Application of petG-tm P sequence to systematic study of Chinese Cupressus species

Chinese Cupressus L.includes five species.The molecular phylogenetic relationships of the Cupressus species and Chamaecyparis L.were determined by comparing 417-479 bp of chloroplast petG-trnP intergenic spacer sequence.In PAUP* analysis,Platycladus orientalis was used as the functional out group.By using the maximum likelihood method 1077 trees were examined and the result showed that one tree had a best score of -Ln=2 232.47.The phylogenetic tree clearly showed that Chamaecyparis nootkatensis was diverged from other Chamaecyparis species.Based on the results,together with evidences from other aspects,we consider that Cupressus funebris and Chamaecyparis nootkatensis should be placed in the genus Cupressus.The use of cpDNA intergenic spacer petG-trnP in Cupressus was also discussed.     

The circumscription and phylogenetic relationships of Callitropsis and the newly described genus Xanthocyparis (Cupressaceae)

A new species of conifer was recently discovered in northern Vietnam. In a preliminary phylogenetic analysis of morphological data a possible sister species, Chamaecyparis nootkatensis (D. Don) Spach, was identified; however, because of the presumed phylogenetic remoteness of these two species to the remainder of the Cupressaceae, a new genus-Xanthocyparis-was described to accommodate both species. Here an analysis of ITS (nrDNA), matK, and rbcL sequence data in combination with 58 informative morphological characters was aimed at testing the monophyly of the remainder of Chamaecyparis and evaluating the placement and monophyly of Xanthocyparis. Chamaecyparis, minus C. nootkatensis, was resolved as a monophyletic group, remote from Cupressus and Xanthocyparis. Cupressus, Juniperus, and Xanthocyparis formed a very highly supported monophyletic group. However, Cupressus was not monophyletic. Instead the Old World species sampled were resolved sister to a clade containing a monophyletic Juniperus, a monophyletic Xanthocyparis, and a clade of New World Cupressus species. If both species of Xanthocyparis are to be treated as members of the same genus, then due to the principal of priority they will have to be recognized in the genus Callitropsis. Research is continuing to resolve the status of New World and Old World Cupressus.     

Correction method for null alleles in species with variable microsatellite flanking regions, a case study of the black-lipped pearl oyster Pinctada margaritifera

In bivalves, heterozygote deficiencies and departures from Hardy-Weinberg equilibrium (HWE) in microsatellite analysis are common and mainly attributed to inbreeding, genetic patchiness (Walhund effect), or null alleles. We checked for the occurrence of null alleles at 3 microsatellite loci in 3 populations of black-lipped pearl oyster, Pinctada margaritifera, using a step-by-step method to re-amplify homozygotes and null individuals with redesigned primer pair combinations. After amplification with original primer pairs, the 3 populations exhibited null alleles, absence of structure, and significant departure from HWE for all 3 loci due to heterozygote deficiencies. After 3 re-amplification steps, with modified primer sets, all loci were corrected for null alleles. Once corrected, all populations appeared at HWE, demonstrating that null alleles were responsible for the initial disequilibrium of the populations. Furthermore, analysis from corrected genotypes demonstrates significant genetic differentiation for one population from the other 2.     

Development of simple sequence repeat markers for the soybean rust fungus, Phakopsora pachyrhizi

Twenty-four simple sequence repeat markers were developed for Phakopsora pachyrhizi, a fungal pathogen of soybean (Glycine max) and other legumes. All 24 of the loci were evaluated on 28 isolates of P. pachyrhizi. Twenty-one loci were polymorphic, with allelic diversity ranging from two to eight alleles, and null alleles were observed for eight of the 24 loci. A preliminary screen with the closely related species, P. meibomiae, indicated that these primer pairs are specific to P. pachyrhizi.     

ent-Daucane and acorane sesquiterpenes from x Cupressocyparis leylandii foliage.

One new ent-daucadiene and enantiomers of four previously reported daucadienes or acoradienes were identified in foliage of the hybrid cypress species xCupressocyparis leylandii. The major compound is (+)-dauca-5,8-diene; minor congeners are (-)-dauca-4,8-diene, (-)-dauca-8,11-diene, (-)-acora-3,7(14)-diene and 1S-dauca-4(11),8-diene. None of these compounds is observed in foliage of either of the parent species, Cupressus macrocarpa and Chamaecyparis nootkatensis. However, we have subsequently found them in some specimens of Cupressus macnabiana.     

Isolation and characterization of polymorphic microsatellite loci in Hemibarbus labeo (Cyprinidae) using PCR‐based isolation of microsatellite arrays (PIMA)

A total of 10 polymorphic microsatellite loci from Hemibarbus labeo were isolated and characterized using an optimized protocol to construct a microsatellite‐enriched genomic library. The analysis of variability was performed in 24 specimens of mainland China. The mean number of alleles across loci was 3.10 ± 1.10 and the level of expected heterozygosity varied from 0.0417 to 0.7482. Frequencies of null alleles of the 10 loci are not significantly greater than zero. No linkage disequilibrium was detected between loci in either population. Five primer pairs cross‐amplify the microsatellites in other species, indicating transportability of the markers within the family Cyprinidae.     

Development and characterization of 30 polymorphic microsatellite markers for the Atlantic surfclam, Spisula solidissima (Dillwyn, 1817)

Thirty polymorphic microsatellite markers were developed for the Atlantic surfclam, Spisula solidissima, from an enriched library and characterized in 24 clams from a wild population. The number of alleles ranged from 3 to 16 per locus. The expected and observed heterozygosities ranged from 0.1942 to 0.9238 and 0.0833 to 0.875 respectively. Six loci showed significant (P < 0.05 after Bonferroni correction) deviation from Hardy–Weinberg equilibrium, probably because of the presence of null alleles. Three primer pairs amplified duplicated loci with two involving tandem mini‐satellite repeats. Most of the microsatellite markers developed here should be useful for genetic studies in this species.     

Characteristics of single- and multi-copy microsatellites from Pinus radiata

 Dinucleotide microsatellites were isolated from Pinus radiata using both a standard genomic library and libraries enriched for microsatellites. Locus-specific primers were designed to amplify 43 unique microsatellites. Thirty two of these loci had interpretable PCR patterns, 11 of which were polymorphic in a screen of 19 P. radiata individuals; all 11 polymorphic loci contained at least 17 repeats in the sequenced plasmid. Six of the eleven primer pairs amplified multiple fragments per individual (3–8), suggesting that these loci were present in multiple copies in the genome. Genotyping a 48-tree P. radiata production population with seven of the most polymorphic microsatellites revealed an average of 17 bands per locus (the multi-copy microsatellites were treated as one locus). When tested on known pedigrees, both single and multi-copy microsatellites exhibited co-dominant inheritance and Mendelian segregation. Two loci had null alleles and one locus had a high frequency of non-parental alleles, suggesting a high mutation rate. Eight of these microsatellites, including five multi-copy loci, were placed on a partially constructed P. radiata genetic map. Four of the five multi-copy microsatellites had two or more sets of alleles that mapped to the same locus, and the fifth mapped to two unlinked loci. All seven tested primer pairs amplified PCR products from other species of hard pine, three amplified products from soft-pine species, and one amplified bands in other conifers. Received: 10 November 1997 / Accepted: 5 January 1998     

Identification of microsatellite sequences in Vitis riparia and their applicability for genotyping of different Vitis species.

A Vitis riparia genomic library was screened for the presence of (GA)n simple sequence repeats (SSR) and 18 primer pairs yielding amplification products of the expected size were designed. Heterologous amplification with the primer pairs in related species (V. rupestris, V. berlandieri, V. labrusca, V. cinerea, V. aestivalis, V. vinifera, and interspecific hybrids) was successful in most primer-species combinations. Therefore, the new markers are applicable to the genotyping of a range of Vitis species. Variations in the SSR flanking sequence were detected between and within the species. The degree of polymorphism and performance of the markers were determined in up to 120 individuals of V. vinifera. Four of fifteen alleles per locus were detected and expected heterozygosity ranged between 0.37 and 0.88. Null alleles were shown to be present at two loci by a lack of heterozygous individuals and by transmission of the null alleles in a controlled cross. Regular Mendelian inheritance is indicated for all but one loci by a preliminary segregation analysis in 36 offspring. Thirteen of the markers were found suitable for the genotyping of grapevines (V. vinifera).     

Isolation and characterization of microsatellites in Brassica rapa L

We report here the isolation and characterization of microsatellites, or simple sequence repeats (SSRs), in Brassica rapa. The size-fractionated genomic library was screened with (GA)(15) and (GT)(15) oligonucleotide probes. A total of 58 clones were identified as having the microsatellite repeats, and specific primer pairs were designed for 38 microsatellite loci. All primer pairs, except two, amplified fragments having the sizes expected from the sequences. Of the 36 primer pairs, 35 amplified polymorphic loci in 19 cultivars of B. rapa, while monomorphism was observed in only one primer pair. A total of 232 alleles was identified by the 36 primer pairs in 19 cultivars of B. rapa, and these primer pairs were examined also in nine Brassicaceae species. Most of the 36 primer pairs amplified the loci in the Brassicaceae species. Segregation of the microsatellites was studied in an F(2) population from a cross of doubled-haploid lines DH27 x G309. The microsatellites segregated in a co-dominant manner. These results indicate that the microsatellites isolated in this study were highly informative and could be useful tools for genetic analysis in B. rapa and other related species.     

Microsatellite markers for eastern hemlock (Tsuga canadensis)

We describe polymerase chain reaction primer pairs and reaction conditions for amplification of 15 microsatellite loci from eastern hemlock (Tsuga canadensis). The primers were tested on 23 individuals from a natural population in southwestern North Carolina, USA. These primers yielded an average of 5.9 alleles per locus (range of 2-14), an average observed heterozygosity of 0.45 (range 0.14-0.73), and an average polymorphic information content of 0.54 (range 0.28-0.86). In addition, eight of the primer pairs were found to amplify microsatellite loci in one or more additional species of Tsuga.     

Characterization of 35 microsatellite loci in the Pacific lion‐paw scallop (Nodipecten subnodosus) and their cross‐species amplification in four other scallops of the Pectinidae family

Four microsatellite‐enriched DNA libraries yielded 35 microsatellite loci from 100 primer pairs designed for Pacific lion‐paw scallop, Nodipecten subnodosus. The number of alleles ranged from four to 28. Three of the 35 loci were not in Hardy–Weinberg equilibrium and linkage disequilibrium was found for one pair of loci. These microsatellites will be used to analyse the population structure of the species in Mexico's Baja Peninsula to propose management strategies for scallop aquaculture development. Twenty‐six primer pairs cross‐amplified in Nodipecten nodosus, whereas none (Argopecten ventricosus) or few cross‐amplified in the Argopecten species.     

Ten new microsatellite loci for the yellowhammer (Emberiza citrinella) and their cross‐species applicability among related taxa

We describe isolation and characterization of the first microsatellite loci specifically developed for a very common European passerine bird, the yellowhammer Emberiza citrinella. Nine out of our 10 loci are polymorphic within the species E. citrinella. Number of alleles ranged from two to 21 per locus and observed heterozygosity between 0.20 and 0.91. Four primer pairs also yielded reproducible results in other species of Emberizidae. These loci comprise a set of molecular markers for various applications, from moderately polymorphic loci suitable for population studies to highly polymorphic loci for pedigree analysis in Emberizidae.     

Isolation,characterization and cross‐species amplification of microsatellite DNA loci in the tropical American yam Dioscorea trifida

We developed microsatellite markers in American yam (Dioscorea trifida). A microsatellite sequence‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Among these, eight primer pairs yielded amplification products that were both interpretable and polymorphic in 24 yam cultivars. The number of alleles per locus ranged from two to 13 and the overall expected heterozygosity was around 0.5. Six of the eight Dioscorea trifida microsatellite loci gave amplification products in other Dioscorea species.     

PERMANENT GENETIC RESOURCES: Microsatellite markers for the threatened Bliss Rapids snail (Taylorconcha serpenticola) and cross-amplification in its congener, T. insperata

We developed and tested microsatellite markers to investigate population structure of a threatened North American freshwater gastropod, Taylorconcha serpenticola. Of the 21 primer pairs that were evaluated, 11 were readily optimized and scored, providing amplification of 12 loci that were screened for 820 specimens from 29 populations. The number of alleles across 11 of these polymorphic loci ranged from three to 20 and the observed heterozygosity varied from 0.0061 to 0.7561. All loci yielded suitable amplification products in the second species of Taylorconcha (T. insperata) and three proved to be diagnostic for these congeners, demonstrating that these markers are also useful for species identification studies.     

Isolation and characterization of nine microsatellite loci in Podarcis bocagei (Squamata: Lacertidae)

Nine dinucleotide microsatellite loci were developed through an enrichment protocol for Bocage's wall lizard, Podarcis bocagei Seoane 1884, a lacertid endemic to the Iberian Peninsula. Nineteen primer pairs were designed and tested. From these, nine loci yielded satisfactory results and were screened on 15–19 individuals. These loci revealed a high level of polymorphism (8–15 alleles) and heterozygosity (0.611–0.947) and will certainly be useful in the study of population structure and evolutionary history of this species.     

Isolation and characterization of microsatellite markers in the East African tree,Acacia brevispica (Fabaceae: Mimosoideae)

We have developed primer pairs for 24 microsatellite loci isolated from Acacia brevispica, which is widespread in woodland savannas of East Africa. The loci were screened for levels of variation using 16–52 individuals from Mpala Research Centre, Kenya. Number of alleles per locus ranged from two to 17, and polymorphic information content ranged from 0.248 to 0.864. Several loci showed the presence null alleles, but many loci will be useful in ongoing studies of genetic structure, gene flow, breeding systems, and natural selection.     

Fourteen new di- and tetranucleotide microsatellite loci for the critically endangered Indian tiger (Panthera tigris tigris)

We describe 11 dinucleotide and three tetranucleotide microsatellite loci for the critically endangered Indian tiger, Panthera tigris tigris. All of them were polymorphic with four to nine alleles per locus and an observed heterozygosity between 0.13 and 1.0. All primers also amplify microsatellite loci in leopard, Panthera pardus, and 12 primer pairs yielded reproducible results in domestic cat, Felis catus. These new microsatellites specifically developed for Indian tiger - in combination with those already available - comprise a reasonable number of loci to genetically analyse wild and captive populations of this illustrative species and might allow for recognition of individual tigers.     

Isolation and characterization of microsatellite loci from the endangered highlands scrub hypericum (Hypericum cumulicola)

We report the isolation of 19 primer pairs for amplification of polymorphic microsatellite loci for Hypericum cumulicola. These markers were evaluated in 24 individuals from one population; two to four alleles were detected per locus, and observed heterozygosity ranged from 0 to 0.5. Two loci demonstrated significant heterozygote deficiencies, possibly due to null alleles, and significant linkage disequilibrium was found between six pairs of loci. The remaining microsatellite loci will help determine if genetic differentiation is responsible for life‐history differences between natural and anthropogenically disturbed populations of H. cumulicola.