Isolation and characterization of nine polymorphic microsatellite markers of the marbled rockfish Sebastiscus marmoratus (Scorpaeniformes, Scorpaenidae)

Sebastiscus marmoratus is a typical sedentary fish and usually lives at the bottom of the coastal zone from Japan to the East China Sea. Next-generation sequencing platform was used to obtain microsatellite loci sequences of S. marmoratus, among which forty-eight perfect microsatellite repeats were suitable for primer design. Nine polymorphic microsatellite markers were exploited and characterized for it. The allele number of all polymorphic microsatellite markers ranged from 4 to 12. Expected and observed heterozygosity varied from 0.302 to 0.909 and from 0.190 to 0.625, respectively. In further studies, the genetic variation and population structure of this species would be investigated using these microsatellite loci.     

Development of Chloroplast Microsatellite Markers and Analysis of Chloroplast Diversity in Chinese Jujube (Ziziphus jujuba Mill.) and Wild Jujube (Ziziphus acidojujuba Mill.)

Ziziphus is an important genus within the family Rhamnaceae. This genus includes several important fruit tree species that are widely planted in China and India, such as the Chinese jujube (Ziziphus jujuba Mill.), the wild jujube (Z. acidojujuba), and the Indian jujube (Z. mauritiana). However, information about their domestication based on the chlorotype diversity of Chinese jujube population is lacking. In this study, chloroplast microsatellite (cpSSR) markers were developed and used to investigate the genetic relationships between and domestication of jujube cultivars and wild jujube populations. Primer sets flanking each of the 46 cpSSR loci in non-coding regions of the chloroplast genome sequence of Z. jujuba Mill. cv. ‘Junzao’ were designed. In total, 10 markers showed polymorphisms from 15 samples (9 jujube cultivars and 6 wild jujube individuals), of which 8 loci were due to variations in the number of mononucleotide (A/T) repeats and 2 were due to indels. Six cpSSR markers were used in further analyses of 81 additional samples (63 jujube cultivars, 17 wild jujube samples, and 1 Indian jujube). Using these cpSSR markers, the number of alleles per locus ranged from two to four. In general, the Shannon Index (I) for each cpSSR ranged from 0.159 to 0.1747, and the diversity indices (h) and uh were 0.061 to 0.435 and 0.062 to 0.439, respectively. Seven chlorotypes were found; the Indian jujube showed distinct chlorotypes, and both the Chinese and wild jujube had four chlorotypes and shared two chlorotypes. A dominant chlorotype (G) accounted for 53 of 72 jujube cultivars and 13 of 23 wild jujube individuals. All chlorotypes were highly localized along the Yellow River, from the mid- to the lower reaches, suggesting a wide origin of jujube. These cpSSR markers can be applied to population and evolution studies of Chinese jujube and wild jujube.     

Development and characterization of genic SSR markers from low depth genome sequence of <Emphasis Type="Italic">Clarias batrachus</Emphasis> (magur)

Indian magur (Clarias batrachus) is an important freshwater catfish, which is listed as endangered under A3cde + 4acde ver. 3.1 categories by the IUCN (2015) due to decreasing population trend. Microsatellites or short sequence repeats (SSRs) tagged to genes have been utilized as gene marker. In the present study, 31,814 SSRs of C. batrachus (magur) were identified using microsatellite identification tool programme from the next generation sequencing data generated on Roche 454 and Ion Torrent platforms. A bioinformatics pipeline, with stringent criteria resulted in selection of 1672 microsatellite loci falling in the genic region. Initially, a total of 30 loci were selected for primer development; and of these 14 were successfully amplified and five were found to be polymorphic in 30 individuals of C. batrachus (magur). The observed as well as expected heterozygosity ranged from 0.038 to 0.526 and 0.434 to 0.784, respectively, and the number of observed alleles ranged from three to five. The study reported the application of next generation sequencing technologies for rapid development of microsatellite loci in Indian catfish species, C. batrachus (magur).     

Development of EST-SSR markers through data mining and their use for genetic diversity study in Indian accessions of Jatropha curcas L.: a potential energy crop

Jatropha curcas L. is gaining importance as a potential energy crop. However, lack of sufficient numbers of molecular markers hinder current research on crop improvement in Jatropha. The expressed sequences tags (EST) sequences deposited in public databases, offers an excellent opportunity to identify simple sequence repeats (SSRs) through data mining, for further research on molecular breeding. In the present study 42,477 ESTs of J. curcas were screened, out of which 5,673 SSRs were identified with 48.8 % simple (excluding mononucleotide repeats) and 52.2 % compound repeat motifs. Amongst these repeat motifs, dinucleotide repeats were abundant (26.5 %), followed by trinucleotide (23.1 %) and tetranucleotide repeats (0.8 %). From these microsatellites, 32 EST-SSR (genic microsatellite) primer pairs were designed. These primers were used to analyze the genetic diversity among 42 accessions collected from different parts of India. Out of the 32 EST-SSR primers, 24 primer pairs exhibited polymorphism among the genotypes, with amplicons varying from one to eight, giving an average of 2.33 alleles per polymorphic marker. Polymorphic information content value ranged from 0.02 to 0.5 with an average of 0.402 indicating moderate level of informativeness within these EST-SSRs markers. The EST-SSR markers developed here will serve as a valuable resource for genetic studies, like linkage mapping, diversity analysis, quantitative trait locus/association mapping, and molecular breeding. The current study also revealed low diversity in the screened Indian Jatropha germplasm. Therefore, the future efforts must be made to broaden the gene pool of Jatropha for the creation of genetic diversity that can be further used for crop improvement through breeding.     

A set of microsatellite markers for population genetics of leopard cat (Prionailurus bengalensis) and cross-species amplification in other felids

We describe the isolation and characterization of novel microsatellite loci from the leopard cat, Prionailurus bengalensis Kerr, 1792 (Family Felidae). Using Illumina HiSeq2500 sequencing technology, we sequenced the leopard cat genome and identified 1.5 million loci of simple sequence repeats with di- to deca-nucleotide motifs. We developed twelve polymorphic markers with tetra-nucleotide motif types after screening 35 loci for amplification and polymorphism. The observed and expected heterozygosities of the markers were 0.438 and 0.423, respectively. The number of alleles per locus ranged from 2 to 7, with a mean polymorphism information content of 0.383. Eleven loci were at Hardy-Weinberg equilibrium and no linkage disequilibrium was detected among any pairs of loci. We tested cross-species amplification of these markers across five other felids (Panthera tigris, P. pardus, P. onca, Acinonyx jubatus, and Felis catus). All loci were transferable to at least one other feline species and four amplified all five species. The microsatellite markers developed in this study will be valuable for estimating ecological parameters of populations and to establish conservation and management strategies for feline species.     

Development,Characterization and Cross Species Amplification of Polymorphic Microsatellite Markers from Expressed Sequence Tags of Turmeric (<Emphasis Type="Italic">Curcuma longa</Emphasis> L.)

Expressed sequence tags (ESTs) from turmeric (Curcuma longa L.) were used for the screening of type and frequency of Class I (hypervariable) simple sequence repeats (SSRs). A total of 231 microsatellite repeats were detected from 12,593 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs accounts to one SSR per 17.96 kb of EST. Mononucleotides were the most abundant class of microsatellite repeat in turmeric ESTs followed by trinucleotides. A robust set of 17 polymorphic EST–SSRs were developed and used for evaluating 20 turmeric accessions. The number of alleles detected ranged from 3 to 8 per loci. The developed markers were also evaluated in 13 related species of C. longa confirming high rate (100%) of cross species transferability. The polymorphic microsatellite markers generated from this study could be used for genetic diversity analysis and resolving the taxonomic confusion prevailing in the genus.     

Comparative sequence analyses reveal sites of ancestral chromosomal fusions in the Indian muntjac genome

Background

Indian muntjac (Muntiacus muntjak vaginalis) has an extreme mammalian karyotype, with only six and seven chromosomes in the female and male, respectively. Chinese muntjac (Muntiacus reevesi) has a more typical mammalian karyotype, with 46 chromosomes in both sexes. Despite this disparity, the two muntjac species are morphologically similar and can even interbreed to produce viable (albeit sterile) offspring. Previous studies have suggested that a series of telocentric chromosome fusion events involving telomeric and/or satellite repeats led to the extant Indian muntjac karyotype.

Results

We used a comparative mapping and sequencing approach to characterize the sites of ancestral chromosomal fusions in the Indian muntjac genome. Specifically, we screened an Indian muntjac bacterial artificial-chromosome library with a telomere repeat-specific probe. Isolated clones found by fluorescence in situ hybridization to map to interstitial regions on Indian muntjac chromosomes were further characterized, with a subset then subjected to shotgun sequencing. Subsequently, we isolated and sequenced overlapping clones extending from the ends of some of these initial clones; we also generated orthologous sequence from isolated Chinese muntjac clones. The generated Indian muntjac sequence has been analyzed for the juxtaposition of telomeric and satellite repeats and for synteny relationships relative to other mammalian genomes, including the Chinese muntjac.

Conclusions

The generated sequence data and comparative analyses provide a detailed genomic context for seven ancestral chromosome fusion sites in the Indian muntjac genome, which further supports the telocentric fusion model for the events leading to the unusual karyotypic differences among muntjac species.     

Phylogeny and Strain Typing of Escherichia coli, Inferred from Variation at Mononucleotide Repeat Loci

Multilocus sequencing of housekeeping genes has been used previously for bacterial strain typing and for inferring evolutionary relationships among strains of Escherichia coli. In this study, we used shorter intergenic sequences that contained simple sequence repeats (SSRs) of repeating mononucleotide motifs (mononucleotide repeats [MNRs]) to infer the phylogeny of pathogenic and commensal E. coli strains. Seven noncoding loci (four MNRs and three non-SSRs) were sequenced in 27 strains, including enterohemorrhagic (six isolates of O157:H7), enteropathogenic, enterotoxigenic, B, and K-12 strains. The four MNRs were also sequenced in 20 representative strains of the E. coli reference (ECOR) collection. Sequence polymorphism was significantly higher at the MNR loci, including the flanking sequences, indicating a higher mutation rate in the sequences flanking the MNR tracts. The four MNR loci were amplifiable by PCR in the standard ECOR A, B1, and D groups, but only one (yaiN) in the B2 group was amplified, which is consistent with previous studies that suggested that B2 is the most ancient group. High sequence compatibility was found between the four MNR loci, indicating that they are in the same clonal frame. The phylogenetic trees that were constructed from the sequence data were in good agreement with those of previous studies that used multilocus enzyme electrophoresis. The results demonstrate that MNR loci are useful for inferring phylogenetic relationships and provide much higher sequence variation than housekeeping genes. Therefore, the use of MNR loci for multilocus sequence typing should prove efficient for clinical diagnostics, epidemiology, and evolutionary study of bacteria.     

Development of Genomic Microsatellite Markers in Carthamus tinctorius L. (Safflower) Using Next Generation Sequencing and Assessment of Their Cross-Species Transferability and Utility for Diversity Analysis

Background

Safflower (Carthamus tinctorius L.), an Asteraceae member, yields high quality edible oil rich in unsaturated fatty acids and is resilient to dry conditions. The crop holds tremendous potential for improvement through concerted molecular breeding programs due to the availability of significant genetic and phenotypic diversity. Genomic resources that could facilitate such breeding programs remain largely underdeveloped in the crop. The present study was initiated to develop a large set of novel microsatellite markers for safflower using next generation sequencing.

Principal Findings

Low throughput genome sequencing of safflower was performed using Illumina paired end technology providing ~3.5X coverage of the genome. Analysis of sequencing data allowed identification of 23,067 regions harboring perfect microsatellite loci. The safflower genome was found to be rich in dinucleotide repeats followed by tri-, tetra-, penta- and hexa-nucleotides. Primer pairs were designed for 5,716 novel microsatellite sequences with repeat length ≥ 20 bases and optimal flanking regions. A subset of 325 microsatellite loci was tested for amplification, of which 294 loci produced robust amplification. The validated primers were used for assessment of 23 safflower accessions belonging to diverse agro-climatic zones of the world leading to identification of 93 polymorphic primers (31.6%). The numbers of observed alleles at each locus ranged from two to four and mean polymorphism information content was found to be 0.3075. The polymorphic primers were tested for cross-species transferability on nine wild relatives of cultivated safflower. All primers except one showed amplification in at least two wild species while 25 primers amplified across all the nine species. The UPGMA dendrogram clustered C. tinctorius accessions and wild species separately into two major groups. The proposed progenitor species of safflower, C. oxyacantha and C. palaestinus were genetically closer to cultivated safflower and formed a distinct cluster. The cluster analysis also distinguished diploid and tetraploid wild species of safflower.

Conclusion

Next generation sequencing of safflower genome generated a large set of microsatellite markers. The novel markers developed in this study will add to the existing repertoire of markers and can be used for diversity analysis, synteny studies, construction of linkage maps and marker-assisted selection.     

Extensive Polymorphism in Cryptosporidium parvum Identified by Multilocus Microsatellite Analysis

Restriction fragment length polymorphism and DNA sequence analysis discern two main types of Cryptosporidium parvum. We present a survey of length polymorphism at several microsatellite loci for type 1 and type 2 isolates. A total of 14 microsatellite loci were identified from C. parvum DNA sequences deposited in public databases. All repeats were mono-, di-, and trinucleotide repeats of A, AT, and AAT, reflecting the high AT content of the C. parvum genome. Several of these loci showed significant length polymorphism, with as many as seven alleles identified for a single locus. Differences between alleles ranged from 1 to 27 bp. Karyotype analysis using probes flanking three microsatellites localized each marker to an individual chromosomal band, suggesting that these markers are single copy. In a sample of 19 isolates for which at least three microsatellites were typed, a majority of isolates displayed a unique multilocus fingerprint. Microsatellite analysis of isolates passaged between different host species identified genotypic changes consistent with changes in parasite populations.     

濒危植物羊踯躅全基因组SSR标记开发与鉴定研究

该研究利用基于全基因组限制性酶切位点简化基因组测序技术（RAD seq技术）,开发濒危植物羊踯躅（Rhododendron molle G. Don）全基因组SSR标记,并对3个群体共63份羊踯躅材料进行验证鉴定,为进一步研究羊踯躅的遗传多样性和群体遗传结构以及保护利用提供技术支持。结果显示：（1）羊踯躅基因组测序获得原始数据7.653G bp,过滤后为7.513G bp;经组装发现,羊踯躅171.534 M bp的基因组分布在498 252 contigs中。（2）通过SSR检测,在11 961 SSR位点中获得了11 687对SSR分子标记,并且二核苷酸为基序的重复类型最丰富,达51.76%。（3）随机选取128对SSR标记在6个羊踯躅株系中进行PCR扩增,获得20对高多态性的SSR标记。（4）用所选的20对多态性SSR标记对3个群体共63份羊踯躅材料进行验证分析发现,这些多态性SSR标记位点的等位基因数为4~16个,期望杂合度（H_e）为0.489~0.908。 研究表明,羊踯躅的SSR丰度适中,且二核苷酸为羊踯躅中最丰富的重复序列,该实验进一步证明RAD seq技术是一种经济有效的基因测序方法,实验中开发的SSR引物将有助于进一步研究羊踯躅和其他近缘种的群体结构和多样性。     

Microsatellite-based evidences of genetic bottlenecks in the cryptic species “Andrographis paniculata Nees”: a potential anticancer agent

Andrographis paniculata (AP) is a medicinal plant species introduced into Malaysia. To address the genetic structure and evolutionary connectedness of the Malaysian AP with the Indian AP, a DNA sequence analysis was conducted based on 24 microsatellite markers. Out of the 24 primer sets, seven novel microsatellite primers were designed and amplified intra-specifically according to the available Indian AP sequences at the National Centre for Biotechnology Information (NCBI), where 17 of them were amplified using the cross-species strategy by employing the primers belonging to Acanthus ilicifolius Linn (Acanthaceae) and Lumnitzera racemosa Wild (Combretaceae). The primers were then applied on the Malaysian AP accessions. Sixteen of the new microsatellite loci were amplified successfully. Analysis of these microsatellite sequences, revealed some significant differences between the Indian and Malaysian AP accessions in terms of the size and type of the repeat motifs. These findings depicted the cryptic feature of this species. Despite identifying several heterozygous alleles no polymorphism was observed in the detected loci of the selected accessions. This situation was in concordance with the presence of “fixed heterozygosity” phenomenon in the mentioned loci. Accordingly, this was fully consistent with the occurrence of the genetic bottleneck and founder effect within Malaysian AP population. Apart from the amplification of new microsatellites in this species, our observations could be in agreement with the risk of genetic depletion and consequently extinction of this precious herb in Malaysia. This issue should be taken into consideration in the future studies.     

Abundance, polymorphism and genetic mapping of microsatellites in rice

Dinucleotide microsatellites have been characterized and used as genetic markers in rice. Screening of a rice genomic library with poly(dG-dA)·(dC-dT) and poly(dG-dT)·(dC-dA) probes indicated that (GA)_n repeats occurred, on average, once every 225 kb and (GT)_n repeats once every 480 kb. DNA sequencing of ten randomly selected microsatellites indicated that the numbers of repeats ranged from 12 to 34 and that the patterns of microsatellites in rice were similar to those of humans and other mammals. Primers to these microsatellite loci as well as to four published microsatellite-containing sequences have been designed and degrees of polymorphism has been examined with 20 rice accessions. Multiple alleles, ranging from 5 to 11, have been observed at all the microsatellite loci in 20 rice accessions. Alleles specific to two cultivated subspecies, indica and japonica, were found in some microsatellite loci. Heterozygosity values of all the microsatellite markers were significantly higher than those of RFLP markers, based upon a parallel comparison. Ten microsatellite loci have been genetically mapped to four rice chromosomes. The genomic distribution of microsatellites appears to be random in rice.