Development of species‐specific markers in an organism with endosymbionts: microsatellites in the scleractinian coral Seriatopora hystrix

We report on the development of microsatellites in Seriatopora hystrix, a coral with algal endosymbionts. In order to obtain a genomic library free of algal DNA, we conducted a whole genome preamplification from minute amounts of symbiont‐free tissue. The resulting fragments were cloned into pUC18, and Escherichia coli were transformed with the recombinant plasmids. Twenty‐nine microsatellites were isolated from a library screen with a fluorescently labelled (CA)₁₅ probe. Five of these yielded reliable polymorphic markers.     

正常和环割条件下不同形态氮素添加对红椎幼苗光合特性的影响

采用盆栽试验和韧皮部环割方法研究了碳水化合物供应、氮素形态及其交互作用对红椎幼苗叶片光合特性的影响。无机氮源采用硝酸铵(AN)、铵态氮(NH_4~+-N)、硝态氮(NO_3~--N),有机氮源采用尿素(Urea)、精氨酸(Arg)和甘氨酸(Gly),氮素施用量均为10g N/m~2,处理时间为10 d。研究结果表明,环割、氮素形态及其交互作用均显著影响了红椎幼苗叶片的净光合速率(Pn);单一环割处理显著降低了红椎幼苗叶片Pn、气孔导度(Gs)、胞间CO_2浓度(Ci)、蒸腾速率(Tr)、相对叶绿素含量和CO2利用效率(CUE),但显著提高了叶片水分利用效率(WUE)和气孔限制值(Ls),其叶片Pn降低主要是由气孔限制和叶绿素含量降低所导致的。正常条件下,所有氮素形态处理均显著降低了红椎幼苗叶片的Pn、Gs、Ci和Tr,但显著升高了叶片Ls和WUE,其叶片Pn降低的主要原因是供氮过高引起的叶片气孔限制。正常条件下,除Arg处理外,其他氮素形态均显著升高了红椎幼苗叶片的CUE,其中以Gly处理的促进作用最大。环割条件下,3种有机态氮素的添加均显著缓解了单一环割处理对红椎幼苗叶片Pn的抑制作用,尤其以精氨酸最为明显,供应精氨酸的叶片Pn值从单一环割处理的强烈抑制中恢复到了对照水平,而无机态氮的供应均未显著改变这种抑制。结果表明短期碳水化合物供应的阻断会显著抑制红椎幼苗叶片的光合能力,适量供应精氨酸、甘氨酸和尿素等有机态氮会有效缓解这种抑制作用,其中以精氨酸的作用最为明显。     

Polymorphic microsatellite markers for Mexican salamanders of the genus Ambystoma

We screened a partial genomic library enriched for microsatellites and characterized nine loci for the Mexican species of Ambystoma for studies of population structure. We tested marker variability in two metamorphic (A. granulosum, A. altamirani) and two paedomorphic (A. andersoni, A. mexicanum) species of the A. tigrinum complex. Our microsatellites were developed from pooled genomic DNA from three species, and may work on all species in the A. tigrinum complex in Mexico. These markers will be important for studies of conservation genetics in this radiation.     

Oviposition preference of swallowtail butterfly,Papilio polytes (Lepidoptera: Papilionidae) on four Rutaceae (Sapindales) host plant species

Abstract Oviposition preference and egg deposition site selection by the butterfly, Papilio polytes L. (Lepidoptera: Papilionidae) on four rutaceous host plants, Citrus aurantofolia (Christm.) Swing., Citrus hystrix DC., Citrus reticulata Blanco, and Murraya koenigii (L.) Sprengel, were assessed in field cages measuring 2 × 2 × 2 m. Simultaneous two-choice and four-choice oviposition tests of whole host plants were conducted. The mean total number of eggs laid per plant on C. reticulata in the two-choice test was significantly higher (P < 0.01) than those on C. aurantifolia, C. hystrix, and M. koenigii. Among the three other host plants, C. aurantifolia was preferred over C. hystrix, and M. koenigii (P < 0.01) and C. hystrix was preferred over M. koenigii (P < 0.01). In the four-choice test, C. reticulata was highly preferred and significantly different from C. hystrix and M. koenigii. However, no oviposition preference was detected with C. aurantifolia. Among the various plant parts in the two- and four-choice tests, eggs on leaves of each plant were the highest, followed by numbers on stems, and negligible numbers on pots. These numbers on leaves of C. reticulata and C. aurantifolia were not significantly different (P > 0.05), but differed significantly (P < 0.01) from those on leaves of C. hystrix and M. koenigii. The quantitative trend of egg-laying on stems was very similar to that observed for the leaves. Papilio polytes showed strong preference to lay eggs on the underside of leaves of all host plants than on the upper side or on the petiole. More eggs were laid on the upper side of each host plant than on its petiole. The four host plants in descending order of preference were C. reticulata≥C. aurantifolia > C. hystrix > M. koenigii. Although M. koenigii was the least preferred, it has the potential to serve as an alternative host plant for P. polytes which can be manipulated when necessary, to alleviate the infestation of this pest to the citrus industry.     

Next‐generation sequencing,FISH mapping and synteny‐based modeling reveal mechanisms of decreasing dysploidy in Cucumis

In the large Cucurbitaceae genus Cucumis, cucumber (C. sativus) is the only species with 2n = 2x = 14 chromosomes. The majority of the remaining species, including melon (C. melo) and the sister species of cucumber, C. hystrix, have 2n = 2x = 24 chromosomes, implying a reduction from n = 12 to n = 7. To understand the underlying mechanisms, we investigated chromosome synteny among cucumber, C. hystrix and melon using integrated and complementary approaches. We identified 14 inversions and a C. hystrix lineage‐specific reciprocal inversion between C. hystrix and melon. The results reveal the location and orientation of 53 C. hystrix syntenic blocks on the seven cucumber chromosomes, and allow us to infer at least 59 chromosome rearrangement events that led to the seven cucumber chromosomes, including five fusions, four translocations, and 50 inversions. The 12 inferred chromosomes (AK1–AK12) of an ancestor similar to melon and C. hystrix had strikingly different evolutionary fates, with cucumber chromosome C1 apparently resulting from insertion of chromosome AK12 into the centromeric region of translocated AK2/AK8, cucumber chromosome C3 originating from a Robertsonian‐like translocation between AK4 and AK6, and cucumber chromosome C5 originating from fusion of AK9 and AK10. Chromosomes C2, C4 and C6 were the result of complex reshuffling of syntenic blocks from three (AK3, AK5 and AK11), three (AK5, AK7 and AK8) and five (AK2, AK3, AK5, AK8 and AK11) ancestral chromosomes, respectively, through 33 fusion, translocation and inversion events. Previous results (Huang, S., Li, R., Zhang, Z. et al., 2009 , Nat. Genet. 41, 1275–1281; Li, D., Cuevas, H.E., Yang, L., Li, Y., Garcia‐Mas, J., Zalapa, J., Staub, J.E., Luan, F., Reddy, U., He, X., Gong, Z., Weng, Y. 2011a, BMC Genomics, 12, 396) showing that cucumber C7 stayed largely intact during the entire evolution of Cucumis are supported. Results from this study allow a fine‐scale understanding of the mechanisms of dysploid chromosome reduction that has not been achieved previously.     

Microsatellites for three distantly related genera in the Brassicaceae

Microsatellites are important genetic markers both in population genetics and for delimitation of closely related species. However, to develop microsatellites for each target organism is expensive and time consuming. In this study, we have therefore developed 65 new microsatellite primers for the species Draba nivalis and tested cross-species and cross-genus transfer success of these primers for two other genera in the Brassicaceae; Cardamine and Smelowskia. Furthermore, 15 previously developed microsatellites were tested for amplification in these three genera. The microsatellite markers that amplify across these genera may be useful for other genera in the Brassicaceae as well.     

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     

Microsatellite markers for species of the genus Dionda (Cyprinidae) from the American southwest

Thirty-eight microsatellite markers were developed from an enriched genomic DNA library of the cyprinid fish (minnow) Dionda episcopa. The microsatellites include 31 perfect-repeat motifs (29 dinucleotide, 1 trinucleotide, and 1 tetranucleotide) and 7 imperfect-repeat dinucleotide motifs. The microsatellite primers were used to amplify microsatellites from five related congeners: D. argentosa, D. diaboli, D. episcopa, D. nigrotaeniata, and D. serena. One species (D. diaboli) is listed as threatened and critically imperiled and two species (D. argentosa and D. serena) are listed as imperiled; the conservation status of D. nigrotaeniata is unknown. The number of experimentally tractable microsatellite markers varied from 28 for D. diaboli to 34 for D. episcopa. The number of polymorphic microsatellites conforming to Hardy–Weinberg expectations (following Bonferroni correction) ranged from 19 (D. diaboli) to 27 (D. argentosa). One pairwise comparison of microsatellites (in D. nigrotaeniata) deviated significantly from expectations of genotypic equilibrium. The microsatellite markers will be useful for conservation and population-genetic studies of these and other species in genus Dionda.     

Characterization of microsatellite markers in the endangered Sinojackia xylocarpa (Styracaceae) and cross‐species amplification in closely related taxa

Twenty‐four polymorphic microsatellite loci were isolated and characterized from an AAG‐enriched genomic library of Sinojackia xylocarpa. The average allele number of these microsatellites was 3.3 per locus, ranging from two to seven. The observed and expected heterozygosities at population level were 0.10–0.83 and 0.10–1.00, respectively. In addition, successful cross‐species amplification of this set of microsatellites in three other species of Sinojackia and a closely related taxon, Changiostyrax dolichocarpa, suggested that this set of microsatellite markers should provide a useful tool for genetic and conservation studies of Sinojackia species and other closely related taxa in the Styracaceae.     

Characterization and cross-species amplification of microsatellites from the endangered Hawksbill turtle (Eretmochelys imbricate)

Twelve novel polymorphic microsatellites were isolated from the endangered Hawksbill turtle (Eretmochelys imbricate). Eight of 12 markers were used to study genetic diversity of two sea turtle species: E. imbricate and green sea turtle (C. mydas). In E. imbricate, the average allele number of the eight microsatellites was 6.25/locus with a range of 3–13. The average expected and observed heterozygosity was 0.66 and 0.63 respectively. In C. mydas, the average allele number of the eight markers was 11.63/locus. The observed heterozyosity (0.68) was lower than the expected heterozyosity (0.79). Most of 12 microsatellites amplified specific and polymorphic PCR products in other six turtle species. Hence, the developed microsatellites would facilitate studies on genetic diversity and population structure of E. imbricate and other marine turtle species.     

PCR primers for nuclear-encoded microsatellites of the groupers <Emphasis Type="Italic">Cephalopholis fulva</Emphasis> (coney) and <Emphasis Type="Italic">Epinephelus guttatus</Emphasis> (red hind)

Twenty-one nuclear-encoded microsatellites were isolated from an enriched genomic DNA library of coney, Cephalopholis fulva, and characterized in both C. fulva and red hind, Epinephelus guttatus. The microsatellites include 16 dinucleotide repeats, two trinucleotide repeats, and three tetranucleotide repeats. An additional 11 microsatellites, isolated originally from an enriched genomic DNA library of E. guttatus, were characterized in both E. guttatus and C. fulva. Both grouper species support important commercial and recreational fisheries in the western Atlantic along the coasts of North, Central, and South America.     

Isolation and characterization of microsatellites in lane snapper (Lutjanus synagris), mutton snapper (Lutjanus analis), and yellowtail snapper (Ocyurus chrysurus)

Polymerase chain reaction primer pairs for a total of 25 nuclear‐encoded microsatellites (loci) were developed from genomic DNA libraries of lane snapper (Lutjanus synagris), mutton snapper (Lutjanus analis), and yellowtail snapper (Ocyurus chrysurus). The microsatellites include 24 perfect (21 dinucleotide and three trinucleotide) and one imperfect (combination tetranucleotide/tetranucleotide) repeat motifs. A total of 32 individuals of each species were assayed for allelic variation at all 25 microsatellites; reliable amplification products were generated for lane snapper (25 loci), mutton snapper (21 loci), and yellowtail snapper (24 loci). Significant deviations from Hardy–Weinberg expectations, following Bonferroni corrections, were found for one microsatellite in lane and yellowtail snappers, and for three microsatellites in mutton snapper. All pairwise comparisons of microsatellites (all three species) did not deviate significantly from genotypic equilibrium.     

四种乡土珍贵阔叶树种叶功能性状的种内和种间变异

植物功能性状种间性状变异反映不同物种的生活史对策,种内性状变异反映了同一物种不同个体应对不同环境的性状应答.人工林均一的环境有利于深入分析不同树种种内和种间变异.该研究以南宁良凤江林场的四种乡土珍贵阔叶树种[观光木(Tsoongiodendron odorum)、红锥(Castanopsis hystrix)、灰木莲(...     

Microsatellites for tree of heaven (Ailanthus altissima)

Nine (CT)_n microsatellites were developed for tree of heaven, Ailanthus altissima, from invasive populations on the Mediterranean islands. These loci had seven to 12 alleles in 96 trees from five islands. Two loci had significant deficits of heterozygotes within islands while the other loci were in Hardy–Weinberg equilibrium, and four pairs of loci had significant linkage disequilibrium within a single island. These loci were also polymorphic in one to three individuals of the tree of heaven varieties, Ailanthus altissima erythrocarpa, Ailanthus altissima sutchuenensis and Ailanthus altissima tanakai, and the related species Ailanthus giraldii and Ailanthus vilmariniana.     

SYNTHETIC HYBRIDS OF HORDEUM BOGDANII WITH ELYMUS CANADENSIS AND SITANION HYSTRIX

Seven viable hybrid seeds were obtained from 48 hand-emasculated Elymus canadensis L., 2n = 28, florets pollinated by Hordeum bogdanii Wilensky, 2n = 14. The hybrids were large, vigorous, and completely sterile plants that bore a closer morphological resemblance to E. canadensis than to H. bogdanii. Chromosome associations in the 21-chromosome hybrids averaged 9.98^I, 5.40^II, and 0.08^III in 264 metaphase-I cells. Chromosome pairing was attributed to allosyndetic pairing between E. canadensis and H. bogdanii chromosomes. The H. bogdanii genome appears to be partially homologous with one of the two E. canadensis genomes. One Sitanion hystrix (Nutt.) J. G. Smith X H. bogdanii hybrid was obtained from a cross involving 37 emasculated S. hystrix florets. This triploid, 2n = 21, hybrid was morphologically intermediate between the parents and totally sterile. Averages of 9.09^I, 5.72^II, and 0.16^III were observed in 106 cells at metaphase I. A modified form of the H. bogdanii genome appears to occur in S. hystrix as well as in E. canadensis. Many allotetraploid Agropyron, Elymus, and Sitanion species apparently contain a genome derived from Hordeum.     

Individual assignment tests proved genetic boundaries in a species complex of Pacific abalone (genus <Emphasis Type="Italic">Haliotis</Emphasis>)

We conducted this study to find genetic evidence to distinguish the members of Pacific abalone species complex (Haliotis discus hannai, H. discus discus, H. madaka, and H. gigantea) based on microsatellite DNA markers, illustrating the potential of microsatellites for species-assignment. First, we addressed the transferability of H. discus hannai microsatellites to the three other members of Pacific abalone and five additional species (H. diversicolor aquatilis, H. midae, H. corrugata, H. fulgens, and H. rubra). Second, using the microsatellites we applied two types of individual assignment testing (the distance-based assignment and Bayesian model-based clustering) to individuals from the Pacific abalone species. A total of 24 microsatellites were subjected to PCR trials for nine Haliotis species, and the cross-species amplification performance of these markers turned out to drop precipitously even for less divergent congeners. Within the Pacific abalone species complex, four of the 24 markers were not transferable to H. gigantea, suggesting a solid genetic boundary between H. gigantea and H. discus hannai, H. discus discus, and H. madaka. Among the three latter abalones, both assignment tests achieved approximately 90% or more success rate of assignment. The feasibility of the microsatellite markers to classify species sheds light on the genetic management of the Pacific abalone species complex. Electronic Supplementary Material  Supplementary material is available in the online version of this article at and is accessible for authorized users.

Identification of microsatellite markers in a neotropical seasonally dry forest tree,Astronium urundeuva (Anacardiaceae)

Nine dinucleotide microsatellites were developed in Astronium urundeuva (Anacardiaceae), a typical tree of the seasonally‐dry tropical forests of South America and characterized on three populations from Paraguay and Argentina. Seven microsatellites were found polymorphic in within population gene diversities ranging from 0.32 to 0.91, and an observed number of alleles varying between four and 20. Despite their relatively low number of alleles, these markers proved valuable tools in detecting genetic structure between three populations in Paraguay and North Argentina.     

广州红锥-马占相思林物种组成与多样性研究

为了解广州市龙眼洞林场红锥(Castanopsis hystrix)-马占相思(Acacia mangium)阔叶混交林的群落结构,对其物种多样性进行了分析.结果表明,红锥-马占相思阔叶混交林的物种丰富度依次为乔木层>灌木层>草本层>层间植物层.乔木层的Simpson和Shannon-Wiener指数分别为0.945和...     

New microsatellite markers for the snow crab Chionoecetes opilio (Brachyura: Majidae)

Five microsatellite markers were developed for the snow crab (Chionoecetes opilio) and polymerase chain reaction conditions and/or primer sequences of three previously developed microsatellites were adapted for fluorescent labelling analysis. All loci were analysed in 449 individuals from 11 sampling sites in the northwest Atlantic. The high degree of polymorphism exhibited by these microsatellites (mean of 34 alleles per locus and mean observed heterozygosity of 0.76) suggests that they will be suitable for spatial and temporal genetic analysis of C. opilio populations.     

Single-copy,species-transferable microsatellite markers developed from loblolly pine ESTs

Microsatellites, or simple sequence repeats (SSRs), are usually regarded as the markers of choice in population genetics research because they exhibit high variability. The development cost of these markers is usually high. In addition, microsatellite primers developed for one species often do not cross-amplify in related species, requiring separate development for each species. However, microsatellites found in expressed sequence tags (ESTs) might better cross-amplify as they reside in or near conserved coding DNA. In this study, we identified 14 Pinus taeda (loblolly pine) EST-SSRs from public EST databases and tested for their cross-species transferability to P. contorta ssp. latifolia, P. ponderosa, and P. sylvestris. As part of our development of a P. contorta microsatellite set, we also compared their transferability to that of 99 traditional microsatellite markers developed in P. taeda and tested on P. contorta ssp. latifolia. Compared to traditional microsatellites, EST-SSRs had higher transfer rates across pine species; however, the level of polymorphism of microsatellites derived from ESTs was lower. Sequence analyses revealed that the frequencies of insertions/deletions and base substitutions were lower in EST-SSRs than in other types of microsatellites, confirming that EST-SSRs are more conserved than traditional SSRs. Our results also provide a battery of 23 polymorphic, robust microsatellite primer pairs for lodgepole pine.Communicated by O. Savolainen