Search

Drought is a major constraint in oil palm (Elaeis guineensis Jacq.) production. As oil palm breeding takes a long time, molecular markers of genes related to drought tolerance characteristics were developed for effective selection. Two methods of gene identification associated with drought, differential display reverse transcription polymerase chain reaction (DDRT-PCR) and pyrosequencing platform, were conducted before developing the EST-SSR marker. By DDRT-PCR, fourteen out of twenty-four primer combinations yielded the polymorphism in leaf as 77.66% and root as 96.09%, respectively. BLASTN and BLASTX revealed nucleotides from 8 out of 236 different banding similarities to genes associated with drought stress. Five out of eight genes gave a similarity with our pyrosequencing sequencing database. Furthermore, pyrosequencing analysis of two oil palm libraries, drought-tolerant, and drought sensitive, found 117 proteins associated with drought tolerance. Thirteen out of sixty EST-SSR primers could be distinguished in 119 oil palm parents in our breeding program. All of our found genes revealed an ability to develop as a molecular marker for drought tolerance. However, the function of the validated genes on drought response in oil palm must be evaluated.

Pseudotaxus chienii, belonging to the monotypic genus Pseudotaxus (Taxaceae), is a relict conifer endemic to China. Its populations are usually small and patchily distributed, having a low capacity of natural regeneration. To gain a clearer understanding of how landscape variables affect the local adaptation of P. chienii, we applied EST‐SSR markers in conjunction with landscape genetics methods: (a) to examine the population genetic pattern and spatial genetic structure; (b) to perform genome scan and selection scan to identify outlier loci and the associated landscape variables; and (c) to model the ecological niche under climate change. As a result, P. chienii was found to have a moderate level of genetic variation and a high level of genetic differentiation. Its populations displayed a significant positive relationship between the genetic and geographical distance (i.e., “isolation by distance” pattern) and a strong fine‐scale spatial genetic structure within 2 km. A putatively adaptive locus EMS6 (functionally annotated to cellulose synthase A catalytic subunit 7) was identified, which was found significantly associated with soil Cu, K, and Pb content and the combined effects of temperature and precipitation. Moreover, P. chienii was predicted to experience significant range contractions in future climate change scenarios. Our results highlight the potential of specific soil metal content and climate variables as the driving force of adaptive genetic differentiation in P. chienii. The data would also be useful to develop a conservation action plan for P. chienii., Landscape and environmental heterogeneity play important roles in the spatial genetic structure and the adaptive evolution of P. chienii. Adaptive gene indeed occurred in genome of P. chienii and made the plant to have adaptive potential in response to severe environmental stress. These factors related to adaptation should be specially considered in ex situ conservation of P. chienii.

Pistacia chinensis Bunge (P. chinensis), a dioecious plant species, has been widely found in China. The female P. chinensis plants are more important than male plants in agricultural production, as their seeds can serve as an ideal feedstock for biodiesel. However, the sex of P. chinensis plants is hard to distinguish during the seedling stage due to the scarcity of available transcriptomic and genomic information. In this work, Illumina paired-end RNA sequencing assay was conducted to unravel the transcriptomic profiles of female and male P. chinensis flower buds. In total, 50,925,088 and 51,470,578 clean reads were obtained from the female and male cDNA libraries, respectively. After quality checks and de novo assembly, a total of 83,370 unigenes with a mean length of 1.3 kb were screened. Overall, 64,539 unigenes (77.48%) could be matched in at least one of the NR, NT, Swiss-Prot, COG, KEGG, and GO databases, 71 of which were putatively related to the floral development of P. chinensis. Additionally, 21,662 simple sequence repeat (SSR) motifs were identified in 17,028 unigenes of P. chinensis, and the mononucleotide motif was the most dominant type of repeats (52.59%) in P. chinensis, followed by dinucleotide (22.29%), trinucleotide (20.15%). The most abundant repeats were AG/CT (13.97%), followed by AAC/GTT (6.75%) and AT/TA (6.10%). Based on these SSR, 983 EST-SSR primers were designed, 151 of which were randomly chosen for validation. Of these validated EST-SSR markers, 25 SSR markers were found to be polymorphic between male and female plants. One SSR marker, namelyPCSSR55, displayed excellent specificity in female plants, which could clearly distinguish between male and female P. chinensis. Altogether, our findings not only reveal that the EST-SSR marker is extremely effective in distinguishing between male and female P. chinensis but also provide a solid framework for sex determination of plant seedlings.

Hypericum triquetrifolium Turra is an ecologically, medicinally and economically important species in Tunisia. Thirty-six Hypericum individuals sampled from 6 northern Tunisian locations were investigated for their diversity and relationships using 10 inter-simple sequence repeats (ISSR) markers and 10 morphological features at vegetative stage. The phylogenetic analysis, using 308 bp of sequenced ITS1 region, identified the Hypericum individuals as H. triquetrifolium that clustered with members of genus Hypericum section 9, 9a, 9b and 27, in agreement with the previous molecular classification of the genus. Among the 10 ISSR markers tested, 7 were scorable and yielded 91 loci with 94.5% of polymorphism. UBC848 and UBC836 were the most polymorphic ISSR markers. The level of genetic diversity (HT = 0.247) and gene flow between the six populations (N (m) = 1.169) were moderate. The structure analysis revealed three genetic subpopulations: individuals of Le Krib location formed a subpopulation divergent from two other subpopulations, probably due to its northwestern and high-altitude geographic barriers, and its sub-humid microclimate. Zaghouan, northeastern location in the lower semi-arid, with the highest genetic (I = 0.370) and morphological (I = 0.631) Shannon's information indices and, regrouping two out of the three genetic subpopulations, is the most probable zone of origin for H. triquetrifolium. In addition, morphological data showed higher diversity than ISSR data; however, no evidence of correlation between genetic and morphologic traits could be suggested in this study. These results on the genetic diversity and phylogenetic analysis will contribute to the conservation of the gene pool of H. triquetrifolium in Tunisia.

Olea europaea subsp. cuspidata (Wall. & G. Don) Cif. is one of the six subspecies important for domestication of olive described as having valuable breeding traits. It is distributed from South Africa to the Middle East and the Mediterranean region to China mainly at mid to high altitudes with adequate precipitation. The currently rather fragmented Olea europaea subsp. cuspidata populations likely represent outposts of a formerly larger and more connected area and are hence vulnerable to additional stresses such as erratic weather extremes and anthropogenic influences. This may result in further fragmentation of stands, shifts in demography, increased clonal growth, lower genetic diversity, and higher differentiation between olive stands. In this study, we present data on Olea europaea subsp. cuspidata growing in the Hajar Mountains, Sultanate of Oman, a large area subjected to many anthropogenic disturbances. We used dendrological traits and genetic parameters to infer its stand and population genetic structure, levels of genetic diversity, and differentiation. To accomplish this, 366 individual trees in total were inventoried and 188 of them genotyped using 12 microsatellite markers. Dendrological measures indicated disturbances mainly due to human activities. Height and crown area of individuals averaged 3.7 m (ranging from 0.2 to 12 m) and 14 m (ranging from 1 to 18 m), respectively. The stand conditional index was mainly 50%. The identified genetic variation within the sampled areas was relatively moderate. The expected and observed heterozygosity equalled 0.55–0.58 and 0.62–0.64, respectively. All individual genotypes were unique indicating the absence of clones. Though significant genetic differentiation was found, there was no evidence of population substructure suggesting effective pollen and long-distance seed dispersal in the past. However, changes in gene flow patterns may be reflected in the next (sibling) generation. Further studies are necessary to detect any changes in the future and to develop sustainable conservation strategies for this species.

Olive is considered one of the oldest and the most important cultivated fruit trees in Albania. In the present study, the genetic diversity and structure of Albanian olive germplasm is represented by a set of 194 olive genotypes collected in-situ in their natural ecosystems and in the ex-situ collection. The study was conducted using 26 microsatellite markers (14 genomic SSR and 12 Expressed Sequence Tag microsatellites). The identity analysis revealed 183 unique genotypes. Genetic distance-based and model-based Bayesian analyses were used to investigate the genetic diversity, relatedness, and the partitioning of the genetic variability among the Albanian olive germplasm. The genetic distance-based analysis grouped olives into 12 clusters, with an average similarity of 50.9%. Albanian native olives clustered in one main group separated from introduced foreign cultivars, which was also supported by Principal Coordinate Analysis (PCoA) and model-based methods. A core collection of 57 genotypes representing all allelic richness found in Albanian germplasm was developed for the first time. Herein, we report the first extended genetic characterization and structure of olive germplasm in Albania. The findings suggest that Albanian olive germplasm is a unique gene pool and provides an interesting genetic basis for breeding programs.

Floral color plays a crucial role in plant life such as plant-pollinator interactions and modifying the abiotic environment of reproductive structures. In the current study, 123 gerbera accessions were divided into six color groups (white, yellow, orange, pink, red, and purple), based on Royal Horticultural Society Color Chart calibration and colorimeter measurement. Partial least squares discriminant analysis showed that the white group was mainly affected by L* value, a* value, C value, and total anthocyanin contents, while the yellow group was positively correlated with L* value, b* value, and total anthocyanin contents. Similarly, the orange group was mainly affected by b* value and total carotenoid contents, whereas the pink group was positively correlated with L* and h values. Furthermore, the red group was affected by L* value, a* value, C value, and total anthocyanin contents, whilst the purple group was mainly distributed by L* value, a* value, b* value, and total anthocyanin contents. Based on ‘Jin Xiang’ transcriptome data, 14,106 expressed sequence tag (EST)-SSR markers were identified and 48 pairs of primers (19 newly developed primers) were screened. Population genetic structure, neighbor-joining clustering, and principal coordinate analysis showed that 123 gerbera accessions could be divided into two groups. EST-SSR-based association analysis showed that 1, 1, 2, 1, 1, 2, and 1 significant loci were related to L*, a*, b*, C, and h, total carotenoid, and total anthocyanin contents, respectively. These results provide an important reference for flower color classification and genetic improvement of gerbera.

Chrysosplenium macrophyllum Oliv., belonging to the family Saxifragaceae, is a traditional and unique Chinese herbal medicine. However, the lack of adequate molecular markers has hampered the progress regarding population genetics and evolution within this species. In this research, we used the DNBSEQ-T7 Sequencer (MGI) sequencing assay to analyze the transcriptome profiles of C. macrophyllum. SSR markers were developed on the basis of transcriptomic sequences and further validated on C. macrophyllum and other Chrysosplenium species. The genetic diversity and structure of the 12 populations were analyzed by using polymorphic expressed sequence tag simple sequence repeat (EST-SSR) markers. A potential pool of 3127 non-redundant EST-SSR markers were identified for C. macrophyllum in this study. The developed EST-SSR markers had high amplification rates and cross-species transferability in Chrysosplenium. Our results also showed that the natural populations of C. macrophyllum had a high level of genetic diversity. Genetic distance, principal component analysis, and popular structure analysis revealed that all 60 samples clustered into two major groups that were consistent with their geographical origins. This study provided a batch of highly polymorphic EST-SSR molecular markers that were developed via transcriptome sequencing. These markers will be of great significance for the study of the genetic diversity and evolutionary history of C. macrophyllum and other Chrysosplenium species.

Kenaf is an important bast fiber crop. In order to diversify the available kenaf simple sequence repeat (SSR) molecular markers and generate markers potentially useful for kenaf breeding, we developed expression sequence tag simple sequence repeat (EST-SSR) molecular markers based on lead-stressed kenaf transcriptome sequencing data and spliced unigene sequences. Additionally, the distribution of the SSRs in the transcriptome and the potential functions of the SSR-containing genes were determined. Moreover, SSR markers in the differentially expressed genes (DEGs) of a protein–protein interaction (PPI) network were analyzed to screen for polymorphic markers, which were used to examine the genetic diversity and population structure of kenaf germplasm resources. The genetic diversity and population structure of 138 kenaf germplasm materials revealed that 22 EST-SSR markers could be used to distinguish the kenaf germplasms. The 22 EST-SSR markers enrich the kenaf molecular markers database and provide an important tool for future genetic improvement of kenaf resistance to lead stress.

In the present study, novel genomic-SSR (g-SSR) markers generated in our laboratory were used to characterize Tinospora cordifolia and related species. The g-SSR marker was also compared with EST-SSR and SCoT markers used earlier in our laboratory to assess the genetic diversity of T. cordifolia. A total of 26 accessions of T. cordifolia and 1 accession each of Tinospora rumphii and Tinospora sinensis were characterized using 65 novel g-SSR markers. A total of 125 alleles were detected with 49 polymorphic g-SSR markers. The number of alleles per locus varied from 1–4 with a mean value of 2.55 alleles per locus. Mean PIC, gene diversity, and heterozygosity were estimated to be 0.33, 0.41, and 0.65, respectively. The two species, namely T. rumphii and T. sinensis, showed cross-species transferability of g-SSRs developed in T. cordifolia. The success rate of cross-species transferability in T. rumphii was 95.3% and 93.8% in T. sinensis, proving the usefulness of this marker in genetic diversity studies of related species. The Tinospora accessions were also used for molecular characterization using SCoT and EST-SSR markers and compared for genetic diversity and cross-species transferability. The PIC, gene diversity, heterozygosity, and principal coordinate analysis showed that g-SSR is the better maker for a genetic diversity study of T. cordifolia. Additionally, high cross-species transferability of g-SSRs was found (95.3% and 93.8%) compared to EST-SSRs (68.8% and 67.7%) in T. rumphii and T. sinensis, respectively.

Colletotrichum fructicola causes strawberry anthracnose, which results in considerable economic losses in quality and production in Asia. However, the molecular mechanism of strawberry infection with C. fructicola is still poorly understood. Here, we employed RNA-Seq technology to mine the strawberry transcriptome for EST-SSRs (Expressed Sequence Tag–Simple Sequence Repeats) following C. fructicola infection. Fortunately, we detected 3019 potential EST-SSRs from the transcriptomic data of cv. Jiuxiang. The seven commonly occurring repeat motifs were A/T (278), GAA/TTC (263), GAG/CTC (215), AAG/CTT(188), GGA/TCC (163), AGA/TCT (153) and AGG/CCT (153). We identified 31, 84 and 129 differentially expressed EST-SSR-containing sequences corresponding to 24, 72 and 96 Hpi, while 13 DE-EST-SSRs -containing sequences were differentially expressed and common for all the three time points. Furthermore, we identified allele polymorphisms for some DE-EST-SSRs among several resistant and susceptible strawberry varieties and wild species. The EST-SSRs in our research were related to the reported QTL resistance loci in strawberry which are differentially expressed after C. fructicola infection and have allele polymorphisms in different strawberry varieties and wild species. These provide a clue for utilisation of the EST-SSRs in marker-assisted breeding to improve anthracnose-resistant breeding in future.

Auxin/Indole-3-Acetic Acid (Aux/IAA) genes are involved in auxin signaling pathway and play an important role in plant growth and development. However, many studies focus on Aux/IAA gene families and much less known in Bletilla striata. In this study, a total of 27 Aux/IAA genes (BsIAA1-27) were cloned from the transcriptome of Bletilla striata. Based on a phylogenetic analysis of the Aux/IAA protein sequences from B. striata, Arabidopsis thaliana and Dendrobium officinale, the Aux/IAA genes of B. striata (BsIAAs) were categorized into 2 subfamilies and 9 groups. While BsIAAs were more closer to those of D. officinale compared to A. thaliana. EST-SSR marker mining test showed that 4 markers could be stably amplified with obvious polymorphisms among 4 landraces. Our results suggested that BsIAAs were involved in the process of tuber development and provided insights into functional roles of Aux/IAA genes in B. striata and other plants., Graphical Abstract In this paper, we identified the 27 members of the Aux/IAA gene family which can be divided into 2 groups and 9 subfamilies. The similar branches of Aux/IAA proteins had the same or similar motifs, such as genes between BsIAA10 and BsIAA12, BsIAA11 and BsIAA13, BsIAA18 and BsIAA20, indicating that Aux/IAA proteins were conserved. Among them, BsIAAs were more closer to those of D. officinale compared to A. thaliana.

Key message Understanding the adaptive mechanisms of forest species is vital to ensure their survival in a climate change scenario. This study aimed at uncovering the relationship between genetic variability and environmental variables in natural Castanea sativa populations, unveiling how different climate scenarios drove local adaption processes using a landscape genomics approach. Our findings provide useful data for future management of this species. o Context Temperate forest species, such as chestnut (Castanea sativa Mill.), are currently threatened by increasing temperature together with disruption and reduction of precipitation due to climate change. In this context, understanding the adaptation processes of species will help to manage and ensure the conservation of forests. o Aims We studied the relationship between genetic variability and climate variables in natural populations of C. sativa using a landscape genomics approach aimed to identify local adaption processes. o Methods Using five genomic SSRs and eight functional EST-SSRs markers, 268 individuals belonging to ten different natural European chestnut populations distributed in contrasting climatic sites were genotyped. In addition, associations between allelic variation and climatic variables (environmental association analyses approach) were performed using Sam?ada and LFMM. o Results Results highlighted a strong inter-relationship between climate variables and evolutionary processes resulting in adaptive variation. STRU CTU RE analysis based on functional markers split the populations in three separate gene pools (K = 3), mostly in agreement with the different climatic conditions existing in the studied areas. Divergent spatial patterns of genetic variation between rainy and arid areas were found. We detected a total of 202 associations with climate among 22 different alleles, 9% of which related with the outlier locus FIR059, known to be implicated in regulatory mechanisms during water stress adaptation processes. o Conclusion Landscape genomics analyses revealed a pattern of adaptive variation, where specific climatic variables influenced the frequencies distribution and fixation of several alleles, resulting in local adaptation processes of the populations in the investigated areas. Our findings underline the close inter-relationship existing between climate and genetic variability and indicate how this approach could provide valuable information for the management of forest species in a rapidly changing environment.

BackgroundTheLycorisgenus includes many ornamentally and medicinally important species. Polyploidization and hybridization are considered modes of speciation in this genus, implying great genetic diversity. However, the lack of effective molecular markers has limited the genetic analysis of this genus.ResultsIn this study, mining of EST-SSR markers was performed using transcriptome sequences ofL. aurea, and 839 primer pairs for non-redundant EST-SSRs were successfully designed. A subset of 60 pairs was randomly selected for validation, of which 44 pairs could amplify products of the expected size. Cross-species transferability of the 60 primer pairs amongLycorisspecies were assessed inL. radiataHreb,L. sprengeriComes ex Baker,L. chinensisTraub andL. anhuiensis, of which between 38 to 77% of the primers were able to amplify products in theseLycorisspecies. Furthermore, 20 and 10 amplification products were selected for sequencing verification inL. aureaandL. radiatarespectively. All products were validated as expected SSRs. In addition, 15 SSRs, including 10 sequence-verified and 5 unverified SSRs were selected and used to evaluate the genetic diversity of sevenL. radiatalines. Among these, there were three sterile lines, three fertile lines and one line represented by the offspring of one fertile line. Unweighted pair group method with arithmetic mean analysis (UPGMA) demonstrated that the outgroup,L. aureawas separated fromL. radiatalines and that the sevenL. radiatalines were clustered into two groups, consistent with their fertility. Interestingly, even a dendrogram with 34 individuals representing the sevenL. radiatalines was almost consistent with fertility.ConclusionsThis study supplies a pool of potential 839 non-redundant SSR markers for genetic analysis ofLycorisgenus, that present high amplification rate, transferability and efficiency, which will facilitate genetic analysis and breeding program inLycoris.

Chrysanthemums (Chrysanthemum morifolium Ramat.) are famous ornamental crops with high medicinal and industrial values. The inflorescence and leaf traits are key factors that affect the yield and quality of chrysanthemum. However, the genetic improvement of those traits is slow within chrysanthemum because of its hexaploidy, high heterozygosity and enormous genome. To study the genetic control of the important traits and facilitate marker-assisted selection (MAS) in chrysanthemum, it is desirable to populate the genetic maps with an abundance of transferrable markers such as microsatellites (SSRs). A genetic map was constructed with expressed sequence tag&ndash, simple sequence repeat (EST-SSR) markers in an F1 progeny of 192 offspring. A total of 1000 alleles were generated from 223 EST-SSR primer pairs. The preliminary maternal and paternal maps consisted of 265 marker alleles arranged into 49 and 53 linkage groups (LGs), respectively. The recombined parental maps covered 906.3 and 970.1 cM of the genome, respectively. Finally, 264 polymorphic loci were allocated to nine LGs. The integrated map spanned 954.5 cM in length with an average genetic distance of 3.6 cM between two neighbouring loci. Quantitative trait loci (QTLs) analysis was performed using the integrated map for inflorescence diameter (ID), central disc flower diameter (CDFD), number of whorls of ray florets (NWRF), number of ray florets (NRF), number of disc florets (NDF), number of florets (NF), ray floret length (RFL), ray floret width (RFW), ray floret length/width (RFL/W), leaf length (LL), leaf width (LW) and leaf length/width (LL/W). Overall, 36 (21 major) QTLs were identified. The successful mapping of inflorescence and leaf traits QTL demonstrated the utility of the new integrated linkage map. This study is the first report of a genetic map based on EST-SSR markers in chrysanthemum. The EST-SSR markers, genetic map and QTLs reported here could be valuable resources in implementing MAS for chrysanthemums in breeding programs.

Background Tea (Camellia sinensis (L). O. Kuntze) is known as the oldest, mild stimulating caffeine containing non-alcoholic beverage. One of the major threats in south Asian tea industry is the blister blight leaf disease (BB), caused by the fungus Exobasidium vexans Masse. SSR DNA marker EST SSR 073 is used as a molecular marker to tag blister blight disease resistance trait of tea. The amino acid sequences were derived from cDNA sequences related to EST SSR 073 of BB susceptible (TRI 2023) and BB resistant (TRI 2043) cultivars. An attempt has been made to understand the structural characteristics and variations of EST SSR 073 locus that may reveal the factors influencing the BB resistance of tea with multiple bioinformatics tools such as ORF finder, ExPasy ProtParam tools, modeler V 9.17, Rampage server, UCSF-Chimera, and HADDOCK docking server. Results The primary, secondary, and tertiary structures of EST SSR 073 coding protein were analyzed using the amino acid sequences of both BB resistant TRI 2043 and BB susceptible TRI 2023 tea cultivars. The coding amino acid sequences of both the cultivars were homologous to photosystem I subunit protein (PsaD I) of Pisum sativum. The predicted 3D structures of proteins were validated and considered as an acceptable overall stereochemical quality. The BB resistant protein showed CT repeat extension and did not involve in topology of the PsaD I subunit. The C terminal truncation of BB resistance caused the formation of hydrogen bonds interacting with PsaD I and other subunits of photosystem I in the modeled three-dimensional protein structure. Conclusions Camellia sinensis EST 073 SSR motif coding protein was identified as the PsaD I subunit of photosystem I. The exact mechanism of PsaD I conferring the resistance for blister blight in tea needs to be further investigated.

Picea neoveitchii Mast. is a rare and endangered evergreen coniferous tree species in China. P. neoveitchii grows within a specific ecological and geographical range. Here, we performed transcriptome sequencing of P. neoveitchii using the Illumina high-throughput deep sequencing technology with the following aims: (i) to perform de novo transcriptome assembly, (ii) to perform transcriptome assembly with functional annotation, (iii) to study the candidate genes involved in the biosynthesis of cellulose and lignin, (iv) to identify and analyse the transcription factors, and (v) to develop potential expressed sequence tag–simple sequence repeat (EST-SSR) markers for future population genomic and genome-wide association studies in P. neoveitchii. This is, to the best of our knowledge, the first study to provide a comprehensive transcriptome of P. neoveitchii. The transcriptome data contained 12.97 gigabase pairs (Gbp) for a total of 120,951 different transcripts belonging to 66,022 unigenes. In addition, we classified enzymes involved in the biosynthesis of cellulose and lignin and identified and analysed the transcription factors involved in stress responses, growth and development. Based on the larger than 1 kb unigenes, 1,814 EST-SSR markers were shown. Our results demonstrated that the transcriptome analysis is a valuable technology for non-model species with large genomes and this dataset will be an essential resource for future genetic and genomic research in P. neoveitchii.

Common buckwheat (Fagopyrum esculentum M.) is known for its adaptability, good nutrition, and medicinal and health care value. However, genetic studies of buckwheat have been hindered by limited genomic resources and genetic markers. In this study, Illumina HiSeq 4000 high-throughput sequencing technology was used to sequence the transcriptome of green-flower common buckwheat (Gr) with coarse pedicels and white-flower Ukrainian daliqiao (UD) with fine pedicels. A total of 118,448 unigenes were obtained, with an average length of 1248 bp and an N50 of 1850 bp. A total of 39,432 differentially expressed genes (DEGs) were identified, and the DEGs of the porphyrins and chlorophyll metabolic pathway had significantly upregulated expression in Gr. Then, a total of 17,579 sequences containing SSR loci were detected, and 20,756 EST-SSR loci were found. The distribution frequency of EST-SSR in the transcriptome was 17.52%, and the average distribution density was 8.21 kb. A total of 224 pairs of primers were randomly selected for synthesis; 35 varieties of common buckwheat and 13 varieties of Tartary buckwheat were verified through these primers. The clustering results well verified the previous conclusion that common buckwheat and Tartary buckwheat had a distant genetic relationship. The EST-SSR markers identified and developed in this study will be helpful to enrich the transcriptome information and marker-assisted selection breeding of buckwheat.

(1) Background and objectives: Cretan chestnut belongs to sweet chestnut (Castanea sativa Mill.) and has been historically associated with the lifestyle of rural communities with great economic importance. However, chestnut genetic resources in Crete have rarely been studied and assessed, while chestnuts are threatened by several anthropogenic factors. This study assessed the genetic variability of the Cretan sweet chestnut using 59 trees corresponding to the four best-known chestnut cultivars (Strovliani, Rogdiani, Koutsakera and Katharokastania). (2) Materials and Methods: The trees were evaluated using seven simple sequence repeat markers (SSRs): three nSSRs and four EST-SSRs. (3) Results: Genomic SSR results revealed notable genetic diversity in terms of expected heterozygosity, level of polymorphism and effective number of alleles. Moreover, in the four chestnut cultivars, twenty-two unique genotypes were identified, deeming each cultivar to be in fact a multiclonal variety. Genetic differentiation among cultivars was relatively low, though highly significant. Four different groups of synonymies were found: two homonymy groups in Katharokastania and Strovliani, six in Rogdiani and eight in Koutsakera. The cluster analysis and PCoA results reveal two main clusters, one corresponding to the Rogdiani cultivar and the other to Katharokastania, while the other two could not be assigned to a particular group. (4) Conclusions: The null hypothesis of single-clone genotype-to-cultivar correspondence was tested and could not be accepted.

Simple sequence repeat (SSR) markers have been shown to be a powerful tool for varieties identification in plants. However, SSR fingerprinting of sweetpotato varieties has been a little reported. In this study, a total of 1294 SSR primer pairs, including 1215 genomic-SSR and 79 expressed sequence tag (EST)-SSR primer pairs, were screened with sweetpotato varieties Zhengshu 20 and Luoxushu 8 and their 2 F1 individuals randomly sampled, and 273 and 38 of them generated polymorphic bands, respectively. Four genomic-SSR and 3 EST-SSR primer pairs, which showed good polymorphism, were selected to amplify 203 sweetpotato varieties and gave a total of 172 bands, 85 (49.42%) of which were polymorphic. All of the 203 sweetpotato varieties showed unique fingerprint patterns, indicating the utility of SSR markers in variety identification of this crop. Polymorphism information content (PIC) ranged from 0.5824 to 0.9322 with an average of 0.8176. SSR-based genetic distances varied from 0.0118 to 0.6353 with an average of 0.3100 among these varieties. Thus, these sweetpotato varieties exhibited high levels of genetic similarity and had distinct fingerprint profiles. The SSR fingerprints of the 203 sweetpotato varieties have been successfully constructed. The highly polymorphic SSR primer pairs developed in this study have the potential to be used as core primer pairs for variety identification, genetic diversity assessment and linkage map construction in sweetpotato and other plants.

Altitude could greatly influence species distribution and even their genetic diversity. However, it is unclear how altitude has affected the genetic diversity and population structure of Rhododendron simsii Planch., an dominant forestry species in north temperate forest. In this research, 22 polymorphic EST-SSR markers were utilized to assess the genetic diversity of R. simsii population distributed at different altitudes of Wujiashan Mountain, a major peak of Dabie Mountains (central China). Totally, 203 alleles were obtained, and each locus gave out 5 to 19 alleles. High genetic diversity existed, as Nei’s gene diversity (h) and Shannon’s Information index (I) ranged from 0.728 to 0.920 and 1.430 to 2.690, with the mean value of 0.821 and 1.916, respectively. In particular, 11.1% of genetic differentiation was maintained between populations, while 88.9% occurred within populations. Moreover, moderate gene flow (2.001) among populations was observed, which could effectively resist genetic drift. The genetic diversity of all these five R. simsii populations varied significantly with elevation, basically showing high-low-high pattern with elevation increase. Without human intervention, genetic diversity of R. simsii populations might increase with the altitude. At the significance level (p < 0.05), negative correlation was found between genetic diversity and attenuation rate of light intensity (r=-0.873). Soil of Wujiashan Mountain was acid (the pH value ranged from 4.33 to 4.70), which was rich in organic matter, available phosphorus, available potassium, and alkali hydrolysable nitrogen, as these soil factors interacted with each other to affect the growth of R. simsii population. This research would contribute a lot to the knowledge of evolutionary history of R. simsii species and benefit subsequent management and conservation actions., Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics, V. 72 N. 3 (2019)

Patagonian toothfish (Dissostichus eleginoides) is a hefty notothenioid fish and a key species within the marine ecosystem with a high migratory capacity across sub-Antarctic and south American Pacific oceans. Transcriptome characterization and molecular markers associated with micro and macro-evolutionary studies are not available, which in turn limits the gain of knowledge about the genetic basis of this species. Therefore, in the present study, a de novo transcriptome from eight tissue and an embryonic state of Patagonian toothfish was developed, using the Illumina Hiseq 4000 platform. A total of 233,424 superTranscripts were assembled and 37,446 annotated against public databases. Moreover, we identified 71,107 expressed sequence tag-simple sequence repeats (EST-SSRs), with an average number of 0.3 SSRs per superTranscripts and one SSR per 1.12kB. The most abundant SSR type was repeated dinucleotide (53.67%), followed by trinucleotide (13.73%) repeats. From the total of EST-SSRs identified, 34,196 primer pairs were properly designed and a subset of twenty-five immune loci were selected for its evaluation as potential EST-SSR population markers. Of this subset, eleven proved to have good technical features and were evaluated in 64 animals from four Patagonian toothfish populations. A number of 63 alleles were identified, with a mean of 4.9 alleles per locus and a polymorphism information content ranging from 0.224 to 0.591, with a mean of 0.50. Significant (FST, range 0.082-0.117 and G´ST, range 0.069-0.291) genetic differentiation (P

Amentotaxus argotaenia (Taxaceae) is a vulnerable coniferous species with preference for shade and moist environment. Accurate estimation of genetic variation is crucial for its conservation, especially in the context of global warming. In this study, we acquired a transcriptome from A. argotaenia leaves using Illumina sequencing and de novo assembled 62,896 unigenes, of which 5510 EST-SSRs were detected. Twenty-two polymorphic EST-SSRs were successfully developed and further used to investigate genetic variation, linkage disequilibrium, and bottleneck signatures of A. argotaenia. The results showed that A. argotaenia had moderate genetic variation and high genetic differentiation, which may provide raw material to protect against climatic changes and accelerate local adaptation, respectively. No bottlenecks were found to occur in A. argotaenia. Our study not only showed that these EST markers are very effective in population genetic analysis but also lay a solid foundation for further investigating adaptive evolution and conservation strategies of A. argotaenia.

Atlantska tuna Thunnus thynnus je pelagična riba s visokim stupnjem eksploatacije i gospodarskog značaja. Kavezni uzgoj ove vrste koji obuhvaća ulov juvenilnih divljih jedinki, njihov transport do uzgajališta i prilagodbu na kavezne uvjete života, može biti stresan za juvenilne ribe i rezultirati neočekivanim mortalitetima. U ovom istraživanju razvijen je novi set od 13 EST-SSR za T. thynnus kako bi se utvrdilo može li se preživljavanje jedinki kroz uzgojni ciklus pratiti na genetičkoj razini i povezati s genima povezanima s imunitetom i odgovorom na uvjete stresa. Zabilježena je niska FST vrijednost (0,005), slična frekvencija alela i niska stopa gubitka alela između uzorkovanih populacija. Dva različita pristupa za identifikaciju lokusa pod selekcijom nisu ukazala na odstupanje novorazvijenih lokusa od neutralnosti, te rezultati istraživanja upućuju na to da se oni ne mogu smatrati informativnim u istraživanom kontekstu. Atlantic bluefin tuna Thunnus thynnus is pelagic fish with a high level of exploitation and economic importance. The capture-based aquaculture that involves the catch of wild individuals, a slow transport to the rearing facilities and adaptation to the captive environment, could be stressful for juvenile fish, consequently resulting in unexpected mortalities. In this study, a new set of 13 EST-SSRs for T. thynnus was developed to explore whether the survival success through the farming cycle might be monitored at the genetic level and linked to several immunity and stress response-associated genes. The observed FST value was low (0.005), showing similar allele frequencies and no major allele loss between tested populations. Two different approaches for the identification of loci under selection did not indicate departure from neutrality for any of the 13 EST-SSRs, suggesting that they may not be considered informative in the studied context.

Mungbean (Vigna radiata L.) is one of important legume in Indonesia. G-SSR and EST-SSR markers had been widely used in mungbean genetic diversity research. DNA isolation and DNA amplification are required to obtain genetic information about mungbean to obtain accurate data on the genetic diversity of mungbean. This study aims to determine the Temperature of annealing (Ta) of the g-SSR and EST-SSR primer pairs. The total DNA was isolated from young leaves mungbean origin Pelalawan and the eight primer pairs of the g-SSR and EST-SSR were optimized. The optimal Ta for G2436, G3598, G2516, G7472, G0483. G1671, G3302, and G3427 were 50,55ºC, 51,15ºC,51,25ºC, 51,2ºC, 51,6ºC, 49,0ºC, 49,8ºC, and 52,8ºC respectively. Meanwhile, the optimal Ta for E51985, E19823, E24080, E22860, E26637, E16266, E11659, and E10675 were 52,2ºC, 54,4ºC, 52,5ºC,51,25ºC, 53,25ºC, 54ºC, 54,35ºC, and 53,6ºC respectively.

Background Sino-Vietnamese limestone karsts (SVLK) are a biodiversity hotspot rich in endemic plant species associated with caves and cave-like microhabitats. Based on phylogenetic studies of Begonia sect. Coelocentrum, a species-rich and characteristic SVLK clade, geographic isolation caused by extensive and continuous karstification was proposed as the major driving force triggering population diversification and geographic speciation. To test this proposition, population genetics and phylogeography of Begonia luzhaiensis were investigated using EST-SSR markers and the chloroplast trnC-ycf6 intergenic spacer. Results F statistics, Bayesian clustering analysis, AMOVA, and PCoA of both data sets all indicated substantial population differentiation and significant isolation by distance. Nested clade phylogeographic analyses inferred that historical fragmentations have been prominent, congruent with Guangxi’s geohistory of karstification as well as suggesting a mountain chain in northeastern Guangxi could have also acted as a major geographic barrier. A Bayesian skyline plot (BSP) indicated a slight decline in effective population size at 75,000 years ago (75 Kya), coinciding with the last glacial period during which the increased aridity in East Asia had retarded karstification, negatively affecting the populations of B. luzhaiensis. However, BSP detected a continuous and further population decline until the present time even though summer monsoons have resumed since the end of the last glacial maximum. Conclusions The microevolution patterns of B. luzhaiensis support that limited gene flow would have greatly enhanced the effects of random genetic drift and has been a major factor promoting diversification in Begonia, highly congruent with previous proposition. Based our study, we further propose that the arrival of Paleolithic Homo sapiens whose activities centered around limestone caves could have had further impacts on the populations of B. luzhaiensis, resulting in additional population decline. Further habitat destruction could have resulted from the transition from hunter gathering to food-producing societies ca. 20–10 Kya and the development of agriculture ca. 10 Kya in South China. Implications of the current study for SVLK plant conservation are also discussed. Electronic supplementary material The online version of this article (10.1186/s40529-019-0267-9) contains supplementary material, which is available to authorized users.

Black locust (Robinia pseudoacacia L.) is an economically and ecologically important tree species which is used for pillar construction, honey production and soil improvement. More EST-SSR (Expressed sequence tag simple sequence repeat) markers of black locust can be used as a complement and improvement of Genomic-SSR markers for the identification of the function of gene and the construction of genetic map. Additionally, currently there is no simple method for identifying black locust cultivars. In this study, we obtained 2702 unigenes from 3095 expressed sequence tags (ESTs) from the National Center of Biotechnology Information (NCBI) database to identify simple sequence repeats (SSRs) in R. pseudoacacia samples. A total of 170 SSR loci were found to be distributed in 162 non-redundant sequences with a frequency of 6.29%. Dinucleotide repeats were the most predominant types among microsatellites (62.35%), followed by tri-nucleotide repeats (25.88%), the remaining SSRs accounted for less than 12%. The repeat motifs AG/TC (29.25%) and CT/GA (29.25%) were the most abundant among dinucleotides, and AAT/TTA (15.91%) was the most common among tri-nucleotides. A total of 62 primer pairs were designed to screen polymorphic and stable SSR loci. The resulting 25 EST-SSR markers capable of amplifying polymorphic, stable, and repeatable products. Eight newly developed EST-SSR markers and four published SSR markers were selected for DNA fingerprinting and genetic diversity analysis of the 123 main R. pseudoacacia cultivars in China. The 12 SSR loci amplified 102 alleles, with an average number of alleles per locus of 8.5 (range 4&ndash, 15). The average polymorphism information content at the 12 SSR loci for the 123 cultivars was 0.670 (range 0.427&ndash, 0.881). The 123 cultivars clustered into six main groups based on similarity coefficients, with most cultivars in one subgroup. Fingerprinting was performed using eight SSR markers, 110 black locust cultivars were distinguished. The results of this study increase the availability of EST-SSR markers in black locust and make it a simple method for checking the collection, the certification, and the correct attribution of clones and cultivars.

Interspecific hybridization is one of the major actuators of evolutionary changes in plants. As the result of allopolyploid hybridization, offspring may gain different ploidy levels in comparison to parental species, which can provide them instant reproductive isolation. Two tetraploid sister species, Centaurium erythraea and C. littorale, readily cross-fertilize, resulting in hybrids of various ploidy. In northern Serbia, two stable populations of a hexaploid taxon C. pannonicum have been documented. It has been proposed previously that this taxon emerged after an interspecific hybridization event between two tetraploid sister-species: C. erythraea and C. littorale subsp. compressum. The existing populations of the hybridogenic taxon, as well as neighboring populations of the two parental taxa were here characterized by both morphometrics and molecular markers (EST-SSR and trnL-F). Three leaf and two flower characteristics were found to be informative in delimitation of the parental taxa and in their discernment from hybrid individuals, the latter having intermediate values. Eight microsatellite markers were found to have good ability to distinguish studied taxa, placing C. pannonicum in closer relationship with C. erythraea. Conversely, trnL-F plastid marker nominated C. littorale subsp. compressum to be the donor of the C. pannonicum plastid DNA. Reproductive isolation of the hexaploid hybrid individuals from the parental species should be examined as the next logical step in describing the new species.

Interspecific hybridization is one of the major actuators of evolutionary changes in plants. As the result of allopolyploid hybridization, offspring may gain different ploidy levels in comparison to parental species, which can provide them instant reproductive isolation. Two tetraploid sister species, Centaurium erythraea and C. littorale, readily cross-fertilize, resulting in hybrids of various ploidy. In northern Serbia, two stable populations of a hexaploid taxon C. pannonicum have been documented. It has been proposed previously that this taxon emerged after an interspecific hybridization event between two tetraploid sister-species: C. erythraea and C. littorale subsp. compressum. The existing populations of the hybridogenic taxon, as well as neighboring populations of the two parental taxa were here characterized by both morphometrics and molecular markers (EST-SSR and trnL-F). Three leaf and two flower characteristics were found to be informative in delimitation of the parental taxa and in their discernment from hybrid individuals, the latter having intermediate values. Eight microsatellite markers were found to have good ability to distinguish studied taxa, placing C. pannonicum in closer relationship with C. erythraea. Conversely, trnL-F plastid marker nominated C. littorale subsp. compressum to be the donor of the C. pannonicum plastid DNA. Reproductive isolation of the hexaploid hybrid individuals from the parental species should be examined as the next logical step in describing the new species.

Digitaria exilis is an important indigenous cereal in West Africa. The first fonio reference transcriptome was released and became a key tool for developing new molecular markers contributing to a better understanding of its genetic diversity. A total of 126 new putative primer pairs were successfully designed in 37,327 unigenes from the D. exilis transcriptome. Thirty-seven primer pairs were randomly selected and tested for their ability to cross-amplify to related species. Clear amplification patterns were observed on 24 primer pairs. Of these, 71, 74 and 35% showed polymorphism in three species: D. exilis, D. longiflora and D. iburua. The transferability from D. exilis was 96% to D. longiflora and 71% to D. iburua. The new SSR markers confirmed the close genetic proximity of D. exilis with D. longiflora and its stronger genetic difference of D. exilis from D. iburua. These markers will be valuable for completing future knowledge on Digitaria evolutionary history, and for testing gene flows between related species.

Background Blumeria graminis f. sp. tritici (Bgt), the causal agent of wheat powdery mildew severely affects yield security wheat production in China. Understanding the virulence structure and genetic variations of this pathogen is important for breeding wheat lines resistant to wheat powdery mildew. However, information related to genes controlling resistance remains elusive. This study analyzes the virulence structure and the genetic diversity of pathogenic Bgt populations isolated from northeastern (Liaoning, Heilongjiang) and northwestern (Gansu) China, two representative wheat producing areas, on 37 wheat cultivars each carrying a known powdery mildew resistance (Pm) gene. Results Bgt isolates from northeastern China show higher frequencies of virulence genes than populations from Gansu Province. Many of the known Pm genes failed to provide resistance in this study. However, Pm21 provided 100% resistance to all isolates from all three provinces, obtained during two consecutive years, while Pm13 provided 100% resistance in Gansu. Pm13, Pm16, Pm18, and Pm22 also showed partial resistance in northeastern China, while Pm16, Pm18, Pm22, Pm5 + 6 and Pm2 + 6 +? maintained some resistance in Gansu. Genetic diversity among populations in different regions was detected by cluster analyses using expressed sequence tag-simple sequence repeat (EST-SSR). When the genetic similarity coefficient is relatively high, populations from the same regional origin are mostly clustered into one group while populations from different regions exhibit large genetic differences. Conclusion Pm21 remains the best choice for breeding programs to maintain resistance to Bgt. Only 58% of the isolates tested show a clear correlation between EST-SSR genetic polymorphisms and frequency of virulence gene data.

Background Sainfoin (Onobrychis viciifolia) is a highly nutritious, tannin-containing, and tetraploid forage legume. Due to the lack of detailed transcriptomic and genomic information on this species, genetic and breeding projects for sainfoin improvement have been significantly hindered. Methods In this study, a total of 24,630,711 clean reads were generated from 14 different sainfoin tissues using Illumina paired-end sequencing technology and deposited in the NCBI SRA database (SRX3763386). From these clean reads, 77,764 unigene sequences were obtained and 6,752 EST-SSRs were identified using de novo assembly. A total of 2,469 primer pairs were designed, and 200 primer pairs were randomly selected to analyze the polymorphism in five sainfoin wild accessions. Results Further analysis of 40 sainfoin individuals from the five wild populations using 61 EST-SSR loci showed that the number of alleles per locus ranged from 4 to 15, and the expected heterozygosity varied from 0.55 to 0.91. Additionally, by counting the EST-SSR band number and sequencing the three or four bands in one sainfoin individual, sainfoin was confirmed to be autotetraploid. This finding provides a high level of information about this plant. Discussion Through this study, 61 EST-SSR markers were successfully developed and shown to be useful for genetic studies and investigations of population genetic structures and variabilities among different sainfoin accessions.

Caper (Capparis spinosa L.) is a xerophytic shrub cultivated for its flower buds and fruits, used as food and for their medicinal properties. Breeding programs and even proper taxonomic classification of the genus Capparis has been hampered so far by the lack of reliable genetic information and molecular markers. Here, we present the first genomic resource for C. spinosa, generated by transcriptomic approach and de novo assembly. The sequencing effort produced nearly 80 million clean reads assembled into 124,723 unitranscripts. Careful annotation and comparison with public databases revealed homologs to genes with a key role in important metabolic pathways linked to abiotic stress tolerance and bio-compounds production, such purine, thiamine and phenylpropanoid biosynthesis, α-linolenic acid and lipid metabolism. Additionally, a panel of genes involved in stomatal development/distribution and encoding for Stress Associated Proteins (SAPs) was also identified. We also used the transcriptomic data to uncover novel molecular markers for caper. Out of 50 SSRs tested, 14 proved polymorphic and represent the first set of SSR markers for the genus Capparis. This transcriptome will be an important contribution to future studies and breeding programs for this orphan crop, aiding to the development of improved varieties to sustain agriculture in arid conditions.

Aiming at accelerating the application of molecular markers in the genetic improvement of quails, six EST-SSR markers were successfully developed using a bioinformatics method. Polymorphisms of three quail populations (Chinese yellow quail, China black quail and Korean quail) were detected. The results showed that there were 2-6 alleles in six EST-SSR markers. Mean polymorphism information contents of Chinese yellow quails, Chinese black quails and Korean quails were determined as 0.5451, 0.4962 and 0.4937, respectively. Average heterozygosity valuesof 0.6134, 0.5759 and 0.5613 were calculated. Among the six EST-SSR markers, three were highly polymorphic, and the other three were moderately polymorphic. The newly-developed six EST-SSR markers may be used to determine the genetic diversity of quails. The six EST-SSR markers identified were related to carbohydrate metabolism and melanin synthesis, but their specific mechanisms need to be further analyzed.

Traditional agroecosystems are considered as main conservatories of landraces biodiversity because they preserve their dynamic processes and farmers' knowledge of selection and management inherent in the development of local cultivars. The Mediterranean region still harbours traditional agroecosystems of particular importance for preserving crop biodiversity, being sweet chestnut (Castanea sativa Miller) a good example. This is one of the multipurpose species of most economic importance in the Mediterranean basin, cultivated extensively for fruit production, and the majority of varieties are the result of the selection of landraces by farmers. In this study, a set of European chestnut traditional varieties were evaluated in order to characterise its genetic resources and detect a possible response in the adaptive potential of this germplasm. Results of Bayesian analysis revealed a clear structure among cultivars from Italy and Spain with a low degree of admixture between them. Furthermore, a strong structure was detected within germplasm from each country congruent with their geographic origin. Statistical analyses did not reveal any significant marker deviating from neutral expectations. Results were discussed considering previous studies carried out on neutral and morphological markers, concluding that information provided by different markers was more powerful in detecting the maximum amount of genetic variation in chestnut varieties, and establishing a baseline to continue identifying markers underlying phenotypic differentiation and/or response to environmental cues among varieties

Chinese hawthorn (Crataegus pinnatifida) is an important ornamental and economic horticultural plant. However, the lack of molecular markers has limited the development and utilization of hawthorn germplasm resources. Simple sequence repeats (SSRs) derived from expressed sequence tags (ESTs) allow precise and effective cultivar characterization and are routinely used for genetic diversity analysis. Thus, we first reported the development of polymorphic EST-SSR markers in C. pinnatifida with perfect repeats using Illumina RNA-Seq technique. In total, we investigated 14,364 unigenes, from which 5091 EST-SSR loci were mined. Di-nucleotides (2012, 39.52%) were the most abundant SSRs, followed by mono- (1989, 39.07%), and tri-nucleotides (1024, 20.11%). On the basis of these EST-SSRs, a total of 300 primer pairs were designed and used for polymorphism analysis in 70 accessions collected from different geographical regions of China. Of 239 (79.67%) pairs of primer-generated amplification products, 163 (54.33%) pairs of primers showed polymorphism. Finally, 33 primers with high polymorphism were selected for genetic diversity analysis and tested on 70 individuals with low-cost fluorescence-labeled M13 primers using capillary electrophoresis genotyping platform. A total of 108 alleles were amplified by 33 SSR markers, with the number of alleles (Na) ranging from 2 to 14 per locus (mean: 4.939), and the effective number of alleles (Ne) ranging from 1.258 to 3.214 (mean: 2.221). The mean values of gene diversity (He), observed heterozygosity (Ho), and polymorphism information content (PIC) were 0.524 (range 0.205&ndash, 0.689), 0.709 (range 0.132&ndash, 1.000), and 0.450 (range 0.184&ndash, 0.642), respectively. Furthermore, the dendrogram constructed based on the EST-SSR separated the cultivars into two main clusters. In sum, our study was the first comprehensive study on the development and analysis of a large set of SSR markers in hawthorn. The results suggested that the use of NGS techniques for SSR development represented a powerful tool for genetic studies. Additionally, fluorescence-labeled M13 markers proved to be a valuable method for genotyping. All of these EST-SSR markers have agronomic potential and constitute a scientific basis for future studies on the identification, classification, and innovation of hawthorn germplasms.

Digitaria exilis is an important indigenous cereal in West Africa. The first fonio reference transcriptome was released and became a key tool for developing new molecular markers contributing to a better understanding of its genetic diversity. A total of 126 new putative primer pairs were successfully designed in 37,327 unigenes from the D. exilis transcriptome. Thirty-seven primer pairs were randomly selected and tested for their ability to cross-amplify to related species. Clear amplification patterns were observed on 24 primer pairs. Of these, 71, 74 and 35% showed polymorphism in three species: D. exilis, D. longiflora and D. iburua. The transferability from D. exilis was 96% to D. longiflora and 71% to D. iburua. The new SSR markers confirmed the close genetic proximity of D. exilis with D. longiflora and its stronger genetic difference of D. exilis from D. iburua. These markers will be valuable for completing future knowledge on Digitaria evolutionary history, and for testing gene flows between related species.

Köpekler, insanlarlaortak bir çevreyi paylaşır ve belirli köpek ırklarının hastalıkları hakkındabilgi, insan kanser çalışmaları için bir model geliştirmede çok yararlıdır.EST'ler, bir organizmanın transkribe edilen genomunun bir parçasını temsil ederve mikrosatellitleri tanımlamak için önemli bir kaynaktır. Tekrarlayan DNAdizilerini içeren Basit Dizi Tekrarları (SSR'ler) veya mikrosatellitler,bilinen en güçlü genetik belirteçler arasındadır. EST-SSR'lerin gelişimi,genomik çalışmalar için hızlı, verimli ve düşük maliyetli bir seçenek halinegelmiştir. Bu çalışmada, 2304 EST içeren Terrier köpeğin meme bezi dokusununEST koleksiyonundan SSR'lerin belirlenmesi için; Web tabanlı sürümleri olan vekolayca erişilebilen SSRIT ve IMEx yazılımları kullanılmıştır. SSRIT, 2 ila 10baz uzunluğundaki motifleri bulur ve tekli nükleotid motiflerini ortadankaldırarak minimum tekrar sayısını ayarlar. IMEx mükemmel ve kusurlu mikrosatellitleriayrı ayrı bulur. Farklı uzunluklarda 1 ile 6 arasındaki motifleri bulabilir veminimum tekrar sayısı ayarlanabilir. Ek olarak, istenen SSR bölgesi için uygunprimer tasarlanabilir. Normal doku EST'leri için 2, 3, 4, 5 ve 6 nükleotidli motiflerbulunurken, tümörlü doku EST'leri için 5 nükleotidli motif bulunamamıştır., Dogs share a commonenvironment with humans and knowledge of the specific dog breed diseases isvery useful in developing a model for human cancer studies. ESTs represent partof the transcribed genome of an organism and are an important resource foridentifying microsatellites. Simple Sequence Repeats (SSRs), ormicrosatellites, which contain repetitive DNA sequences, are among the mostpowerful genetic markers known. The development of EST-SSRs has become a fast,efficient, and low-cost option for genomic studies. In this study, to determineSSRs from EST libraryof mammary gland tissue of the Terrier dog that has 2304ESTs; SSRIT and IMEx software, which have web-based versions and are easilyaccessible, were used. SSRIT finds motifs from 2 to 10 base lengths and adjuststhe minimum number of repeats by eliminating single nucleotide motifs. IMExfinds perfect and imperfect microsatellites separately. It can find motifs ofdifferent lengths from 1 to 6 and the minimum number of repeats can be set. Inaddition, the appropriate primer for the desired SSR region can bedesigned. The 2, 3, 4, 5 and 6nucleotide motifs were found for normal tissue ESTs whereas 5 nucleotide motifswere not found for tumoral tissue ESTs.

As one of the earliest domesticated species, Cucurbita pepo (including squash and pumpkin) is rich in phenotypic polymorphism and has huge economic value. In this research, using 1660 expressed sequence tags-simple sequence repeats (EST-SSRs) and 632 genomic simple sequence repeats (gSSRs), we constructed the highest-density EST-SSR-based genetic map in Cucurbita genus, which spanned 2199.1 cM in total and harbored 623 loci distributed in 20 linkage groups. Using this map as a bridge, the two previous gSSR maps were integrated by common gSSRs and the corresponding relationships around chromosomes in three sets of genomes were also collated. Meanwhile, one large segmental inversion that existed between our map and the C. pepo genome was detected. Furthermore, three Quantitative Trait Loci (QTLs) of the dwarf trait (gibberellin-sensitive dwarf type) in C. pepo were located, and the candidate region that covered the major QTL spanned 1.39 Mb, which harbored a predicted gibberellin 2-&beta, oxidase gene. Considering the rich phenotypic polymorphism, the important economic value in the Cucurbita genus species and several advantages of the SSR marker were identified, thus, this high-density EST-SSR-based genetic map will be useful in Pumpkin and Squash breeding work in the future.

The common walnut (Juglans regia L.) and iron walnut (J. sigillata Dode) are well-known economically important species cultivated for their edible nuts, high-quality wood, and medicinal properties and display a sympatric distribution in southwestern China. However, detailed research on the genetic diversity and introgression of these two closely related walnut species, especially in southwestern China, are lacking. In this study, we analyzed a total of 506 individuals from 28 populations of J. regia and J. sigillata using 25 EST-SSR markers to determine if their gene introgression was related to sympatric distribution. In addition, we compared the genetic diversity estimates between them. Our results indicated that all J. regia populations possess slightly higher genetic diversity than J. sigillata populations. The Geostatistical IDW technique (HO, PPL, NA and PrA) revealed that northern Yunnan and Guizhou provinces had high genetic diversity for J. regia while the northwestern Yunnan province had high genetic diversity for J. sigillata. AMOVA analysis revealed that significant genetic variation was mainly distributed within population as 73% in J. regia and 76% in J. sigillata. The genetic differentiation (FST) was 0.307 between the two walnut species (p &lt, 0.0001), which was higher than FST values within populations (J. regia FST = 0.265 and J. sigillata FST = 0.236). However, the STRUCTURE analysis of the J. regia and J. sigillata populations revealed two genetic clusters in which gene introgression exists, therefore, the boundary of separation between these two walnut species is not clear. Moreover, these results were validated by NJ and UPGMA analysis with additional conformation from the PCoA. Based on the SSR data, our results indicate that J. sigillata is an ecotype of J. regia. Taken together, these results reveal novel information on population genetics and provide specific geographical regions containing high genetic diversity of the Juglans species sampled, which will assist in future conservation management.

Understanding the genetic diversity and differentiation of the genetic resources of a species is important for the effective use and protection of forest tree resources. Ex situ development is a common method for the protection of genetic diversity and an essential resource for users who require ready access to a species&rsquo, germplasm. In this study, we collected seeds of black locust (Robinia pseudoacacia L.) from 19 provenances, covering most of its natural distribution, we randomly selected 367 tender leaves with well-grown and different maternal strains from this group for further analysis. Forty-eight simple sequence repeat (SSR) primers were successfully selected from 91 pairs of SSR primers using native-deformation polyacrylamide gel electrophoresis. In addition, we identified identical genotypes among all individuals and evaluated the quality of the markers. From this, 35 loci were confirmed for analyses of genetic diversity and differentiation of the black locust provenances, which contained 28 expressed sequence tag-derived simple sequence repeats (EST-SSRs) and 7 genomic DNA-derived simple sequence repeats (G-SSRs). We observed high genetic diversity among the native black locust provenances, from which Wright&rsquo, s fixation index and molecular variance suggested that a majority of the genetic differentiation variation could be attributed to within-provenance differences. The genetic distance and identity results indicated that geographic distance was not a dominating factor influencing the distribution of black locust. This is the first study to evaluate provenance genetic variation in native black locust samples using two types of SSR markers, which provides a comprehensive theoretical basis for ex situ conservation and utilization of genetic resources, with an emphasis on breeding applications.

TEZ13088 Tez (Yüksek Lisans) -- Çukurova Üniversitesi, Adana, 2018. Kaynakça (s. 43-50) var. XIII, 51 s. :_tablo ; c29 cm. Bu çalışmada farklı orijinli antepfıstığı (Pistacia vera L.) çeşit ve genotiplerinin moleküler olarak karakterize edilmesi amaçlanmıştır. Karakterizasyon çalışmasında antepfıstığı çeşitlerinden elde edilen transkriptom dizileri kullanılarak SSR primerleri dizayn edilmiştir. Dizayn edilen 100 adet genik SSR’lar arasından polimorfik olan 18 adet primer bu çalışma kapsamında geliştirilmiştir. Geliştirilen genik SSR ile daha önceden geliştirilen 18 adet genomik SSR primerleri toplam 72 adet antepfıstığının karakterizasyonunda kullanılmıştır. Elde edilen sonuçlara göre, toplam 192 allel elde edilmiş ve ortalama allel sayısı 5.3 olarak hesaplanmıştır. Polimorfizm bilgi içeriği (PIC) 0.07 ile 0.79 arasında değişim göstermiş ve ortalaması 0.52 olmuştur. Ortalama gözlenen ve beklenen heterozigotluk değerleri sırasıyla 0.51 ve 0.57 olarak hesaplanmıştır. Filogenetik analiz sonuçlarına göre, antepfıstığı çeşit ve genotipleri temelde İran ve Akdeniz ülkeleri çeşitleri olmak üzere iki ana grubta toplamıştır. Bu iki grup arasında ise Siirt grubu yer almıştır. Bu çalışma sonucunda geliştiirlen EST-SSR primerleri ile çeşitlerin karakterizasyonu sonucu elde edilen veriler antepfıstığında bundan sonra yapılacak çeşit tanımlama, genetik kaynakların karakterizasyonu, markör destekli seleksyon ve ıslah programları gibi çalışmalarda kullanılabilecektir. This study focused on the characterization of different cultivars and genotypes of pistachio (Pistacia vera L.). The SSR primers were designed by transcriptome sequenced from pistachio cultivars for characterization study. Totally 18 EST-SSRs were developed from 100 EST-SSR primer pairs in this study. Developed genic SSR in this study and previously developed 18 genomic SSR markers were used in 72 pistachio cultivars and genotypes for characterization. According to the results, a total of 192 alleles were predicted and the average allele number was 5.3. The Polymorphic Information Content (PIC) was between 0.07 and 0.79, and the average was 0.52. The observed heterozygosity (Ho) and the expected heterozygosity (He) were showed as 0.51 and 0.57. According to the phylogenetic analysis, pistachio cultivars and genotypes are grouped in two main classes of their origin, namely Iran and Mediterranean. ‘Siirt’ group is placed between Iran and the Mediterranean groups. The EST-SSR primers developed in this study along with the results of characterization will be used in further studies of pistachio in order for cultivar identification, germplasm characterization, marker-assisted selection, and molecular breeding studies. Bu Çalışma Ç.Ü. Bilimsel Araştırma Projeleri Birimi Tarafından Desteklenmiştir. Proje no: FYL-2018-10667.

Le previsioni dei possibili cambiamenti climatici nelle regioni del bacino del Mediterraneo ipotizzano, nel corso di questo secolo, un innalzamento delle temperature nell'ordine di 2° - 4°, unitamente ad una costante diminuzione delle precipitazioni (IPCC 2007; Giorgi and Lionello, 2008). La sopravvivenza degli organismi viventi ai cambiamenti ambientali è strettamente legata alla loro variabilità genetica e plasticità adattativa; per questo motivo, la comprensione della base genetica dei meccanismi coinvolti nel processo di adattamento ai cambiamenti climatici potrebbe rivelarsi di primaria importanza per la gestione e conservazione delle risorse genetiche (Homolka et al, 2012). Castanea sativa (Fagaceae), specie ampiamente diffusa nel bacino del Mediterraneo, dalle regioni del Caucaso fino all'Europa occidentale, ha un'elevata valenza economica, legata alla produzione di legno ed al consumo dei frutti a fini alimentari. Ricerche precedenti condotte su popolamenti di naturali castagno Europeo hanno evidenziato variabilità sia a livello neutrale che adattativo, (Villani et al. 1991; Martin et al., Machon et al. 1996; Mattioni et al. 2009). In questo studio, si è valutata la diversità neutrale ed adattativa di 4 popolamenti di castagno situati in 4 regioni Spagnole (Cataluna, Galicia, Extremadura ed Andalucia) con caratteristiche ambientali differenti. Due sono stati gli obiettivi (1) indagare la struttura e la diversità genetica tramite l'utilizzo di marcatori microsatellitari neutrali e EST-SSR ed (2) indentificare ed evidenziare la differenza a livello nucleotidico in geni candidati potenzialmente legati alla riposta ad eventi di stress idrico. Mediante il software GenAlEx 6.5 è stato possibile calcolare i principali indici di diversità genetica. L'analisi della struttura della popolazione è stata condotta utilizzando il software STRUCTURE 2.3.4. L'analisi con i marcatori funzionali ha evidenziato una diversa struttura delle popolazioni rispetto a quella osservata con i marcatori SSR neutrali. I marcatori funzionali evidenziano differenti pool genetici nelle popolazioni del Nord rispetto a quello del Sud indicando una possibile congruenza con le differenti condizioni climatiche.

Premise of the study: Brainea insignis (Aspleniaceae) is an endangered tree fern in China whose wild populations have been seriously damaged due to overexploitation. Expressed sequence tag–simple sequence repeat (EST-SSR) primers were developed to investigate its genetic diversity and provide resources for future conservation studies. Methods and Results: We obtained 72,897 unigenes of B. insignis using transcriptome sequencing and detected 15,006 SSRs in 12,058 unigenes. Based on these results, we designed 100 EST-SSR primer pairs and successfully amplified 52 of them in six individuals; 27 demonstrated polymorphisms after amplification against 72 individuals across three populations. Allele numbers ranged from three to 10, and the observed and expected heterozygosities ranged from 0.105 to 1.000 and from 0.523 to 0.865, respectively, in the tested populations. Most of these primers could be successfully amplified in two other fern species (Blechnum orientale and Chieniopteris harlandii). Conclusions: These selected EST-SSRs are valuable for genetic diversity and conservation studies in B. insignis and other related fern species.

As important antioxidants and secondary metabolites in peanut seeds, flavonoids have great nutritive value. In this study, total flavonoid contents (TFC) were determined in seeds of 57 peanut accessions from the province of Hebei, China. A variation of 0.39 to 4.53 mg RT g-1 FW was found, and eight germplasm samples containing more than 3.5 mg RT g-1 FW. The TFC of seed embryos ranged from 0.14 to 0.77mg RT g-1 FW. With a view to breeding high-quality peanut varieties with high yields and high TFC, we analyzed the correlations between TFC and plant and pod characteristics. The results of correlation analysis indicated that TFC was significantly negatively correlated with pod number per plant (P/P) and soluble protein content (SPC). We used 251 pairs of expressed sequence tag - simple sequence repeat (EST-SSRs) primers to sequence all germplasm samples and found four EST-SSR markers that were significantly related to TFC.

To develop a high density linkage map in faba bean, a total of 1,363 FBES (Faba bean expressed sequence tag [EST]-derived simple sequence repeat [SSR]) markers were designed based on 5,090 non-redundant ESTs developed in this study. A total of 109 plants of a 'Nubaria 2' × 'Misr 3' F2 mapping population were used for map construction. Because the parents were not pure homozygous lines, the 109 F2 plants were divided into three subpopulations according to the original F1 plants. Linkage groups (LGs) generated in each subpopulation were integrated by commonly mapped markers. The integrated 'Nubaria 2' × 'Misr 3' map consisted of six LGs, representing a total length of 684.7 cM, with 552 loci. Of the mapped loci, 47% were generated from multi-loci diagnostic (MLD) markers. Alignment of homologous sequence pairs along each linkage group revealed obvious syntenic relationships between LGs in faba bean and the genomes of two model legumes, Lotus japonicus and Medicago truncatula. In a polymorphic analysis with ten Egyptian faba bean varieties, 78.9% (384/487) of the FBES markers showed polymorphisms. Along with the EST-SSR markers, the dense map developed in this study is expected to accelerate marker assisted breeding in faba bean.

Plant Breed.ISI Document Delivery No.: AJ4WPTimes Cited: 0Cited Reference Count: 31Cited References: Argout X, 2008, BMC GENOMICS, V9, DOI 10.1186/1471-2164-9-512 BESSE P, 1994, THEOR APPL GENET, V88, P199, DOI 10.1007/BF00225898 Conesa A, 2005, BIOINFORMATICS, V21, P3674, DOI 10.1093/bioinformatics/bti610 Cubry P, 2013, BMC GENOMICS, V14, DOI 10.1186/1471-2164-14-10 Ellis JR, 2007, HEREDITY, V99, P125, DOI 10.1038/sj.hdy.6801001 Feng SP, 2009, MOL BREEDING, V23, P85, DOI 10.1007/s11032-008-9216-0 Garcia D, 2011, PHYSIOL MOL PLANT P, V76, P126, DOI 10.1016/j.pmpp.2011.07.006 Gupta PK, 2005, PLANT MOL BIOL, V57, P461, DOI 10.1007/s11103-005-0257-z Gupta S, 2013, PLANT BREEDING, V132, P367, DOI 10.1111/pbr.12070 Kumar Yadav H., 2010, TREE GENET GENOMES, V7, P207 Le Guen V., 2008, EXPLORATION DIVERSIT Le Guen V, 2011, PLANT BREEDING, V130, P294, DOI 10.1111/j.1439-0523.2010.01774.x Le Guen V, 2009, TREE GENET GENOMES, V5, P673, DOI 10.1007/s11295-009-0218-9 Lespinasse D, 2000, THEOR APPL GENET, V100, P127, DOI 10.1007/s001220050018 Li DJ, 2012, BMC GENOMICS, V13, DOI 10.1186/1471-2164-13-192 Liu KJ, 2005, BIOINFORMATICS, V21, P2128, DOI 10.1093/bioinformatics/bti282 Ohyama A, 2009, MOL BREEDING, V23, P685, DOI 10.1007/s11032-009-9265-z Perovic D, 2009, MOL BREEDING, V23, P641, DOI 10.1007/s11032-009-9262-2 Perrier Xavier, 2003, P31 Perrier X., 2006, DARWIN SOFTWARE Pootakham W, 2012, PLANT BREEDING, V131, P555, DOI 10.1111/j.1439-0523.2012.01982.x Rafalski JA, 2010, CURR OPIN PLANT BIOL, V13, P174, DOI 10.1016/j.pbi.2009.12.004 Rozen S, 2000, Methods Mol Biol, V132, P365 SAITOU N, 1987, MOL BIOL EVOL, V4, P406 Seguin Marc, 2003, P277 Souza LM, 2009, CONSERV GENET RESOUR, V1, P75, DOI 10.1007/s12686-009-9018-7 Triwitayakorn K, 2011, DNA RES, V18, P471, DOI 10.1093/dnares/dsr034 Tyrka M, 2008, J APPL GENET, V49, P127, DOI 10.1007/BF03195605 Varshney RK, 2005, TRENDS BIOTECHNOL, V23, P48, DOI 10.1016/j.tibtech.2004.11.005 Vigouroux Y, 2005, GENETICS, V169, P1617, DOI 10.1534/genetics.104.032086 Wen Mingfu, 2010, BMC Res Notes, V3, P42, DOI 10.1186/1756-0500-3-42Cubry, Philippe Pujade-Renaud, Valerie Garcia, Dominique Espeout, Sandra Le Guen, Vincent Granet, Francoise Seguin, MarcFrench National Research Agency (Agence Nationale pour la Recherche; contrat ANR/Genoplante) [GPLA07017C]This work was performed as part of the CIRAD - Michelin collaborative project 'Genesalb' supported by a grant from the French National Research Agency (Agence Nationale pour la Recherche; contrat ANR/Genoplante no GPLA07017C). We thank the Montpellier Languedoc-Roussillon Grand plateau technique regional for hosting the SSR genotyping activities. ESTtik database was made available by the SouthGreen bioinformatics platform at http://southgreen.cirad.fr/. We thank Ronan Rivallan for technical help, Xavier Argout for computational procedures and data retrieving, Gerald Oliver for keeping some vegetal material in a greenhouse in Montpellier, Jean-Marc Thevenin and Fabien Doare for providing us with some fresh material from CIRAD's Pointe Combi Centre (French Guyana) collection.Wiley-blackwellHoboken; Despite its economic importance and recent genome release, the need for molecular tools for Hevea brasiliensis is high. In the frame of a disease resistance study, EST sequences were retrieved from public database or generated by sequencing SSH libraries. Sequences were trimmed and microsatellite motifs searched using an ad hoc bioinformatic pipeline, and pairs of primers for the amplification of candidate markers were generated. We found a total of 10499 unigenes from both sources of sequences, and 673 microsatellites motifs were detected using the default parameters of the pipeline. Two hundred sixty-four primer pairs were tested and 226 (85.6%) successfully amplified. Out of the amplified candidate markers, 164 exhibited polymorphism. Relationships based on dendrograms using simple matching index and diversity statistics based on EST-SSRs were compared with Genomic SSRs, showing the potentialities of EST-derived microsatellites for resistance studies but also for population genetics approaches.

Celery (Apium graveolens L.) is one of the most economically important vegetables worldwide, but genetic and genomic resources supporting celery molecular breeding are quite limited, thus few studies on celery have been conducted so far. In this study we made use of simple sequence repeat (SSR) markers generated from previous celery transcriptome sequencing and attempted to detect the genetic diversity and relationships of commonly used celery accessions and explore the efficiency of the primers used for cultivars identification. Analysis of molecular variance (AMOVA) of Apium graveolens L. var. dulce showed that approximately 43% of genetic diversity was within accessions, 45% among accessions, and 22% among horticultural types. The neighbor-joining tree generated by unweighted pair group method with arithmetic mean (UPGMA), and population structure analysis, as well as principal components analysis (PCA), separated the cultivars into clusters corresponding to the geographical areas where they originated. Genetic distance analysis suggested that genetic variation within Apium graveolens was quite limited. Genotypic diversity showed any combinations of 55 genic SSRs were able to distinguish the genotypes of all 30 accessions.

High-quality simple sequence repeat (SSR) markers are invaluable tools for revealing genetic variability which could be utilized for many purposes, such as breeding new varieties or the identifying current ones, among other applications. Based on the analysis of 3.7 million EST sequences and 15 genomic sequences from bacterial artificial chromosome (BAC) libraries, 200 trinucleotide genic (EST)-SSR and three genomic (gSSR) markers were tested, where 17 of them fulfilled all criteria for quality markers. Moreover, the reproducibility of these new markers was verified by two genetics laboratories, with a mean error rate per allele and per locus equal to 0.17%. These markers were tested on 38 accessions of Papaver somniferum and nine accessions of another five species of the Papaver and Argemone genera. In total, 118 alleles were detected for all accessions (median = 7, three to ten alleles per locus) and 88 alleles (median = 5, three to nine alleles per locus) within P. somniferum alone. Multivariate methods and identity analysis revealed high resolution capabilities of the new markers, where all but three pair accessions (41 out of 47) had a unique profile and opium poppy was distinguished from other species.