SSR Loci Research Articles

Microsatellite analysis and polymorphic marker development based on the full-length transcriptome of Camellia chekiangoleosa

Camellia chekiangoleosa is a popular variety of Oil-camellia that has high oil production and ornamental value. Microsatellite (SSR) markers are the preferred tool for the molecular marker-assisted breeding of C. chekiangoleosa. By focusing on the problems of the low development efficiency of polymorphic SSR markers and the lack of available functional markers in Oil-camellia, we identified 97,510 SSR loci based on the full-length transcriptome sequence of C. chekiangoleosa. An analysis of SSR characteristics showed that mononucleotide (51.29%) and dinucleotide (34.36%) SSRs were the main repeat types. The main SSR distribution areas based on proportion covered were ordered as follows: 5'UTR > 3'UTR > CDS. By comparing our data with those in databases such as GO and KEGG, we obtained functional annotations of unigene sequences containing SSR sites. The data showed that the amplification efficiency of the SSR primers was 51.72%, and the development efficiency of polymorphic SSR primers was 26.72%. Experiments verified that dinucleotide and pentanucleotide SSRs located in UTR regions could produce more polymorphic markers. An investigation into the genetic diversity of several C. chekiangoleosa populations also suggested that the developed SSR markers had higher levels of polymorphism. This study will provide a reference and high-quality markers for the large-scale development of functional SSR markers and genetic research in Oil-camellia.

De novo transcriptome assembly using Illumina sequencing and development of EST-SSR markers in a monoecious herb Sagittaria trifolia Linn.

BackgroundSagittaria trifolia Linn. is a widespread macrophyte in Asia and southeast Europe and cultivated in parts of Asia. Although a few genomic studies have been conducted for S. trifolia var. sinensis, a crop breed, there is limited genomic information on the wild species of S. trifolia. Effective microsatellite markers are also lacking.ObjectiveTo assemble transcriptome sequence and develop effective EST-SSR markers for S. trifolia.MethodsHere we developed microsatellite markers based on tri-, tetra-, penta-, and hexa-nucleotide repeat sequences by comparatively screening multiple transcriptome sequences of eleven individuals from ten natural populations of S. trifolia.ResultsA total of 107,022 unigenes were de novo assembled, with a mean length of 730 bp and an N50 length of 1,378 bp. The main repeat types were mononucleotide, trinucleotide, and dinucleotide, accounting for 55.83%, 23.51%, and 17.56% of the total repeats, respectively. A total of 86 microsatellite loci were identified with repeats of tri-, tetra-, penta-, and hexa-nucleotide. For SSR verification, 28 polymorphic loci from 41 randomly picked markers were found to produce stable and polymorphic bands, with the number of alleles per locus ranging from 2 to 11 and a mean of 5.2. The range of polymorphic information content (PIC) of each SSR locus varied from 0.25 to 0.80, with an average of 0.58. The expected heterozygosity ranged from 0.29 to 0.82, whereas the observed heterozygosity ranged from 0.25 to 0.90.ConclusionThe assembled transcriptome and annotated unigenes of S. trifolia provide a basis for future studies on gene functions, pathways, and molecular mechanisms associated with this species and other related. The newly developed EST-SSR markers could be effective in examining population genetic structure, differentiation, and parentage analyses in ecological and evolutionary studies of S. trifolia.

Molecular characterization of Kinnow mutants

The present experiment was conducted during 2016-18 at Division of Fruits and Horticultural Technology, ICARIARI, New Delhi. A total of 40 Kinnow mutants and 1 wild type were selected for molecular characterization and diversity analyses. Thirty-four SSR markers were screened for polymorphism, of which only 2 were found informative. Twelve alleles were detected among 2 SSRs with an average of 6 alleles per locus and the highest number of alleles (7) was recorded in SSR locus AMB2. The average diversity indices of SSR, viz. allele frequency, gene diversity, observed heterozygosity and PIC were 0.415, 0.672, 0.415 and 0.611 respectively. The N-J tree was constructed based on the 2 SSRs data which clustered the mutants into 2 major groups. Subsequently, clusters were simplified into 6 clades which distinguished gamma-irradiated and EMS derived mutants and results were reconfirmed through principal coordinate analysis (PCoA). First three axes of PCoA contributed 80.88% of the cumulative variation among the Kinnow mutants. The analysis of molecular variance (AMOVA) explained 42% variation among the populations, 48 and 10% variation within and among the individuals respectively. The present investigation genetically characterized the Kinnow mutants and deciphered the genetic diversity among them. Thus, the variability generated through induced mutagenesis could be used as valuable genetic material for Kinnow improvement.

Mining and validating novel SSR markers based on coconut (Cocos nucifera L.) whole genome and their use for phylogenetic analysis

Abstract. Hatta ANNL, Sukma D, Maskromo I, Sudarsono. 2022. Mining and validating novel SSR markers based on coconut (Cocos nucifera L.) whole genome and their use for phylogenetic analysis. Biodiversitas 23: 5122-5131. The coconut (Cocos nucifera L.) genome sequences were available at the chromosome level in 2019, and they can be used to develop the whole genome SSR markers. This study aimed to conduct in silico analysis of the whole coconut genome to mine SSR sequences, design SSR markers distributed across coconut chromosomes, validate the designed primers to generate SSR markers, and use them for the phylogenetic analysis of coconuts. The SSR loci were mined from publicly available coconut genomes. Targeted primers for amplifying 20 dinucleotide SSR loci were selected and used for phylogenetic analysis of coconuts. The SSR mining identified 543,774 dinucleotide repeat motifs across the coconut genome, and most of the dinucleotide SSRs are AG (46.18%) and AT (40.03%) repeats. Primer pairs targeted to amplify 876 SSR loci and distributed in 16 coconut chromosomes were successfully designed and deposited in a public data repository for public access. The 237 SSR primer loci were novel and could generate new SSR markers. Among 20 selected and tested primers, 18 produced SSR markers, and 15 pairs yielded polymorphic markers when used to evaluate 25 coconut accessions. The analysis showed that the designed SSR primers were informative for evaluating coconut genetic diversity and phylogenetic analysis.

Determining the genetic and agronomic variations in lines from Samsun tobacco growing areas

Tobaccos grown in Samsun are known all over the world. Samsun tobaccos are grown using nonregistered populations traditionally maintained by local farmers. The present study was conducted to determine the genetic and agronomic variations in Samsun tobaccos. Fifty-eight lines were collected from Samsun in tobacco growing area. These lines were analyzed using eighteen SSR markers. Polymorphic information content (PIC) values of markers ranged between 0.0 and 0.702. Forty-two alleles were obtained from 18 SSR markers. Average number of alleles per SSR locus was 2.33. Forty-eight lines were different for at least one SSR locus, indicating a high level of variation. The forty-eight lines were evaluated in two field trials along with local varieties Nail and Canik for agronomic characteristics such as plant height, number of leaves, leaf width, leaf length, leaf yield, grade index, leaf sugar and nicotine contents. High level of variation was also evident for agronomic characters. Lines No: 2, 11, 13, 21, 28 and 41 were notable for their superior agronomic characteristics. Some of these lines could be directly registered as new cultivars, but they should be further evaluated in future field trials in multiple environments. These lines could also be used as parents for the development of new cultivars.

Gene flow between wild trees and cultivated varieties shapes the genetic structure of sweet chestnut (Castanea sativa Mill.) populations

Gene flow between cultivated and wild gene pools is common in the contact zone between agricultural lands and natural habitats and can be used to study the development of adaptations and selection of novel varieties. This is likely the case in the northern Adriatic region, where centuries-old cultivated orchards of sweet chestnut (Castanea sativa Mill.) are planted within the natural distribution area of the species. Thus, we investigated the population structure of several orchards of sweet chestnuts. Furthermore, the genetic background of three toponymous clonal varieties was explored. Six genomic simple sequence repeat (gSSR) and nine EST-derived SSR (EST-SSR) loci were utilized in this research, and both grafted and non-grafted individuals were included in this study. Five closely related clones were identified, which represent a singular, polyclonal marron variety, found in all three cultivation areas. Furthermore, many hybrids, a result of breeding between cultivated and wild chestnuts, have been found. Analyzed semi-wild orchards defined by a diverse genetic structure, represent a hotspot for further selection and could result in creation of locally adapted, high-yielding varieties.

Changes in the Genetic Structure of Lithuania's Wild Boar (Sus scrofa) Population Following the Outbreak of African Swine Fever.

The emergence of African swine fever (ASF) in Lithuania and its subsequent persistence has led to a decline in the population of wild boar (Sus scrofa). ASF has been spreading in Lithuania since its introduction, therefore it is important to understand any genetic impact of ASF outbreaks on wild boar populations. The aim of this study was to assess how the propensity for an outbreak has shaped genetic variation in the wild boar population. A total of 491 wild boar samples were collected and genotyped using 16 STR markers. Allele richness varied between 15 and 51, and all SSR loci revealed a significant deviation from the Hardy–Weinberg equilibrium. Fixation indices indicated a significant reduction in heterozygosity within and between subpopulations. PCoA and STRUCTURE analysis demonstrated genetic differences between the western region which had had no outbreaks (restricted zone I) and the region with ASF infection (restricted zones II and III). It is concluded that environmental factors may play a particular role in shaping the regional gene flow and influence the genetic structure of the wild boar population in the region with ASF outbreaks.

Genetic Map Construction, QTL Mapping, and Candidate Genes Screening of Grain Size Traits in Common Buckwheat (Fagopyrum esculentum M.)

Common buckwheat (Fagopyrum esculentum M.) is an important pseudo-cereal crop and contains an abundance of nutrients and bioactive compounds. However, the yield of buckwheat is low compared to that of other major crops. QTL mapping and candidate gene screening of grain size are very important for increasing production in common buckwheat through molecular breeding in the future. In the present study, an F1 segregating population with 217 individuals was established using a cross between Ukraine daliqiao (UD) and Youqiao 2 (YQ2) that showed a significant difference in grain size. The InDel and SSR primers were developed based on transcriptome sequencing between parents in the previous study. We constructed a genetic linkage map, including 39 SSR loci and 93 InDel loci, with a total length of 1398.33 cM and an average spacing of 10.59 cM. Combined with the grain size phenotype data of the F1 population, a total of 14 QTL were detected, including 6-grain length QTL, 3-grain width QTL, and 5 hundred-grain-weight QTL. QTL of grain width and hundred-grain weight were all detected near SWU_Fe_InDel086 and SWU_Fe_InDel076. Some putative candidate genes with the ex1pression or InDel difference between parents were screened within the QTL interval. This study would lay the foundation for map-based cloning and molecular mechanism of grain size and ultimately improvement of yield in common buckwheat.

Molecular characterization and genetic diversity analysis in Indian mustard (Brassica juncea L. Czern & Coss.) varieties using SSR markers.

In this study, we evaluated genetic diversity in a panel of 87 Indian mustard varieties using 200 genomic-SSR markers. A total of 189 SSRs resulted into positive amplification with 174 (92.06%) SSRs generating polymorphic products and 15 (7.94%) SSRs producing monomorphic amplicons. A total of 552 alleles were obtained and allele number varied from 2–6 with an average number of 3.17 alleles per SSR marker. The major allele frequency ranged from 0.29 (ENA23) to 0.92 (BrgMS841) with an average value of 0.58 per SSR locus. The polymorphic information content (PIC) value ranged from 0.10 (BrgMS841) to 0.68 (BrgMS519) with 0.39 as mean PIC value. The gene diversity per locus ranged from 0.13 (BrgMS841) to 0.72 (ENA23 & BrgMS519) with a mean value of 0.48 per SSR primer pair. Both Unweighted Neighbor Joining-based dendrogram and population structure analysis divided all the 87 varieties into two major groups/subpopulations. Analysis of molecular variance (AMOVA) inferred the presence of more genetic variation (98%) among individuals than among groups (2%). A total of 31 SSRs produced 36 unique alleles for 27 varieties which will serve as unique DNA-fingerprints for the identification and legal protection of these varieties. Further, the results obtained provided a deeper insight into the genetic structure of Indian mustard varieties in India and will assist in formulating future breeding strategies aimed at Indian mustard genetic improvement.

Comparative and Phylogenetic Analysis of Complete Chloroplast Genomes in Leymus (Triticodae, Poaceae).

Leymus is a perennial genus that belongs to the tribe Triticeae (Poaceae) which has an adaptive capacity to ecological conditions and strong resistance to cold, drought, and salinity. Most Leymus species are fine herbs that can be used for agriculture, conservation, and landscaping. Due to confusion taxonomy within genera, the complete chloroplast (cp) genome of 13 Leymus species was sequenced, assembled, and compared with those of three other previously published Leymus species (Leymus condensatus, Leymus angustus, and Leymus mollis) to clarify the issue. Overall, the whole cp genome size ranged between 135,057 (L. condensatus) and 136,906 bp (Leymus coreanus) and showed a typical quadripartite structure. All studied species had 129 genes, including 83 protein-coding genes, 38 transfer RNAs, and 8 ribosomal RNAs. In total, 800 tandem repeats and 707 SSR loci were detected, most of which were distributed in the large single-copy region, followed by the inverted repeat (IR) and small single-copy regions. The sequence identity of all sequences was highly similar, especially concerning the protein-coding and IR regions; in particular, the protein-coding regions were significantly similar to those in the IR regions, regardless of small sequence differences in the whole cp genome. Moreover, the coding regions were more conserved than the non-coding regions. Comparisons of the IR boundaries showed that IR contraction and expansion events were reflected in different locations of rpl22, rps19, ndhH, and psbA genes. The close phylogenetic relationship of Leymus and Psathyrostachys indicated that Psathyrostachys possibly is the donor of the Ns genome sequence identified in Leymus. Altogether, the complete cp genome sequence of Leymus will lay a solid foundation for future population genetics and phylogeography studies, as well as for the analysis of the evolution of economically valuable plants.

Comparative analysis of chloroplast genomes of three medicinal Carpesium species: Genome structures and phylogenetic relationships.

Carpesium (Asteraceae) is a genus that contains many plant species with important medicinal values. However, the lack of chloroplast genome research of this genus has greatly hindered the study of its molecular evolution and phylogenetic relationship. This study used the Illumina sequencing platform to sequence three medicinal plants of the Carpesium genus: Carpesium abrotanoides, Carpesium cernuum, and Carpesium faberi, obtaining three complete chloroplast genome sequences after assembly and annotation. It was revealed that the three chloroplast genomes were typical quadripartite structures with lengths of 151,389 bp (C. abrotanoides), 151,278 bp (C. cernuum), and 151,250 bp (C. faberi), respectively. A total of 114 different genes were annotated, including 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Abundant SSR loci were detected in all three chloroplast genomes, with most composed of A/T. The expansion and contraction of the IR region indicate that the boundary regions of IR/SC are relatively conserved for the three species. Using C. abrotanoides as a reference, most of the non-coding regions of the chloroplast genomes were significantly different among the three species. Five different mutation hot spots (trnC-GCA-petN, psaI, petA-psbJ, ndhF, ycf1) with high nucleotide variability (Pi) can serve as potential DNA barcodes of Carpesium species. Additionally, phylogenetic evolution analysis of the three species suggests that C. cernuum has a closer genetic relationship to C. faberi than C. abrotanoides. Simultaneously, Carpesium is a monophyletic group closely related to the genus Inula. Complete chloroplast genomes of Carpesium species can help study the evolutionary and phylogenetic relationships and are expected to provide genetic marker assistance to identify Carpesium species.

First report of the NA2 clonal lineage of the sudden oak death pathogen, Phytophthora ramorum, infecting tanoak in Oregon forests.

Phytophthora ramorum Werres, de Cock & Man in't Veld, causal agent of sudden oak death (SOD) and ramorum leaf blight, is comprised of four clonal lineages in its invasive ranges of North America and Europe (Grünwald et al. 2012, Van Poucke et al. 2012). Of these, three - the NA1, NA2, and EU1 lineages - are found in U.S. nurseries, but only two, the NA1 and EU1 lineages, have been found infecting trees in North American forests (Grünwald et al. 2012, 2016). In the spring of 2021, tanoak (Notholithocarpus densiflorus Manos, Cannon & Oh) displaying symptoms consistent with SOD were detected north of Port Orford (Curry County, Oregon). Symptoms were canopy dieback and blackened petiole and stem lesions on tanoak sprouts. The pathogen isolated on PAR (CMA plus 200 ml/L ampicillin, 10 mg/L rifamycin, 66.7 mg/L PCNB) selective media was determined to be P. ramorum based on characteristic morphology of hyphae, sporangia, and chlamydospores (Werres et al. 2001). Positive identification as P. ramorum was obtained with a lineage-specific LAMP assay targeting an NA2 orphan gene, indicating the presence of the NA2 lineage. NA2 was confirmed by sequencing a portion of the cellulose binding elicitor lectin (CBEL) gene using CBEL5U and CBEL6L primers (Gagnon et al. 2014). Sequences (GenBank accessions MZ733981 and MZ733982) were aligned against reference sequences for all lineages (Gagnon et al. 2014) confirming the presence of NA2. Lineage determination as NA2 was further confirmed at eleven SSR loci (ILVOPrMS145, PrMS39, PrMS9C3, ILVOPrMS79, KI18, KI64, PrMS45, PrMS6, ILVOPrMS131, KI82ab, and PrMS43) using the methods of Kamvar et al. (2015). We completed Koch's postulates using potted tanoaks, wound-inoculated at the midpoint of 1-year old stems with either hyphal plugs or non-colonized agar (n=4 per treatment). Tanoaks were maintained in a growth chamber (20°C-day / 18°C-night temperatures) with regular watering and an 18-photoperiod using F32T8 fluorescent bulbs (Phillips, Eindhoven, The Netherlands). After 7 days, brown to black lesions 1.2 to 2.9 cm in length were observed on the inoculated stems, from which P. ramorum was subsequently re-isolated; no symptoms were observed on the controls, and no pathogens were recovered when plating the wound sites in PAR. This is the first detection of the NA2 lineage causing disease in forests worldwide. The outbreak was found on private and public lands in forests typical to the SOD outbreak in Oregon (mixed conifer and tanoak), and was 33 km north of the closest known P. ramorum infestation. Follow-up ground surveys on adjacent lands have identified over 100 P. ramorum-positive tanoak trees, from which additional NA2 isolates have been recovered from bole cankers. NA2 is thought to be more aggressive than the NA1 lineage (Elliott et al. 2011), which has been present in Curry County since the mid-1990s (Goheen et al. 2017). Eradication of the NA2 lineage is being pursued to slow its further spread and prevent overlap with existing NA1 and EU1 populations. The repeated introductions of novel lineages into the western United States native plant communities highlights the vulnerability of this region to Phytophthora establishment, justifying continued monitoring for P. ramorum in nurseries and forests. References • Elliott, M, et al. 2011. For. Path. 41:7. https://doi.org/10.1111/j.1439-0329.2009.00627.x • Gagnon, M.-C., et al. 2014. Can. J. Plant Pathol. 36:367. https://doi.org/10.1080/07060661.2014.924999 • Goheen, E.M., et al. 2017. For. Phytophthoras 7:45. https://doi: 10.5399/osu/fp.7.1.4030 • Grünwald, N. J., et al. 2012. Trends Microbiol. 20:131. https://doi.org/10.1016/j.tim.2011.12.006 • Grünwald, N. J., et al. 2016. Plant Dis. 100:1024. https://doi.org/10.1094/PDIS-10-15-1169-PDN • Kamvar, Z.N. et al. 2015. Phytopath. 105:982. https://doi.org/10.1094/PHYTO-12-14-0350-FI • Van Poucke, K., et al. 2012. Fungal Biol. 116:1178. https://doi.org/10.1016/j.funbio.2012.09.003 • Werres, S., et al. 2001. Mycol. Res. 105: 1155. https://doi.org/10.1016/S0953-7562(08)61986-3.

Full-length transcriptome sequencing analysis and characterization, development and validation of microsatellite markers in Kengyilia melanthera.

As a typical psammophyte of the Triticeae, Kengyilia melanthera possesses high feeding potential and great utilization values in desertification control in the Qinghai-Tibet Plateau. However, few gene function and genetic studies have been performed in K. melanthera. In this study, single-molecule real-time sequencing technology was used to obtain the full-length transcriptome sequence of K. melanthera, following the functional annotation of transcripts and prediction of coding sequences (CDSs), transcription factors (TFs), and long noncoding RNA (lncRNA) sequences. Meanwhile, a total of 42,433 SSR loci were detected, with 5′-UTRs having the most SSR loci and trinucleotide being the most abundant type. In total, 108,399 SSR markers were designed, and 300 SSR markers were randomly selected for diversity verification of K. melanthera. A total of 49 polymorphic SSR markers were used to construct the genetic relationships of 56 K. melanthera accessions, among which 21 SSR markers showed good cross-species transferability among the related species. In conclusion, the full-length transcriptome sequence of the K. melanthera will assist gene prediction and promote molecular biology and genomics research, and the polymorphic SSR markers will promote molecular-assisted breeding and related research of K. melanthera and its relatives.

High genetic diversity and low population differentiation of a medical plant Ficus hirta Vahl., uncovered by microsatellite loci: implications for conservation and breeding

BackgroundWuzhimaotao (Radix Fici Hirtae) originates from the dry root of Ficushirta (Moraceae), which is widely known as a medical and edible plant distributed in South China. As the increasing demand for Wuzhimaotao, the wild F.hirta has been extremely reduced during the past years. It is urgent to protect and rationally develop the wild resources of F.hirta for its sustainable utilization. However, a lack of genetic background of F.hirta makes it difficult to plan conservation and breeding strategies for this medical plant. In the present study, a total of 414 accessions of F.hirta from 7 provinces in southern China were evaluated for the population genetics using 9 polymorphic SSR markers.ResultsA mean of 17.1 alleles per locus was observed. The expected heterozygosity (He) varied from 0.142 to 0.861 (mean = 0.706) in nine SSR loci. High genetic diversity (He = 0.706, ranged from 0.613 to 0.755) and low genetic differentiation among populations (G’ST = 0.147) were revealed at population level. In addition, analysis of molecular variance (AMOVA) indicated that the principal molecular variance existed within populations (96.2%) was significantly higher than that among populations (3.8%). Meanwhile, the three kinds of clustering methods analysis (STRUCTURE, PCoA and UPGMA) suggested that the sampled populations were clustered into two main genetic groups (K = 2). Mantel test showed a significant correlation between geographic and genetic distance among populations (R2 = 0.281, P < 0.001). Pollen flow, seed flow and/or geographical barriers might be the main factors that formed the current genetic patterns of F.hirta populations.ConclusionsThis is a comprehensive study of genetic diversity and population structure of F.hirta in southern China. We revealed the high genetic diversity and low population differentiation in this medicinal plant and clarified the causes of its current genetic patterns. Our study will provide novel insights into the exploitation and conservation strategies for F.hirta.

Assessing genetic structure of Eugenia uniflora L. populations along an environmental gradient using a novel set of SSR markers

Gradients in biodiversity are often considered a result of environmental variables like temperature, precipitation, ecological disturbance regimes, and species coexistence. This study aimed at investigating the genetic diversity and structure of natural populations of Eugenia uniflora growing in two different biomes in southern Brazil (Atlantic Forest and Pampa biomes), as well as in the transition zone between them. A novel set of 11 SSR loci from E. uniflora was validated and employed to test the hypothesis that the allelic composition of the populations changes gradually along the environmental gradient sampled, with a characteristic genetic structure within each biome and an intermediary allelic composition in the transition zone. The results revealed high polymorphism of the validated markers and significant genetic structure of the different populations. The main source of genetic variation observed is the individual samples, but there was a considerable amount of variation among populations, and regions. We suggested that the genetic structure of the studied populations presents distinct patterns which may be related to adaptation to local environmental conditions, or at least related to the transition among them. Our study provides evidence that even environments neglected regarding the information on their biodiversity and the transition zones may hold important levels of genetic diversity. Thus, the distribution of genetic diversity should be interpreted in the light of the life traits of the species and the local environment since valuable diversity may be found both in a hotspot of diversity and in neglected forest formations.

The Diversity of Melia azedarach L. from China Based on Transcriptome-Developed SSR Marker

Melia azedarach L. is a native tree species that can be used in a comprehensive way and is widely distributed in all provinces south of the Yellow River in China. Genetic diversity analysis of different M. azedarach germplasm sources is an important basic work for the selection, evaluation, and genetic improvement of M. azedarach germplasm resources. In this study, 100 pairs of SSR primers were designed and synthesized based on M. azedarach transcriptome data, and 16 pairs of reliable SSR primers were finally selected. The developed primers were used to analyze the genetic diversity of M. azedarach from 15 sources in 10 provinces in East, Central, and South China. The results showed that the frequency of the M. azedarach transcriptome SSR loci was high, and the distribution density was high. There were 15 sources of M. azedarach genetic diversity at a moderate level, and genetic variation was mainly present within the sources. The present study further enriches the existing SSR marker database of the M. azedarach family and can provide a reference for genetic diversity analysis and molecularly assisted breeding of M. azedarach plants at the genomic level.

Comparative chloroplast genomes and phylogenetic analyses of Pinellia

BackgroundPinellia Tenore (Araceae) is a genus of perennial herbaceous plants, all of which have medicinal value. The chloroplast (cp) genome data of Pinellia are scarce, and the phylogenetic relationship and gene evolution remain unclear.Methods and resultsWe sequenced and annotated the Pinellia pedatisecta cp genome and combined it with previously published genomes for other Pinellia species. We used bioinformatics methods to analyse the genomic structure, repetitive sequences, interspecific variation, divergence hotspots, phylogenetic relationships, divergence time estimation and selective pressure of four Pinellia plastomes. Results showed that the cp genomes of Pinellia varied in length between 168,178 (P. pedatisecta MN046890) and 164,013 bp (P. ternata KR270823). A total of 68–111 SSR loci were identified as candidate molecular markers for further genetic diversity study. Eight mutational hotspot regions were determined, including psbI-trnG-UCC, psbM-rpoB, ndhJ-trnT-UGU, trnP-UGG-trnW-CCA, ndhF-trnN-GUU, ndhG-ndhE, ycf1-rps15 and trnR-ycf1. Gene selection pressure suggested that four genes were subjected to positive selection. Phylogenetic inferences based on the complete cp genomes revealed a sister relationship between Pinellia and Arisaema plants whose divergence was estimated to occur around 22.48 million years ago. All Pinellia species formed a monophyletic evolutionary clade in which P. peltata, rather than P. pedatisecta, earlier diverged, indicating that P. pedatisecta is not the basal taxon of Pinellia but P. peltata may be.ConclusionsThe cp genomes of Pinellia will provide valuable information for species classification, identification, molecular breeding and evolutionary exploration of the genus Pinellia.

Comparative Analysis of the Complete Chloroplast Genomes of Eight Ficus Species and Insights into the Phylogenetic Relationships of Ficus.

The genus Ficus is an evergreen plant, the most numerous species in the family Moraceae, and is often used as a food and pharmacy source. The phylogenetic relationships of the genus Ficus have been debated for many years due to the overlapping phenotypic characters and morphological similarities between the genera. In this study, the eight Ficus species (Ficus altissima, Ficus auriculata, Ficus benjamina, Ficus curtipes, Ficus heteromorpha, Ficus lyrata, Ficus microcarpa, and Ficus virens) complete chloroplast (cp) genomes were successfully sequenced and phylogenetic analyses were made with other Ficus species. The result showed that the eight Ficus cp genomes ranged from 160,333 bp (F. heteromorpha) to 160,772 bp (F. curtipes), with a typical quadripartite structure. It was found that the eight Ficus cp genomes had similar genome structures, containing 127 unique genes. The cp genomes of the eight Ficus species contained 89–104 SSR loci, which were dominated by mono-nucleotides repeats. Moreover, we identified eight hypervariable regions (trnS-GCU_trnG-UCC, trnT-GGU_psbD, trnV-UAC_trnM-CAU, clpP_psbB, ndhF_trnL-UAG, trnL-UAG_ccsA, ndhD_psaC, and ycf1). Phylogenetic analyses have shown that the subgenus Ficus and subgenus Synoecia exhibit close affinities and based on the results, we prefer to merge the subgenus Synoecia into the subgenus Ficus. At the same time, new insights into the subgeneric classification of the Ficus macrophylla were provided. Overall, these results provide useful data for further studies on the molecular identification, phylogeny, species identification and population genetics of speciation in the Ficus genus.

Optimization of marker system for genotyping oil flax varieties from the collection of VNIIMK

Relevance. Currently, in Russia, oil flax (Linum usitatissimum L.) crop acreage is increasing, therefore, expansion of the range of varieties and rapid introduction of new, highly adaptive varieties into production are important tasks. Identification methods based on the use of genetic passports are useful both at the stage of studying parent material and when protecting breeders' copyrights. An optimal system for identification should cover evenly the entire genome. Therefore, the purpose of this work is the optimization of the existing marker system for genotyping oil flax varieties from the collection of All-Russian Research Institute of Oil Crops named after V.S. Pustovoit (VNIIMK) by increasing the number of the used polymorphic microsatellite loci. Materials and methods. 17 samples of oil flax from the VNIIMK collection were used as object of the research. Eight pairs of primers flanking the microsatellite loci were used as tools for the research. Localization of the studied primers in the reference genome of L. usitatissimum was determined using the web version of Primer-BLAST. DNA was extracted from two-week-old seedlings with CTAB buffer. The discriminative power of the marker system was determined using parameters such as polymorphic information content (PIC), frequency, the observed and effective number of alleles. Cluster analysis and graphical composition of dendrograms were carried out using Statistica 6.0 software package.Results. We determined the localization of the studied primers on seven chromosomes. Three pairs of primers were localized simultaneously on two chromosomes. Testing of the primers revealed 6 polymorphic loci. The number of observed alleles per locus ranged from 2 to 3 (average of 2.1 alleles per locus). The effective number of alleles ranged from 1.13 to 1.99 (average value —1.62), and the value of polymorphic information content (PIC) ranged from 0.111 to 0.498 (average value — 0.358). The extension of the set of polymorphic SSR loci allowed the differentiation of all used 17 genotypes.

Investigation of the genetic structure of some common bean (Phaseolus vulgaris L.) commercial varieties and genotypes used as a genitor with SSR and SNP markers

Common bean is a species belonging to the Phaseolus genus of the Leguminosae family. It has economic importance due to being rich in protein, vitamin A and C, and minerals. Being one of the most cultivated species of legumes, the determination of genetic diversity in bean genotypes or populations has an important role in terms of our genetic resources. The objective of this study was to evaluate the genetic structure of 94 genotypes which were cultivated in different parts of the world and our country with SSR and SNP markers. 10 SSR loci and 73 SNP primers were used for the determination of genetic structure in commercial cultivars and breeding lines. All of the SSR and SNP loci used in the study were found to be polymorphic. A total of 89 alleles were identified for 10 SSR loci. Mean number of alleles per locus (Na = 8.9), effective allele number (Ne = 3.731), Shannon information index (I = 1.468), observed heterozygosity (Ho = 0.023), and expected heterozygosity (He = 0.654) were calculated based on SSR analysis. According to the results of Bayesian-based STRUCTURE analysis using SSR and SNP data, 94 bean genotypes were genetically divided into three main clusters. According to genetic distance based UPGMA dendrogram obtained from SNP analysis, 94 bean genotypes were divided into 2 main clusters corresponding Mesoamerican and Andean gene pools. The obtained results provide important information about the genetic structures of the studied bean cultivars and breeding lines. With the obtained results, it will be possible to develop breeding programs to develop new cultivars by using our gene resources.