EST-SSR Markers Research Articles

Genetic Diversity of Phyllanthus emblica From Two Different Climate Type Areas.

Phyllanthus emblica L. is a well-known medicinal and edible plant species. Various medicinal compounds in the fruit make it an important medicinal and promising economic material. The plant is widely distributed in Southwestern and Southern China. However, due to massive deforestation and land reclamation as well as deterioration of its natural habitat in recent years, the wild resources of this species have been sharply reduced, and it is rare to see large-scale wild P. emblica forests so far. In order to effectively protect and rationally utilize this species, we investigated the genetic diversity, genetic structure, and population dynamics of 260 individuals from 10 populations of P. emblica sampled from the dry climate area in Yunnan and wet climate area in Guangxi using 20 polymorphic EST-SSR markers. We found high genetic diversity at the species level (He = 0.796) and within populations (He = 0.792), but low genetic differentiation among populations (FST = 0.084). In addition, most genetic variation existed within populations (92.44%) compared with variation among the populations (7.56%). Meanwhile, the NJ tree, STRUCTURE, and hierarchical analysis suggested that the sampled individuals were clustered into two distinct genetic groups. In contrast, the genetic diversity of the dry climate group (He = 0.786, Na = 11.790, I = 1.962) was higher than that of the wet climate group (He = 0.673, Na = 9.060, I = 1.555), which might be attributed to the combined effects of altitude, precipitation, and geographic distance. Interestingly, only altitude and precipitation had significant pure effects on the genetic diversity, and the former was slightly stronger. In addition, DIYABC analysis suggested the effective population size of P. emblica might have contracted in the beginning of the Last Glacial Maximum. These genetic features provided vital information for the conservation and sustainable development of genetic resources of P. emblica, and they also provided new insights and guidelines for ecological restoration and economic development in dry-hot valleys of Yunnan and karst areas in Guangxi.

Development of EST-SSR markers for genetic diversity analysis in coconut (Cocos nucifera L.).

Genetic improvement in coconut relies on exploiting the vast existing diversity among coconut accessions. Robust molecular markers are a pre-requisite for efficient characterization of genetic diversity. Microsatellites or simple sequence repeats (SSRs), mined from expressed sequence tags (ESTs), constitute an important resource for analysis of genetic diversity as they are abundant, polymorphic and represent function regions of the genome. We have identified a total of 318,528 putative EST-SSRs from 130,942 unigenes utilizing a leaf transcriptome dataset of coconut. Among the EST-SSRs, dinucleotide repeats were abundant (219,912; 69.04%) followed by trinucleotide (70,722; 22.2%) and tetra-nucleotide repeats (6281; 1.9%). Among the dinucleotide repeat motifs, the dominant repeat was AG/CT (35.87%), followed by AT/AT (18.59%), while the dominant trinucleotide repeat was AAG/CTT (4.59%). One hundred and twenty EST-SSR primer pairs were designed and utilized to amplify six DNA samples of coconut accessions. Fifty primers (41.7%) produced reproducible polymorphic fragments of expected sizes, from which a total of 10 primers were selected for the diversity assessment in 186 palms of 50 coconut accessions, comprising of 25 each of tall and dwarf accessions. A total of 137 alleles were detected with an average of 13.7 alleles per SSR locus. The number of alleles observed at each locus in the data set ranged from 7 to 22. All the loci showed 100% polymorphism with respect to the samples screened. The average observed heterozygosity was 0.46. The PIC values ranged from 0.79 (CnKGDEST129 and CnKGDEST100) to 0.91 (CnKGDEST117 andCnKGDEST122) with a mean value of 0.85, indicating the capacity of the EST-SSR markers to detect high levels of polymorphism. The cluster analysis revealed that accessions were generally clustered based on their relative similarity and irrespective of their geographic origins. The present study demonstrates the usefulness of transcriptome sequencing as a rapid and cost-effective methodology for the development of molecular markers. The EST-SSR markers generated through this study constitute useful and reliable tools for assessment of genetic diversity and marker-assisted selection in coconut.

Characterization, validation, and cross-species transferability of EST-SSR markers developed from Lycoris aurea and their application in genetic evaluation of Lycoris species

BackgroundThe Lycoris genus 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 of L. 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 among Lycoris species were assessed in L. radiata Hreb, L. sprengeri Comes ex Baker, L. chinensis Traub and L. anhuiensis, of which between 38 to 77% of the primers were able to amplify products in these Lycoris species. Furthermore, 20 and 10 amplification products were selected for sequencing verification in L. aurea and L. radiata respectively. 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 seven L. radiata lines. 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. aurea was separated from L. radiata lines and that the seven L. radiata lines were clustered into two groups, consistent with their fertility. Interestingly, even a dendrogram with 34 individuals representing the seven L. radiata lines was almost consistent with fertility.ConclusionsThis study supplies a pool of potential 839 non-redundant SSR markers for genetic analysis of Lycoris genus, that present high amplification rate, transferability and efficiency, which will facilitate genetic analysis and breeding program in Lycoris.

The influence of breeding history, origin and growth type on population structure of barley as revealed by SSR markers

Natural and mass selection during domestication and cultivation favored particular traits of interest in barley. In the present study, population structure, and genetic relationships among 144 accessions of barley landraces and breeding materials from various countries were studied using a set of 77 and 72 EST-SSR and gSSR markers, respectively distributed on seven chromosomes of barley. In total, 262 and 429 alleles were amplified in 77 EST-SSRs and 72 gSSR loci, respectively. Out of which, 185 private/group-specific alleles were identified in the landraces compared with 14 in "cultivar and advanced breeding lines", indicating the possibility to introgress favorite alleles from landraces into breeding materials. Comparative analysis of genetic variation among breeding materials, Iranian landraces, and exotic landraces revealed higher genetic diversity in Iranian landraces compared with others. A total of 37, 15, and 14 private/group-specific alleles were identified in Iranian landraces, exotic landraces, and breeding materials, respectively. The most likely groups for 144 barley genotypes were three as inferred using model- and distance-based clustering as well as principal coordinate analysis which assigned the landraces and breeding materials into separate groups. The distribution of alleles was found to be correlated with population structure, domestication history and eco-geographical factors. The high allelic richness in the studied set of barley genotype provides insights into the available diversity and allows the construction of core groups based on maximizing allelic diversity for use in barley breeding programs.

Development of EST-SSR markers for Pongamia pinnata by transcriptome database mining: cross-species amplification and genetic diversity.

EST-SSR markers were developed from Pongamia pinnata transcriptome libraries. We have successfully utilised EST-SSRs to study the genetic diversity of Indian P. pinnata germplasms and transferability study on legume plants. P. pinnata is a non-edible oil, seed-bearing leguminous tree well known for its multipurpose benefits and acts as a potential source for medicine and biodiesel preparation. Moreover, the plant is not grazable by animal and wildly grown in different agro climatic condition of India. Recently, it is much used in reforestation and rehabilitation of marginal and coal mined land in different part of India. Due to increasing demand for cultivation, understanding of the genetic diversity is important parameter for further breeding and cultivation program. In this investigation, an attempt has been undertaken to develop novel EST-SSR markers by analyzing the assembled transcriptome from previously published Illumina libraries of P. pinnata, which is cross transferrable to legume plants. Twenty EST-SSR markers were developed from oil yielding and secondary metabolite biosynthesis genes. To our knowledge, this is the first EST-SSR marker based genetic diversity study on Indian P. pinnata germplasms. The genetic diversity parameter analysis of P. pinnata showed that the Gangetic plain and Eastern India are highly diverse compared to the Central Deccan and Western germplasms. The lowest genetic diversity in the Western region may be due to the pressure of lower precipitation, high-temperature stress and reduced groundwater availability. Nevertheless, the highest genetic diversity of Gangetic plain and Eastern India may be due to the higher groundwater availability, high precipitation, higher temperature fluctuations and growing by the side of glacier-fed river water. Thus, our study shows the evidence of natural selection on the genetic diversity of P. pinnata germplasms of the Indian subcontinent.

Functional description and development of polymorphic EST-SSR markers in bread wheat and their gene interactions network

The tolerance of abiotic stress is considered to be one of the key features of crop breeding under climate changes. For any crop improvement program, the selection of suitable gene pools among the germplasms is first needed. Drought and salinity traits are the most difficult ones to monitor for being polygenic. Bread wheat (Triticum aestivum) is the most commonly grown cereal grain. To predict new EST-SSR markers, 6717 expressed sequence tags (ESTs) expressed in drought and salinity stress conditions were collected from the National Center for Biotechnology Information (NCBI) then clustered and assembled. Simple sequence repeats (SSRs) have been researched from all unigenes, and then characterization and functional annotation (GO, KEGG, enzymes, TFs, and conserved motifs) for SSR-containing unigenes have been achieved. Fifty primer pairs were selected and synthesized. A polymorphism comparison between the in-silico and real PCR results was conducted. High drought and salinity-tolerant genotypes were grouped in cluster B. Interaction networks between the predicted genes were explored. Out of the polymorphic primer pairs, two primers were identified as unique primers, which may be more related to drought and salinity tolerance and could be further used in quantitative trait locus (QTLs) mapping and marker-assisted selection (MAS). The transferability of the EST-SSR markers for related wheat species ranged between 90% in some Triticum L. species and 50% in Hordeum vulgare L. In addition, some SSR-containing unigenes were highly conserved with the Zea mays genome.

Full-length transcriptome sequencing analysis and development of EST-SSR markers for the endangered species Populus wulianensis

Populus wulianensis is an endangered species endemic to Shandong Province, China. Despite the economic and ornamental value of this species, few genomics and genetic studies have been performed. In this study, we performed a relevant analysis of the full-length transcriptome sequencing data of P. wulianensis and obtained expressed sequence tag (EST)-simple sequence repeat (SSR) markers with polymorphisms that can be used for further genetic research. In total, 8.18 Gb (3,521,665) clean reads with an average GC content of 42.12% were obtained. From the corrected 64,737 high-quality isoforms, 42,323 transcript sequences were obtained after redundancy analysis with CD-HIT. Among these transcript sequences, 41,876 sequences were annotated successfully. A total of 23,539 potential EST-SSRs were identified from 16,057 sequences. Excluding mononucleotides, the most abundant motifs were trinucleotide SSRs (47.80%), followed by di- (46.80%), tetra- (2.98%), hexa- (1.58%) and pentanucleotide SSRs (0.84%). Among the 100 designed EST-SSRs, 18 were polymorphic with high PIC values (0.721 and 0.683) and could be used for analyses of the genetic diversity and population structure of P. wulianensis. These full-length transcriptome sequencing data will facilitate gene discovery and functional genomics research in P. wulianensis, and the novel EST-SSRs developed in our study will promote molecular-assisted breeding, genetic diversity and conservation biology research in this species.

Molecular markers in the genetic analysis of crossability of bread wheat with rye

Мягкая пшеница (Triticum aestivum L.), сорта которой широко используются в мировом про- изводстве зерна, плохо скрещивается с видами других родов Triticeae Dum., что ограничивает возмож- ности введения чужеродного генетического материала в ее генофонд и создания новых сортов, хорошо адаптированных к различным неблагоприятным абиотическим и биотическим факторам внешней среды. Известно, что скрещиваемость мягкой пшеницы с представителями других родов контролируется генами Kr1–Kr4 (Crossability with Rye, Hordeum and Aegilops spp.) и геном SKr (Suppressor of crossability). Из названных генов наиболее сильное влияние на признак оказывают SKr и Kr1. В рецессивном состоянии, когда гены не функционируют, может завязываться более 50 % зерновок от числа цветков в колосе при опылении пыль- цой чужеродного вида. Оба гена локализованы в хромосоме 5B. Расположение гена SKr в коротком плече хромосомы 5B ограничено маркерами GBR0233 и Xgwm234 в тесном сцеплении с маркерами Xcfb341, TGlc2 и gene12. Ген Kr1 расположен в длинном плече хромосомы 5B, проксимальнее гена Ph1, между EST-SSR- маркерами Xw5145 и Xw9340. Маркеры, разработанные для гена SKr, применяли для контроля переноса его рецессивного аллеля skr в другие генотипы мягкой пшеницы, что позволило получать формы с высокой за- вязываемостью гибридных зерновок при скрещивании с рожью. Однако в целом использование маркеров генов SKr и Kr1 в практической маркер-ориентированной селекции и молекулярном скрининге образцов ex situ коллекций изучено недостаточно. Большие перспективы в этом плане открывает определение пол- ной нуклеотидной последовательности гена Kr1 у контрастных по скрещиваемости сортов мягкой пшени- цы, это дает возможность создания внутригенных аллель-специфичных маркеров. В представленном обзо- ре рассмотрены генетические ресурсы, созданные посредством гибридизации мягкой пшеницы с рожью; вопросы географического распространения легко скрещивающихся форм пшеницы и генетического кон- троля совместимости пшеницы и ржи; достижения в использовании молекулярных маркеров в картирова- нии Kr-генов и контроле их передачи.

Mining and Validation of Polymorphic EST-SSR Markers for Analysing Genetic Diversity among Interspecific Hybrids of Tea

Molecular markers are useful tools for studying gene flow and genetic diversity within a population. With the establishment of online databases, sequence can be now be downloaded for identification of Expressed Sequence Tags- Simple Sequence Repeat (SSR) markers designed to amplify the microsatellite loci present in the genes. Of the 170 microsatellites detected from the downloaded 789 ESTs belonging to the nine Camellia spp, only fourteen showed functional EST-SSR markers. Among them, five polymorphic markers specific to tri-nucleotide SSRs were randomly selected and screened alongside five established primers on four tea genotypes (TRFK 570/2, TRFK 688/1, TRFK 83/1, and TRFK 6/8,) The polymorphism information content (PIC) of the SSR markers had mean of 0.33 that ranged between 0.16 and 0.53, whereas discriminating power (D) had mean value of 0.23 ranging between 0.00 and 0.75 with. A total of 64 alleles were detected with an average of 6.4 alleles per SSR locus. Based on a PIC greater than or equal to 0.40, D value greater than or equal to 0.20 and number of polymorphic bands greater than or equal to 1, five functional SSR markers comprising two novel EST-SSRs (Camjap A1, Camjap A4) and three adapted microsatellite markers (TM 134, A37, and A47) were recommended for use in discriminating interspecific hybrids of tea.

Identification of novel QTLs for late leaf spot resistance and validation of a major rust QTL in peanut (Arachis hypogaea L.).

Co-occurrence of two devastating foliar-fungal diseases of peanut, viz., late leaf spot (LLS), and rust may cause heavy yield loss besides adversely affecting the quality of kernel and fodder. This study reports the mapping of seven novel stress-related candidate EST-SSRs in a region having major QTLs for LLS and rust diseases using an F2 mapping population (GJG17 × GPBD4) consisting of 328 individuals. The parental polymorphism using 1311 SSRs revealed 84 SSRs (6.4%) as polymorphic and of these 70 SSRs could be mapped on 14 linkage groups (LG). QTL analysis has identified a common QTL (LLSQTL1/RustQTL) for LLS and rust diseases in the map interval of 1.41cM on A03 chromosome, explaining 47.45% and 70.52% phenotypic variations, respectively. Another major QTL for LLS (LLSQTL1), explaining a 29.06% phenotypic variation was also found on LG_A03. A major rust QTL has been validated which was found harboring R-gene and resistance-related genes having a role in inducing hypersensitive response (HR). Further, 23 linked SSRs including seven novel EST-SSRs were also validated in 177 diverse Indian groundnut genotypes. Twelve genotypes resistant to both LLS and rust were found carrying the common (rust and LLS) QTL region, LLS QTL region, and surrounding regions. These identified and validated candidate EST-SSR markers would be of great use for the peanut breeding groups working for the improvement of foliar-fungal disease resistance.

Development of EST-SSR markers and association mapping with floral traits in Syringa oblata

BackgroundLilac (Syringa oblata) is an important woody plant with high ornamental value. However, very limited genetic marker resources are currently available, and little is known about the genetic architecture of important ornamental traits for S. oblata, which is hindering its genetic studies. Therefore, it is of great significance to develop effective molecular markers and understand the genetic architecture of complex floral traits for the genetic research of S. oblata.ResultsIn this study, a total of 10,988 SSRs were obtained from 9864 unigene sequences with an average of one SSR per 8.13 kb, of which di-nucleotide repeats were the dominant type (32.86%, 3611). A set of 2042 primer pairs were validated, out of which 932 (45.7%) exhibited successful amplifications, and 248 (12.1%) were polymorphic in eight S. oblata individuals. In addition, 30 polymorphic EST-SSR markers were further used to assess the genetic diversity and the population structure of 192 cultivated S. oblata individuals. Two hundred thirty-four alleles were detected, and the PIC values ranged from 0.23 to 0.88 with an average of 0.51, indicating a high level of genetic diversity within this cultivated population. The analysis of population structure showed two major subgroups in the association population. Finally, 20 significant associations were identified involving 17 markers with nine floral traits using the mixed linear model. Moreover, marker SO104, SO695 and SO790 had significant relationship with more than one trait.ConclusionThe results showed newly developed markers were valuable resource and provided powerful tools for genetic breeding of lilac. Beyond that, our study could serve an efficient foundation for further facilitate genetic improvement of floral traits for lilac.

Primer development and functional classification of EST-SSR markers in Ulmus species

At present, the available SSR markers of elm (Ulmus) have been seriously deficient, which can not meet the needs of molecular marker-assisted breeding and evaluation of germplasm resources. In this study, transcriptome data of Ulmus pumila were used as materials to develop EST-SSR markers of elms. EST-SSR markers were also classified according to gene function for the first time. In this study, 8288 perfect and 569 compound SSR loci were detected from 17,624 EST sequences (36,609,384 bp). Most of the perfect SSR sequences were based on short sequence lengths of 10–22 bp. The most common SSR repeat units were A/T (3330, 40.18%), AG/CT (1211, 14.61%), and AAG/CTT (568, 6.85%). All EST-SSR loci were function classified based on the Cluster of Orthologous Groups (COG) database. There were substantial differences in the type and proportion of SSR repeat units among the functional classes. Most EST-SSR loci were located in gene-coding regions. Ninety pairs were randomly selected and tested for validation using polymerase chain reaction amplification. Forty-nine primers were verified with clear bands. The number of alleles per locus (Na) ranged from 2 to 6; the number of effective alleles (Ne) ranged from 1.061 to 3.261; the observational heterozygosity (Ho) and expected heterozygosity (He) ranged from 0 to 0.968 and 0.112 to 0.651, respectively. The clustering pattern is consistent with traditional taxonomy that provides a molecular basis for the classification of elm. There were differences in the effective amplification and percentage of polymorphic loci among different functional SSR loci. The analysis of 880 primer combinations showed that markers within a single COG group were less effective than across groups. This study clarified the importance of SSR primer selection for clustering results. This study developed a large number of SSR markers in elm, providing effective markers for molecular marker-assisted breeding, genetic mapping, and other related research.

Characterization of new microsatellite markers from sugarcane (Saccharum officinarum) transcriptome

Microsatellites, or simple sequence repeats (SSR), developed by expressed sequence tag (EST) databases is an economical and efficient tools that can be used to perform genetic investigations at a functional level. Here, a new sugarcane database of transcriptome from our variety, GT35, was examined for the presence of SSRs. To test the utility of EST-derived SSR markers, a total of 51 new EST-SSRs were identified for possible use as potential genetic markers from no redundant SSR-positive ESTs, which were unmapped with the sequences available in the NCBI’EST database of sugarcane by BLASTN. Polymorphisms of the identified 51 EST-SSR markers were evaluated in 21 sugarcane genotypes, planted and collected in 2013 at Sugarcane Research Institute of Guangxi Academy of Agricultural Sciences (SRI-GXAAS) in China. High polymorphisms were detected in terms of number of alleles ranging from 5-36 with an average of 16.8 per locus and polymorphism information content values ranging from 0.74 to 0.95 with a mean of 0.92. Average transferability to Erianthus arundinaceus and Narenga porphyrocoma was 23.9% and 34.4%, respectively. The ability to establish genetic relationship was analyzed by cluster analysis, the result of which revealed that the major grouping was in accordance with taxonomical classification. The development of new EST-SSR markers presented in this work will have important implications for genetic analysis and breeding.

Genetic Diversity and Structure of Japanese Endemic Genus Thujopsis (Cupressaceae) Using EST-SSR Markers

The genus Thujopsis (Cupressaceae) comprises monoecious coniferous trees endemic to Japan. This genus includes two varieties: Thujopsis dolabrata (L.f.) Siebold et Zucc. var. dolabrata (southern variety, Td) and Thujopsis dolabrata (L.f.) Siebold et Zucc. var. hondae Makino (northern variety, Th). The aim of this study is to understand the phylogeographic and genetic population relationships of the genus Thujopsis for the conservation of genetic resources and future breeding. A total of 609 trees from 22 populations were sampled, including six populations from the Td distribution range and 16 populations from the Th distribution range. The genotyping results for 19 expressed sequence tag (EST)-based simple sequence repeat (SSR) markers, followed by a structure analysis, neighbor-joining tree creation, an analysis of molecular variance (AMOVA), and hierarchical F statistics, supported the existence of two genetic clusters related to the distribution regions of the Td and Th varieties. The two variants, Td and Th, could be defined by their provenance, in spite of the ambiguous morphological differences between the varieties. The distribution ranges of both variants, which have been defined from their morphology, was confirmed by genetic analysis. The Th populations exhibited relatively uniform genetic diversity, most likely because Th refugia in the glacial period were scattered throughout their current distribution area. On the other hand, there was a tendency for Td’s genetic diversity to decrease from central to southern Honshu island. Notably, the structure analysis and neighbor-joining tree suggest the hybridization of the two varieties in the contact zone. More detailed studies of the genetic structure of Td are required in future analyses.

Development of EST-SSR markers and their application in the genetic diversity of persimmon (Diospyros kaki Thunb.)

SSRs related to parent selection of D. kaki were validated by transcriptome sequencing, and genetic diversity of these markers among 76 persimmon germplasm resources was additionally investigated. The selection of hybrid parents is critical in cross-breeding plants for germplasm innovation and cultivating improved varieties. This study used a published RNA-Seq database of a persimmon cultivar ‘Zenjimaru’ to develop simple sequence repeat (SSR) markers to assist efficiently selection of appropriate persimmon cultivars for cross-breeding. A total of 154,741 unigenes with 41,999 SSR loci at a frequency of one per 2.30 kb were identified. Of the different SSR motif types, mononucleotide repeats (47.63%) were the most abundant, followed by dinucleotide repeats (38.23%). The main repeat motifs were A/T (42.26%) and AG/CT (24.72%). A total of 17,111 SSR primer pairs were designed, from which 100 selected loci were synthesized and preliminary screened with capillary electrophoresis by testing PCR products from 15 persimmon germplasm resources. The effectiveness of 13 primer pairs that produced products with distinct peaks at the predicted sizes was subsequently determined using 76 persimmon germplasm resources. A total of 111 alleles were identified by using 13 SSR markers, ranging from 2 to 17 with an average of 8.54 bands per locus. Additionally, different hybridized combinations could be selected according to a dendrogram based on these newly developed 13 SSR markers. And two androecious persimmons (“Yunjia-9” and “Luotian Yeshi-1”) were genetically closely related to Chinese pollination-constant non-astringent (C-PCNA) persimmons, which was significant for the cultivation of PCNA offspring. The identified SSR markers will not only promote persimmon cross-breeding programs by increasing the efficiency of parent selection but also assist in genetic resource identification and conservation.

Application of EST-SSR markers for analysis of polymorphism of lettuce varieties (Lactuca sativa L.) of domestic breeding

Purpose. Determination of molecular genetic polymorphism of lettuce cultivars of Ukrainian breeding by EST-SSR markers. Methods. Molecular genetic analysis, statistical methods. Results. The results of molecular genetic polymorphism study of 7 lettuce cultivars by 7 EST-SSR markers are presented. As a result of the analysis by studied markers, 37 alleles were detected with an average 5.29 alleles per locus. The KSL-92 marker, which identified 7 alleles, proved to be the most polymorphic (PIC – Polymorphism Information Content 0.98). The lowest PIC value (0.57) was noted for the KSL-37 marker by which 3 alleles were identified. For assessing the genetic diversity of lettuce cultivars by EST-SSR markers, genetic distances between cultivars were determined based on Jaccard’s similarity coefficient. It was determined that the most similar cultivars with genetic distances 0.17 were ‘Zorepad’ and ‘Malakhit’, ‘Malakhit’ and ‘Dublianskyi’, ‘Dublianskyi’ and ‘Smuhlianka’, ‘Krutianskyi’ and ‘Smuhlianka’. The most distant cultivar was ‘Skarb’ with a genetic distance 0.00 compared to other studied cultivars. According to the calculated genetic distances, the cultivars ‘Zorepad’ and ‘Malakhit’, ‘Malakhit’ and Dublianskyi’, which belong to the same variety Lactuca sativa L. var. secalina have a strong genetic similarity. The cultivars‘Skarb’ and ‘Pohonych’, which belong to the varieties Lactuca sativa L. var. longifolia and Lactuca sativa L. var. angustana, respectively, were genetically distant, what was confirmed by genetic distances values. It was determined that the Shannon index within the lettuce variety by 7 EST-SSR markers is 0.61, between varieties – 0.96, the average value is 1.57. Conclusions. According to the results of studies of 7 lettuce cultivars, it was found that the highest polymorphism was determined by the KSL-92 marker, the minimum of alleles number (3 alleles) was identified by KSL-37 marker. Based on calculated genetic distances, it was noticed that lettuce cultivars, genetic distances between which are 0.17, were the most similar. The most distant cultivar based on 7 EST-SSR markers was ‘Skarb’ cultivar.

Enhanced polycross breeding of tall fescue through marker-based paternity identification and estimation of combining ability

Half-sib (HS) mating is the most important breeding method of open-pollinated sexual forage grasses such as tall fescue. Progeny testing procedures of half-sib families can only measure general combining ability (GCA) due to the unknown pollen donor. In tall fescue, genetic studies using HS mating and application of molecular markers for paternity identification in polycross progenies are so far limited. In this study, 960 genotypes from sixteen half-sib families of tall fescue derived from polycrosses, along with their corresponding parental genotypes were evaluated for agro-morphological traits in the field during 2016 and 2017. Genetic diversity of the parental genotypes was assessed using twenty EST-SSR markers and then a minimal set of primers was used to identify the most likely male parent in the polycross progenies. Considerable genetic variation was observed among the parental genotypes based on both morphological and molecular assessments. Moderate to high estimates of narrow sense heritability (0.53–0.84) for morphological traits indicated that phenotypic selection can be successful to achieve genetic progress. The EST-SSR markers allowed to identify a unique paternal parent in 177 of the offspring which were divided into 66 full-sib families. The number of genotypes per full-sib family varied from 1 to 11. Marker data used to identify full-sib families, estimate specific combining ability (SCA), and select siblings based on the genetic value of both parents. No clear association was found between GCA of parents and the corresponding SCA for forage yield, indicating that both additive and dominant gene actions may play a role in the genetic control of this trait. The obtained results highlighted the importance of marker-based paternity identification to increase gain from selection in polycross breeding programs of tall fescue.

Genetic diversity in tomato accessions [Solanum lycopersicum (L.) H. Karst] from Nigeria employing morphological and SSR markers

The level of genetic diversity among 40 accessions of tomato collected from Nigeria was assessed employing 10 quantitative traits and 13 EST-SSR markers. There were significant differences among accessions as revealed by the quantitative traits. Meanwhile, the level of polymorphism displayed by the EST-SSR markers was low with mean PIC of 0.13. The clusters formed by morphological traits were completely different from the ones established by EST-SSR markers, as supported by the weak correlation (r = 0.143) between morphological and molecular distances by Mantel test. PCA and bi-plot analyses indicate that fruit yield, average fruit weight, number of days to first flowering and first fruit set and plant height, number of branches and number of fruits per cluster had the highest discriminating potentials. The best accessions T5, T17 and T12 were vertex accessions in the fruit yield, plant height, number of fruits per cluster and number of main branches sector. Accessions T11, T32 and T38 were vertex accessions in the number of days to first flowering and number of days to fruit set sector, hence were late maturing accessions, poor in yield and can however be improved by crossing them with the accessions in the first sector.

De novo assembly and Transcriptome characterization of an endemic species of Vietnam, Panax vietnamensis Ha et Grushv., including the development of EST-SSR markers for population genetics

BackgroundUnderstanding the genetic diversity in endangered species that occur inforest remnants is necessary to establish efficient strategies for the species conservation, restoration and management. Panax vietnamensis Ha et Grushv. is medicinally important, endemic and endangered species of Vietnam. However, genetic diversity and structure of population are unknown due to lack of efficient molecular markers.ResultsIn this study, we employed Illumina HiSeq™ 4000 sequencing to analyze the transcriptomes of P. vietnamensis (roots, leaves and stems). Raw reads total of 23,741,783 was obtained and then assembled, from which the generated unigenes were 89,271 (average length = 598.3191 nt). The 31,686 unigenes were annotated in different databases i.e. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Nucleotide Collection (NR/NT) and Swiss-Prot for functional annotation. Further, 11,343 EST-SSRs were detected. From 7774 primer pairs, 101 were selected for polymorphism validation, in which; 20 primer pairs were successfully amplified to DNA fragments and significant amounts of polymorphism was observed within population. The nine polymorphic microsatellite loci were used for population structure and diversity analyses. The obtained results revealed high levels of genetic diversity in populations, the average observed and expected heterozygosity were HO = 0.422 and HE = 0.479, respectively. During the Bottleneck analysis using TPM and SMM models (p < 0.01) shows that targeted population is significantly heterozygote deficient. This suggests sign of the bottleneck in all populations. Genetic differentiation between populations was moderate (FST = 0.133) and indicating slightly high level of gene flow (Nm = 1.63). Analysis of molecular variance (AMOVA) showed 63.17% of variation within individuals and 12.45% among populations. Our results shows two genetic clusters related to geographical distances.ConclusionOur study will assist conservators in future conservation management, breeding, production and habitats restoration of the species.

Discovery of simple sequence repeat (SSR) markers in hazelnut (Corylus avellana L.) by transcriptome sequencing and SSR-based characterization of hazelnut cultivars

ABSTRACT Current cultivation and breeding strategy based on selections from local and wild vegetation does not meet the hazelnut demand. This situation would benefit from the development of high-yielding cultivars with greater tolerance to abiotic and biotic stress conditions. Molecular markers are useful tools to support these breeding efforts. In the current study, EST-SSR markers were developed from RNA-sequencing data obtained from three hazelnut varieties. The transcriptome sequences contained 86,542 unigenes and a total of 25,391 SSRs, most of which (50.9%) were di-nucleotide repeats. A set of 40 primer pairs were randomly selected to validate their usefulness for investigating genetic diversity in 19 C. avellana cultivars and one accession of Corylus colurna. Of the 40, 28 were highly polymorphic with average values of observed heterozygosity, polymorphism information content and number of alleles per locus of 0.45, 0.67 and 6.11, respectively. The principal coordinate and dendrogram analysis separated the C. colurna accession from the C. avellana cultivars, and separated the cultivars for date of leaf bud burst. These new EST-SSRs and others that could be developed from the transcriptome sequences increase the number of markers for cultivar fingerprinting and may be useful for identifying marker-trait associations and further genetic diversity analyses.