Genetic effects on microsatellite diversity in wild emmer wheat (Triticum dicoccoides) at the Yehudiyya microsite,Israel

This study investigated allele size constraints and clustering, and genetic effects on microsatellite (simple sequence repeat, SSR) diversity at 28 loci comprising seven types of tandem repeated dinucleotide motifs in a natural population of wild emmer wheat, Triticum dicoccoides, from a shade vs sun microsite in Yehudiyya, northeast of the Sea of Galilee, Israel. It was found that allele distribution at SSR loci is clustered and constrained with lower or higher boundary. This may imply that SSR have functional significance and natural constraints. Genetic factors, involving genome, chromosome, motif, and locus significantly affected SSR diversity. Genome B appeared to have a larger average repeat number (ARN), but lower variance in repeat number (sigma(ARN)(2)), and smaller number of alleles per locus than genome A. SSRs with compound motifs showed larger ARN than those with perfect motifs. The effects of replication slippage and recombinational effects (eg, unequal crossing over) on SSR diversity varied with SSR motifs. Ecological stresses (sun vs shade) may affect mutational mechanisms, influencing the level of SSR diversity by both processes.     

SSR allelic variation in almond (Prunus dulcis Mill.)

Sixteen SSR markers including eight EST-SSR and eight genomic SSRs were used for genetic diversity analysis of 23 Chinese and 15 international almond cultivars. EST- and genomic SSR markers previously reported in species of Prunus, mainly peach, proved to be useful for almond genetic analysis. DNA sequences of 117 alleles of six of the 16 SSR loci were analysed to reveal sequence variation among the 38 almond accessions. For the four SSR loci with AG/CT repeats, no insertions or deletions were observed in the flanking regions of the 98 alleles sequenced. Allelic size variation of these loci resulted exclusively from differences in the structures of repeat motifs, which involved interruptions or occurrences of new motif repeats in addition to varying number of AG/CT repeats. Some alleles had a high number of uninterrupted repeat motifs, indicating that SSR mutational patterns differ among alleles at a given SSR locus within the almond species. Allelic homoplasy was observed in the SSR loci because of base substitutions, interruptions or compound repeat motifs. Substitutions in the repeat regions were found at two SSR loci, suggesting that point mutations operate on SSRs and hinder the further SSR expansion by introducing repeat interruptions to stabilize SSR loci. Furthermore, it was shown that some potential point mutations in the flanking regions are linked with new SSR repeat motif variation in almond and peach. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

水稻蜡质基因引导区的两个SSR序列与直链淀粉含量的相关性(英文)

分析了蜡质基因引导区的两个简单重复序列 (SSR) (CT) n 和 (AATT) n 在 74份水稻材料中的多态性及其与直链淀粉含量 (AC)的关系。这些材料包括了籼稻 (OryzasativaL .ssp .indica)、粳稻 (O .sativassp .japonica)和普通野生稻 (O .rufipogon) ,其AC值覆盖了栽培稻AC分布的整个范围。以 (CT) n 作标记检测到 8个等位基因 ,粳稻品种趋于含有重复数目较多 (n≥ 16 )的等位基因 ,重复次数较少 (n≤ 14)的等位基因只出现在籼稻中。 (AATT)n检测到 2个等位基因 ,野生稻中少数植株表现出杂合性。分析表明AC与这两个SSR序列基因型高度相关 ,高AC (>2 2 .0 % )品种具有 (CT)重复次数较少 (n≤ 14)的等位基因 ;相反 ,除了糯米外 ,所有低或者中等AC的品种都有 (CT)重复数较多 (n≥ 16 )的等位基因。具有重复次数较多的 (AATT) 6等位基因的品种多为高AC ,具有重复次数较少的(AATT) 5等位基因的品种多为低或中等AC。不同SSR基因型品种间AC差异极显著。虽然目前还不能确定这两个SSR序列在直链淀粉合成中的直接功能 ,SSR变异与AC间近乎完全的相关性可作为分子标记直接用于水稻的品质改良。     

Correlation of Simple Sequence Repeat (SSR) Variants in the Leader Sequence of the waxy Gene with Amylose Content of the Grain in Rice

Variation of two simple sequence repeats (SSRs) in the leader region of the waxy gene was analyzed in a sample of 74 accessions, including Oryza sativa L. ssp. indica, japonica and wild rice ( O.rufipogon) representing a wide distribution range of amylose content (AC) in cultivated rice. Eight alleles were detected in the (CT)n motif and two alleles were resolved in the (AATT)n motif. The distribution of the alleles of the two SSRs was quite uneven as detected by the (CT)n motif. The repeat numbers of the two SSR motifs, (CT)n and (AATT)n, appeared to be inversely related such that the total length of this region was maintained. AC of the varieties was highly correlated with the length of SSRs. Differences in AC among the various SSR genotypes were statistically highly significant as analyzed using genotypes of both SSR motifs. Although the SSR variation did not seem to have obvious function in the synthesis of the starch synthase encoded by the waxy gene, the almost perfect correlation between the two SSRs and AC level could be used for quality improvement in rice breeding programs.     

Distribution and localization of microsatellites in the Perigord black truffle genome and identification of new molecular markers

The level of genetic diversity and genetic structure in the Perigord black truffle (Tuber melanosporum Vittad.) has been debated for several years, mainly due to the lack of appropriate genetic markers. Microsatellites or simple sequence repeats (SSRs) are important for the genome organisation, phenotypic diversity and are one of the most popular molecular markers. In this study, we surveyed the T. melanosporum genome (1) to characterise its SSR pattern; (2) to compare it with SSR patterns found in 48 other fungal and three oomycetes genomes and (3) to identify new polymorphic SSR markers for population genetics. The T. melanosporum genome is rich in SSRs with 22,425 SSRs with mono-nucleotides being the most frequent motifs. SSRs were found in all genomic regions although they are more frequent in non-coding regions (introns and intergenic regions). Sixty out of 135 PCR-amplified mono-, di-, tri-, tetra, penta, and hexa-nucleotides were polymorphic (44%) within black truffle populations and 27 were randomly selected and analysed on 139 T. melanosporum isolates from France, Italy and Spain. The number of alleles varied from 2 to 18 and the expected heterozygosity from 0.124 to 0.815. One hundred and thirty-two different multilocus genotypes out of the 139 T. melanosporum isolates were identified and the genotypic diversity was high (0.999). Polymorphic SSRs were found in UTR regulatory regions of fruiting bodies and ectomycorrhiza regulated genes, suggesting that they may play a role in phenotypic variation. In conclusion, SSRs developed in this study were highly polymorphic and our results showed that T. melanosporum is a species with an important genetic diversity, which is in agreement with its recently uncovered heterothallic mating system.     

An analysis of genetic diversity across the maize genome using microsatellites

How domestication bottlenecks and artificial selection shaped the amount and distribution of genetic variation in the genomes of modern crops is poorly understood. We analyzed diversity at 462 simple sequence repeats (SSRs) or microsatellites spread throughout the maize genome and compared the diversity observed at these SSRs in maize to that observed in its wild progenitor, teosinte. The results reveal a modest genome-wide deficit of diversity in maize relative to teosinte. The relative deficit of diversity is less for SSRs with dinucleotide repeat motifs than for SSRs with repeat motifs of more than two nucleotides, suggesting that the former with their higher mutation rate have partially recovered from the domestication bottleneck. We analyzed the relationship between SSR diversity and proximity to QTL for domestication traits and observed no relationship between these factors. However, we did observe a weak, although significant, spatial correlation for diversity statistics among SSRs within 2 cM of one another, suggesting that SSR diversity is weakly patterned across the genome. Twenty-four of 462 SSRs (5%) show some evidence of positive selection in maize under multiple tests. Overall, the pattern of genetic diversity at maize SSRs can be explained largely by a bottleneck effect with a smaller effect from selection.     

基于转录组测序信息的水生植物莕菜SSR标记开发

利用软件MISA对水生植物莕菜转录组测序所获得的79536条EST序列进行分析,共检测出12319个EST-SSR位点。在莕菜的EST-SSR中,二核苷酸和三核苷酸重复单元是主导类型,分别占总SSR的57.31%和30.87%,其中AG/CT、AAG/CTT分别是二、三重复单元类型的优势重复基元,分别占总SSR的29.76%和8.66%。随机挑选了130对EST-SSR引物对莕菜两个居群进行遗传多样性检测,结果发现：78对引物能扩增出清晰可分辨的条带,其中37对能成功检测出多态性,引物多态率为 47.44%。这些多态性引物共检测出114个等位基因,每个位点2~6个,平均3.08个。观测杂合度（H_o）及预期杂合度（H_e）分别在0.229~1.000和0.351~0.756之间,多态信息含量PIC值在0.286~0.698之间,平均达0.495。以上研究结果表明,通过莕菜转录组测序产生的EST数据来开发SSR标记是一种简单而高效的途径,这些新的SSR分子标记为研究莕菜的居群遗传多样性及其遗传结构提供了工具。     

Development and Application of Microsatellites in Candidate Genes Related to Wood Properties in the Chinese White Poplar (Populus tomentosa Carr.)

Gene-derived simple sequence repeats (genic SSRs), also known as functional markers, are often preferred over random genomic markers because they represent variation in gene coding and/or regulatory regions. We characterized 544 genic SSR loci derived from 138 candidate genes involved in wood formation, distributed throughout the genome of Populus tomentosa, a key ecological and cultivated wood production species. Of these SSRs, three-quarters were located in the promoter or intron regions, and dinucleotide (59.7%) and trinucleotide repeat motifs (26.5%) predominated. By screening 15 wild P. tomentosa ecotypes, we identified 188 polymorphic genic SSRs with 861 alleles, 2–7 alleles for each marker. Transferability analysis of 30 random genic SSRs, testing whether these SSRs work in 26 genotypes of five genus Populus sections (outgroup, Salix matsudana), showed that 72% of the SSRs could be amplified in Turanga and 100% could be amplified in Leuce. Based on genotyping of these 26 genotypes, a neighbour-joining analysis showed the expected six phylogenetic groupings. In silico analysis of SSR variation in 220 sequences that are homologous between P. tomentosa and Populus trichocarpa suggested that genic SSR variations between relatives were predominantly affected by repeat motif variations or flanking sequence mutations. Inheritance tests and single-marker associations demonstrated the power of genic SSRs in family-based linkage mapping and candidate gene-based association studies, as well as marker-assisted selection and comparative genomic studies of P. tomentosa and related species.     

山地虎耳草转录组SSR信息分析

利用MISA（MicroSatellite）软件对山地虎耳草转录组拼接序列进行微卫星位点信息分析,为后期SSR标记的开发和物种遗传多样性检测提供候选序列。结果发现,在拼接得到的63 763条Unigene序列中含有4 622个SSR,发生频率为7.25%,有110种重复基元,平均每10.00 kB出现一个SSR位点。山地虎耳草转录组序列的SSR主要集中在三核苷酸重复（55.50%）,其次为二核苷酸重复（30.23%）。二核苷酸重复和三核苷酸重复中的优势重复基元分别为AG/TC和AAG/TTC。二核苷酸重复基元的重复次数类型最多,跨度最大,具有更高的多态性,三核苷酸次之,而四、五、六核苷酸重复类型很少。山地虎耳草转录组SSR以5~9次重复为主,且SSR数量随着重复次数的增加逐渐减少,基序长度主要集中于12~30 bp,多态性均在中等以上。     

Characterization of tomato SSR markers developed using BAC-end and cDNA sequences from genome databases

We developed nearly 700 non-redundant 2- or 3-base simple sequence repeat (SSR) markers from tomato using sequence data obtained from open genome databases. Among various types of core motifs, AT was most abundant in SSRs derived from cDNAs (~53%) and bacterial artificial chromosome (BAC) ends (~72%). There was a positive correlation between the rate of detection of polymorphic alleles (heterozygosity value; Hv) and the repeat number of the core motif in all markers showing polymorphisms among at least one pair of six cultivars or lines tested (r = 0.566**). The average Hv of BAC-end-derived SSR markers (~0.5) was higher than that of cDNA-derived markers (~0.3). These characteristics of BAC-end-derived SSRs are useful for genetic studies using closely related cultivars and lines. However, BAC-end-derived SSRs tended to cluster in centromeric regions (~80%). A scheme for the construction of a high-density linkage map of tomato is discussed.     

Genetic diversity in European chestnut populations by means of genomic and genic microsatellite markers

Microsatellite or simple sequence repeats (SSRs) are one of the most used markers in population genetic studies. SSR markers developed from expressed sequence tags (EST) have proved useful to examine functional diversity in relation to adaptive variation. The information provided by both genomic and genic microsatellite markers could offer more accurate indication on the distribution of the genetic diversity among and within populations assuming different evolutionary drivers. This is the first study on chestnut (Castanea sativa Mill.) in which the genetic diversity was evaluated by means of genomic (SSRs) and genic (EST-SSRs) microsatellite markers. We genotyped nine natural European chestnut populations distributed throughout representative areas of contrasting climatic conditions in the Mediterranean basin. Genomic SSRs showed significantly higher levels of diversity in terms of number of alleles, effective number of alleles, expected heterozygosity and level of polymorphism. Furthermore, there were significant differences in the level of differentiation among populations. The UPGMA analysis revealed different clustering pattern between populations, being the grouping according to geographic distances in the case of genomic SSRs and two differentiated groups based on the northern–southern distribution of the populations for EST-SSRs. Furthermore, the EST-SSR transferability among related Castanea and Quercus species was stated. Our results confirm that combining genomic SSRs and EST-SSRs is a useful tool to give complementary information to explain the genetic and adaptive diversity in chestnut.     

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     

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。     

Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

A software tool was developed for the identification of simple sequence repeats (SSRs) in a barley ( Hordeum vulgare L.) EST (expressed sequence tag) database comprising 24,595 sequences. In total, 1,856 SSR-containing sequences were identified. Trimeric SSR repeat motifs appeared to be the most abundant type. A subset of 311 primer pairs flanking SSR loci have been used for screening polymorphisms among six barley cultivars, being parents of three mapping populations. As a result, 76 EST-derived SSR-markers were integrated into a barley genetic consensus map. A correlation between polymorphism and the number of repeats was observed for SSRs built of dimeric up to tetrameric units. 3'-ESTs yielded a higher portion of polymorphic SSRs (64%) than 5'-ESTs did. The estimated PIC (polymorphic information content) value was 0.45 +/- 0.03. Approximately 80% of the SSR-markers amplified DNA fragments in Hordeum bulbosum, followed by rye, wheat (both about 60%) and rice (40%). A subset of 38 EST-derived SSR-markers comprising 114 alleles were used to investigate genetic diversity among 54 barley cultivars. In accordance with a previous, RFLP-based, study, spring and winter cultivars, as well as two- and six-rowed barleys, formed separate clades upon PCoA analysis. The results show that: (1) with the software tool developed, EST databases can be efficiently exploited for the development of cDNA-SSRs, (2) EST-derived SSRs are significantly less polymorphic than those derived from genomic regions, (3) a considerable portion of the developed SSRs can be transferred to related species, and (4) compared to RFLP-markers, cDNA-SSRs yield similar patterns of genetic diversity.     

Mining and survey of simple sequence repeats in wheat rust Puccinia sp

The abundance and inherent potential for extensive allelic variations in simple sequence repeats (SSRs) or microsatellites resulted in valuable source for genetic markers in eukaryotes. In this study, we analyzed and compared the abundance and organisation of SSR in the genome of two important fungal pathogens of wheat, brown or leaf rust (Puccinia triticina) and black or stem rust (Puccinia graminis f. sp. tritici). P. triticina genome with two fold genome size as compared to P. graminis tritici has lower relative abundance and SSR density. The distribution pattern of different SSR motifs provides the evidence of greater accumulation of dinucleotide followed by trinucleotide repeats. More than two-hundred different types of repeat motifs were observed in the genomes. The longest SSR motifs varied in both genomes and some of the repeat motifs are found in higher frequency. The information about survey of relative abundance, relative density, length and frequency of different repeat motifs in Puccinia sp. will be useful for developing SSR markers that could find several applications in analysis of fungal genome such as genetic diversity, population genetics, race identification and acquisition of new virulence.     

基于转录组测序信息的水生植物蕃菜SSR标记开发

利用软件MISA对水生植物蓄菜转录组测序所获得的79536条EST序列进行分析,共检测出12319个EST—SSR位点。在蓄菜的EST—SSR中,二核苷酸和三核苷酸重复单元是主导类型,分别占总SSR的57．31％和30．87％,其中AG／CT、AAG／C1_r分别是二、三重复单元类型的优势重复基元,分别占总SSR的29．76％和8．66％。随机挑选了130对EST-SSR引物对蓄菜两个居群进行遗传多样性检测,结果发现：78对引物能扩增出清晰可分辨的条带,其中37对能成功检测出多态性,引物多态率为47．44％。这些多态性引物共检测出114个等位基因,每个位点2—6个,平均3．08个。观测杂合度（心）及预期杂合度（He）分别在0．229～1．000和0．351-0．756之间,多态信息含量PIC值在0．286～O．698之间,平均达0．495。以上研究结果表明,通过萏菜转录组测序产生的EsT数据来开发SSR标记是一种简单而高效的途径,这些新的SSR分子标记为研究苔菜的居群遗传多样性及其遗传结构提供了工具。     

Analysis of simple sequence repeats (SSRs) in wild barley from the Fertile Crescent: associations with ecology,geography and flowering time

Wild barley, Hordeum spontaneum C. Koch, is the progenitor of cultivated barley, Hordeum vulgare. The centre of diversity is in the Fertile Crescent of the Near East, where wild barley grows in a wide range of conditions (temperature, water availability, day length, etc.). The genetic diversity of 39 wild barley genotypes collected from Israel, Turkey and Iran was studied with 33 SSRs of known map location. Analysis of molecular variance (AMOVA) was performed to partition the genetic variation present within from the variation between the three countries of origin. Using classification tree analysis, two (or three) specific SSRs were identified which could correctly classify most of the wild barley genotypes according to country of origin. Associations of SSR variation with flowering time and adaptation to site-of-origin ecology and geography were investigated by two contrasting statistical approaches, linear regression based on SSR length variation and linear regression based on SSR allele class differences. A number of SSRs were significantly associated with flowering time under four different growing regimes (short days, long days, unvernalised and vernalised). Most of the associations observed could be accounted for by close linkage of the SSR loci to earliness per se genes. No associations were found with photoperiodic and vernalisation response genes known to control flowering in cultivated barley suggesting that different genetic factors may be active in wild barley. Novel genomic regions controlling flowering time in wild barley were detected on chromosomes 1HS, 2HL, 3HS and 4HS. Associations of SSRs with site-of-origin ecological and geographic data were found primarily in genomic regions determining plant development. This study shows that the analyses of SSR variation by allele class and repeat length are complementary, and that some SSRs are not necessarily selectively neutral.     

Molecular characterization and genetic diversity analysis of soybean (Glycine max (L.) Merr.) germplasm accessions in India

Molecular characterization and genetic diversity among 82 soybean accessions was carried out by using 44 simple sequence repeat (SSR) markers. Of the 44 SSR markers used, 40 markers were found polymorphic among 82 soybean accessions. These 40 polymorphic markers produced a total of 119 alleles, of which five were unique alleles and four alleles were rare. The allele number for each SSR locus varied between two to four with an average of 2.97 alleles per marker. Polymorphic information content values of SSRs ranged from 0.101 to 0.742 with an average of 0.477. Jaccard’s similarity coefficient was employed to study the molecular diversity of 82 soybean accessions. The pairwise genetic similarity among 82 soybean accessions varied from 0.28 to 0.90. The dendrogram constructed based on genetic similarities among 82 soybean accessions identified three major clusters. The majority of genotypes including four improved cultivars were grouped in a single subcluster IIIa of cluster III, indicating high genetic resemblance among soybean germplasm collection in India.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-014-0266-y) contains supplementary material, which is available to authorized users.     

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs

Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different- colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were sampled. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating independently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once. Received: 3 October 2000 / Accepted: 19 February 2001