Ploidy Level Research Articles

A naturalized diploid Fragaria sp. (Rosaceae) found in southern Chile as revealed by morphological, ploidy and cytogenetic analyses

Fragaria genus has a worldwide distribution and different ploidy levels. The only wild Fragaria species described in Chile is the octoploid Fragaria chiloensis. In a group of Fragaria accessions from southern Chile including Fragaria chiloensis subsp. chiloensis f. patagonica individuals, an accession named Fragaria-MEN, caught our attention because it shows notable phenotypic differences compared to the rest of the accessions. Fragaria-MEN was collected near the Menetue hot springs (Araucania Region, Chile) and differs from the other F. chiloensis plants mainly in leaf morphology. Due to these differences, we carried out a full morphology description of Fragaria-MEN individuals and then a characterization of the chromosomes and genome size to verify the ploidy level. Morphological analysis reveals close vegetative and reproductive characteristics shared with the non-native diploid Fragaria vesca ‘Hawaii’ species. We also observed a DNA amount according to the diploidy (2n = 2× = 14) revealed by cytological analysis of chromosomes. We propose Fragaria-MEN as F. vesca population that should have been introduced and now is naturalized in the Araucania Region of Chile.

Polyploid Genome Assembly Provides Insights into Morphological Development and Ascorbic Acid Accumulation of Sauropus androgynus.

Sauropus androgynus (S. androgynus) (2n = 4x = 52) is one of the most popular functional leafy vegetables in South and Southeast Asia. With its rich nutritional and pharmaceutical values, it has traditionally had widespread use for dietary and herbal purposes. Here, the genome of S. androgynus was sequenced and assembled, revealing a genome size of 1.55 Gb with 26 pseudo-chromosomes. Phylogenetic analysis traced back the divergence of Sauropus from Phyllanthus to approximately 29.67 million years ago (Mya). Genome analysis revealed that S. androgynus polyploidized around 20.51 Mya and shared a γ event about 132.95 Mya. Gene function analysis suggested that the expansion of pathways related to phloem development, lignin biosynthesis, and photosynthesis tended to result in the morphological differences among species within the Phyllanthaceae family, characterized by varying ploidy levels. The high accumulation of ascorbic acid in S. androgynus was attributed to the high expression of genes associated with the L-galactose pathway and recycling pathway. Moreover, the expanded gene families of S. androgynus exhibited multiple biochemical pathways associated with its comprehensive pharmacological activity, geographic adaptation and distinctive pleasurable flavor. Altogether, our findings represent a crucial genomic asset for S. androgynus, casting light on the intricate ploidy within the Phyllanthaceae family.

The species problem in Artemia Leach, 1819 (Crustacea: Anostraca), a genus with sexual species and obligate parthenogenetic lineages

Abstract Parthenogenesis is an asexual reproduction mode characterized by the development of a female oocyte without fertilization. From an evolutionary perspective, parthenogenesis seems less successful than the predominant sexual mode, though there are groups in which both reproductive types exist, an example of which is the genus Artemia Leach, 1819. This salt-tolerant crustacean inhabiting hypersaline environments contains regionally endemic sexual species and obligate parthenogenetic groups with different ploidy levels, collectively referred to as Artemia parthenogenetica. Here, we discuss the difficulties of using a common species concept in Artemia Leach, 1819. While sexual species are widespread and fit the Biological Species Concept (BSC), which emphasizes reproductive isolation to maintain species genetic integrity or cohesiveness, it does not apply to uniparental organisms originating from sexual species with major meiotic changes. We show that different ploidy levels of parthenogenetic Artemia groups with uniform nuclear gene pools are maternally independent genetic entities (or cohesive), collectively and wrongly referred to as Artemia parthenogenetica. Thus, we conclude that ‘Artemia parthenogenetica’ is an invalid nominal specific name. Additionally, parthenogenetic Artemia groups cannot be considered a form of Artemia species (A. urmiana and/or A. sinica). In conclusion, we recommend using the term ‘parthenogenetic lineage(s)’ instead of ‘parthenogenetic population(s)’ to describe asexual Artemia group(s), because in modern population genetics and systematics, the term ‘population’ refers to interbreeding individuals with sexual reproduction. Furthermore, it clarifies that parthenogenetic lineages of Artemia are native to Mediterranean biodiversity.

Chemical Diversity of Wild-Growing and Cultivated Common Valerian (Valeriana officinalis L. s.l.) Originating from Poland.

Common valerian is a medicinal plant. The underground organs of this species are used as a mild sedative and sleeping aid. Poland is one of the largest producers of this raw material in Europe, with local cultivar 'Lubelski' as a primary cultivated form. Although valerian is the subject of more or less deliberate selection carried out by farmers, it is still genetically unstable. The aim of this study was to determine the diversity of the 'Lubelski' cultivar originating from four regions of Poland (forms: L1-L4) in relation to wild-growing populations of the species. The plants were assessed in terms of the mass of underground organs and the content of valerenic acids and essential oils (EOs). The content of valerenic acids was determined using HPLC, whereas the content of EOs was determined using hydrodistillation. The composition of EOs was assessed using GC-MS GC-FID. The ploidy level of the analyzed objects was determined as well. Wild-growing populations (diploids) were characterized by lower masses of underground organs and lower contents of valerenic acid than cultivated forms (tetraploids). However, they produced higher contents of EOs. All the cultivated forms were strongly diversified with respect to the analyzed traits, including the mass of the roots (CV 49-75%), the content of valerenic acids (CV 18-55%), and the content of EOs (CV 28-57%). A total of 44 compounds were identified in the EOs. The dominant compound of both wild-growing populations and the 'Lubelski' forms were: α-fenchene, bornyl acetate, and valerenal. Among 'Lubelski' forms, the most interesting seems to be the L2 form, which was characterized by a relatively high yield and high content of valerenic acids and EOs. Thus, it appears to be a promising source of objects for further valerian cultivar improvement.

Species Delimitation in a Polyploid Group of Iberian Jasione (Campanulaceae) Unveils Coherence between Cryptic Speciation and Biogeographical Regionalization.

Groups with morphological stasis are an interesting framework to address putative cryptic species that may be hidden behind traditional taxonomic treatments, particularly when distribution ranges suggest disjunct and environmentally heterogeneous biogeographic patterns. New hypotheses of delimitation of evolutionary independent units can lead to the identification of different biogeographic processes, laying the foundation to investigate their historical and ecological significance. Jasione is a plant genus with a distribution centered in the Mediterranean basin, characterized by significant morphological stasis. Within the western Mediterranean J. gr. crispa species complex, J. sessiliflora s.l. and allied taxa form a distinct group, occupying environmentally diverse regions. At least two ploidy levels, diploid and tetraploid, are known to occur in the group. The internal variability is assessed with phylogenetic tools, viz. GMYC and ASAP, for species delimitation. The results are compared with other lines of evidence, including morphology and cytology. The fitting of distribution patterns of the inferred entities to chorological subprovinces is also used as a biogeographical and environmental framework to test the species hypothesis. Despite the scarcity of diagnostic morphological characters in the group, phylogenetic delimitation supports the description of at least one cryptic species, a narrow endemic in the NE Iberian Peninsula. Moreover, the results support the segregation of a thermophilic group of populations in eastern Iberia from J. sessiliflora. Ploidy variation from a wide geographical survey supports the systematic rearrangement suggested by species delimitation. Taxonomic reorganization in J. sessiliflora s.l. would allow ecological interpretations of distribution patterns in great accordance with biogeographical regionalization at the subprovince level, supporting geobotanical boundaries as a framework to interpret species ecological coherence of cryptic lineages. These results suggest that species differentiation, together with geographic isolation and polyploidization, is associated with adaptation to different environments, shifting from more to less thermophilic conditions. Thus, the recognition of concealed evolutionary entities is essential to correctly interpret biogeographical patterns in regions with a complex geologic and evolutionary history, such as the Mediterranean basin, and biogeographical units emerge as biologically sound frameworks to test the species hypothesis.

Synthetic polyploid induction influences morphological, physiological, and photosynthetic characteristics in Melissa officinalis L.

Melissa officinalis L., a well-known herb with diverse industrial and ethnopharmacological properties. Although, there has been a significant lack in the breeding attempts of this invaluable herb. This study aimed to enhance the agronomical traits of M. officinalis through in vitro polyploidization. Nodal segments were micropropagated and subjected to oryzalin treatment at concentrations of 20, 40, and 60 mM for 24 and 48 hours. Flow cytometry, chromosome counting, and stomatal characteristics were employed to confirm the ploidy level of the surviving plants. The survival rate of the treated explants decreased exponentially with increasing oryzalin concentration and duration. The highest polyploid induction rate (8%) was achieved with 40 mM oryzalin treatment for 24 hours. The induced tetraploid plants exhibited vigorous growth, characterized by longer shoots, larger leaves, and a higher leaf count. Chlorophyll content and fluorescence parameters elucidated disparities in photosynthetic performance between diploid and tetraploid genotypes. Tetraploid plants demonstrated a 75% increase in average essential oil yield, attributed to the significantly larger size of peltate trichomes. Analysis of essential oil composition in diploid and tetraploid plants indicated the presence of three major components: geranial, neral, and citronellal. While citronellal remained consistent, geranial and neral increased by 11.06% and 9.49%, respectively, in the tetraploid population. This effective methodology, utilizing oryzalin as an anti-mitotic agent for polyploid induction in M. officinalis, resulted in a polyploid genotype with superior morpho-physiological traits. The polyploid lemon balm generated through this method has the potential to meet commercial demands and contribute significantly to the improvement of lemon balm cultivation.

Differentially methylated genes involved in reproduction and ploidy levels in recent diploidized and tetraploidized Eragrostis curvula genotypes.

Epigenetics studies changes in gene activity without changes in the DNA sequence. Methylation is an epigenetic mechanism important in many pathways, such as biotic and abiotic stresses, cell division, and reproduction. Eragrostis curvula is a grass species reproducing by apomixis, a clonal reproduction by seeds. This work employed the MCSeEd technique to identify deferentially methylated positions, regions, and genes in the CG, CHG, and CHH contexts in E. curvula genotypes with similar genomic backgrounds but with different reproductive modes and ploidy levels. In this way, we focused the analysis on the cvs. Tanganyika INTA (4x, apomictic), Victoria (2x, sexual), and Bahiense (4x, apomictic). Victoria was obtained from the diploidization of Tanganyika INTA, while Bahiense was produced from the tetraploidization of Victoria. This study showed that polyploid/apomictic genotypes had more differentially methylated positions and regions than the diploid sexual ones. Interestingly, it was possible to observe fewer differentially methylated positions and regions in CG than in the other contexts, meaning CG methylation is conserved across the genotypes regardless of the ploidy level and reproductive mode. In the comparisons between sexual and apomictic genotypes, we identified differentially methylated genes involved in the reproductive pathways, specifically in meiosis, cell division, and fertilization. Another interesting observation was that several differentially methylated genes between the diploid and the original tetraploid genotype recovered their methylation status after tetraploidization, suggesting that methylation is an important mechanism involved in reproduction and ploidy changes.

Triploid Hybrids of 2x Lingonberry (Vaccinium vitis-idaea) by 2x Black Highbush Blueberry (V. fuscatum) and 2x Elliott’s Blueberry (V. elliottii) as Evidence of a Genome Balance Requirement for Hybridization Success

Hybridizations were made between a 2x V. vitis-idaea (sect. Vitis-idaea, lingonberry) and 2x V. fuscatum (sect. Cyanococcus, Black Highbush Blueberry) as part of a project aimed at understanding the crossability and compatibility of these but specifically aimed at assessing the possibilities for improvement and utilization of lingonberry. The crosses succeeded at a low level, and six hybrids were produced and genetically verified. When tested for ploidy level, five of the six hybrids were found to be triploids and one was found to be a tetraploid. Hybrids were intermediate in morphology and only fertile at very low levels, largely due to their triploid nature. Several of these hybrids produced progeny when used as females and pollinated with 6x V. virgatum-derived males, indicating viable unreduced 2n ovules. Similar crosses were made between lingonberry and 2x V. elliottii (sect. Cyanococcus, Elliott’s Blueberry). These crosses produced two genetically verified hybrids, which were also determined to be triploids. These hybrids were effectively sterile. The production of triploids from 2x × 2x crosses indicates that there is a natural selection for a reproductive genome balance of two V. vitis-idaea:one Cyanococcus. The success of secondary hybridizations with hexaploid materials suggests that the triploid hybrids may be used to advance the utilization and recombination of lingonberry germplasm.

Poiploidy induction of Indonesian Black Rice Oryza sativa L. Var. Cempo Ireng with Bio-catharantine

High demand on black rice Oryza sativa L. due to their high antioxydant content and better nutrition than the white rice. On the other hand this high price rice still less interest for the farmet to cultivate it. Therefore, improvement of plant characters was carried out through polyploid induction using the natural anti-mitotic compound bio-catharanthine. The purpose of this study was to test the effectiveness of bio-catharanthine in inducing polyploidy in Cemp ireng black rice. This study used a two-factor completely randomized design (CRD) method with three replications. The treatment factor of bio-catharanthine concentration was 1%, 1.5 %, 2 %, 2 %, 2.5 % and 3 % with the soaking time of 48 hours. The germination rate, ploidy level, stomatal size and density, and antioxidant were measuret to observed the characteristic of the mutan. The results showed that the treatments of 3 % bio-catharanthine enhanced the chromosome number of black rice Cempo ireng. Biocatharantine did not affected the germination rate through the treatments. Out mutants shows alternation on the stomatal size and density. The antioxidant of leaf sample did not changed after the treatment. Bio-catharanthine on high concentration migh be possible to be applied on polyploidy induction in black rice

A novel visual marker to distinguish haploids from doubled haploids in rice (Oryza sativa, L) at early growth stages

Doubled haploid technology, which enables the generation of homozygous lines in a single step, is one of the modern tools being employed for accelerating breeding processes in different crops. In rice, a globally important staple food crop, doubled haploid production through androgenesis is increasingly being employed in breeding programs. Amongst the androgenic rice lines, doubled haploids are formed spontaneously at about 50–60%, while the remaining 40–50% of plants remain as haploids. As haploids cannot be easily identified, it is routine to grow all the rice androgenic lines till maturity and harvest the seeds from the fertile doubled haploids. Therefore, the methods that facilitate easy identification of haploids at an early developmental stage in rice would enable treatment of such haploid lines with colchicine, to increase the efficiency of doubled haploid production. Further, it would also help in eliminating the operational cost involved in maintaining them till maturity. In the above context, a systematic study to identify easily observable physiological and morphological differences between haploid and doubled haploid rice lines was undertaken. Rice haploids were found to be noticeably different from doubled haploids in photosynthetic rate, transpiration rate, stomatal conductance, and morphology of lodicules, stigma and style, features which have not been reported before. Most importantly, rice haploids invariably have acute leaf apex which is easily distinguishable from the doubled haploids that have attenuated leaf apex shape. Very high per cent accuracy in the prediction of ploidy level was observed when haploids were identified at an early developmental stage based on leaf apex shape, and the results verified with flow cytometry perfectly matches with leaf apex shape. The study establishes ‘acute leaf apex’ shape as an accurate visual marker to rapidly identify haploid rice lines at an early developmental stage in a cost-effective manner.

Comparative Analysis of Transposable Elements in Strawberry Genomes of Different Ploidy Levels

Transposable elements (TEs) make up a large portion of plant genomes and play a vital role in genome structure, function, and evolution. Cultivated strawberry (Fragaria x ananassa) is one of the most important fruit crops, and its octoploid genome was formed through several rounds of genome duplications from diploid ancestors. Here, we built a pan-genome TE library for the Fragaria genus using ten published strawberry genomes at different ploidy levels, including seven diploids, one tetraploid, and two octoploids, and performed comparative analysis of TE content in these genomes. The TEs comprise 51.83% (F. viridis) to 60.07% (F. nilgerrensis) of the genomes. Long terminal repeat retrotransposons (LTR-RTs) are the predominant TE type in the Fragaria genomes (20.16% to 34.94%), particularly in F. iinumae (34.94%). Estimating TE content and LTR-RT insertion times revealed that species-specific TEs have shaped each strawberry genome. Additionally, the copy number of different LTR-RT families inserted in the last one million years reflects the genetic distance between Fragaria species. Comparing cultivated strawberry subgenomes to extant diploid ancestors showed that F. vesca and F. iinumae are likely the diploid ancestors of the cultivated strawberry, but not F. viridis. These findings provide new insights into the TE variations in the strawberry genomes and their roles in strawberry genome evolution.

Stomata variation in the process of polyploidization in Chinese chive (Allium tuberosum).

Stomatal variation, including guard cell (GC) density, size and chloroplast number, is often used to differentiate polyploids from diploids. However, few works have focused on stomatal variation with respect to polyploidization, especially for consecutively different ploidy levels within a plant species. For example, Allium tuberosum, which is mainly a tetraploid (2n = 4x = 32), is also found at other ploidy levels which have not been widely studied yet. We recently found cultivars with different ploidy levels, including those that are diploid (2n = 2x = 16), triploid (2n = 3x = 24), pseudopentaploid (2n = 34-42, mostly 40) and pseudohexaploid (2n = 44-50, mostly 48). GCs were evaluated for their density, size (length and width) and chloroplast number. There was no correspondence between ploidy level and stomatal density, in which anisopolyploids (approximately 57 and 53 stomata/mm2 in triploid and pseudopentaploid, respectively) had a higher stomatal density than isopolyploids (approximately 36, 43, and 44 stomata/mm2 in diploid, tetraploid and pseudohexaploid, respectively). There was a positive relationship between ploidy level and GC chloroplast number (approximately 44, 45, 51, 72 and 90 in diploid to pseudohexaploid, respectively). GC length and width also increased with ploidy level. However, the length increased approximately 1.22 times faster than the width during polyploidization. This study shows that GC size increased with increasing DNA content, but the rate of increase differed between length and width. In the process of polyploidization, plants evolved longer and narrower stomata with more chloroplasts in the GCs.

Newly Identified Roles for PIEZO1 Mechanosensor in Controlling Normal Megakaryocyte Development and in Primary Myelofibrosis

Introduction: Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix (ECM) to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Primary Myelofibrosis (PMF) is a Chromosome Philadelphia-negative Myeloproliferative Neoplasm (MPN), characterized by altered stiffer ECM and megakaryocytosis, often driven by the gain-of-function mutation in JAK2 V617F. Here, we study a role for PIEZO1 in megakaryopoiesis and proplatelet formation under normal physiological conditions and in the context of PMF. Methods: Bone marrow Mks from C57BL/6J control and myelofibrotic mice carrying the human JAK2 V617F+ mutation, or Mks derived from stem cells of patients carrying the same mutation were analyzed at mRNA and protein levels for Piezo1 expression. Effects of Piezo1 activation or inhibition on Mk maturation and ploidy were assessed by flow cytometry, and proplatelet formation by fluorescent microscopy. Human peripheral blood samples from PMF patients, diagnosed according to established criteria, or healthy individuals were used to compare the two categories for Piezo1 relative expression on mRNA and protein levels and platelet biogenesis. Mk-specific double knockout mice of Piezo1/2 were generated to compare platelet counts under normal conditions or bone marrow ablation with 5-FU challenge. Culture of mouse and human Mks in different 3D microenvironments was used to demonstrate in vitro association of Piezo1 expression levels with different ECM stiffness substrates. Results: Mouse Mks express PIEZO1 and negligible levels of PIEZO2. Pharmacological activation of mouse PIEZO1 increases the number of immature CD41 + Mks, while the number of mature CD41 +CD42 + Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its pharmacological activation reduces Mk size, ploidy, maturation, and proplatelet development. Resulting effects of PIEZO1 activation on Mks resemble the profile in PMF. Intriguingly, Mks derived from PMF mice bearing the Jak2 V617F mutation show significantly elevated PIEZO1 expression, compared to wild-type controls. Pharmacological activation of PIEZO1 in these cells significantly augments the number of immature CD41 + Mks, and sharply reduces the number of mature CD41 +CD42 + cells and ploidy level. Accordingly, Mks isolated from bone marrow aspirates of JAK2 V617FPMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Additionally, PIEZO1 was overexpressed in 3D culture of human and mouse Mks in stiffer microenvironments as compared to softer ones and liquid medium. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2 V617FPMF patients are rescued upon PIEZO1 inhibition with GsMTx4. Discussion: Our data show that PIEZO1 might serve as a break of Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease. Our finding that inhibition of PIEZO1 activity partially rescues the aberrant Mk phenotype associated with PMF suggests that GsMTx4 holds potential as a therapeutic strategy to mitigate the pathological effects observed in Mk development in PMF patients.

Adaptive differentiation on serpentine soil in diploid versus autotetraploid populations of Biscutella laevigata (Brassicaceae)

Serpentine soils exhibit extreme properties (e.g. high magnesium content) influencing plant growth and survival, and have been repeatedly documented to promote adaptive edaphic differentiation in plants. Individuals from four pairs of nearby diploid and autotetraploid populations of Biscutella laevigata sampled on serpentine versus non‐serpentine soils in a factorial design are used to assess the genetic and phenotypic changes associated with edaphic origin and ploidy level. Individual samples from natural populations were subjected to soil elemental analysis and genotyping using restriction site‐associated DNA sequences (RAD‐seq) to link genetic variation with contrasting soils and ploidy levels. In diploids, genetic variation was consistent with demographic contraction and a pattern of isolation by environment with respect to the ratio of calcium/magnesium concentrations, whereas tetraploids presented evidence of expansion with limited edaphic differentiation. The genetic basis of tolerance and adaptation to serpentine was further assessed experimentally on seed‐grown individuals from all populations subjected to high (serpentine‐like) versus low (control) concentrations of magnesium in hydropony. Fitness‐related phenotypic traits under experimental cultivation were consistent with adaptive differentiation among diploid ecotypes but not among the tetraploids that similarly grow in both habitats and consistently present higher investment in roots. Further work comparing experimentally resynthesized polyploids to natural diploids and polyploids would help to tease the role of whole genome duplication apart from the effects of post‐polyploidy evolution.

Identifying biomass yield potential of tetra‐, hexa‐, and octoploid prairie cordgrass populations grown on marginal lands

AbstractAs the demand for renewable energy sources with reduced carbon emissions to the atmosphere increases, bioenergy crops remain an attractive alternative to fossil fuels. Another appeal of bioenergy crops is the potential utilization of marginal lands not suitable for food crops. Prairie cordgrass is a perennial warm‐season polyploid grass species that is considered a bioenergy crop that also exhibits a high tolerance for salinity and flooding. The ploidy level of prairie cordgrass has previously been suggested to correlate with agronomic traits. However, little is known about the agricultural performance of prairie cordgrass on marginal lands and if a correlation of ploidy level and stress tolerance exists. In this experiment, the productivity of tetra‐, hexa‐, and octoploids was investigated when grown on three different marginal sites, including waterlogged, saline, and low‐nutrient gravelly soils. Here, we found that prairie cordgrass populations annually produced dry biomass on a wet marginal site comparable to normal cropland. However, the productivity was remarkedly decreased at both a salt‐affected site and a limestone mine reclamation site. We were unable to conclude that different ploidy levels affected the biomass yield of prairie cordgrass grown under abiotic stress. However, there was a differential response of populations to various types of marginal sites. These results indicate potential genetic resources for developing breeding targets and identifying mechanisms of abiotic stress tolerance. Moreover, we highlight the need to generate diverse ploidy levels of prairie cordgrass in the same genetic backgrounds for future testing.

Physiological and Biochemical Evaluation of Salt Stress Tolerance in a Citrus Tetraploid Somatic Hybrid

Somatic hybridization has emerged as a valuable tool for developing novel genetic combinations in citrus breeding programs, including the creation of salt-tolerant rootstocks. In this study, the performance of a tetraploid somatic hybrid, obtained by fusing protoplasts derived from salt-tolerant Cleopatra mandarin (Citrus reshni hort. ex Tanaka) and salt-sensitive Carrizo citrange (Citrus sinensis L. Osbeck × Poncirus trifoliata L. Raf), was assessed under in vitro salt stress. Hybrid plants were characterized by leaf morphology, and ploidy level by flow cytometry and molecular markers. In vitro shoots were generated from the micropropagation of mature stem pieces of the somatic hybrid and its parents, and these were challenged by exposure to NaCl (0, 50, or 100 mM) supplemented to the media for three weeks to induce salt stress. The leaves of the somatic hybrid display intermediate morphology compared to the parental Cleopatra mandarin and Carrizo citrange rootstocks. All molecular markers successfully amplified DNA from the three cultivars; however, only 11 of 14 unequivocally confirmed somatic hybridity. The physiological and biochemical parameters, including chlorophyll content, lipid peroxidation, total phenolic compounds, antioxidants activity and proline content, were measured in the leaves. The somatic hybrid exhibited superior salt stress tolerance compared to the parent varieties, as evidenced by the reduced cellular membrane damage indicated by the lower levels of malondialdehyde and electrolyte leakage, particularly under 100 mM NaCl treatment. The somatic tetraploid hybrid also displayed higher total phenolic content than either parent, while Cleopatra mandarin exhibited the highest proline levels under 50 mm NaCl. These results demonstrate the enhanced salinity stress tolerance of the somatic hybrid compared to its parent lines, highlighting its potential as a valuable candidate for developing salt-tolerant citrus rootstocks.

Oat species and interspecific amphiploids show predominance of diploid nuclei in the syncytial endosperm

Apart from apomictic types, the Polygonum-type eight-nuclear embryo sac is considered to be dominant in grasses. A triploid endosperm is formed as a result of double fertilisation. This study showed, for the first time, the dominance of diploid nuclei in the syncytial stage of the central cell of embryo sac in oat species and amphiploids. The dominance of diploid nuclei, which were the basis for the formation of polyploid nuclei, was weaker in amphiploids due to aneuploid events. The genomic in situ hybridisation method applied in the study did not distinguish the maternal and paternal haploid nuclei of embryo sac. However, this method demonstrated the lack of a set of genomes of one haploid nucleus. Embryological analyses of the initial stages of oat endosperm development revealed a fertilised egg cell, and two polar nuclei differing in size. It can be assumed that the formation of diploid oat endosperm occurred after the fusion of one polar nucleus and the nucleus of a male gamete, while the second polar nucleus gave rise to 1n nuclei. The levels of ploidy of syncytial nuclei were not influenced by both aneuploid events and correlated with pollen developmental anomalies. The differences in the analysed cytogenetic events distinguished amphiploids and their parental species in the ordination space.

Revealing genetic diversity of wild Phalaenopsis orchids in Thailand through Random Amplified Polymorphic DNA markers

Abstract. Kate-ngam S, Jantasuriyarat C, Lakote P, Promchot T, Chueakaew C. 2023. Revealing genetic diversity of wild Phalaenopsis orchids in Thailand through Random Amplified Polymorphic DNA markers. Biodiversitas 24: 5409-5417. Wild Phalaenopsis orchids are widely distributed in Thailand, especially along the banks of the Mekong River. The tremendous decrease in the population of these orchids is a matter of concern for maintaining and preventing genetic loss in the natural ecosystem. This study aimed to determine the genetic diversity of 50 wild Phalaenopsis accessions collected from the northeastern of Thailand using random amplified polymorphic DNA (RAPD) markers. The 204 polymorphic bands generated from 27 RAPD primers were employed for fingerprinting these orchid accessions. Dice’s similarity coefficients ranged from 0.43 to 0.98, with an average of 0.65. In general, a dendrogram constructed based on the unweighted pair group method with arithmetic average (UPGMA) grouped these wild Phalaenopsis accessions into two clusters, namely P. pulcherrima and P. ubonensis clusters. The UPGMA clustering pattern and principal component analysis (PCA) corresponded well with their morphological classification and ploidy level. Our results suggest that this wild Phalaenopsis orchid exhibits a moderate level of relatedness. This might be a consequence of habitat destruction and human over-exploitation which limit gene flow and cause genetic drift among populations. The present finding supports the urgent need for a systematic conservation effort of this orchid species in Thailand.

Genetic enhancement of okra [Abelmoschus esculentus (L.) Moench] germplasm through wide hybridization.

The introgression of genetic material from one species to another through wide hybridization and repeated back-crossing, plays an important role in genetic modification and enriching the cultivated gene-pool with novel genetic variations. Okra (Abelmoschus esculentus [(L.) Moench)] is a popular vegetable crop with high dietary fibre and protein, rich in essential amino acids, lysine and tryptophan. The wild Abelmoschus genepool has many desirable traits like ornamental value, short internodal length, more number of productive branches, extended bearing, perennation tendency, reduced fruit length (more consumer preferred trait), high mucilage content (medicinal value), abiotic stress tolerances such as drought, high temperature and biotic stress resistances such as okra Yellow Vein Mosaic Virus (YVMV) and Enation Leaf Curl Virus (ELCV) diseases. The repeated use of elite breeding lines led to narrowing of the genetic base of the okra crop, one of the major factors attributed to breakdown of resistance/ tolerance to biotic stresses. YVMV and ELCV are the two major diseases, causing significant yield loss in okra. Hence, wide hybridization was attempted to transfer tolerance genes from wild species to the cultivated genepool to widen the genetic base. The screening of germplasm of wild Abelmoschus species at hotspots led to the identification of tolerant species (Abelmoschus pungens var. mizoramensis, A. enbeepeegeearensis, A. caillei, A. tetraphyllus and A. angulosus var. grandiflorus), which were further used in a wide-hybridization programme to generate interspecific hybrids with the cultivated okra. Presence of pre- and post-zygotic barriers to interspecific geneflow, differences in ploidy levels and genotype specific variations in chromosome numbers led to varying degrees of sterility in F1 plants of interspecific crosses. This was overcome by doubling the chromosome number of interspecific hybrids by applying Colchicine at the seedling stage. The 113 cross derivatives generated comprising amphidiploids in the F1 generation (30), F3 (14), one each in F2 and F4 generations, back cross generation in BC1F2 (03), BC1F3 (25), and BC2F3 (02), crosses between amphidiploids (27), multi-cross combinations (07) and inter-specific cross (between A. sagittifolius × A. moschatus subsp. moschatus) selfed derivatives at F8 generation (03) were characterized in the present study. Besides they were advanced through selfing and backcrossing. The amphidiploids were found to possess many desirable genes with a considerable magnitude of linkage drag. Majority of the wide cross derivatives had an intermediate fruit morphology and dominance of wild characters viz., hispid fruits, stem, leaves, tough fruit fibre, vigorous perennial growth habit and prolonged flowering and fruiting. The fruit morphology of three BC progenies exhibited a high morphological resemblance to the cultivated okra, confirming successful transfer of useful genes to the cultivated okra genepool. The detailed morphological characteristics of the various combinations of Abelmoschus amphidiploids and the genetic enhancement of the genepool achieved in this process is reported here.

Genetic and phenotypic diversity of native potatoes (Solanum spp.) from the Central Andes of Peru

The diversity of native potatoes cultivated above 3500 masl in the Pasco region (Central Andes of Peru) has not been fully characterized. It is currently subject to constant genetic erosion caused by biotic and abiotic factors. The research aimed to characterize phenotypically and genotypically 40 native potato landraces representative of 4 Solanum species. Twenty phenotypic descriptors and 10 microsatellites were used for genetic evaluation. Likewise, the ploidy level was evaluated based on the number of chloroplasts in the stomata. The clustering analysis was performed using the Infostat software and the R program with the Adegenet and Polysat libraries. The phenotypic characterization allowed to obtain five groups with a distance coefficient of 9.5. The molecular characterization found seven groups and 58 alleles in total. The average number of alleles per microsatellite was 5.5. 13.2 % of duplicates were identified. The microsatellites STG001, STM1106, ST0032, and STM5127 with an average He of 0.8 and a polymorphism information content (PIC) of 0.5 - 0.8 were the most informative. Finally, the ploidy results were 13 % diploid, 35 % triploid, and 52 % tetraploid. It was evidenced low diversity when using a set of 10 SSR markers, which indicates limited applicability for studying the genetic diversity of local potato landraces. It is necessary to involve a broader range of markers and a more diverse set of genotypes from the Pasco region for further studies.