Utility of EST-derived SSRs as population genetics markers in a beetle

Microsatellite, or simple sequence repeat (SSR), loci can be identified by mining expressed sequence tag (EST) databases, and where these are available, marker development time and expense can be decreased considerably over conventional strategies of probing the entire genome. However, it is unclear whether they provide information on population structure similar to that generated by anonymous genomic SSRs. We performed comparative population genetic analyses between EST-derived SSRs (EST-SSRs) and anonymous SSRs developed from genomic DNA for the same set of populations of the insect Diabrotica virgifera, a beetle in the family Chrysomelidae. Compared with noncoding, nontranscribed regions, EST-SSRs were generally less polymorphic but had reduced occurrence of null alleles and greater cross-species amplification. Neutrality tests suggested the loci were not under positive selection. Across all populations and all loci, the genomic and EST-SSRs performed similarly in estimating genetic diversity, F(IS), F(ST), population assignment and exclusion tests, and detection of distinct populations. These findings, therefore, indicate that the EST-SSRs examined can be used with confidence in future genetic studies of Diabrotica populations and suggest that EST libraries can be added as a valuable source of markers for population genetics studies in insects and other animals.     

Comparative analysis of population genetic structure in Athyrium distentifolium (Pteridophyta) using AFLPs and SSRs from anonymous and transcribed gene regions

To examine the performance and information content of different marker systems, comparative assessment of population genetic diversity was undertaken in nine populations of Athyrium distentifolium using nine genomic and 10 expressed sequence tag (EST) microsatellite (SSR) loci, and 265 amplified fragment length polymorphism (AFLP) loci from two primer combinations. In range-wide comparisons (European vs. North American populations), the EST-SSR loci showed more reliable amplification and produced more easily scorable bands than genomic simple sequence repeats (SSRs). Genomic SSRs showed significantly higher levels of allelic diversity than EST-SSRs, but there was a significant correlation in the rank order of population diversities revealed by both marker types. When AFLPs, genomic SSRs, and EST-SSRs are considered, comparisons of different population diversity metrics/markers revealed a mixture of significant and nonsignificant rank-order correlations. However, no hard incongruence was detected (in no pairwise comparison of populations did different marker systems or metrics detect opposingly significant different amounts of variation). Comparable population pairwise estimates of F(ST) were obtained for all marker types, but whilst absolute values for genomic and EST-SSRs were very similar (F(ST) = 0.355 and 0.342, respectively), differentiation was consistently higher for AFLPs in pairwise and global comparisons (global AFLP F(ST) = 0.496). The two AFLP primer combinations outperformed 18 SSR loci in assignment tests and discriminatory power in phenetic cluster analyses. The results from marker comparisons on A. distentifolium are discussed in the context of the few other studies on natural plant populations comparing microsatellite and AFLP variability.     

Expressed sequence tag-simple sequence repeat-based molecular variance in two Salicornia (Amaranthaceae) populations

Salicornia spp is one of the most salt-tolerant vascular plants and is native to salt marshes and estuaries. We developed expressed sequence tag derived-simple sequence repeat (EST-SSR) markers for estimating genetic diversity and marker-assisted Salicornia breeding. Six polymorphic EST-SSRs of 40 detected 27 alleles, ranging from three to five alleles per locus. The average number of alleles per locus was 4.33 and 4.17, and the major allele frequency at locus DY529765 was high, being 0.859 and 0.857 in S. bigelovii and S. europea, respectively. Gene diversity, heterozygosity and polymorphism information content were highest at locus DY529950 and similar in these two species. Gene diversity increased with increase in the number of alleles that had a low major allele frequency at a locus. Six polymorphic loci effectively discriminated 46 taxa into three clusters via different analyses. Significant deviation of F(ST) from zero in three suggested populations for six loci indicated population differentiation and limited gene flow among them. A reduced median network established that taxon SB65 is primitive. SMART (simple modular architecture research tool) analysis of peptide sequences of six EST-SSRs showed that loci DY529765, DY529950 and EC906203 contained transmembrane, TLC, AgrB and NTR domains and might be involved in salinity stress tolerance. These EST-SSRs are a valuable resource for marker development and may be useful in marker-assisted Salicornia breeding.     

Outlier Loci and Selection Signatures of Simple Sequence Repeats (SSRs) in Flax (Linum usitatissimum L.)

Genomic microsatellites (gSSRs) and expressed sequence tag-derived SSRs (EST-SSRs) have gained wide application for elucidating genetic diversity and population structure in plants. Both marker systems are assumed to be selectively neutral when making demographic inferences, but this assumption is rarely tested. In this study, three neutrality tests were assessed for identifying outlier loci among 150 SSRs (85 gSSRs and 65 EST-SSRs) that likely influence estimates of population structure in three differentiated flax sub-populations (F _ST?=?0.19). Moreover, the utility of gSSRs, EST-SSRs, and the combined sets of SSRs was also evaluated in assessing genetic diversity and population structure in flax. Six outlier loci were identified by at least two neutrality tests showing footprints of balancing selection. After removing the outlier loci, the STRUCTURE analysis and the dendrogram topology of EST-SSRs improved. Conversely, gSSRs and combined SSRs results did not change significantly, possibly as a consequence of the higher number of neutral loci assessed. Taken together, the genetic structure analyses established the superiority of gSSRs to determine the genetic relationships among flax accessions, although the combined SSRs produced the best results. Genetic diversity parameters did not differ statistically (P?>?0.05) between gSSRs and EST-SSRs, an observation partially explained by the similar number of repeat motifs. Our study provides new insights into the ability of gSSRs and EST-SSRs to measure genetic diversity and structure in flax and confirms the importance of testing for the occurrence of outlier loci to properly assess natural and breeding populations, particularly in studies considering only few loci.     

Genetic structure of Quercus rubra L. and Quercus ellipsoidalis E. J. Hill populations at gene-based EST-SSR and nuclear SSR markers

Sympatric hybridizing oak species provide a model system for studying local adaptation. Disjunct populations of Quercus rubra L. and Quercus ellipsoidalis E. J. Hill at the northern edge of their distribution may harbor important reservoirs of adaptive genetic variation. Genic (expressed sequence tag- simple sequence repeat?=?EST-SSR) and non-genic nuclear microsatellite (nuclear SSR?=?nSSR) markers were used to estimate neutral and potentially adaptive genetic variation in these two supposedly interfertile oak species showing different adaptations to drought. Eleven populations of putative Q. rubra and Q. ellipsoidalis located in the Western Upper Peninsula of Michigan were characterized using seven EST-SSRs and eight nSSRs. Bayesian cluster analysis revealed two distinct groups corresponding to each species with evidence of low levels of potential introgression. A comparison of the genetic structure of adult trees and seedlings revealed no evidence for selection against hybrids. Overall, similar levels of genetic variation and differentiation between populations and species were found at both EST-SSRs and nSSRs indicating that most EST-SSRs chosen reflect neutral variation. Two loci, 3A05 (nSSR) and GOT021 (EST-SSR, putative histidine kinase 4-like), were identified as putative outlier loci between species showing largely reduced variation in Q. ellipsoidalis. Future analyses of an increased number of EST-SSRs located in functional genes will allow the identification of genes involved in the reproductive isolation between both species.     

Development of polymorphic EST-derived SSR markers for the shrimp, Fenneropenaeus chinensis

In our study, we evaluated simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) of Fenneropenaeus chinensis. Characteristics of 11 EST-SSR loci were investigated using 34 F. chinensis individuals. The number of alleles per locus ranged from 4 to 9. The observed heterozygosity ranged from 0.118 to 0.826, while the expected heterozygosity ranged from 0.623 to 0.847. Two loci (T2 and T12) conformed to the Hardy–Weinberg equilibrium. There was no LD observed between all pairs of EST-SSRs loci. The PIC values of 11 microsatellite loci were higher than 0.5. These loci and markers will be useful for population genetics and systemic evolution of F. chinensis.     

Exploiting EST databases for the development and characterization of EST-SSRs in the Pacific oyster (Crassostrea gigas)

A total of 147 microsatellite-containing expressed sequence tags (ESTs) (3.63%) were detected from 4053 ESTs of the Pacific oyster (Crassostrea gigas) in GenBank. The average density of simple sequence repeats (SSRs) was 1 per 8.25 kb of EST after redundancy elimination. Dinucleotide repeat motifs appeared to be the most abundant type. Sixteen new polymorphic EST-SSRs were developed. The number of alleles per locus varied from 3 to 12, with an average of 5.9 alleles per locus. Marker transferability was tested on 2 other Crassostrea species, and 14 loci gave successful amplifications in both species. Twenty EST-SSRs were tested on 3 families of C. gigas for examination of inheritance mode of EST-SSRs. Thirty-five tests of segregation ratios revealed 5 significant departures from expected Mendelian ratios, 4 of which confirmed Mendelian expectations when accounting for the presence of null alleles. Null alleles were detected at 3 loci (15.0%) of the 20 loci, and the frequency of null alleles at EST-SSRs was lower than that in genomic SSRs in C. gigas. The results obtained in this study suggest that C. gigas EST-SSRs will complement the currently available genomic SSR markers and may be useful for comparative mapping, marker-assisted selection, and evolutionary studies.     

Exploring the genetic diversity of Ethiopian grass pea (Lathyrus sativus L.) using EST-SSR markers

Expressed sequence tags (ESTs) in public databases and cross-species transferable markers are considered to be a cost-effective means for developing sequence-based markers for less-studied species. In this study, EST-simple sequence repeat (SSR) markers developed from Lathyrus sativus L. EST sequences and cross-transferable EST-SSRs derived from Medicago truncatula L. were utilized to investigate the genetic diversity among grass pea populations from Ethiopia. A total of 45 alleles were detected using eleven EST-SSRs with an average of four alleles per locus. The average polymorphism information content for all primers was 0.416. The average gene diversity was 0.477, ranging from 0.205 for marker Ls942 to 0.804 for MtBA32F05. F(ST) values estimated by analysis of molecular variance were 0.01, 0.15, and 0.84 for among regions, among accessions and within accessions respectively, indicating that most of the variation (84%) resides within accessions. Model-based cluster analysis grouped the accessions into three clusters, grouping accessions irrespective of their collection regions. Among the regions, high levels of diversity were observed in Gojam, Gonder, Shewa and Welo regions, with Gonder region showing a higher number of different alleles. From breeding and conservation aspects, conducting a close study on a specific population would be advisable for genetic improvement in the crop, and it would be appropriate if future collection and conservation plans give due attention to under-represented regions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9662-y) contains supplementary material, which is available to authorized users.     

Development of EST-SSR markers for the study of population structure in lettuce (Lactuca sativa L.)

A set of 61 simple sequence repeat (SSR) markers was developed from the 19,523 Lactuca sativa and Lactuca serriola unigenes. Approximately 4.5% of the unigenes contained a perfect SSR at least 20 bp long, corresponding to roughly 1 perfect SSR per 14.7 kb. Marker polymorphism was tested on a set comprising 96 accessions representing all major horticultural types and 3 wild species (L. serriola, Lactuca saligna, and Lactuca virosa). Both the average marker heterozygosity (UHe = 0.32) and the number of different alleles per locus (Na = 3.56) were significantly reduced in expressed sequence tag (EST)-SSRs as compared with anonymous SSRs (UHe = 0.59, Na = 5.53). Marker transfer rate to the wild species corresponded to the decreasing sexual compatibility with L. sativa and was higher for EST-SSRs (100% L. serriola, 87% L. saligna, and 75% L. virosa) than for anonymous SSRs (93%, 66%, and 42%, respectively). Assessment of population structure among 90 L. sativa cultivars with SSRs was in good agreement with classification into the horticultural types. The average marker heterozygosity was smallest in iceberg (0.097), Latin (0.140), and romaine-type (0.151) cultivars while highest in leaf (green leaf 0.208 and red leaf 0.240) lettuces. The level of marker heterozygosity is in accord with morphological variability observed in different horticultural types.     

SSR and EST-SSR-based genetic linkage map of cassava (Manihot esculenta Crantz)

Simple sequence repeat (SSR) markers provide a powerful tool for genetic linkage map construction that can be applied for identification of quantitative trait loci (QTL). In this study, a total of 640 new SSR markers were developed from an enriched genomic DNA library of the cassava variety 'Huay Bong 60' and 1,500 novel expressed sequence tag-simple sequence repeat (EST-SSR) loci were developed from the Genbank database. To construct a genetic linkage map of cassava, a 100 F(1) line mapping population was developed from the cross Huay Bong 60 by 'Hanatee'. Polymorphism screening between the parental lines revealed that 199 SSRs and 168 EST-SSRs were identified as novel polymorphic markers. Combining with previously developed SSRs, we report a linkage map consisted of 510 markers encompassing 1,420.3?cM, distributed on 23 linkage groups with a mean distance between markers of 4.54?cM. Comparison analysis of the SSR order on the cassava linkage map and the cassava genome sequences allowed us to locate 284 scaffolds on the genetic map. Although the number of linkage groups reported here revealed that this F(1) genetic linkage map is not yet a saturated map, it encompassed around 88% of the cassava genome indicating that the map was almost complete. Therefore, sufficient markers now exist to encompass most of the genomes and efficiently map traits in cassava.     

重要食药同源植物余甘子转录组微卫星特征分析

为全面了解余甘子转录组SSR位点的分布特征和变异规律,本研究利用Illumina Hiseq 4000平台对余甘子叶片转录组进行测序,通过MISA软件对获得的Unigenes进行SSR位点搜索和统计分析。结果发现9 538条包含SSR位点的Unigenes,共检测到9 991个SSR位点,平均每5.49 kB出现1个SSR。单碱基和二碱基为余甘子转录组SSR主要重复类型,分别占SSR总数的42.3%和30.79%。位于基因编码区的SSR位点共有1 731个,出现频率为0.039 SSRs/kB,优势重复类型为三碱基重复。余甘子转录组SSR中共有169种重复基元,其中所占比例最高的是A/T（42.10%）,其次是AG/CT（22.91%）和AAG/CTT（5.02%）。SSR各基元的重复次数波动于4~75次,且多数集中于4~20次。重复片段长度≥ 20 bp的SSR占21.20%,且SSR发生频率与片段长度呈显著负相关（P<0.01）,相关系数为-0.561。本研究获得的余甘子转录组SSR位点出现频率较高、分布密度较大、低级重复基元较多,重复次数较高、长片段较多,大多数SSR位点的多态性潜能较高,用于余甘子遗传多样性分析的潜力较大,为下一步余甘子转录组SSR标记的大规模开发和群体遗传学研究提供了重要的数据信息,进而为余甘子野生资源的保护和合理开发利用提供了参考依据。     

Selection of high heterozygosity popcorn varieties in Brazil based on SSR markers

We analyzed genetic structure and diversity among eight populations of popcorn, using SSR loci as genetic markers. Our objectives were to select SSR loci that could be used to estimate genetic diversity within popcorn populations, and to analyze the genetic structure of promising populations with high levels of heterozygosity that could be used in breeding programs. Fifty-seven alleles (3.7 alleles per locus) were detected; the highest effective number of alleles (4.21) and the highest gene diversity (0.763) were found for the Umc2226 locus. A very high level of population differentiation was found (F(ST) = 0.3664), with F(ST) for each locus ranging from 0.1029 (Umc1664) to 0.6010 (Umc2350). This analysis allowed us to identify SSR loci with high levels of heterozygosity and heterozygous varieties, which could be selected for production of inbred lines and for developing new cultivars.     

Validation and comparison of microsatellite markers derived from Senegalese sole (Solea senegalensis, Kaup) genomic and expressed sequence tags libraries

In this work, we tested 100 potential new microsatellites (SSRs) equally derived from expressed sequence tag (EST) and enriched genomic-DNA libraries from Senegalese sole (Solea senegalensis, Kaup), a valuable cultured flatfish species. A final set of 69 new polymorphic microsatellites were validated after a population analysis, 37 of which corresponded to the first EST library constructed for Senegalese sole (EST-SSR). Although differences were not significant, EST sequences provided a higher proportion of quality markers (74%) than anonymous ones (64%). Most of the rejected anonymous SSRs (17 loci) were discarded because they did not generate PCR products; only one was monomorphic. On the contrary, all EST-SSRs gave PCR products, although monomorphism was more frequent (26%). Altogether, the number of alleles per locus was fairly similar in both SSR types, ranging from 2 to 19. The observed and expected heterozygosities varied from 0.105 to 1 and from 0.108 to 0.937, respectively. The main difference between the two sets was the percentage of annotated loci, being higher in EST-SSRs, as expected. Within the EST-SSRs, 46% of them showed flanking regions that significantly matched with EST sequences from other three flatfish species; however, the microsatellite itself was present only on half of these cases. These two new SSR sets constitute a suitable tool for fingerprinting, gene flow, genetic diversity, genome mapping studies and molecular-assisted breeding in this species.     

Assessment of genetic diversity and population structure of Chinese wild almond, Amygdalus nana, using EST- and genomic SSRs

Amygdalus nana L., commonly known as wild almond, is an endangered wild relative of cultivated almond, which has great potential in almond crop breeding. In this study, we used microsatellite (SSR) loci derived from both expressed sequence tag (EST) and anonymous genomic sequence to explore the genetic diversity and population structure of A. nana in Xinjiang of China. Seven natural populations were collected across the whole distribution of A. nana in China, including populations from both inside (four populations) and outside (three populations) the established protected areas. A total of 22 and 19 alleles were detected from the seven pairs of EST and genomic SSR loci, respectively. Generally, the genomic SSRs showed lower levels of variation than EST-SSRs, which may partially due to the higher cross-species transferability in EST-SSRs than in genomic SSRs. The population-level genetic diversity (A = 1.84, P = 50.00%, H_o = 0.3491, H_E = 0.2271) was lower than cultivated almond and several wild fruit species with similar breeding system. Most of the genetic variation (82.16%) was partitioned within populations. In particular, the population collected from Tacheng County (outside the protected areas) had the highest levels of genetic diversity and had significantly different genetic constitution from other populations.     

Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat

Genetic variation present in 64 durum wheat accessions was investigated by using three sources of microsatellite (SSR) markers: EST-derived SSRs (EST-SSRs) and two sources of SSRs isolated from total genomic DNA. Out of 245 SSR primer pairs screened, 22 EST-SSRs and 20 genomic-derived SSRs were polymorphic and used for genotyping. The EST-SSR primers produced high quality markers, but had the lowest level of polymorphism (25%) compared to the other two sources of genomic SSR markers (53%). The 42 SSR markers detected 189 polymorphic alleles with an average number of 4.5 alleles per locus. The coefficient of similarity ranged from 0.28 to 0.70 and the estimates of similarity varied when different sources of SSR markers were used to genotype the accessions. This study showed that EST-derived SSR markers developed in bread wheat are polymorphic in durum wheat when assaying loci of the A and B genomes. A minumum of ten EST-SSRs generated a very low probability of identity (0.36×10⁻¹²) indicating that these SSRs have a very high discriminatory power. EST-SSR markers directly sample variation in transcribed regions of the genome, which may enhance their value in marker-assisted selection, comparative genetic analysis and for exploiting wheat genetic resources by providing a more-direct estimate of functional diversity. Received: 19 December 2000 / Accepted: 17 April 2001     

Selection mapping of loci for quantitative disease resistance in a diverse maize population

The selection response of a complex maize population improved primarily for quantitative disease resistance to northern leaf blight (NLB) and secondarily for common rust resistance and agronomic phenotypes was investigated at the molecular genetic level. A tiered marker analysis with 151 simple sequence repeat (SSR) markers in 90 individuals of the population indicated that on average six alleles per locus were available for selection. An improved test statistic for selection mapping was developed, in which quantitative trait loci (QTL) are identified through the analysis of allele-frequency shifts at mapped multiallelic loci over generations of selection. After correcting for the multiple tests performed, 25 SSR loci showed evidence of selection. Many of the putatively selected loci were unlinked and dispersed across the genome, which was consistent with the diffuse distribution of previously published QTL for NLB resistance. Compelling evidence for selection was found on maize chromosome 8, where several putatively selected loci colocalized with published NLB QTL and a race-specific resistance gene. Analysis of F(2) populations derived from the selection mapping population suggested that multiple linked loci in this chromosomal segment were, in part, responsible for the selection response for quantitative resistance to NLB.     

Genetic analysis of Phytophthora infestans populations in the Nordic European countries reveals high genetic variability

Late blight, caused by the oomycete Phytophthora infestans, is the most important disease of potato (Solanum tuberosum). The pathogen is highly adaptable and to get an overview of the genetic variation in the Nordic countries, Denmark, Finland, Norway and Sweden we have analyzed 200 isolates from different fields using nine simple-sequence repeat (SSR) markers. Forty-nine alleles were detected among the nine SSR loci and isolates from all four Nordic countries shared the most common alleles across the loci. In total 169 multilocus genotypes (based on seven loci) were identified among 191 isolates. The genotypic diversities, quantified by a normalized Shannon's diversity index (H(s)), were 0.95 for the four Nordic countries. The low F(ST) value of 0.04 indicates that the majority of variation is found within the four Nordic countries. The large number of genotypes and the frequency distribution of mating types (60% A1) support the hypothesis that sexual reproduction is contributing notably to the genetic variation of P. infestans in the Nordic countries.     

Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling

Background

Genetic markers and linkage mapping are basic prerequisites for marker-assisted selection and map-based cloning. In the case of the key grassland species Lolium spp., numerous mapping populations have been developed and characterised for various traits. Although some genetic linkage maps of these populations have been aligned with each other using publicly available DNA markers, the number of common markers among genetic maps is still low, limiting the ability to compare candidate gene and QTL locations across germplasm.

Results

A set of 204 expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers has been assigned to map positions using eight different ryegrass mapping populations. Marker properties of a subset of 64 EST-SSRs were assessed in six to eight individuals of each mapping population and revealed 83% of the markers to be polymorphic in at least one population and an average number of alleles of 4.88. EST-SSR markers polymorphic in multiple populations served as anchor markers and allowed the construction of the first comprehensive consensus map for ryegrass. The integrated map was complemented with 97 SSRs from previously published linkage maps and finally contained 284 EST-derived and genomic SSR markers. The total map length was 742 centiMorgan (cM), ranging for individual chromosomes from 70 cM of linkage group (LG) 6 to 171 cM of LG 2.

Conclusions

The consensus linkage map for ryegrass based on eight mapping populations and constructed using a large set of publicly available Lolium EST-SSRs mapped for the first time together with previously mapped SSR markers will allow for consolidating existing mapping and QTL information in ryegrass. Map and markers presented here will prove to be an asset in the development for both molecular breeding of ryegrass as well as comparative genetics and genomics within grass species.     

基于RAD-seq技术的异型花SSR信息分析

用RAD-seq（restriction-site associated DNA sequencing）对异型花（Sinoswertia tetraptera（Maxiowicz） T.N.Ho,S.W.Liu & J.Q.Liu）进行简化基因组测序,并借此分析异型花的SSR（simple sequence repeats）信息。利用SR search软件甄别所得序列中的SSR,得到了双端各有至少100 bp的SSR位点5 844个,其中5 339个（91.38%）成功设计引物,而三核苷酸SSR位点最多（3 323个）;在能成功设计引物的SSR位点中,重复序列长度包括17种（12~36 bp）;重复序列的基序共277种,其中五核苷酸基序种类最多（106种）;随机挑选10对SSR引物,用4个异型花居群的32个个体检测检测其可用性和多态性,经PCR和聚丙烯酰氨凝胶电泳检测,有4对（ST2、ST3、ST6和ST10）成功扩增并表现出多态性;经GENEPOP 4.4对4个位点分析,显示其等位基因数量均值为6,多态性较高且不连锁（P<0.01）;4个位点在多数居群中偏离哈迪温伯格平衡（P<0.01）且存在较高的纯合子数量（观测杂合度均值0.023）,该结果归因于异型花主要进行自花授粉,在自然界中很难形成进行自由交配的居群;此外,ST2和ST6可在椭圆叶花锚（Halenia elliptica D.Don）中成功扩增,具有潜在通用性。本研究将为日后基于异型花SSR标记的相关研究提供数据库支持。