Cytogenetics of selected Solanum L. species within a phylogenetic framework

Genome size can affect the phenotype of plants by a simple physical effect of the DNA material at the cellular level. Pollen contains the bare necessities to initiate and sustain pollen tube growth and carries the haploid genome. This work investigates the extent to which the nuclear DNA content affects pollen size in an evolutionary context within Streptocarpus (Gesneriaceae), by correlating genome size with pollen size of 38 samples representing 36 taxa in a phylogenetic framework. Streptocarpus was found to possess an average genome size among diploid species of 0.82pg (1C). Significant genome downsizing of up to 44.4% was observed among the polyploid species which are exclusively found in Madagascar. The pollen size ranged between 11.27μm and 25.55μm at the diploid level, but 1C values were not found to drive pollen size. On the other hand, 1C values in most polyploids showed a strong positive correlation with pollen size, near linear in species of sect. Parasaintpaulia. In a phylogenetic context, polyploidy has evolved at least twice in the genus, and contrary to pollen size, genome size was strongly lineage-specific rather than adaptive in Streptocarpus. Repeated parallel increases and decreases in genome size (1C, and 1Cx) during the evolution of the genus were inferred. Overall, in Streptocarpus at least, pollen size is a limited predictor of genome size and only partly reflecting ploidy level, but may be of taxonomic value. The study demonstrates that the relationship between pollen size and genome size is not straightforward, and their evolutionary trajectories unlinked.

We investigated the effects of clinical and laboratory properties at the time of the initial application of patients recently diagnosed and presenting metabolic indicators of diabetic ketoacidosis who were given disease prognoses in years 1 and 2 after discharge. A total of 94 patients admitted to Bakirkoy Maternity and Children's Diseases Training and Research Hospital with diabetic ketoacidosis and recently diagnosed with type 1 diabetes mellitus were investigated. Patient files were examined within 2 years following discharge. All 94 study patients (53.2% male and 46.8% female) presented acidosis, ketonuria and hyperglycemia. While a moderate correlation was detected between the prodromal period and HbA(1c) values in year 1, only a slight correlation was seen in HbA(1c) values in year 2. In addition, a slight correlation was observed between the prodromal period and the number of hospitalizations due to diabetic ketoacidosis in the first year. Again, while a moderate correlation was observed between HbA(1c) values and the number of hospitalizations due to diabetic ketoacidosis in year 1, only a slight correlation was seen in year 2. The prodromal period was directly proportional to patient age. Hospital admissions may be reduced through appropriate treatment, follow-up and metabolic control of patients with type 1 diabetes mellitus. In addition, we report a relationship between the prodromal period and HbA(1c) values in type 1 diabetes patients.

Comparative studies of insect genome size show that Orthoptera is a unique group of Insecta with a significantly enlarged genome. To determine a suitable internal standard for a large genome and to compare the effects of different internal standards on estimates of genome size, we used four internal standards to estimate nuclear DNA content in nine insect species with large genomes. The results showed that the combination of two internal standards, Locusta migratoria (♂1C = 6.20 pg, ♀1C = 6.60 pg) and Periplaneta americana♂ (1C = 3.41 pg), was suitable for estimating large genome of Caelifera by flow cytometry. Using these two internal standards, we estimated the genome sizes of 17 species of Caelifera (12 genera in Acrididae, 2 genera in Pamphagidae, 1 genus in Pyrgomorphidae) using flow cytometry. Genomes ranged from 6.57 pg (Shirakiacris shirakii) to 18.64 pg (Bryodemella holdereri), the largest described in insects to date. These species showed significant genomic dimorphism based on sex: females had a 0.56 pg larger genome than males on average, which might be due to the sex chromosome determinism mechanism of X0(♂)/XX(♀). To test the results obtained by flow cytometry, we used k-mers of Illumina sequencing data to gauge the C-value of Calliptamus abbreviatus and Haplotropis brunneriana. The results of the two methods are slightly different. Genomes were estimated to be about 0.28 and 0.26 pg smaller, respectively, than the flow cytometry values. Furthermore, we also reconstructed the evolutionary relationships of these taxa and discuss the genome size evolution in a phylogenetic framework.

Recent studies have proposed a monophyletic circumscription of Filago and a new subgeneric treatment for this genus. The aim of this study was to analyse the nuclear genome size in a phylogenetic framework in order to evaluate the systematic significance of this trait to provide insights into the dynamics of genome size evolution and to assess relationships among DNA content, specific life and ecological features within the study group. A holoploid genome size of 76 samples corresponding to 27 taxa was determined using flow cytometry, which represents the first estimates of genome size in Bombycilaena, Filago, Ifloga and Logfia. Chromosome counts were performed for six species. Parsimony and Bayesian analysis of ITS, ETS and rpl32-trnL intergenic spacer sequence data were used to construct molecular phylogenetic trees. The evolution of genome size was investigated troughout the Brownian motion model with the three scaling parameters λ, κ and δ. The mean 2C-value in the Filago group is relatively low (1.3644 ± 0.0079 pg) and homogeneous among species. A high degree of congruence was found between genome size distribution and the major phylogenetic lineages obtained. The generally accepted assumption that annual, ephemeral and autogamous species show low genome sizes was confirmed. Also the relatively high DNA contents found for a couple of species could be correlated with their highly specific ecological requirements. Phylogeny seems to represent the most important factor explaining the pattern of DNA amount variation in the Filago group. The DNA amount does not seem to be strongly influenced by selection.

The study of genome size evolution in a phylogenetic context in related polyploid and diploid lineages can help us to understand the advantages and disadvantages of genome size changes and their effect on diversification. Here, we contribute 199 new DNA sequences and a nearly threefold increase in genome size estimates in polyploid and diploid Veronica (Plantaginaceae) (to 128 species, c. 30% of the genus) to provide a comprehensive baseline to explore the effect of genome size changes. We reconstructed internal transcribed spacer (ITS) and trnL-trnL-trnF phylogenetic trees and performed phylogenetic generalized least squares (PGLS), ancestral character state reconstruction, molecular dating and diversification analyses. Veronica 1C-values range from 0.26 to 3.19 pg. Life history is significantly correlated with 1C-value, whereas ploidy and chromosome number are strongly correlated with both 1C- and 1Cx-values. The estimated ancestral Veronica 1Cx-value is 0.65 pg, with significant genome downsizing in the polyploid Southern Hemisphere subgenus Pseudoveronica and two Northern Hemisphere subgenera, and significant genome upsizing in two diploid subgenera. These genomic downsizing events are accompanied by increased diversification rates, but a ‘core shift’ was only detected in the rate of subgenus Pseudoveronica. Polyploidy is important in the evolution of the genus, and a link between genome downsizing and polyploid diversification and species radiations is hypothesized. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178, 243–266.

The genus Cheirolophus constitutes one of the most striking cases of species radiation in Macaronesia, where it diversified into a lineage of ca. 20 endemic species at a rate that is amongst the fastest reported for oceanic islands. Whilst the cytogenetic dynamics of many of the Macaronesian Cheirolophus species have been comparatively well studied, an overall vision of chromosome and genome evolution has been hampered by the lack of data for the earliest‐diverging species, Ch. crassifolius. In this study, we have completed the cytogenetic survey of Cheirolophus to investigate how different cytogenetic traits may have contributed to the dramatic radiation of the genus in Macaronesia. We provide new cytogenetic data (i.e., chromosome counts, genome size estimates and physical mapping of 35S rDNA loci) for several key species, including Ch. crassifolius, and then model trait evolution within a phylogenetic context. Our results reveal a trend of genome downsizing accompanied by a dramatic increase in number of 35S rDNA loci which started early in the evolutionary history of the genus, before its radiation in Macaronesia. It is notable that the increasing number of 35S rDNA loci has not been driven by polyploidisation, in contrast to the more typical trend observed in many angiosperms. In addition, the number of 35S rDNA loci was observed to negatively correlate with genome size, which is also very unusual in angiosperms. It is suggested that non‐homologous and unequal homologous recombination are the most likely mechanisms to explain these observations and we discuss whether the unique genomic architectures of Cheirolophus could have predisposed the genus to its successful and rapid speciation in Macaronesia.

Polyploidy is a key factor in the evolution of higher plants and plays an important role in the variation of plant genomes, leading to speciation in some cases. During polyploidisation, different balancing processes take place at the genomic level that can promote variation in nuclear DNA content. We estimated genome size using flow cytometry in 84 populations of 67 Artemisia species and one population of Crossostephium chinense. A total of 73 sequences of nrDNA ITS and 3'-ETS were newly generated and analysed, together with previously published sequences, to address the evolution of genome size in a phylogenetic framework. Differences in 2C values were detected among some lineages, as well as an increase of genome size heterogeneity in subgenera whose phylogenetic relationships are still unclear. We confirmed that the increase in 2C values in Artemisia polyploids was not proportional to ploidy level, but 1Cx genome size tended to decrease significantly when high ploidy levels were reached. The results lead us to hypothesise that genome size in polyploids tends to a maximum as it follows saturation behaviour, in agreement with the Michaelis-Menten model. We tested different arithmetic functions with our dataset that corroborated a non-linear relationship of genome size increase in polyploids, allowing us to suggest a theoretical upper limit for the DNA content of this genus.

Karyotype evolution in species with non-localised centromeres (holocentric chromosomes) is usually very dynamic and associated with recurrent fission and fusion (also termed agmatoploidy/symploidy) events. In Rhynchospora (Cyperaceae), one of the most species-rich sedge genera, all analysed species have holocentric chromosomes and their numbers range from 2n=4 to 2n=84. Agmatoploidy/symploidy and polyploidy were suggested as the main processes in the reshuffling of Rhynchospora karyotypes, although testing different scenarios of chromosome number evolution in a phylogenetic framework has not been attempted until now. Here, we used maximum likelihood and model-based analyses, in combination with genome size estimation and ribosomal DNA distribution, to understand chromosome evolution in Rhynchospora. Overall, chromosome number variation showed a significant phylogenetic signal and the majority of the lineages maintained a karyotype of 2n=10 (~48% of the species), the most likely candidate for the ancestral number of the genus. Higher and lower chromosome numbers were restricted to specific clades, whilst polyploidy and/or fusion/fission events were present in specific branches. Variation in genome size and ribosomal DNA site number showed no correlation with ploidy level or chromosome number. Although different mechanisms of karyotype evolution (polyploidy, fusion and fission) seem to be acting in distinct lineages, the degree of chromosome variation and the main mechanisms involved are comparable to those found in some monocentric genera and lower than expected for a holocentric genus.

In previous studies, we developed a wheat-Psathyrostachys huashanica Keng disomic addition line 3-8-10-2, which exhibited high stripe rust resistance and could be used as a donor source for introducing novel disease resistance gene(s) into wheat in future breeding programs. It was identified using cytology, genomic in situ hybridization (GISH), EST-SSR, EST-STS and morphological analyses. However, these techniques are not suitable for breeding programs that require the rapid screening of large numbers of genotypes because they are highly technical and time-consuming. In this study, three Ns genome-specific SCAR markers were developed via random amplified polymorphic DNA (RAPD) markers. These SCAR markers were further validated using a complete set of wheat-P. huashanica disomic addition lines, which segregated the 5Ns disomic addition line individuals. Our results indicated that the SCAR markers associated with the 5Ns chromosome of P. huashanica and they provide a low cost, high efficiency, alternative tool for screening 5Ns chromosomes in a wheat background. These newly developed SCAR markers that species-specificity of the markers was proved by analysis of a wide range of cereal species, and specific for 5Ns chromosome, which should be useful in marker-assisted selection for wheat breeders who want to screen genotypes that may contain 5Ns chromatin.

Development of cultivars resistant to powdery mildew instigated by Erysiphe polygoni DC. is an important objective of breeding mungbean (Vigna radiata L. Wilczek) worldwide. The obligate parasitic nature of the pathogen, diverse host range and quantitative inheritance pattern obscure breeding progress towards identification of resistance sources in this crop. Moreover, environment and genotype × environment interaction (GEI) effects also complicate identification of sources of durable resistance against powdery mildew. The present study is an attempt to unravel the GEI interaction for the purpose of evaluating sources of durable resistance, followed by identification of testing locations based on decisive parameters (discrimination power, representativeness and desirability index) as well as mega-environment delineation using the heritability-adjusted GGE (HA-GGE) biplot method. Initial studies with 236 mungbean genotypes revealed 20 promising genotypes, which were assessed further in multi-location and multi-year trials. Integration of the HA-GGE biplot and other statistical parameters revealed significant influence of environment and GEI on the dynamics of the disease, advocating the efficacy of multi-locational trials. HA-GGE biplot detected IPM-312-19 as an ideal genotype and Pusa-0672 and MH-2-15 as desirable, having durable resistance and genetic buffering capacity against this disease. These genotypes could be utilised in future resistance breeding program in mungbean. Enumeration of bootstrapping at a 95% confidence interval validated the genotype recommendation. Mega-environment delineation and desirability index suggest precise testing of material with resource optimisation in future breeding programs.

Once thought to be a minor disease, foliar blast disease of pearl millet, caused by Magnaporthe grisea, has recently emerged as an important biotic constraint for pearl millet production in India. The presence of a wider host range as well as high pathogenic heterogeneity complicates host–pathogen dynamics. Furthermore, environmental factors play a significant role in exacerbating the disease severity. An attempt was made to unravel the genotype-by-environment interactions for identification and validation of stable resistant genotypes against foliar blast disease through multi-environment testing. A diversity panel consisting of 250 accessions collected from over 20 different countries was screened under natural epiphytotic conditions in five environments. A total of 43 resistant genotypes were found to have high and stable resistance. Interestingly, most of the resistant lines were late maturing. Combined ANOVA of these 250 genotypes exhibited significant genotype-by-environment interaction and indicated the involvement of crossover interaction with a consistent genotypic response. This justifies the necessity of multi-year and multi-location testing. The first two principal components (PCs) accounted for 44.85 and 29.22% of the total variance in the environment-centered blast scoring results. Heritability-adjusted genotype plus genotype × environment interaction (HA-GGE) biplot aptly identified “IP 11353” and “IP 22423, IP 7910 and IP 7941” as “ideal” and “desirable” genotypes, respectively, having stable resistance and genetic buffering capacity against this disease. Bootstrapping at a 95% confidence interval validated the recommendations of genotypes. Therefore, these genotypes can be used in future resistance breeding programs in pearl millet. Mega-environment delineation and desirability index suggested Jaipur as the ideal environment for precise testing of material against the disease and will increase proper resource optimization in future breeding programs. Information obtained in current study will be further used for genome-wide association mapping of foliar blast disease in pearl millet.

<p style="text-align: justify;"><strong>Aim</strong>: Assessing levels and patterns of population genetic variation in combination with morphological characterization represents an important step for evaluating rare or endangered species and determining appropriate conservation strategies. This is particularly important for ensuring the preservation of valuable genetic variation in wild relatives of crops, which could provide beneficial alleles for plant breeding and improvement.</p><p style="text-align: justify;"><strong>Methods and results</strong>: A survey of the relict wild grapevine population was carried out in the province of Burgos, in the Castilian and Leon region (Spain). Genetic diversity, inbreeding extent and possible hybridization with cultivated grapevine were investigated using molecular markers. Results showed that overall, genetic diversity was low but inbreeding was not present. Private alleles were found in the Burgos wild genotypes, suggesting the potential value of these accessions. Comparisons of morphological traits and molecular features among the wild population showed a close genetic relationship among them and no genetic relationship to Castilian grapevine cultivars.</p><p style="text-align: justify;"><strong>Conclusion</strong>: The genetic differentiation observed between wild and domesticated forms of grapevine points out the interest to characterize and conserve the existing populations as a source of novel alleles for future grapevine breeding programs. At the same time, the observed enological differences between wild and domesticated grapevine populations could be interesting for the wine industry. Finally, given that these populations are at severe risk of extinction, we recommend that this population be prioritized for <em>ex situ</em> and possibly on-farm conservation as well as <em>in</em> <em>situ</em> protection.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: This work showed the existence of novel alleles and enological characters in natural wild grapevine populations, which represents a first step in the potential contribution of these natural populations to viticulture, suggesting a possible role in future breeding programs.</p>

Genetic parameters of linear conformation type traits and their relationship with milk yield throughout lactation in mixed-breed dairy goats

Nuclear male sterility (NMS) conditioned by recessive genes provides a useful tool for sunflower (Helianthus annuus L.) breeding and genetics programs. The objectives of this study were to evaluate seven induced NMS mutants derived from inbred line HA 89 and two lines, B11A3 and P21, for their mode of inheritance, allelic relationships, and agronomic characteristics. Self‐pollinated F2 progenies from heterozygous F1 male‐fertile (MF) plants and BC1F1 progenies from crossing male‐sterile (MS) with F1 heterozygous MF plants segregated in ratios of three MF to one MS and one MF to one MS, respectively, indicating single recessive gene control of male sterility. Half‐diallel crosses of MS with heterozygous F, MF plants indicated that the seven NMS mutant lines represented mutations at four loci that differ from that of P21 or B11A3. Male‐fertile segregates had normal pollen production, as that of HA 89, and MS segregates lacked anther extrusion and pollen production in both field and greenhouse evaluations. B11A3 had a few partially fertile florets at the center of the heads in summer greenhouse plantings, perhaps due to high temperature. The shorter plant height and associated female sterility in NMS 747 (ms8) make it less useful. B11A3 (mslO) and P21 (msll) are agronomically less desirable because of their taller height and lower self‐compatibility. NMS 360 (ms9), 552 (ms7), and 872 (ms6), representing three of the four mutant genes, performed similarly to agronomically adapted HA 89, were completely male‐sterile, and should be considered for use in future genetics and breeding programs as replacements for B11A3 and P21.

Chromosome data are fundamental in evolution. However, there has been no attempt to synthesize and evaluate the significance of such information from a phylogenetic perspective in the giant genus Solanum, which was the aim of this work. New and published information of the main cytotaxonomic features (chromosome number, polyploidy, total length of the haploid complement, mean chromosome length, mean arm ratio, karyotype formula, nuclear DNA amount, number/position of rDNA sites) was compiled and mapped onto an embracing Solanaceae phylogeny, performing Ancestral States Reconstruction. There were 506 Solanum species with chromosome counts (49.7% from an estimated total of 1,018 spp.), with x = 12 being the most frequent number (97%). Species with karyotypes represent 18.8%, while 8% have been studied with any molecular cytogenetic technique. Chromosome characters showed transitions associated with supported nodes, some of which have undergone fewer transitions than others. The common ancestor of all Solanum was a diploid with 2n = 24, a karyotype with st and/or t chromosomes, 2C DNA content of 1–1.2 pg, one locus of 18–5.8–26S rDNA and one of 5S, both loci being asyntenic. The chromosomal variables behave as homoplastic, with reversions in all branches. The analysed characters were sorted from more to less conserved: asynteny of rDNA loci; number of sites of 18–5.8–26S; chromosome number; karyotype formula; number of 5S loci. This pattern of chromosomal evolution distinguishes Solanum from closely related genera and from genera from other families with a similar number of species.