Diploid Accessions Research Articles

Polyploidy in Odontarrhena bertolonii (Brassicaceae) in relation to seed germination performance and plant phenotype, with taxonomic implications

Odontarrhena bertolonii is an endemic serpentine species of Tuscany and Liguria and a model system for nickel-hyperaccumulation research in plants. Phenotypic, genetic and karyological variability between populations was previously detected, but existence and distribution of cytotypes was unknown. Accordingly, we examined ploidy level in eight populations from the species range and tested the polyploid fitness hypothesis by analysing relationships with plant phenotype and seed germination capacity. Accessions from central-western Tuscany and Liguria resulted diploid, while those from the upper Tiber valley were tetraploid. Autopolyploidy via unreduced gametes is likely the mechanism for the origin of tetraploid plants. Size and mass were higher in tetraploid seeds, which germinated faster and to higher percentages than diploid ones. Tetraploid plants were more robust and taller, with more richly branched inflorescences. Silicle shape and size did not differ, but style was longer in tetraploids and seeds had a larger wing. Overall, these results supported a higher fitness in polyploids. Multivariate analysis showed continuous variation but consistent differentiation between diploid and tetraploid accessions. Taxonomically, the tetraploid populations at the eastern limit of the species range in the upper Tiber valley can be referred to a new subspecies, here described as O. bertolonii subsp. cesalpina.

RNA-Seq-based DNA marker analysis of the genetics and molecular evolution of Triticeae species.

The release of high-quality chromosome-level genome sequences of members of the Triticeae tribe has greatly facilitated genetic and genomic analyses of important crops such as wheat (Triticum aestivum) and barley (Hordeum vulgare). Due to the large diploid genome size of Triticeae plants (ca. 5 Gbp), transcript analysis is an important method for identifying genetic and genomic differences among Triticeae species. In this review, we summarize our results of RNA-Seq analyses of diploid wheat accessions belonging to the genera Aegilops and Triticum. We also describe studies of the molecular relationships among these accessions and provide insight into the evolution of common hexaploid wheat. DNA markers based on polymorphisms within species can be used to map loci of interest. Even though the genome sequence of diploid Aegilops tauschii, the D-genome donor of common wheat, has been released, the diploid barley genome continues to provide key information about the physical structures of diploid wheat genomes. We describe how a series of RNA-Seq analyses of wheat relatives has helped uncover the structural and evolutionary features of genomic and genetic systems in wild and cultivated Triticeae species.

The Evolutionary Dynamics of Repetitive DNA and Its Impact on the Genome Diversification in the Genus Sorghum

Polyploidization is an evolutionary event leading to structural changes of the genome(s), particularly allopolyploidization, which combines different genomes of distinct species. The tetraploid species, Sorghum halepense, is assumed an allopolyploid species formed by hybridization between diploid S. bicolor and S. propinquum. The repeat profiles of S. bicolor, S. halepense, and their relatives were compared to elucidate the repeats’ role in shaping their genomes. The repeat frequencies and profiles of the three diploid accessions (S. bicolor, S. bicolor ssp. verticilliflorum, and S. bicolor var. technicum) and two tetraploid accessions (S. halepense) are similar. However, the polymorphic distribution of the subtelomeric satellites preferentially enriched in the tetraploid S. halepense indicates drastic genome rearrangements after the allopolyploidization event. Verified by CENH3 chromatin immunoprecipitation (ChIP)-sequencing and fluorescence in situ hybridization (FISH) analysis the centromeres of S. bicolor are mainly composed of the abundant satellite SorSat137 (CEN38) and diverse CRMs, Athila of Ty3_gypsy and Ty1_copia-SIRE long terminal repeat (LTR) retroelements. A similar centromere composition was found in S. halepense. The potential contribution of S. bicolor in the formation of tetraploid S. halepense is discussed.

Deciphering ploidal levels of Lippia alba by using proteomics

Lippia alba (Mill.) N.E. Brown (Verbenaceae), popularly known as “lemon balm” or “bushy matgrass”, is widely used in folk medicine due to its anti-inflammatory, antispasmodic, analgesic, and digestive properties. It was described as an autopolyploid complex with five cytotypes (2n = 30, 38, 45, 60 and 90). To enhance our understanding of the biological variation of the species, we investigated, comparatively, the proteomic profile of all ploidal levels (diploid, aneuploid, triploid, tetraploid, and hexaploid). Leaf proteins were extracted with subsequent separation by two-dimensional electrophoresis, spot analysis, and protein identification by mass spectrometry. By comparing the proteomic profile of diploid accession to the profile of the other ploidal levels we identified differential expression between the analysed spots. We identified 34 proteins with differential expression between the ploidal levels in comparison with the diploid. The identified proteins seem to play relevant roles in the primary metabolism of L. alba suggesting that a specific set of proteins was selected during the polyploidization process, being the triploid the most different one. Given that protein composition can substantially affect the desired therapeutic effect, we posit that further combination of proteomic and metabolomic studies may help to unravel genetic variations and phenotypic profiles in L. alba.

Allele mining in diverse accessions of tropical grasses to improve forage quality and reduce environmental impact.

The C4Urochloa species (syn. Brachiaria) and Megathyrsus maximus (syn. Panicum maximum) are used as pasture for cattle across vast areas in tropical agriculture systems in Africa and South America. A key target for variety improvement is forage quality: enhanced digestibility could decrease the amount of land required per unit production, and enhanced lipid content could decrease methane emissions from cattle. For these traits, loss-of-function (LOF) alleles in known gene targets are predicted to improve them, making a reverse genetics approach of allele mining feasible. We therefore set out to look for such alleles in diverse accessions of Urochloa species and Megathyrsus maximus from the genebank collection held at the CIAT. We studied allelic diversity of 20 target genes (11 for digestibility, nine for lipid content) in 104 accessions selected to represent genetic diversity and ploidy levels of U. brizantha, U. decumbens, U. humidicola, U. ruziziensis and M. maximum. We used RNA sequencing and then bait capture DNA sequencing to improve gene models in a U. ruziziensis reference genome to assign polymorphisms with high confidence. We found 953 non-synonymous polymorphisms across all genes and accessions; within these, we identified seven putative LOF alleles with high confidence, including those in the non-redundant SDP1 and BAHD01 genes present in diploid and tetraploid accessions. These LOF alleles could respectively confer increased lipid content and digestibility if incorporated into a breeding programme. We demonstrated a novel, effective approach to allele discovery in diverse accessions using a draft reference genome from a single species. We used this to find gene variants in a collection of tropical grasses that could help reduce the environmental impact of cattle production.

Classifying Cider Apple Germplasm Using Genetic Markers for Fruit Acidity

The organic acid concentration in apple (Malus ×domestica) juice is a major component of hard cider flavor. The goal of this study was to determine if the malic acid markers, Ma1 and Q8, could classify the titratable acidity concentration in cider apple accessions from the United States Department of Agriculture Malus germplasm collection into descriptive classifications. Our results indicate that for diploid genotypes, the Ma1 marker alone and the Ma1 and Q8 markers analyzed together could be used to predict cider apple acidity (P < 0.0001). Alone, the Ma1 marker categorized acidity into low (<2.4 g⋅L−1), medium (2.4–5.8 g⋅L−1), and high (>5.8 g⋅L−1) groups. The combination of Ma1 and Q8 markers provided more specificity, which would be useful for plant breeding applications. This work also identified a significant difference (P = 0.0132) in acidity associated with ploidy. On average, the triploids accessions had 0.33 g⋅L−1 higher titratable acidity than the diploid accessions. Based on the results of this work, we propose a genetics-based classification system for cider apples with the acidity component defined by the Ma1 and Q8 markers.

Sources of resistance to Pseudocercospora fijiensis, the cause of black Sigatoka in banana.

Black Sigatoka, caused by Pseudocercospora fijiensis, is one of the most devastating diseases of banana. In commercial banana‐growing systems, black Sigatoka is primarily managed by fungicides. This mode of disease management is not feasible for resource‐limited smallholder farmers. Therefore, bananas resistant to P. fijiensis provide a practical solution for managing the disease, especially under smallholder farming systems. Most banana and plantain hybrids with resistance to P. fijiensis were developed using few sources of resistance, which include Calcutta 4 and Pisang Lilin. To broaden the pool of resistance sources to P. fijiensis, 95 banana accessions were evaluated under field conditions in Sendusu, Uganda. Eleven accessions were resistant to P. fijiensis. Black Sigatoka symptoms did not progress past Stage 2 (narrow brown streaks) in the diploid accessions Pahang (AA), Pisang KRA (AA), Malaccensis 0074 (AA), Long Tavoy (AA), M.A. Truncata (AA), Tani (BB), and Balbisiana (BB), a response similar to the resistant control Calcutta 4. These accessions are potential sources of P. fijiensis resistance and banana breeding programmes can use them to broaden the genetic base for resistance to P. fijiensis.

Development and Exploitation of KASP Assays for Genes Underpinning Drought Tolerance Among Wheat Cultivars From Pakistan.

High-throughput genotyping for functional markers offers an excellent opportunity to effectively practice marker-assisted selection (MAS) while breeding cultivars. We developed kompetitive allele-specific PCR (KASP) assays for genes conferring drought tolerance in common wheat (Triticum aestivum L.). In total, 11 KASP assays developed in this study and five already reported assays were used for their application in wheat breeding. We investigated alleles at 16 loci associated with drought tolerance among 153 Pakistani hexaploid wheat cultivars released during 1953–2016; 28 diploid wheat accessions (16 for AA and 12 for BB) and 19 tetraploid wheat (AABB) were used to study the evolutionary history of the studied genes. Superior allelic variations of the studied genes were significantly associated with higher grain yield. Favored haplotypes of TaSnRK2.3-1A, TaSnRK2.3-1B, TaSnRK2.9-5A, TaSAP-7B, and TaLTPs-1A predominated in Pakistani wheat germplasm indicating unconscious pyramiding and selection pressure on favorable haplotypes during selection breeding. TaSnRK2.8-5A, TaDreb-B1, 1-feh w3, TaPPH-7A, TaMOC-7A, and TaPARG-2A had moderate to low frequencies of favorable haplotype among Pakistani wheat germplasm pointing toward introgression of favorable haplotypes by deploying functional markers in marker-assisted breeding. The KASP assays were compared with gel-based markers for reliability and phenotypically validated among 62 Pakistani wheat cultivars. Association analyses showed that the favorable allelic variations were significantly associated with grain yield-contributing traits. The developed molecular marker toolkit of the genes can be instrumental for the wheat breeding in Pakistan.

English

Paspalum notatum Flüggé (bahiagrass) is the most important forage grass in the native grasslands of southern Brazil; the native germplasm is tetraploid and apomictic. Breeding to produce cultivars has been possible due to chromosome doubling of sexual diploid accessions, allowing hybridizations with apomictic genotypes, generating progenies with variability for agronomic traits and segregating for reproduction mode. The aim of this study was to determine the reproduction mode of selected hybrids derived from intraspecific crosses through cytoembryological analysis and to evaluate the level of apospory expres¬sivity. Eighty one tetraploid hybrids corresponding to 21 families of sexual and apomictic parents were generated and 28 most productive plants based on their superior agronomic performance in the field were evaluated. Sixteen hybrids were reproduced sexually and 12 of them were highly apomictic or facultative apomictic. Sexual hybrids may be used for further crosses in the breeding program while highly apomictic plants produce uniform progeny and become candidates of new cultivars to promote pasture diversification. Key words: Aposporous apomixis, bahiagrass, intraspecific crosses, plant breeding.

Reproductive mode and DNA content of Paspalum accessions from Plicatula group: Reproduction and DNA content of Paspalum

This study aims the reproductive and DNA content characterization of ten different Paspalum species from the Plicatula group (P. atratum, P. compressifolium, P. guenoarum, P. lenticulare, P. lepton, P. oteroi, P. plicatulum, P. rhodopedum, P. rojasii and P. wrightii), contributing to the determination of the best breeding and conservation method to be used. The DNA content of 150 accessions was estimated by flow cytometry using foliar tissues. The reproductive mode of 135 accessions was characterized by flow cytometric seed screening, and five accessions were determined by cytoembryological ovary analysis. DNA content ranged from 1.50 pg (BGP 380) to 4.27 pg (BGP 193), both from P. compressifolium species. Most of the accessions were compatible to the tetraploid level, except for BGP 281 (P. lenticulare), BGP 272 (P. rojasii), and BGP 380 (P. compressifolium), which were diploids. Accessions with tetraploid DNA content presented apomictic reproduction, whereas the three diploid accessions were sexual. These results contribute to the reproductive knowledge of species of the Plicatula group. The diploid sexual accessions identified represent important material to overcome the apomictic barrier in crosses and can be used after polyploidization as female parents in crosses with apomictic accessions in Paspalum breeding programs.

Genetic diversity of pear germplasm in Bosnia and Herzegovina, as revealed by SSR markers

Bosnia and Herzegovina (BIH) pear germplasm in the ex situ field collection at the University of Banja Luka was characterised with simple sequence repeat (SSR) markers in order to reveal redundancies, determine genetic diversity and confirm uniqueness. European pear (Pyrus communis L.) 67 accessions of the BIH collection and 7 reference cultivars of the ex situ collection at SLU in Balsgard, Sweden were analysed using a set of 10 SSR markers. All markers resulted in good amplification and easy scoring of marker alleles One-third of accessions appeared to be triploid. A total of 112 alleles were amplified in unique diploid genotypes with on average 11.2 alleles per marker. Mean observed heterozygosity (Ho) was 0.72, mean expected heterozygosity (He) – 0.80 and mean Shannon index (I) – 1.96. Inbreeding coefficient (FIT ) for diploid accessions ranged from −0.06 to 0.28 (mean 0.02) and Wright’s inbreeding coefficient (FIS ) – from −0.27 to 0.17 (mean −0.03). In the BIH pear collection, mean value of genetic differentiation (FST ) between the reference cultivars and pear accessions was 0.05. Principal coordinate analysis (PCoA) divided the 74 pear accessions into three groups. The first group consisted of diploid, reference and possible triploid pear accessions, while the second and third groups contained only BIH possible triploid accessions. Overall, the results revealed high levels of polymorphism and uniqueness, indicating that BIH pear germplasm represents very diverse and valuable material for future breeding programmes.

Genomics Analyses Reveal Unique Classification, Population Structure and Novel Allele of Neo-Tetraploid Rice

BackgroundNeo-tetraploid rice (NTR) is a useful new germplasm that developed from the descendants of the autotetraploid rice (ATR) hybrids. NTR showed improved fertility and yield potential, and produced high yield heterosis when crossed with indica ATR for commercial utilization. However, their classification, population structure and genomic feature remain elusive.ResultsHere, high-depth genome resequencing data of 15 NTRs and 18 ATRs, together with 38 publicly available data of diploid rice accessions, were analyzed to conduct classification, population structure and haplotype analyses. Five subpopulations were detected and NTRs were clustered into one independent group that was adjacent to japonica subspecies, which maybe the reason for high heterosis when NTRs crossed with indica ATRs. Haplotype patterns of 717 key genes that associated with yield and other agronomic traits were revealed in these NTRs. Moreover, a novel specific SNP variation was detected in the first exon of HSP101, a known heat-inducible gene, which was conserved in all NTRs but absent in ATRs, 3KRG and RiceVarMap2 databases. The novel allele was named as HSP101–1, which was confirmed to be a heat response factor by qRT-PCR, and knockout of HSP101–1 significantly decreased the thermotolerance capacity of NTR. Interestingly, HSP101–1 was also specifically expressed in the anthers of NTR at pre-meiotic and meiosis stages under optimal environment without heat stress, and its loss-of-function mutant showed significant decrease in fertility of NTR.ConclusionThe construction of first genomic variation repository and the revelation of population structure provide invaluable information for optimizing the designs of tetraploid rice breeding. The detection of specific genomic variations offered useful genomic markers and new directions to resolve high fertility mechanism of NTR.

Genetic Similarity Among Accessions of Paspalum notatum Flügge (Poaceae): a Potential to Parental Selection

HIGHLIGHTS Low genetic similarity in Paspalum notatum accessions. High genetic distance among diploid accessions. The accessions have good potential to breeding program.

Evaluation of Indian Musa Germplasm for Resistance to Radopholus similis in Greenhouse

Fifty nine wild and cultivated banana genotypes (Musa spp.) including 10 diploids (AA, AB and BB genome) and 49 triploids (AAA, AAB, ABB) belonging to the Eumusa section, were evaluated for their host reaction against the burrowing nematode (Radopholus similis) under greenhouse conditions. Reduction in plant growth parameters was comparatively higher in susceptible genotypes when inoculated with nematodes. Root studies indicated higher number of roots, root fresh and dry weight in resistant and tolerant accessions even after nematode inoculation. Based on the corm grade, root necrosis percentage and the reproduction rate of nematode, the level of resistance to R. similis was assessed. The experiment revealed that the diploid accessions, Kanai Bansi (AA), Elakkiebale (AB), M. balbisiana (BB), Bhimkol (BB), Athiakol (BB) and Aittakola (BB) were resistant. Among triploids, Kothia (ABB) and Ankur II (ABB) were resistant to R. similis.

Molecular characterisation of the oldest domesticated Turkish einkorn wheat landraces with simple sequence repeat (SSR) markers

Einkorn (Triticum monococcum L. ssp. monococcum) is an ancient diploid wheat species with many useful traits and used as a wheat gene discovery model. In this research, a total of 41 diploid and tetraploid wheat accessions were tested using simple sequence repeat (SSR) markers. A total of 33 genotypes of T. monococcum along with four genotypes each of tetraploid wheat (T. dicoccon and T. durum) were used as plant material. The analysis utilized 10 polymorphic markers, including a total number of 41 alleles with an average frequency of 4.1 alleles per locus during exploration of the level of genetic variations. Various diversity analyses, which are the effective number of alleles (Ne), gene diversity (h), Polymorphic Information Content (PIC), and Shannon’s information index (I), were performed for 10 ‘A’ genome wheat SSR markers. The results showed a narrow variation in einkorn genotypes, supported by Analysis of Molecular Variance (AMOVA), with 66% maximum variation in all genotypes. The structure analysis divided the whole germplasm into two populations. A dendrogram was constructed to determine the genetic similarities using the unpaired group method with arithmetic averages (UPGMA), which separated tetraploid wheat from other genotypes/accessions. Principal Coordinate Analysis (PCoA) co-supported the clustering of UPGMA and structure by differentiating the diploid and tetraploid wheat. These findings will help understand the genetic relationships among these wheat accessions and their use in breeding programs in the future works.

Contrasting response of two Lotus corniculatus L. accessions to combined waterlogging-saline stress.

Waterlogging and salinity impair crop growth and productivity worldwide, with their combined effects being larger than the additive effects of the two stresses separately. Here, a common forage tetraploid Lotus corniculatus (cv. San Gabriel) and a diploid L. corniculatus accession, collected from a coastal area with high frequency of waterlogging-saline stress events, were evaluated for tolerance to waterlogging, salinity and these two stresses combined. We hypothesize that, due to its environmental niche, the diploid accession would show better adaptation to combined waterlogging-saline stress compared to the tetraploid L. corniculatus. Plants were evaluated under control conditions, waterlogging, salinity and a combined waterlogging-saline treatment for 33 days. Shoot and root growth were assessed, together with chlorophyll fluorescence and gas exchange measurements. Results showed that salinity and waterlogging effects were more severe for the tetraploid accession, with a larger effect being observed under the combined stress condition. Concentrations of Na+ , Cl- and K+ were measured in apical and basal leaves, and in roots. A larger accumulation of Na+ and Cl- was observed under both saline and combined stress treatments for the tetraploid L. corniculatus, for which ion toxicity effects were evident. The expression of CLC gene, coding for a Cl- transporter, was only increased in diploid L. corniculatus plants in response to the combined stress condition, suggesting that ion compartmentalization mechanisms were induced in this accession. Thus, this recently characterized L. corniculatus could be used for the introduction of new tolerance traits in other Lotus species used as forage.

Natural epialleles of Arabidopsis SUPERMAN display superwoman phenotypes

Epimutations are heritable changes in gene function due to loss or gain of DNA cytosine methylation or chromatin modifications without changes in the DNA sequence. Only a few natural epimutations displaying discernible phenotypes are documented in plants. Here, we report natural epimutations in the cadastral gene, SUPERMAN(SUP), showing striking phenotypes despite normal transcription, discovered in a natural tetraploid, and subsequently in eleven diploid Arabidopsis genetic accessions. This natural lois lane(lol) epialleles behave as recessive mendelian alleles displaying a spectrum of silent to strong superwoman phenotypes affecting only the carpel whorl, in contrast to semi-dominant superman or supersex features manifested by induced epialleles which affect both stamen and carpel whorls. Despite its unknown origin, natural lol epialleles are subjected to the same epigenetic regulation as induced clk epialleles. The existence of superwoman epialleles in diverse wild populations is interpreted in the light of the evolution of unisexuality in plants.

Application of genome-wide insertion/deletion markers on genetic structure analysis and identity signature of Malus accessions

BackgroundApple (Malus ssp.), one of the most important temperate fruit crops, has a long cultivation history and is economically important. To identify the genetic relationships among the apple germplasm accessions, whole-genome structural variants identified between M. domestica cultivars ‘Jonathan’ and ‘Golden Delicious’ were used.ResultsA total of 25,924 insertions and deletions (InDels) were obtained, from which 102 InDel markers were developed. Using the InDel markers, we found that 942 (75.3%) of the 1251 Malus accessions from 35 species exhibited a unique identity signature due to their distinct genotype combinations. The 102 InDel markers could distinguish 16.7–71.4% of the 331 bud sports derived from ‘Fuji’, ‘Red Delicious’, ‘Gala’, ‘Golden Delicious’, and other cultivars. Five distinct genetic patterns were found in 1002 diploid accessions based on 78 bi-allele InDel markers. Genetic structure analysis indicated that M. domestica showed higher genetic diversity than the other species. Malus underwent a relatively high level of wild-to-crop or crop-to-wild gene flow. M. sieversii was closely related to both M. domestica and cultivated Chinese cultivars.ConclusionsThe identity signatures of Malus accessions can be used to determine distinctness, uniformity, and stability. The results of this study may also provide better insight into the genetic relationships among Malus species.

A new one-tube reaction kit for the SSR genotyping of apple (Malus × domestica Borkh.)

Though apple genotyping is mainly used for scientific and breeding purposes, it can also be adopted by national authorities to control the authenticity of apple cultivars. To facilitate the introduction of routine apple genotyping into practice, a new apple simple sequence repeat (SSR) genotyping kit was developed (called the Ap17 in. SSR Genotyping Kit). The kit combines 17 SSR markers including those recommended by the Working Group of the European Cooperative Programme for Plant Genetic Resources (ECPGR), covering all apple linkage groups in a one-tube reaction format, using a fragment analysis method to simplify the genotyping procedure. The kit was successfully tested using 880 unique diploid apple germplasm accessions; the kit can also readily discriminate triploid and tetraploid samples. The total probability of identity for the kit and the sample collection used was calculated to be 1.73 × 10-22. Tables for converting results to enable genotype comparisons between currently-used genotyping systems and the Ap17 in. kit are provided. The kit is ideally suited for validation in laboratories genotyping a large number of apple samples, saving time, costs, and labor, while minimizing technical and human errors.

Osmotic stress induced root growth and regulation of root development related genes in progenitors of wheat

Maintenance of root growth during water deficit can significantly contribute to yield stability. In this study, total nine accessions, three each of Triticum monococcum, Aegilops speltoides and Aegilops tauschii were analysed for root traits under two levels of osmotic stress at seedling stage. T. monococcum accession (A2) showed highest increase in total root growth while 17% and 34% increase in primary root length was recorded in T. monococcum (A2) and Ae. speltoides (B3) under osmotic stress. Expression of BREVIS RADIX (BRX) and NAM/ATAF/CUC 1(NAC1) was analysed in three diploid accessions with contrasting root phenotype. BREVIS RADIX, a transcription factor modulating root length was up regulated in T. monococcum A2 accession while NAC1 expression was up regulated in three of the accessions under osmotic stress. The accessions and genes analysed in the study can be good resource to explore the molecular-genetic mechanism of root growth under stress.