SSR Loci Research Articles

Basic Characteristics of Flower Transcriptome Data and Derived Novel EST-SSR Markers of Luculia yunnanensis, an Endangered Species Endemic to Yunnan, Southwestern China.

Luculia yunnanensis (Rubiaceae), an evergreen shrub or small tree, is endemic to China and confined to Nujiang Prefecture, Yunnan Province. This plant is of high ornamental value owing to its attractive pink flowers, sweet fragrance, and long flowering period. Due to the influence of climate change and human factors, the distribution range of L. yunnanensis has exhibited a significant shrinking trend, and it has become a vulnerable species that is in urgent need of conservation and rational utilization research. In this study, the flower transcriptome sequencing of L. yunnanensis was conducted using an Illumina HiSeq platform. We designed and developed a series of EST-SSR primers based on the flower transcriptome data of L. yunnanensis. The results showed that 98,389 unigenes were obtained from the L. yunnanensis flower transcriptome, all of which were aligned with sequences in public databases. Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KEGG, and GO annotated 31,859, 13,853, 22,684, 10,947, 21,416, 9722, and 23,390 unigenes, respectively. The MISA (Microsatellite) tool was used to identify SSR loci from all unigenes, and a total of 15,384 SSRs were identified. Repeat motifs were given priority with mononucleotides, dinucleotides, and trinucleotides. The 81 primer pairs were synthesized randomly, of which 44 pairs showed effective amplification. A total of 17 primers showed stable amplification, and rich polymorphism was observed in 6 populations. We concluded via genetic diversity analysis that the average effective number of alleles (Ne), Shannon’s information index (I), and polymorphism information content (PIC) were 1.925, 0.837, and 0.403, respectively. In conclusion, 17 EST-SSR primers can be used for subsequent population genetic diversity analysis and molecular-marker-assisted breeding, which is of great significance for formulating resource conservation and utilization strategies for L. yunnanensis.

Diversity of Tartary Buckwheat (Fagopyrum tataricum) Landraces from Liangshan, Southwest China: Evidence from Morphology and SSR Markers

Tartary buckwheat (Fagopyrum tataricum) has been cultivated for over one thousand years in the Liangshan Prefecture of Sichuan, China. Growing population pressures, economic modernization pressures, and the erosion of traditional culture have led to the rapid loss of area covered by Tartary buckwheat landraces. Morphological and molecular characterization of 112 Tartary buckwheat accessions from 29 populations were assessed based on 10 morphological traits of seeds and 10 SSR markers, respectively. The coefficient of variation and Shannon index showed diversity within the morphological characteristics of the seeds. All accessions were divided into three categories according to phylogenetic dendrogram analysis, which was consistent with folk nomenclature and taxonomy. Genetic analysis using SSR markers identified 45 alleles with a mean value of 4.5 alleles per locus. The high average PIC value (0.459) indicated polymorphism of the SSR markers. The genetic similarity coefficient of the 112 Tartary buckwheat accessions showed a high level of genetic diversity ranging from 0.130 to 0.978. The genetic structure analysis revealed high genetic differentiation (Nei = 0.255). The folk nomenclature, folk taxonomy, and sociocultural norms may also contribute to a significant influence on the diversity of folk nomenclature and taxonomy. The assessment of the genetic diversity of Tartary buckwheat landraces and detection of SSR loci associated with traits could be used as scientific guidance for selecting Tartary buckwheat seed for improved production relative to local farmers and consumer preferences. Local traditional knowledge (seed exchange network) and culture also contribute to breeding and the maintenance of the genetic diversity of Tartary buckwheat.

Analysis of the structure of linseed flax varieties based on polymorphic microsatellite loci

High profitability of linseed flax production is an incentive for developing new varieties with high yield performance and for expansion of cultivation areas. The restricted genetic diversity of Linum usitatissimum L. due to selfpollination and of initial forms used by developing the new linseed flax varieties limits the efficiency of the breeding work. The purpose of the research was to determine the genetic structure of linseed flax varieties using a system of 10 microsatellite loci. Four varieties bred by the V. S. Pustovoit All-Russian Research Institute of Oil Crops were used as the research material. DNA was isolated by the CTAB method. Electrophoretic separation of amplicons was performed in 8 % polyacrylamide gel followed by ethidium bromide staining. The determination of the main indicators of genetic variability, analyzed molecular variance (AMOVA), the Wright F-statistics indicators, and the principal coordinates analysis (PCoA) was carried out using the GenAlEx 6.5 program. A total of 53 alleles have been identified. The main indicators of genetic diversity were calculated for each variety. According to statistical characteristics (Na, Ne, I, F), the variety Nilin is the source of the greatest genetic diversity. For all varieties, the ratio of expected heterozygosity (He) and observed heterozygosity (Ho) corresponds to inbred populations with a minimum proportion of random crossing. By the allelic state of the 10 SSR loci, the variety Snegurok belongs to the linear type, which corresponds to its origin. The genetic passport of this variety has been compiled according to these loci. According to the results of the analysis of molecular variance (AMOVA), 71 % of the total dispersion is due to genetic differences between the varieties, and 29 % is due to intra-varietal differences.

Comparison of genetic diversity between ancient and common populations of Docynia delavayi (Franch.) Schneid

Docynia delavayi (Franch.) Schneid. (D. delavayi), is a wild fruit tree which combines edible, medicinal, ecological and ornamental uses. In this study, ancient and common populations of D. delavayi were examined for genetic diversity and structure using SSR markers. As a result, a total of 136 alleles were detected at 18 SSR loci, with the mean of 7.56 alleles. The value of Na, Ne, I, He and Nm of the ancient populations were lower than those of the common populations except for Ho and Fst. It indicates that the genetic diversity of the common populations is higher than that in ancient populations. The genetic differences between ancient populations were slightly greater than those between common populations, which demonstrated less gene flow between ancient populations. According to the analysis of molecular variance (AMOVA), the genetic variation within the common population was greater than that in the ancient population, indicating that there was a higher genetic diversity within the common population. Also, the clustering heatmap results are partially consistent with the principal coordinate analysis (PCoA) results. Moreover, the mantel test showed an extremely significant correlation between genetic and geography distance (r = 0.214, p < 0.0001). Based on this work, we proposed strategies for protecting, which offers a theoretical basis for their effective utilization and conservation of D. delavayi ancient tree resources.

Validation of genome-data based simple sequence repeats for ascertaining hybridity in mango (Mangifera indica) hybrids

The present study was carried to identify polymorphic SSRs derived from the whole draft genome data of mango (Mangifera indica L.) for their polymorphism between Amrapali and Sensation genotypes (parents) and their validation on the derived hybrid progenies. Fifty new hypervariable mango SSRs (HMSSRs) were screened against the parental genotypes. Out of which, 11 HMSSRs were polymorphic, 18 were monomorphic and 21 did not amplify at all. These polymorphic HMSSRs were then used for ascertaining the hybridity of the hybrid progenies. The genetic diversity indices of each SSR locus among mango hybrids were calculated using Power Marker ver. 3.25. The genetic diversity indices such as the number of alleles ranged from 2 (HMSSR35) to 5 (HMSSR57), gene diversity from 0.36 (HMSSR54) to 0.71 (HMSSR57 &amp; HMSSR58), and PIC values from 0.35 (HMSSR69) to 0.66 (HMSSR57). The dendrogram generated using Neighbor joining tree divided the 22 genotypes into three distinct clades, viz. Clade ‘A’, Clade ‘B’ and Clade ‘C’. The hybrids, viz. H-3-1, H-3-2, and H-7-4 were identified as true hybrids, since they were genetically more close to female parent (Amrapali), while hybrids H-3-3, H-3-11, H-11-1, H-12-5, H-1-6, H-1-13, H-12-8, H-12-10, H-3-12, H-1-1 and H-4-10 were more close to the male donor parent (Sensation) and also had red peel colour. In the present study, the 20 hybrids were confirmed to be the true hybrids since the bands unique to the two parents were present in them.

Development and characterization analysis of potato SSR primers and the amplification research in colored potato materials

<p id="C3">Compared with commonly cultivated potato, colored potato is rich in nutrition, especially in anthocyanins, which is the focus of breeding research in recent years. To date, the number of potato SSR primers developed is limited, especially those related to colored potato. In this study, SSR loci were analyzed using MISA software based on the whole genome. The results were as follows: a total of 218,997 SSR loci were obtained from potato genome with an average of 3.39 kb. Mononucleotide was the main repeat type, accounting for 62.05% of the total SSR, followed by dinucleotide and trinucleotide, accounting for 22.39% and 13.11%, respectively. The repeats of the six nucleotide types ranged from 5 to 746, mainly from 5 to 10, accounting for 60.9% of the total SSRs. A total of 215 motif types were obtained in all detected SSRs. The dominant motifs of the other five nucleotide repeat types were mainly A/T-containing motifs, except for hexanucleotide repeat type. The length of SSR motifs ranged from 12 bp to 20 bp, accounting for 43.19% of all SSRs. A total of 100 SSR primers were designed by Primer 5 software, among which 48 primers were preliminarily screened using parents’ genomic DNA of the colored potato, with an effective amplification rate of 48%. Then, six F₂ individuals were randomly selected for PCR amplification, and 26 primers with clear and stable bands and high polymorphism were finely screened, with the average polymorphism rate of 72.52%. In summary, the number and types of SSR loci in potato genome was abundant and diverse, and the polymorphism was moderate. The developed primers were highly polymorphic, which can be used in the development of SSR markers, genetic diversity analysis, and SSR fingerprint of colored potato, providing a scientific basis for further mining genes related to anthocyanin content.

Allelic diversity of a panel ofAegilops muticaBoiss (Amblyopyrym muticum(Boiss.) Eig) from Turkey

AbstractThe members of the Aegilops genus serve as a vast pool of allele discovery for wheat improvement in abiotic and biotic stress responses.Aegilops muticaBoiss (Amblyopyrym muticum(Boiss) Eig) is an unexplored candidate with significant potential. Even though it has been used in cytogenetics applications within the last century, natural population diversity and allele discovery have been neglected. As an endemic species for Anatolia and the lower Caucasian region, it has an unexplored population structure. Here, seventy-five genotypes from five different newly collected populations from central Anatolia were evaluated with 29 polymorphic SSR loci. Significant diversity within (83%) and between (17%) the populations was obtained. Three of the populations were clearly separated, while two had some level of the mixture. Relatively easy cross-species hybridization and introgressions makeAe. muticaa good candidate for novel allele discovery and pre-breeding. Here, for the first time, representative natural populations ofAe. muticawere compared and population structures were revealed with SSR markers which may clear the misty vision that geneticists might have regardingAe. mutica. This could be exploited in genetic resource conservation and breeding programs and maybe a point for further studies.

Evaluation of genetic diversity of ginseng fruit color germplasm resources: based on SSR analysis

Illumina Xten was employed for shallow sequencing of Panax ginseng(ginseng) samples, MISA for screening of SSR loci, and Primer 3 for primer design. Polymorphic primers were screened from 180 primers. From the successfully amplified polymorphic primers, 15 primers which featured clear peak shape, good polymorphism, and ease of statistics were selected and used to evaluate the genetic diversity and germplasm resources of 36 ginseng accessions with different fruit colors from Jilin province. The results showed that red-fruit ginseng population had high genetic diversity with the average number of alleles(N_a) of 1.031 and haploid genetic diversity(h) of 0.172. The neighbor-joining cluster analysis demonstrated that the germplasms of red-fruit and yellow-fruit ginseng populations were obviously intermixed, and pick-fruit ginseng germplasms clustered into a single clade. The results of STRUCTURE analysis showed high proportion of single genotype in pick-fruit ginseng germplasm and abundant genotypes in red-fruit and yellow-fruit ginseng germplasms with obvious germplasm mixing. AMOVA revealed that genetic variation occurred mainly within populations(62.00%, P&lt;0.001), and rarely among populations(39%, P&lt;0.001), but homogenization was obvious among different populations. In summary, pink-fruit ginseng population may contain rare genotypes, which is the basis for breeding of high-quality high-yield, and multi-resistance varieties, genetic improvement of varieties, and sustainable development and utilization of ginseng germplasm resources.

Diversity and genetic structure of natural populations of the palm tree Euterpe precatoria (Arecaceae)

Introduction: The natural ecosystems of northern Mato Grosso, Brazil, are in process of fragmentation, mainly due to population growth and the expansion of agriculture. This endangers the palm Euterpe precatoria (locally known as açaí), used for construction, palm hearts, juices and ice cream. Objective: To evaluate the local diversity and genetic structure in native populations of E. precatoria. Methods: We collected leaves from 106 fruiting palms from five populations in Mato Grosso State, for analysis of microsatellite markers with Polymerase Chain Reaction. Results: The five SSR loci revealed a total of 30 alleles, ranging from 5 (EE23 and EE43) to 7 (EE2 and EE15), with an average of 6 alleles per locus. The mean PIC was 0.74 and confirmed low heterozygosity and inbreeding. The UPGMA dendrogram produced two groups and molecular variance revealed greater genetic differentiation within populations. The high levels of homozygous microsatellite loci indicate low genetic diversity. Conclusions: These populations have low gene diversity, high average number of alleles per locus, and rare and exclusive alleles. We recommend the establishment of permanent conservation units with corridors among them.

Characterization of Microsatellites in the Akebia trifoliata Genome and Their Transferability and Development of a Whole Set of Effective, Polymorphic, and Physically Mapped Simple Sequence Repeat Markers.

Akebia trifoliata is a perennial climbing woody liana plant with a high potential for commercial exploitation and theoretical research. Similarly, microsatellites (simple sequence repeats, SSRs) also have dual roles: as critical markers and as essential elements of the eukaryotic genome. To characterize the profile of SSRs and develop molecular markers, the high-quality assembled genome of A. trifoliata was used. Additionally, to determine the potential transferability of SSR loci, the genomes of Amborella trichopoda, Oryza sativa, Vitis vinifera, Arabidopsis thaliana, Papaver somniferum, and Aquilegia coerulea were also used. We identified 434,293 SSRs with abundant short repeats, such as 290,868 (66.98%) single-nucleotide repeats (SNRs) and 113,299 (26.09%) dinucleotide repeats (DNRs) in the A. trifoliata genome. 398,728 (91.81%) SSRs on 344,283 loci were physically mapped on the chromosomes, and a positive correlation (r = 0.98) was found between the number of SSRs and chromosomal length. Additionally, 342,916 (99.60%) potential SSR markers could be designed from the 344,283 physically mapped loci, while only 36,160 could be viewed as high-polymorphism-potential (HPP) markers, findings that were validated by PCR. Finally, SSR loci exhibited broad potential transferability, particularly DNRs such as the “AT/AT” and “AG/CT” loci, among all angiosperms, a finding that was not related to the genetic divergence distance. Practically, we developed a whole set of effective, polymorphic, and physically anchored molecular markers and found that, evolutionarily, DNRs could be responsible for microsatellite origin and protecting gene function.

Design of microsatellite markers for Schizophyllum commune (Agaricales, Basidiomycota) based on analysis of its genome

Simple sequence repeats of DNA (SSRs) are the most popular source of genetic markers used in population genetics, phylogenetics, and genetic mapping. A large number of nucleotide repeats enriched in G and C were identified. 336 mononucleotide motifs with more than ten repeats were recorded. 2020 nucleotide repeats were identified, of which 97.4% are di- (68.2%) and trinucleotides (29.2%). The total number of unique SSR loci, to which primers pairs were developed, was 1920. PCR primer sequences for unique SSR loci of the S. commune genome are presented. Of the twenty-two SSR markers synthesized for the S. commune genome, amplicons formed 64% on freshly isolated DNA samples.

Genetic variability and population genetic structure in autotriploid saffron using allelic phenotypes of microsatellite markers

A comprehensive effort was made to characterize potential inter- and intra-population genetic variation governing on the saffron ecotypes originating from 18 distinct geographical regions of Iran (90 accessions) using “allelic phenotypes” of 16 microsatellite markers. The 11 SSR loci (69%) that were polymorphic across the 90 saffron genotypes produced 30 alleles, of which ABRII/CS2 was the most informative locus. Both PCoA and UPGMA phylogenetic tree, alongside Bayesian model-based clustering grouped the 18 ecotypes into three major groups, with similar classification patterns, and highly consistent with geographical distribution. The assignment test accurately assigned 83 out of 90 saffron accessions (92.22%) to the corresponding populations, while the remainder were placed in adjacent populations. The AMOVA results revealed a high level of genetic variation within saffron populations (>80%), with a small fraction among populations (<20%). For the entire 90 accessions, significant correlation was detected between genetic and geographical distances (Mantel test, r = 0.173; P = 0.038). The results, overall, indicated the efficacy of allelic phenotypes-assisted DNA fingerprinting approach in making discrimination and unraveling potential genetic diversity among various Iranian saffron ecotypes, and possibly other autopolyploid crops.

Inflorescence Transcriptome Sequencing and Development of New EST-SSR Markers in Common Buckwheat (Fagopyrum esculentum).

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.

SSR Loci Analysis in Transcriptome and Molecular Marker Development in Polygonatum sibiricum.

To study the SSR loci information and develop molecular markers, a total of 435,858 unigenes in transcriptome of Polygonatum sibiricum were used to explore SSR. The distribution frequency of SSR and the basic characteristics of repeat motifs were analyzed using MISA software, and SSR primers were designed by Primer 3.0 software and then validated by PCR. Moreover, the gene function analysis of SSR Unigene was obtained by Blast. The results showed that 112,728 SSR loci were found in the transcriptome of Polygonatum sibiricum, which distributed in 435,858 unigenes with a distribution frequency of 25.86%. Mo-nucleotide and Di-nucleotide repeat were the main types, accounted for 83.83% of all SSRs. The repeat motifs of A/T and AC/GT were the predominant repeat types of Mo-nucleotide and Di-nucleotide, respectively. A total of 113,305 pairs of SSR primers with the potential to produce polymorphism were designed for maker development. One hundred and fifty-four of the 500 randomly selected primers not only produced fragments with expected molecular size but also had high polymorphism, which could accurately separate the tested varieties. The gene function of unigenes containing SSR was mostly related to the molecular function of Polygonatum sibiricum. The SSR markers in transcriptome of Polygonatum sibiricum show rich type, strong specificity, and high potential of polymorphism, which will benefit the candidate gene mining and marker-assisted breeding. The developed markers can also provide technical methods for molecular identification of intraspecific species of Polygonatum Mill. and maker-assisted breeding of superior varieties of Polygonatum Mill.

Genetic Diversity Analysis of Rice (Oryzae sativa L.) Landraces using SSR Markers in Bangladesh

It is necessary to assess genetic diversity and a molecular characteristic among Bangladesh’s local rice cultivars. The purpose of the study was to assess the genetic diversity and molecular characterization of 83 rice landraces in Bangladesh using nine (9) SSR markers. A total of 262 allels were identified using nine (9) polymorphic markers. The highest number alleles 34 were identified by RM336 while the lowest was 13 by RM262. Polymorphism information content (PIC) value of each marker was evaluated on the basis of the number of alleles and it varied greatly for all the SSR loci tested. The PIC value ranged from 0.951 to 0.766 and marker RM336 was found to be the most appropriate marker to discriminate among the rice genotypes owing to the highest PIC value of 0.951. The allele frequency ranged from 37.35% (RM262) to 10.84% (RM585, RM336) with an average of 18.47%. The genotypes G82, G77, G68, G50, and G1; G65, G37, and G10; G71 and G11; G25 and G14; G39 and G27 have 100% genetic similarity according to the pair wise genetic similarity indexes. Genotype G10 had the least similarity (0.44 percent) to genotype G9, G16 with G17, G22 with G29, G28 with G30, and so on. The dendrogram based on UPGMA and Nei’s genetic distance classified the 83 rice landraces into 5 clusters with a similarity coefficient of 0.6 Cluster 2 had maximum thirty-two genotypes followed by cluster 4. The landraces that were derivatives of genetically similar types were clustered together on the dendrogram. These landraces is showed wide genetic divergence among the constituent in it and in future it will be useful for hybridization programme in plant breeding. SAARC J. Agric., 19(2): 13-25 (2021)

Genetic diversity and population structure of invasive and native populations of Erigeron canadensis L.

Abstract Invasive alien plants threaten biodiversity across the world. Erigeron canadensis (horseweed) is one of the most problematic agricultural weeds and represents a classic example of intercontinental invasion. Here, we studied the genetic diversity and population structure of invasive alien populations from the Jiangsu and Zhejiang Provinces in China and native populations from Alabama, in the USA. We used 10 polymorphic SSR loci to genotype 312 individuals from 5 native and 5 invasive populations to estimate the genetic diversity and structure. Invasive populations from Jiangsu and Zhejiang Provinces showed, on average, similar genetic diversity to native populations from Alabama, indicating no severe genetic bottlenecks during the invasion. STRUCTURE revealed that low population differentiation occurred, and that only two genetic groupings were detected in both native and invaded ranges. The high diversity observed in the invasive populations suggested multiple introductions and/or the introduction of genetically diverse propagules during initial colonization. Our study provides new insights toward understanding the invasion dynamics of this globally noxious weed in Eastern China. Preventing gene flow via seed dispersal between invasive and native populations should be examined to prevent the introduction and dispersal of herbicide-resistant individuals and inform management practices.

Genome survey of Misgurnus anguillicaudatus to identify genomic information, simple sequence repeat (SSR) markers, and mitochondrial genome.

The dojo loach Misgurnus anguillicaudatus is an important economic species in Asia because of its nutritional value and broad environmental adaptability. Despite its economic importance, genomic data for M. anguillicaudatus is currently unavailable. In the present study, we conducted a genome survey of M. anguillicaudatus using next-generation sequencing technology. Its genome size was estimated to be 1105.97Mb by using K-mer analysis, and its heterozygosity ratio, repeat sequence content, GC content were 1.45%, 58.98%, and 38.03%, respectively. A total of 376,357 microsatellite motifs were identified, and mononucleotides, with a frequency of 42.57%, were the most frequently repeated motifs, followed by 40.83% dinucleotide, 7.49% trinucleotide, 8.09% tetranucleotide, and 0.91% pentanucleotide motifs. The AC/GT, AAT/ATT, and ACAG/CTGT repeats were the most abundant motifs among dinucleotide, trinucleotide, and tetranucleotide motifs, respectively. Besides,the complete mitochondrial genome was sequenced. Based on the Maximum Likelihood and Bayesian inference analyses, M. anguillicaudatus yingde in this study was the "introgressed" mitochondrial type. Seventy microsatellite loci were randomly selected from detected SSR loci to test polymorphic, of which, 20 microsatellite loci were assessed in 30 individuals from a wild population. The number of alleles (Na), observed heterozygosity (Ho), and expected heterozygosity (He) per locus ranged from 7 to 19, 0.400 to 0.933, and 0.752 to 0.938, respectively. All 20 loci were highly informative (PIC > 0.700). Eight loci deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.05). This is the first report of genome survey sequencing in M. anguillicaudatus, genome information, mitochondrial genome, andmicrosatellite markers will be valuable for further studies on population genetic analysis, natural resource conservation, and molecular marker-assisted selective breeding.

BigFiRSt: A Software Program Using Big Data Technique for Mining Simple Sequence Repeats From Large-Scale Sequencing Data.

BackgroundSimple Sequence Repeats (SSRs) are short tandem repeats of nucleotide sequences. It has been shown that SSRs are associated with human diseases and are of medical relevance. Accordingly, a variety of computational methods have been proposed to mine SSRs from genomes. Conventional methods rely on a high-quality complete genome to identify SSRs. However, the sequenced genome often misses several highly repetitive regions. Moreover, many non-model species have no entire genomes. With the recent advances of next-generation sequencing (NGS) techniques, large-scale sequence reads for any species can be rapidly generated using NGS. In this context, a number of methods have been proposed to identify thousands of SSR loci within large amounts of reads for non-model species. While the most commonly used NGS platforms (e.g., Illumina platform) on the market generally provide short paired-end reads, merging overlapping paired-end reads has become a common way prior to the identification of SSR loci. This has posed a big data analysis challenge for traditional stand-alone tools to merge short read pairs and identify SSRs from large-scale data.ResultsIn this study, we present a new Hadoop-based software program, termed BigFiRSt, to address this problem using cutting-edge big data technology. BigFiRSt consists of two major modules, BigFLASH and BigPERF, implemented based on two state-of-the-art stand-alone tools, FLASH and PERF, respectively. BigFLASH and BigPERF address the problem of merging short read pairs and mining SSRs in the big data manner, respectively. Comprehensive benchmarking experiments show that BigFiRSt can dramatically reduce the execution times of fast read pairs merging and SSRs mining from very large-scale DNA sequence data.ConclusionsThe excellent performance of BigFiRSt mainly resorts to the Big Data Hadoop technology to merge read pairs and mine SSRs in parallel and distributed computing on clusters. We anticipate BigFiRSt will be a valuable tool in the coming biological Big Data era.

Evolution of fruit and seed traits during almond naturalization

Abstract Cultivated plant species often naturalize and enter wild communities in a process known as feralization. To successfully feralize, crops must overcome ecological barriers and may undergo selection on certain traits, diverging phenotypically and genetically from their crop ancestors. In spite of the agronomic and ecological relevance of crop feralization, the eco‐evolutionary dynamics driving it remain understudied. In this paper, we evaluated phenotypic and genotypic differentiation in fruit and seed traits during the naturalization of the almond tree (Prunus dulcis (Mill.) D.A. Webb) in SE Iberia and evaluated the potential role of natural selection in this process. To do so, we investigated the patterns of genetic divergence between cultivated and feral populations using functional (the cyanogenesis Sk gene) and neutral (17 SSR loci) markers and analysed morphological and biochemical traits in kernels of 342 individuals from 15 cultivated and 24 feral populations. We detected very little genetic differentiation in neutral markers between cultivated and feral populations. The majority of the observed genetic variation was due to differences within each type. Conversely, the recessive allele sk responsible for seed toxicity was significantly more frequent in feral populations. Phenotypic differentiation between cultivated and naturalized almond populations was also significant. Feral almond kernels were smaller and lighter, had denser and more resistant shells (endocarps) and more toxic seeds. Selection analyses indicated that these genetic and phenotypic patterns might be driven by directional selection on fruit and seed traits, potentially linked to defence against predation. Synthesis. Our findings indicate that almond naturalization is consistent with strong directional selection on fruits and seeds, leading to smaller and more toxic seeds encased in harder endocarps. Accordingly, we propose that feralization of this crop is, at least to some degree, driven by adaptive evolution of dispersal and recruitment traits.

Automating microsatellite screening and primer design from multi-individual libraries using Micro-Primers

Analysis of intra- and inter-population diversity has become important for defining the genetic status and distribution patterns of a species and a powerful tool for conservation programs, as high levels of inbreeding could lead into whole population extinction in few generations. Microsatellites (SSR) are commonly used in population studies but discovering highly variable regions across species’ genomes requires demanding computation and laboratorial optimization. In this work, we combine next generation sequencing (NGS) with automatic computing to develop a genomic-oriented tool for characterizing SSRs at the population level. Herein, we describe a new Python pipeline, named Micro-Primers, designed to identify, and design PCR primers for amplification of SSR loci from a multi-individual microsatellite library. By combining commonly used programs for data cleaning and microsatellite mining, this pipeline easily generates, from a fastq file produced by high-throughput sequencing, standard information about the selected microsatellite loci, including the number of alleles in the population subset, and the melting temperature and respective PCR product of each primer set. Additionally, potential polymorphic loci can be identified based on the allele ranges observed in the population, to easily guide the selection of optimal markers for the species. Experimental results show that Micro-Primers significantly reduces processing time in comparison to manual analysis while keeping the same quality of the results. The elapsed times at each step can be longer depending on the number of sequences to analyze and, if not assisted, the selection of polymorphic loci from multiple individuals can represent a major bottleneck in population studies.