Characterization of nuclear microsatellites in New Caledonian Araucaria species

Thirteen of the world's 19 species of Araucaria are endemic to the Pacific Ocean island of New Caledonia. In order to investigate the evolutionary biological processes underlying the radiation of the genus on the island we have developed a set of nuclear microsatellite loci. Using a membrane enrichment procedure, five loci have been developed (four derived from A. subulata DNA and one from A. rulei DNA) which amplified in A. columnaris. PCR products of the expected size were also produced in a very limited sample of eight other New Caledonian Araucaria species tested.     

由国分类学文献中南洋杉属两种之订正

通过调查,对Araucaria cunninghamii和A.heterophylla的选模式和中国分类学文献(如<广州植物志>、<中国树木学>、<中国高等植物图鉴>、<中国植物志>、<中国树木志>、、<中国高等植物>)的A.cunninghamii和A.heterophylla(或A.excelsa)作比较,证实中国分类学文献中的A.cunninghamii和A.heterophylla(或A.excelsa)分别是A.heterophylla(Salisb.)Franco和A.cunninghamii Aiton ex A.Cunn..这两种植物的叶形变化较大,但有明显的区别:诺福克南洋杉A.heterophylla幼树的末级小枝的叶细长呈线形、叶尖急尖,牛长角小于45°,腹面无明显的脊,成龄株的末级小枝的叶扁平呈鳞片状;南洋杉A.cunninghamii幼树的末级小枝的叶呈两侧扁的钻形、叶尖短刺状,生长角45°-90°,腹背两面具明显的脊,成龄株的末级小枝的叶呈四棱状钻形.     

Development and polymorphism of microsatellite markers for Fagus crenata and the closely related species, F. japonica

 We have developed microsatellite markers (SSRs) applicable to Fagus crenata using the RAHM method and investigated their polymorphisms. We also applied the SSRs in an analysis of a closely related species, F. japonica. Here we describe the isolation and characterization of nine polymorphic microsatellite markers, of which eight are applicable to both species. Among 30 individuals of each of F. crenata and F. japonica we detected a total of 79 and 77 alleles, respectively, with an average of 9.9 and 8.6 alleles per locus. The mean expected heterozygosity (He) was 0.615 (range: 0.216–0.925) in F. crenata and 0.660 in F. japonica (range: 0.259–0.827). The He values were considerably higher than those previously found for isozymes. Paternity exclusion probabilities for multiple loci, calculated over all loci, were extremely high (0.999 and 0.998 in F. crenata and F. japonica, respectively): sufficiently high to study pollen flow in both species. Received: 5 December 1998 / Accepted: 28 December 1998     

Characterisation and analysis of microsatellite loci in a mangrove species, Avicennia marina (Forsk.) Vierh. (Avicenniaceae)

An enriched microsatellite library of the mangrove species Avicennia marina was constructed, in which 85.8% of the clones contained microsatellite sequences. Of the microsatellite repeat sequences isolated, 55.0% were di-nucleotides, 34.2% were tri-nucleotides, 50.0% were perfect, 24.2% were imperfect, and 15.0% were compound. Four different di-nucleotide repeats were isolated with repeat lengths ranging from 5 to 33; ten different tri-nucleotide repeats were isolated with repeat lengths ranging from 3 to 25. The most common di-nucleotide was the AC/TG repeat; the most common tri-nucleotide was the CCG/GGC repeat. Sixteen microsatellite sequences were selected for primer design, and 6 primers were selected to investigate the polymorphism detected among 15 individuals of A. marina from three natural populations in Australia. A total of 40 alleles were detected at 6 microsatellite loci. The number of alleles per microsatellite locus ranged from 5 to 13. On average, 7 alleles were detected per locus. All microsatellite loci showed high levels of gene diversity (heterozygosity), with values ranging from 0.53 to 0.88; the mean value of gene diversity was 0.70. Microsatellite loci were also tested for conservation across Avicennia species. There was a decline in amplification success with increasing divergence between Avicennia species. The results indicate that microsatellites are abundant in the Avicennia genome and can be valuable genetic markers for assessing the effects of deforestation and forest fragmentation in mangrove communities, which is an important issue for mangrove conservation and afforestation schemes. Received: 8 June 1999 / Accepted: 21 September 1999     

Development of microsatellite markers for white spruce (Picea glauca) and related species

We report the development of 13 primer pairs that allow the unambiguous amplification of 15 microsatellite (SSR) loci in white spruce (Picea glauca). Fourteen of these loci were polymorphic in trees sampled at three geographically separated regions of western Canada. Segregation analysis carried out on these loci confirmed a Mendelian inheritance pattern for all except two, which showed significant segregation distortion. All of these primer pairs amplified SSR loci in at least one of the other Picea species tested [black spruce (P. mariana), red spruce (P. rubens), Norway spruce (P. abies), Colorado spruce (P. pungens), sitka spruce (P. sitchensis) and Engelmann spruce (P. engelmannii)]. Given the important commercial and ecological roles of these species, this set of markers will be invaluable for their management, the improvement of commercially important traits, and the study of their ecology and genetics. Received: 18 August 2000 / Accepted: 28 September 2000     

Highly polymorphic microsatellites of rice consist of AT repeats, and a classification of closely related cultivars with these microsatellite loci

Microsatellites consisting of AT repeats are highly polymorphic in rice genomes and can be used to distinguish between even closely related japonica cultivars in Japan. Polymorphisms of 20 microsatellite loci were determined using 59 japonica cultivars, including both domestic and modern Japanese cultivars. Although the polymorphisms of these 20 microsatellite loci indicated that the Japanese cultivars were genetically quite similar, microsatellites consisting of AT repeats showed high gene diversity even among such closely related cultivars. Combinations of these hypervariable microsatellites can be employed to classify individual cultivars, since the microsatellites were stable within each cultivar. An identification system based on these highly polymorphic microsatellites could be used to maintain the purity of rice seeds by eliminating contamination. A parentage diagnosis using 17 polymorphic microsatellite loci clearly demonstrated that plants which carried desired chromosome regions had been selected in breeding programs. Thus, these hypervariable microsatellites consisting of AT repeats should promote the selection of plants which carry desired chromosomes from genetically similar parents. Backcrossing could also help to eliminate unnecessary chromosome regions with microsatellite polymorphisms at an early stage in breeding programs. Received: 8 July 1996 / Accepted: 12 July 1996     

Characterization of 12 microsatellite loci of the human MHC in a panel of reference cell lines

 The human genome contains a large number of interspersed microsatellite repeats which exhibit a high degree of polymorphism and are inherited in a Mendelian fashion, making them extremely useful genetic markers. Several microsatellites have been described in the HLA region, but allele nomenclature, a set of broadly distributed controls, and typing methods have not been standardized, which has resulted in discrepant microsatellite data between laboratories. In this report we present a detailed protocol for genotyping microsatellites using a semi-automated fluorescence-based method. Twelve microsatellites within or near the major histocompatibility complex (MHC) were typed in the 10th International Histocompatibility Workshop homozygous typing cell lines (HTCs) and alleles were designated based on size. All loci were sequenced in two HTCs providing some information on the level of complexity of the repeat sequence. A comparison of allele size obtained by genotyping versus that obtained by direct sequencing showed minor discrepancies in some cases, but these were not unexpected given the technical differences in the methodologies. Fluorescence-based typing of microsatellites in the MHC described herein is highly efficient, accurate, and reproducible, and will allow comparison of results between laboratories. Received: 10 May 1997 / Revised: 1 August 1997     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

Characterization and analysis of microsatellite loci in Elymus caninus (Triticeae: Poaceae)

 Microsatellites are highly variable DNA sequences that can be used as markers for the genetic analysis of plants. The potential of microsatellite markers for use in a genetic diversity study in Elymus species was evaluated. Genomic libraries of Elymus caninus were constructed. The libraries were screened with two dinucleotide, (GA)n and (GT)n, and two trinucleotide repeats, (TCT)n and (CAC)n. A total of 19 positive clones were found for the two dinucleotide repeats; no positive clone was found for the trinucleotide repeats. Positive clones were sequenced to confirm the presence of microsatellites and to generate polymerase chain reaction (PCR) primers based on the sequences flanking the microsatellite. All sequenced (GA)n clones have repeats of n>10; over half of the (GT)n microsatellites have n<10 repeats. Primer pairs were designed and evaluated for 8 selected microsatellites. PCR products were amplified from 15 Elymus caninus accessions. The number of alleles found for the eight loci varied from 1 for ECGA89 and ECGT35 to 13 for ECGA22, as determined by non-denaturing polyacrylamide electrophoresis. Six microsatellite loci were found to be polymorphic in E. caninus. The eight primer pairs were tested on three other species; seven were successful in amplifying DNA from Elymus alaskanus and E. mutabilis, and four amplified DNA from E. caucasicus. Based on these results, microsatellites appear to be useful markers in detecting variation in E. caninus. Received: 8 September 1997/Accepted: 6 October 1997     

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG) _n , (AG/TC) _n or (AAG/TTC) _n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars. Received: 1 November 1999 / Accepted: 14 April 2000     

Characterization of novel microsatellite loci in the great leaf-nosed bat, Hipposideros armiger and cross-amplification in other related species

Eight microsatellite loci were isolated from an enriched genomic library of the great leaf-nosed bat, Hipposideros armiger. The polymorphism of these loci was tested on a population of 48 individuals from Anhui Province, China. All loci revealed the polymorphism ranging from three to 12 alleles. The observed heterozygosity values were from 0.213 to 0.875 and expected heterozygosity from 0.232 to 0.820. No significant linkage disequilibrium was detected. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction. In addition, successful cross-amplification also suggested that these microsatellite markers will facilitate research on the population genetics and gene flow of H. armiger and other related species.     

棕果蝠微卫星位点的筛选及其对近缘种的通用性

棕果蝠在我国热带和亚热带地区分布广、种群数量大,对龙眼、荔枝等水果种植业造成巨大危害。近年来,棕果蝠在国内被大范围捕杀,造成其种群数量急剧下降。为了获得用于研究棕果蝠的种群结构和遗传多样性的分子标记,我们利用探针富集法构建了棕果蝠部分微卫星基因组文库,筛选出棕果蝠的4 个高度多态性位点;并结合前期本研究组已经发表的棕果蝠微卫星位点,共检测了10 个棕果蝠特异的微卫星标记在其它4 种果蝠(短耳犬蝠、犬蝠、大长舌果蝠、小长舌果蝠)内的通用性,为不同果蝠种群遗传学研究提供了更多的微卫星分子标记。     

Development and characterization of microsatellite markers in black poplar (Populus nigra L.)

Using an enrichment procedure, we have cloned and sequenced microsatellite loci from black poplar (Populus nigra L.) and developed primers for sequence-tagged microsatellite (STMS) analysis. Twelve primer pairs for dinucleotide repeats produced fragments of sufficient quality which were polymorphic in P. nigra. Some of them also showed amplification in other Populus species (P. deltoides, P. tricocarpa, P. tremula, P. tremuloides, P. candicans, and/or P. lasiocarpa). The best nine and (GT) (GA) microsatellite markers were tested on a set of 23 P. nigra genotypes from all over Europe. The microsatellites were highly polymorphic, with 10–19 different alleles per microsatellite locus among these 23 genotypes. WPMS08 sometimes amplified three fragments. Using the other eight marker loci, the level of heterozygosity among the plants was on average 0.71 (range 0.25–1.00). The microsatellite markers developed will be useful for screening the genetic diversity in natural populations and in gene bank collections. Received: 21 October 1999 / Accepted: 24 November 1999     

Nuclear DNA contents of all species of Helleborus (Ranunculaceae) discriminate between species and sectional divisions

 Genome size (C-values) and pollen viability staining were applied as new criteria to investigate the species of the genus Helleborus Linnaeus (Ranunculaceae). All species have the same chromosome number (2n=32). However, the nuclear DNA content, as measured by flow cytometry with propidium iodide, could be demonstrated to range between 19 pg to 35.7 pg. The different genome sizes of the species coincided to a large extent with earlier determined section boundaries based on morphology. Flow cytometry can be a convenient method to discriminate between some species. Received April 17, 2001 Accepted May 7, 2001     

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     

Sequence variations at a complex microsatellite locus in rice and its conservation in cereals

In an attempt to study changes associated with microsatellites in rice, the DNAs of cultivated rice, including indica and japonica varieties, and wild rice genotypes were amplified by the polymerase chain reaction with primers flanking the (GATA) _n and (AC) _n repeats at a microsatellite-containing locus OS1E6 (Genebank accession number AFO16647) previously reported from a PstI rice (var. Malkolam) genomic library in pUC18. Eight alleles of varying sizes were obtained which were cloned and sequenced. Sequencing data indicated that the size variations of the different alleles were due to differences in the repeat number as well as to sequence variations in the region flanking the microsatellite motifs. In order to study the presence of this complex microsatellite-containing locus of rice in different cereals, their DNAs were amplified using primers flanking the OS1E6 locus. It was found that this locus was present in the various cereal genotypes analyzed, indicating its conservation across different cereal members. Received: 10 March 2000 / Accepted: 14 April 2000     

Chimerism in grapevines: implications for cultivar identity,ancestry and genetic improvement

In the course of DNA profiling of grapevine cultivars using microsatellite loci we have occasionally observed more than two alleles at a locus in some individuals and have identified periclinal chimerism as the source of such anomalies. This phenomenon in long-lived clonally propagated crops, such as grapevine, which contains historically ancient cultivars, may have a role in clonal differences and affect cultivar identification and pedigree analysis. Here we show that when the two cell layers of a periclinal chimera, Pinot Meunier, are separated by passage through somatic embryogenesis the regenerated plants not only have distinct DNA profiles which are different from those of the parent plant but also have novel phenotypes. Recovery of these phenotypes indicates that additional genetic differences can exist between the two cell layers and that the Pinot Meunier phenotype is due to the interaction of genetically distinct cell layers. It appears that grapevine chimerism can not only modify phenotype but can also impact on grapevine improvement as both genetic transformation and conventional breeding strategies separate mutations in the L1 and L2 cell layers. Received: 14 March 2001 / Accepted: 22 May 2001     

Characterization of 13 polymorphic microsatellite loci in the Japanese land leech

We developed 13 polymorphic microsatellite loci of the Japanese land leech (Haemadipsa japonica; Haemadipsidea) using an Illumina MiSeq sequencing approach. A total of 42,064 nuclear DNA contigs were filtered for microsatellite motifs, among which 30,873 simple sequence repeat loci were identified. From these sequences, we selected 30 primer sets, and 13 of these loci were successfully amplified. Polymorphism of the 13 loci was tested using 16 individuals sampled from sixteen populations across Japan. The number of alleles and polymorphism information content varied from 5 to 17 and 0.335 to 0.883, respectively, and observed and expected heterozygosity values ranged from 0.143 to 0.875 and 0.349 to 0.893, respectively, indicating that these loci are polymorphic. Furthermore, we established useful multiplex PCR using these loci. The 13 microsatellite loci described in this paper are the first nuclear microsatellite markers for a land leech species.