Development of cost-effective single nucleotide polymorphism marker assays for genetic diversity analysis in <Emphasis Type="Italic">Brassica rapa</Emphasis>

Competitive allele-specific PCR (KASPar) assay is a user-friendly system that provides flexibility in the numbers of single nucleotide polymorphisms (SNPs) and genotypes. Based on Illumina-GA-IIx genomic data from 10 genotypes with a broad genetic background, 3183 SNPs were selected for KASPar assays development, and 568 were finally converted and selected for Brassica rapa germplasm characterization (17.8%) on the basis of reproducibility, missing data rate, and uniform genetic distribution. High levels of polymorphism of these markers across 231 B. rapa genotypes were verified, illustrating by high polymorphic information content (averaged 0.34), minor allele frequency (0.37), genetic diversity (0.45), and the low observed heterozygosity (0.10). Based on the SNP dataset, structure and principal coordinates analysis, and neighbor-joining phylogenetic methods were used to examine the population structure and gave highly consistent results. The 231 accessions were divided into the four primary subspecies, representing 99 accessions from B. rapa ssp. pekinensis, 85 from B. rapa ssp. chinensis, 30 from B. rapa ssp. rapifera, and 17 from B. rapa ssp. oleifera and were further subdivided into 12 lower-order clusters according to different morphotypes. The genetic variability and pairwise fixation index analysis revealed that the ssp. pekinensis accessions possess the most extensive genetic variation among the four subspecies. The KASPar system is highly useful for validating SNPs and will be valuable for genetics research and breeding applications in B. rapa.     

Screening of rice landraces (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) for seedling stage salinity tolerance using morpho-physiological and molecular markers

Traditional rice landraces of coastal area in Bangladesh are distinct regarding their phenotype, response to salt stress and yield attributes. With characterization of these landraces, suitable candidate genes for salinity tolerance could be identified to introgress into modern rice varieties. Therefore, the aim of this experiment was to uncover prospective rice landraces tolerant to salinity. Relying on morphological, biochemical and molecular parameters 25 rice genotypes were tested for salt tolerance at germination and seedling stage. At germination stage 0 and 12 dSm^?1 salinity were imposed on rice genotypes. Ward’s cluster analysis divided rice genotypes into three clusters (susceptible, moderately tolerant and tolerant) based on the physiological indices. The tolerant rice landraces to salinity were Sona Toly, Nakraji and Komol Bhog. At seedling stage screening was performed following IRRI standard protocol at 12 dSm^?1 salinity level. Based on all morphological and biochemical parameters Komol Bhog was identified as the highly salinity tolerant landrace while Bolonga, Sona Toly, Dud Sail, Tal Mugur and Nakraji were found as tolerant to salinity. Molecular characterization using two simple sequence repeats (SSR) markers, viz. RM121 and RM337 displayed Bolonga, Til Kapor, Panbra, Sona Toly, Bina Sail, Komol Bhog, Nakraji, Tilkapur, Gajor Goria and Gota were tolerant landraces through genetic similarity in dendrogram. These identified salt-resistant landraces can be used as promising germplasm resources for breeding salt-tolerant high-yielding rice varieties in future.     

Easy strategy used to detect the genetic variability in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

A priority in the management and use of elite plant materials for breeding has been based on molecular markers or DNA sequencing of entire genomes, in order to perform genetic differentiation which is still quite costly. Chickpea (Cicer arietinum) is one of the species with genomic monotony and very low polymorphism, and its detection even with DNA markers has not been easy. In germplasm banks, the genetic distinction is a priority in order to use properly selected lines. In this study, 57 chickpea accessions from a germplasm bank were analyzed by using nrRAMP (non-radioactive Random Amplified Microsatellite Polymorphism) markers, and their genetic variability was determined. Our results showed DNA polymorphisms, which are enough to differentiate between the accessions and between C. arietinum and Cicer reticulatum (out-group); this last wild species is closely related to chickpea. We concluded that the nrRAMP technique was an effective and a highly useful method to assess the genetic diversity and variability among closely related plants, such as chickpea; in addition, this technique can be easily implemented in laboratories.     

Unravelling genetic diversity and cultivar parentage in the Danish apple gene bank collection

Characterization of apple germplasm is important for conservation management and breeding strategies. A set of 448 Malus domestica accessions, primarily of local Danish origin, were genotyped using 15 microsatellite markers. Ploidy levels were determined by flow cytometry. Special emphasis was given to pedigree reconstruction, cultivar fingerprinting and genetic clustering. A reference set of cultivars, mostly from other European countries, together with a private nursery collection and a small set of Malus sieversii, Malus sylvestris and small-fruited, ornamental Malus cultivars, was also included. The microsatellite markers amplified 17–30 alleles per loci with an average degree of heterozygosity at 0.78. We identified 104 (23%) duplicate genotypes including colour sports. We could infer first-degree relationships for many cultivars with previously unknown parentages. STRUCTURE analysis provided no evidence for a genetic structure but allowed us to present a putative genetic assembly that was consistent with both PCA analysis and parental affiliation. The Danish cultivar collection contains 10% duplicate genotypes including colour sports and 22% triploids. Many unique accessions and considerable genetic diversity make the collection a valuable resource within the European apple germplasm. The findings presented shed new light on the origin of Danish apple cultivars. The fingerprints can be used for cultivar identification and future management of apple genetic resources. In addition, future genome-wide association studies and breeding programmes may benefit from the findings concerning genetic clustering and diversity of cultivars.     

Characterization of hawthorn (<Emphasis Type="Italic">Crataegus</Emphasis> spp.) genotypes by SSR markers

Hawthorn (Crataegus spp.) is an edible wild fruit that is used in traditional medicine, landscape studies, and food and beverage industries in many countries. It is an important wild plant species in Turkey and is numerous in the Yozgat Province. Genetic and breeding studies on hawthorn are very limited. Therefore, we aimed to characterize 91 hawthorn genotypes using simple sequence repeat (SSR) markers. The SSRs were developed from apple and pear and were screened in hawthorn for amplification and polymorphisms. A total of 265 alleles were detected from thirty-two SSR primer pairs, and those were used to identify genetic relationships. The number of alleles ranged from 2 to 21 alleles per locus with a mean value of 8.28. The Hi05b09 locus showed the highest allele number (Na?=?21). The polymorphism information content (PIC) values ranged from 0.16 (CH03d10) to 0.89 (C6554) with a mean value of 0.60. An Unweighted Pair Group Method with Arithmetic Average method was used to cluster the genotypes, and four major clusters were obtained from the amplification of the SSRs. STRUCTURE software identified four populations (ΔK?=?4) and eight sub-populations (ΔK?=?8), and four major clusters similar results to UPGMA analysis. Our study showed that the SSR markers could be utilized as a reliable tool for the determination of genetic variations and relationships of hawthorn genotypes. A basic molecular analysis on the hawthorn genotypes identified in this study will promote the collection of germplasm collection and the selection of parents’ in future cross-breeding studies.     

Analysis of genetic diversity and population structure in <Emphasis Type="Italic">Capsicum</Emphasis> landraces from North Eastern India using TE-AFLP markers

North eastern (NE) India harbours a precious germplasm repository of Capsicum in the form of various landraces. The present study was undertaken to characterise the extent of genetic variation present in different Capsicum landraces from north eastern India. A set of 171 Capsicum accessions were characterised using three-endonuclease amplified fragment length polymorphism (AFLP) markers. Out of 416 bands obtained from six primer combinations, 254 (61 %) were polymorphic. The pairwise genetic dissimilarity among accessions ranged from 0.03 to 0.97. Cluster analysis based on neighbour joining showed two major clusters. Cluster I contained most of the bhut jolokia accessions whereas cluster II contained all of the Capsicum annuum genotypes. Similar grouping was observed with population STRUCTURE analysis as well as principle coordinate analysis. Analysis of molecular variance (AMOVA) revealed 45 and 54 % variation among and within populations, respectively. This information on population structure analysis and molecular characterisation will be helpful for effective utilisation of this germplasm in Capsicum improvement programs.     

First insights into the biochemical and molecular response to cold stress in <Emphasis Type="Italic">Cicer microphyllum</Emphasis>, a crop wild relative of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis>)

Identifying a potential crop wild relative (CWR) of legumes, especially one with high abiotic stress tolerance, has been a priority of plant breeders for many decades. Traditionally CWRs have been selected based on biometrical traits observed in the field, however this methodology is insufficient for research into nonmorphological traits such as stress tolerance. Biochemical and molecular analysis of potential CWRs allows for more informed selection. Specifically, we focus on Cicer microphyllum Benth, a CWR of cultivated chickpea Cicer arietinum L., which is distributed in Trans Himalayan ranges adjacent to glaciers of India and Pakistan at the alpine altitude gradient between 2700 to 6000 m. The objective of this study is to begin characterization of the biochemical and molecular bases of adaptation of C. microphyllum to cold stress and compare it to its cultivated relative (Cold susceptible genotype ILC533). Significant differences were recorded in terms of malondialdehyde (MDA) concentration, electrolyte leakage and proline accumulation in C. microphyllum, as compared to C. arietinum, upon cold exposure (4°C/24h). C. microphyllum exhibits more membrane stability under cold stress. Furthermore, proline overaccumulation and an increase in the enzymatic activities of antioxidants including superoxide dismutase, catalase, and ascorbate peroxidase were also observed in C. microphyllum under cold stress treatment. Expression of pyrroline-5-carboxylate synthetase, chalcone reductase, flavonoid 3',5'-hydroxylase and flavonoid 3'-monooxygenase are all upregulated under cold treatment in C. microphyllum. The characteristics recommend C. microphyllum both as a model for plant response to cold stress and as a potential source for abiotic stress resistant germplasm for chickpea breeding programs.     

Genetic Diversity,Genotype Discrimination,and Population Structure of Mexican <Emphasis Type="Italic">Opuntia</Emphasis> sp., Determined by SSR Markers

The Opuntia (prickly pear) genus, an important horticultural crop in Mexico, is essentially a fruit crop with two variants: sweet (“tunas”) or acid (“xoconostles”) fruits; it is also a source of vegetables “nopalitos” or fodder for livestock, among other uses. Its taxonomical classification has been reported as complex, although few studies on the genetic structure of Mexican Opuntia are available, and genetic differences between the two types of fruits are unknown. Opuntia genotype identification and classification are still mainly based on morphological characters. In this study, the genetic diversity of Mexican Opuntia germplasm with agronomic and economic importance was revealed, using 88 accessions and 13 SSR markers, in an attempt to explore the genetic relationships among them. A total of 159 alleles were detected ranging from 7 to 23 per locus with an average of 12.2. The SSR markers generated unique fingerprints for each Opuntia accession confirming their usefulness for genetic analysis. The accessions’ grouping was defined by several complementary clustering methods, and the moderate incongruences between the different methods did not influence the overall clustering. DAPC and STRUCTURE analyses grouped the accessions into five groups, thus confirming the incorrect delimitation of species in this genus. The following species had no clear boundaries: Opuntia ficus-indica, Opuntia albicarpa, Opuntia megacantha, Opuntia streptacantha, Opuntia lasiacantha, and Opuntia hyptiacantha. However, Opuntia robusta was separated from the rest of the species. Opuntia joconostle and Opuntia matudae, which produce acid fruits, tended to differ from the others. Median-joining simulation classified all genotypes into a complex network, and both linear and reticular ties between Mexican Opuntia genotypes were revealed. The genetic distance revealed in the present study shows the importance of Mexican accessions for conservation and use in breeding programs.     

New microsatellite markers developed from <Emphasis Type="Italic">Urochloa humidicola</Emphasis> (Poaceae) and cross amplification in different <Emphasis Type="Italic">Urochloa</Emphasis> species

Background

Urochloa humidicola is a forage grass that grows in tropical regions and is recognized for its tolerance to seasonal flooding. It is a polyploid and apomictic species with high phenotypic plasticity. As molecular tools are important in facilitating the development of new cultivars and in the classification of related species, the objectives of this study were to develop new polymorphic microsatellite markers from an enriched library constructed from U. humidicola and to evaluate their transferability to other Urochloa species.

Findings

Microsatellite sequences were identified from a previously constructed enriched library, and specific primers were designed for 40 loci. Isolated di-nucleotide repeat motifs were the most abundant followed by tetra-nucleotide repeats. Of the tested loci, 38 displayed polymorphism when screened across 34 polyploid Urochloa sp. genotypes, including 20 accessions and six hybrids of U. humidicola and two accessions each from U. brizantha, U. dictyoneura, U. decumbens and U. ruziziensis. The number of bands per Simple Sequence Repeat (SSR) locus ranged from one to 29 with a mean of 11.5 bands per locus. The mean Polymorphism Information Content (PIC) of all loci was 0.7136, and the mean Discrimination Power (DP) was 0.7873. Six loci amplified in all species tested. STRUCTURE analysis revealed six different allelic pools, and the genetic similarity values analyzed using Jaccard's coefficient ranged from 0.000 to 0.913.

Conclusions

This work reports new polymorphic microsatellite markers that will be useful for breeding programs for Urochloa humidicola and other Urochloa species as well as for genetic map development, germplasm characterization, evolutionary and taxonomic studies and marker-assisted trait selection.

     

<Emphasis Type="Italic">Wsi18</Emphasis> promoter from wild rice genotype, <Emphasis Type="Italic">Oryza nivara</Emphasis>, shows enhanced expression under soil water stress in contrast to elite cultivar, <Emphasis Type="Italic">IR20</Emphasis>

Identification and characterization of plant promoters from wild rice genotypes showing inducible expression under soil water stress (SWS) is desirable for transgene expression to generate stress tolerant rice cultivars. A comparative expression profiling of Wsi18, a group 3 LEA gene, revealed differential response under SWS conditions between modern cultivated rice (IR20) and its wild progenitor (Oryza nivara). Wsi18 promoter from O. nivara showed enhanced inducible expression of the reporter gusA gene, encoding β-glucuronidase, in transgenic rice plants in comparison to similar promoter from IR20. Deletion analysis unravelled the cis-acting regulatory elements minimally required for optimal expression of Wsi18 promoter from O. nivara under SWS condition. This is the first report of characterization of an inducible promoter from a wild rice genotype to drive the gene expression under water stress conditions. The Wsi18 promoter element from the wild rice genotype can be used in future genetic manipulation strategies for the generation of SWS tolerant rice cultivars with improved yield characteristics.     

Resistance to <Emphasis Type="Italic">Rhynchosporium commune</Emphasis> in a collection of European spring barley germplasm

Key message

Association analyses of resistance to Rhynchosporium commune in a collection of European spring barley germplasm detected 17 significant resistance quantitative trait loci. The most significant association was confirmed as Rrs1.

Abstract

Rhynchosporium commune is a fungal pathogen of barley which causes a highly destructive and economically important disease known as rhynchosporium. Genome-wide association mapping was used to investigate the genetic control of host resistance to R. commune in a collection of predominantly European spring barley accessions. Multi-year disease nursery field trials revealed 8 significant resistance quantitative trait loci (QTL), whilst a separate association mapping analysis using historical data from UK national and recommended list trials identified 9 significant associations. The most significant association identified in both current and historical data sources, collocated with the known position of the major resistance gene Rrs1. Seedling assays with R. commune single-spore isolates expressing the corresponding avirulence protein NIP1 confirmed that this locus is Rrs1. These results highlight the significant and continuing contribution of Rrs1 to host resistance in current elite spring barley germplasm. Varietal height was shown to be negatively correlated with disease severity, and a resistance QTL was identified that co-localised with the semi-dwarfing gene sdw1, previously shown to contribute to disease escape. The remaining QTL represent novel resistances that are present within European spring barley accessions. Associated markers to Rrs1 and other resistance loci, identified in this study, represent a set of tools that can be exploited by breeders for the sustainable deployment of varietal resistance in new cultivars.

     

Taxonomic genetics of methylotrophic yeast genus <Emphasis Type="Italic">Komagataella</Emphasis>: New biological species <Emphasis Type="Italic">K. kurtzmanii</Emphasis>

Genetic hybridization analysis revealed that industrially important species Komagataella kurtzmanii has reproductive postzygotic isolation from K. pastoris, K. phaffii, K. populi, K. pseudopastoris, and K. ulmi. Therefore, it represents a new biological species of the genus Komagataella. The genetic data are in perfect agreement with the molecular taxonomy of the genus Komagataella.     

Molecular and cytological characterization of the global <Emphasis Type="Italic">Musa</Emphasis> germplasm collection provides insights into the treasure of banana diversity

Bananas (Musa spp.) are one of the main fruit crops grown worldwide. With the annual production reaching 144 million tons, their production represents an important contribution to the economies of many countries in Asia, Africa, Latin-America and Pacific Islands. Most importantly, bananas are a staple food for millions of people living in the tropics. Unfortunately, sustainable banana production is endangered by various diseases and pests, and the breeding for resistant cultivars relies on a far too small base of genetic variation. Greater diversity needs to be incorporated in breeding, especially of wild species. Such work requires a large and thoroughly characterized germplasm collection, which also is a safe depository of genetic diversity. The largest ex situ Musa germplasm collection is kept at the International Transit Centre (ITC) in Leuven (Belgium) and currently comprises over 1500 accessions. This report summarizes the results of systematic cytological and molecular characterization of the Musa ITC collection. By December 2015, 630 accessions have been genotyped. The SSR markers confirmed the previous morphological based classification for 84% of ITC accessions analyzed. The remaining 16% of the genotyped entries may need field verification by taxonomist to decide if the unexpected classification by SSR genotyping was correct. The ploidy level estimation complements the molecular data. The genotyping continues for the entire ITC collection, including newly introduced accessions, to assure that the genotype of each accession is known in the largest global Musa gene bank.     

Genetic structure of cultivated <Emphasis Type="Italic">Zanthoxylum</Emphasis> species investigated with SSR markers

Zanthoxylum is an economically and ecologically important genus of the Rutaceae family, of which Z. bungeanum and Z. armatum have a long history of cultivation in China. However, how the natural processes such as selection and drift and agriculture practices have influenced the genetic variation of cultivated Zanthoxylum species during long-term domestication remains elusive. Herein, we determined the population genetic structure of current widely cultivated Zanthoxylum species, Z. bungeanum and Z. armatum. Microsatellite markers revealed a high level of genetic variation and significant genetic differentiation for both species despite Z. bungeanum showed higher genetic diversity than Z. armatum. AMOVA indicated that most of the genetic variation exists within individuals rather than among provenances for both species. Population structure analyses generated three distinct groups within the entire accessions. All Z. bungeanum accessions were distinguished into two major geographic groups, north and south groups, with Qinling Mountains as the main geographic barrier to gene flow while a significant genetic differentiation was observed between cultivated and wild Z. armatum accessions. Mantel test of Z. bungeanum displayed a significant correlation between genetic and geographic distances within each inferred group but no correlation between genetic and geographic distance was observed when comparing genetic and geographic distances focusing only on pairwise of north vs. south provenances, ruling out the hypothesis that gene flow between north and south provenances followed an isolation-by-distance model. Our research provided a fundamental genetic profile that will improve the conservation and responsible exploitation of the extant germplasm of Zanthoxylum.     

Genetic Diversity,Population Structure,Parentage Analysis,and Construction of Core Collections in the French Apple Germplasm Based on SSR Markers

In-depth characterization of apple genetic resources is a prerequisite for genetic improvement and for germplasm management. In this study, we fingerprinted a very large French collection of 2163 accessions with 24 SSR markers in order to evaluate its genetic diversity, population structure, and genetic relationships, to link these features with cultivar selection date or usage (old or modern, dessert or cider cultivars), and to construct core collections. Most markers were highly discriminating and powerful for varietal identification, with a probability of identity P _(ID) over the 21 retained SSR loci close to 10^?28. Pairwise comparisons revealed 34 % redundancy and 18.5 % putative triploids. The results showed that the germplasm is highly diverse with an expected heterozygosity H _e of 0.82 and observed heterozygosity H _o of 0.83. A Bayesian model-based clustering approach revealed a weak but significant structure in three subgroups (F_ST?=?0.014–0.048) corresponding, albeit approximately, to the three subpopulations defined beforehand (Old Dessert, Old Cider, and Modern Cultivars). Parentage analyses established already known and yet unknown relationships, notably between old cultivars, with the frequent occurrence of cultivars such as “King of Pippin” and “Calville Rouge d’Hiver” as founders. Finally, core collections based on allelic diversity were constructed. A large dessert core collection of 278 cultivars contained 90 % of the total dessert allelic diversity, whereas a dessert subcore collection of 48 cultivars contained 71 % of diversity. For cider apples, a 48-cultivar core collection contained 83 % of the total cider allelic diversity.     

Characterization of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) lectin for biological activity

Lectins are proteins that are subject of intense investigations. Information on lectin from chickpea (Cicer arietinum L.) with respect to its biological activities are very limited. In this study, we purified lectin from the seeds of chickpea employing DEAE-cellulose and SP-Sephadex ion exchange chromatography and identified its molecular subunit mass as 35 kDa. The free radical scavenging activity of lectin measured by the DPPH assay has IC₅₀ of 0.88 µg/mL. Lectin exerted antifungal activity against Candida krusei, Fusarium oxysporium oxysporium, Saccharomyces cerevisiae and Candida albicans, while antibacterial activity against E. coli, B. subtilis, S. marcescens and P. aeruginosa. The minimum inhibitory concentrations were 200, 240, 160 and 140 µg for C. krusei, F. oxysporium, S. cerevisiae and C. albicans respectively. Lectin was further examined for its antiproliferative potential against cancerous cell line. The cell viability assay indicated a high inhibition activity on Ishikawa, HepG2, MCF-7 and MDA-MB-231 with IC₅₀ value of 46.67, 44.20, 53.58 and 37.46?µg/mL respectively. These results can provide a background for future research into the benefits of chickpea lectin to pharmacological perspective.