 9locations in Bangladesh. The measurement of genetic variation at enzyme loci was carried out on the seed embryo, on the early leaves of seedlings and on the mature leaves at the vegetative stage. A total of 592individuals, including 240 seeds, 200 seedlings and152 mature leaves were investigated. Using electrophoretic data, chickpea was found to express higher percentages of polymorphic loci at the seed stage (36–64%) than at seedling (22–56%) or the vegetative stage (11–44%). The proportion of mean number of alleles and the average mean observed heterozygosity also were higher at the seed stage when compared to the seedling and vegetative stages. Unique alleles were absent, and only differences infrequencies could be noticed. Positive values of the fixation index were noted for pgm-1 and 6pg-1 for all stages and in both mnr loci for the seed embryo's. A trend towards lower genetic distances of all possible pairs of populations could be observed when comparing those of seed embryo's with seedlings or mature leaves. This trend was even more pronounced when pooling the data of 9 populations into their 3regions. Slight differences in genetic distances caused a separative clustering of population 3 at seed embryo, of population 2 at seedling and of population5 at vegetative stages. It is suggested that careful examination of enzyme polymorphisms at different developmental stages is a prerequisite before drawing conclusions on the genetic distance between germplasm collections from different origins since small differences in the data entry for clustering results in ties that may affect tree topologies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Allozyme variation and conservation genetics of common wild rice (Oryza rufipogon Griff.) in Yunnan, China

In order to reveal levels and distribution of genetic variation within Oryza rufipogon Griff. of Yunnan, China, where one of the centers of genetic diversity for Asian cultivated rice O. sativa L. is located, allozyme variation encoded by 22 loci was electrophoretically analyzed in 149 individuals of all three existing populations as well as five from other regions (Guangxi, Hainan and Jiangxi provinces) of China. As compared to the level of genetic diversity (the mean A = 1.2, P = 24.1%, Ho = 0.045 and He = 0.079) for the populations from other regions, a rather low genetic diversity (the mean A = 1.1, P = 7.6%, Ho = 0.007 and He = 0.011) was found in Yunnan, which may originate from marginal nature of these populations, recent reduction of populations and consequent drift. The result suggests that the current center of genetic diversity for O. rufipogon fail to agree with that for cultivated rice in China. The genetic differentiation for all the eight populations(F_ST = 0.254) was slightly lower than that for three populations from Yunnan (F_ST = 0.302), indicating a fairly high genetic differentiation in the region. Finally, a conservation plan for sampling/preserving fewer populations but more individuals from each population for the species was given, and an appropriate strategy for conserving the three surviving populations from Yunnan was proposed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Estimation of Outcrossing Rate in Chickpea (Cicer Arietinum L.) Sown in Autumn

Gene flow via outcrossing from transgenic plants to relatives will be one of the most important concerns to grow of the transgenic chickpea (Cicer arietinum L.) in European Union (EU). This report is therefore focused on spontaneous outcrossing rate in chickpea. A total of 39 kabuli type mutants with white flower and one desi type with pink flower were grown to estimate spontaneous outcrossing rate. Outcrossing rate ranged from 0.0 to 1.25% in mutant materials. Since labelling threshold for transgenic contamination in food and feed in European Union (EU) is 0.9%, outcrossing rate of 1.25% is higher than threshold of 0.9% in EU, and this result suggests that cultivation of transgenic chickpea will be under high risk to be contaminated chickpeas in neighbourhood fields.     

Natural outcrossing rate in Vernonia galamensis

Vernonia galamensis is a potential new industrial crop growing wild in Ethiopia. The seed oil is rich in vernolic acid, an epoxy fatty acid, which is of interest for oleochemical uses. Basic information on the reproductive system of Vernonia is still very limited. The amount of natural outcrossing was estimated at two locations in Ethiopia (Alemaya and Babile) using flower colour as a marker. Single plants with white flowers, which is a monogenic recessive trait, were planted in plots with normal pink flowers and the outcrossing rate was estimated from the frequency of pink‐flowered plants in the progeny of the white‐flowered plants. Estimates of the natural outcrossing rate ranged between individual plants from 3.5 to 16% at Alemaya and 2.5 to 12% at Babile. Vernonia galamensis can be classified as a mainly self‐pollinated species.     

Molecular diversity and association mapping of quantitative traits in Tibetan wild and worldwide originated barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) germplasm

Molecular diversity of 40 accessions of Tibetan wild barley (TB), 10 Syrian (SY), 72 North American (NA), 36 European (EU), 9 South American (SA) and 8 Australian (AU) varieties were characterized using multiple microsatellite loci. The 42 SSR primers amplified 278 alleles across the 175 barley accessions tested in the present study. The average gene diversity for the whole sample was 0.3387 whereas the mean value for the each population was as follows: TB = 0.3286, SY = 0.2474, EU = 0.299, AU = 0.2867, NA = 0.3138, SA = 0.2536. Clustering analysis based on Nei’s original genetic distance showed that the EU and NA barley populations were grouped together. The TB population was well separated from the other 5 barley populations. Associations between microsatellite markers and 14 quantitative traits were also investigated. Significant associations were found for 18 microsatellite marker loci. The number of marker loci associated with each trait ranged from one (stem diameter, filled grains per plant, grain weight per plant, length of main spike and awn length) to seven (plant height). The percentage of the total variation explained by each marker ranged from 4.59% (HVM2 associated with plant height) to 17.48% (Bmac90 associated with density of main spike). This study provides candidate markers for further QTL mapping of these traits and for marker-assisted selection.     

The Rate of Outcrossing in Grain Amaranths

Studies of intra specific and inter specific out crossing rates were carried out for grain amaranths using two populations of A. hypochondriacus (populations 1008 and 1024) and two populations of A. cruentus (populations 1034 and 434). The studies were conducted at Kabete Campus, University of Nairobi and National Horticultural Research Station, Thika, during the months April to August 1988 and October 1988 to February 1989. The mean intra specific out crossing rate estimates for A. hypochondriacus and A. cruentus were 10.4 % and 10.9 % respectively. The mean inter specific out crossing rate involving population 1024 as the dominant marker parent and 1034 as the recessive marker was 1.2 percent. The study involving population 434 as the dominant marker parent gave the mean inter specific out crossing rate as 6.5 percent. The intra specific and inter specific out crossing rate estimates showed substantial variation. Significant differences in out crossing rate estimates were noted due to locational and seasonal variations. The factors probably contributing to the variations were believed to be locational and seasonal differences in pollinator density and differences among the populations.     

Estimation of between and within accession variation in selected Spanish melon germplasm using RAPD and SSR markers to assess strategies for large collection evaluation

The population structure of 15 Spanish melon (C. melo L.)accessions, mostly of Group Inodorus, was assessed by the analysis of 16individuals of each accession using 100 random amplified polymorphic DNA (RAPD) bands produced by 36 primers, and allelic variation at 12microsatellite (SSR) loci (23 alleles). A relatively high level of polymorphism (25.6%) was detected using RAPD markers, and eight SSR loci (66.7%) were useful in discriminating accessions. Cluster analysis using RAPD- and SSR-based genetic distance estimates resulted in similar and consistent groupings of most of the accessions studied. The mean genetic distance and standard error among accessions estimated by RAPD variation was 0.421 ± 0.099, and mean SSR-based genetic distance estimate was 0.285 ± 0.141. Albeit many dominant markers examined were fitted to a 3:1 test ratio, deviation from this ratio and from Hardy-Weinberg expectations for many SSR loci suggests that some populations were in genotypic disequilibrium. Moreover, a higher level of genetic variation was observed between Cassaba market classes than within accessions, suggesting that, depending upon the accession, allelic fixation has occurred in these populations. The relatively high level of heterogeneity observed (different band morphotypes and cluster grouping within a particular market class), however, indicates that the Spanish melons examined possess a relatively broad genetic background. An appraisal of accession population structure such as the one reported herein indicates that bulk sampling techniques coupled with molecular analysis techniques that employ a unique array of discriminating markers can provide information leading to effective strategies for diversity analyses of large collections. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of the inbreeding depression in progeny fitness of runner bean (Phaseolus coccineus L.) and it is implications for breeding

Moderate levels of selfing despite high inbreeding depression (ID) make runner bean an excellent model for mixed-mating reproductive biology studies in legumes. This work assesses the extent of the ID variation and consistency at different plant growth stages through selfing generations in seven runner bean populations. Field-collected populations after previous isolated multiplication were hand-pollinated in an isolated greenhouse during five generations to produce progeny. Generations were compared for inbreeding effects (δ) on seed germination, survival to flower, and seed weight and yield. The outcrossing rates of the founder populations and the genetic variation and Wright’s ID at the population and generation level were estimated by using 35 polymorphic microsatellite loci. Neutral microsatellite loci were analyzed through generations and populations using different outlier tests to identify loci directly associated with adaptation to inbreeding. Our study revealed patterns of genetic diversity (H _e  = 0.36) and outcrossing rates (ranged from 24 to 44 %) that are consistent with a mixed-mating system. Selfing-pollination procedure significantly affected germination and survival rates, yield, and to a lesser extent seed weight. Three loci had significant hits to genes related to embryonic development when performing BLAST searches to Phytozome database. Results showed a general inconsistency in δ across plant growth stages and populations, suggesting that different deleterious loci are acting at different stages. Inbreeding tended to purge individuals of deleterious recessive alleles to reduce ID. Variation among individuals within populations may lead to the development of inbreeding lineages with lower levels of ID. Several lines that have been self-pollinated for many generations became homozygous at almost all gene loci and produced a uniform population of true breeding progeny and acceptable performance.     

Determination of the outcrossing rate of Phaseolus vulgaris L. using seed protein markers

The outcrossing rates of four varieties of Phaseolus vulgaris in Asturias (Northern Spain) were studied using seed protein polymorphisms as genetic markers. No evidence of outcrossing was obtained, and the outcrossing rate of this species at Asturias was estimated, with a confidence of 95%, as being less than 0.74%. The usefulness of seed proteins as genetic markers for obtaining estimates of outcrossing is also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Esterase 1 and Hordein C allelic and genotypic frequencies in CC XXXI,a male sterile barley population

Summary Seeds from the 5th, 6th, 12th and 18th generation of enforced outcrossing in CCXXXXI-B, a barley (Hordeum vulgare L.) population containing genetic male sterility, were assayed for genotypic distribution of Est 1 and Hor C alleles. The result suggest that natural selection was effective in altering allelic frequencies at both loci and that genotypic frequencies were often different from what would be expected if allelic frequencies solely determined genotypic frequencies. Departures of actual from expected genotypic frequencies occurred in that shifts in allelic frequencies were detected.Joint contribution of the USDA-ARS and published with the approval of the Director of the Montana Agric. Exp. Sta. Journal Series No. 2281.     

Evaluation of three sources of pollen fertility restoration genes and natural outcrossing of cytoplasmic male-sterile wheat

Summary Fertility restoration genes in Triticum aestivum L. in Texas Restorer Composite (TRC), D6301, and four CIMMYT restorer lines were studied, and selection was made for higher fertility in TRC. Mean-while, outcrossing percentages of seed set for 27 spring habit cytoplasmic male sterile (cms) varieties were evaluated for 3 to 5 years at Davis. The winter-habit TRC material did not restore reasonably good fertility, and the response to selection for higher fertility seemed to be slow. This poor fertility could be partly due to its late winter growth habit causing flowering at a period of high temperature and low humidity at Davis. The highest F₁ fertility was 46.6% in the cross cms Ramona x TRC-6, and its F₂ segregated into the ratio of 15 fertile to 1 sterile, with fertility ranging from 3.2 to 100%. Suggested for its improvement was intensive selection in the original TRC material and in the segre-gating F₂ population, followed by intercrossing. D6301 has 2 fertility restoration genes with different strengths which restore fertility up to 45.2% when both genes are heterozygous. D6301 is quite likely heterogeneous for these genes. Four CIMMYT restorer lines, D7464, D7465, D7466, and D7467, had satisfactory F₁ fertility restoration after crossing with cms Ramona 50. In 1975, the fertilities of the F₁'s ranged from 71 to 85% and were over 90% in 1976. The F₂ population of the cross cms Ramona 50 × D7464 segregated into a ratio of 3 fertile to 1 sterile, indicating that D7464 has a single dominant gene for fertility restoration. The F₂'s of crosses cms Ramona 50 × D7465, cms Ramona 50 × D7466, and cms Ramona 50 × D7467 gave a ratio of 15 fertile to 1 sterile, indicating that two gene pairs in these three lines were responsible for the fertility restoration. The best of this group was D7467 which restored fertility fully after being crossed with cms Ramona 50 (T. timopheevi cytoplasm).The early-flowering cms male-sterile varieties had higher outcrossing rates (16 to 38%) than late varieties (6 to 30%) over a 5-year period. This was due to hot and dry weather during the late growing season as well as to the rarity of windborne pollen. In 1970, 1971, 1972, and 1976, the variation among varieties was rather great. Some of them such as Roque 66 and Bajio 67, had consistently high outcrossing rates. This outcrossing ability seemed to be inherited and probably associated with the open-flowering characteristics of each variety.     

New strategies for increasing heterozygosity in crops: Vicia faba mating system as a study case

Our research assesses the feasibility of using artificial selection on pre-mating floral traits to modify the mating system of faba bean (Vicia faba). This analysis considered two synthetic populations, which were derived from different genetic pools and had undergone five years of multiplication. For these populations, we identified floral and inflorescence traits that influence outcrossing per plant and examined the relative importance of these traits in governing yield. Codominant isozyme loci and the mixed-mating model were used to estimate the multilocus female outcrossing rate. A maternal half-sib design was used to evaluate the additive genetic component of floral and inflorescence traits, yield and yield components. Multiple regression was used to assess the effects of floral and inflorescence traits on outcrossing and yield and components of yield. The two populations exhibited mixing mating. Self-fertilization appears to result from the action of pollinating bees, so that its incidence could be modified by selection on floral and inflorescence traits that affect pollination. Floral and inflorescence traits affected individual differences in outcrossing unequally, with most variation being associated with the numbers of displayed flowers and inflorescences. Variation among plants in reward traits and in shape, although statistically significant, had limited and inconsistent influences on individual differences in outcrossing. Yield and its components varied strongly with aspects of floral display and, to a lesser extent, floral design, except for seed weight. Overall, our results imply that both outcrossing and yield could be enhanced by selection for plants that produce more inflorescences, each with relatively few flowers.