Development of polymorphic markers from expressed sequence tags of Manihot esculenta Crantz

In this study, 49 primers were designed from sequences containing di-, tri-, tetra-, penta- and hexanucleotide motifs with a minimum of four repeats and presence of motif size polymorphisms (insertion/deletion) from cassava (Manihot esculenta Crantz) expressed sequence tags deposited in public sequence database. Each locus was subsequently screened on 29 M. esculenta Crantz obtained from 15 different countries. Cross-amplification was tested with M. esculenta Crantz (ssp. flabellifolia) and four different Manihot species, M. chlorosticta, M. carthaginensis, M. filamentosa and M. tristis. Of these, nine loci showed polymorphic profiles within M. esculenta Crantz, which revealed two to four alleles per locus. The average unbiased and direct count heterozygosities were 0.4901 and 0.5674, respectively.     

Cross-species amplification of cassava (Manihot esculenta) (Euphorbiaceae) microsatellites: allelic polymorphism and degree of relationship

Microsatellite amplification was performed on cassava (Manihot esculenta) and six other different species (all wild) of the Manihot genus. We used ten pairs of microsatellite primers previously developed from cassava, detecting 124 alleles in a sample of 121 accessions of the seven species. The number of alleles per locus ranged from four to 21 alleles, and allelic diversity was greater in the wild species than in cassava. Seventy-nine alleles, including unique ones, were detected in the wild species but were not found in the crop. The lower level of heterozygosity in some wild species probably resulted from a combination of fine-scale differentiation within the species and the presence of null alleles. Overall, microsatellite primers worked across the genus, but, with increasing genetic distance, success in amplifying loci tended to decrease. No accession of M. aesculifolia, M. carthaginensis, and M. brachyloba presented a banding pattern at locus Ga-140; neither did one appear for M. aesculifolia at locus Ga-13. Previous work with amplified fragment length polymorphism (AFLP) markers and this microsatellite analysis show that these three wild taxa are the most distant relatives of the crop, whereas the wild forms M. esculenta subsp. flabellifolia and M. esculenta subsp. peruviana appear to be the closest.     

Microsatellite variation in cassava (Manihot esculenta, Euphorbiaceae) and its wild relatives: further evidence for a southern Amazonian origin of domestication

Genetic variation at five microsatellite loci was used to investigate the evolutionary and geographical origins of cassava (Manihot esculenta subsp. esculenta) and the population structure of cassava's wild relatives. Two hundred and twelve individuals were sampled, representing 20 crop accessions, 27 populations of cassava's closest wild relative (M. esculenta subsp. flabellifolia), and six populations of a potentially hybridizing species (M. pruinosa). Seventy-three alleles were observed across all loci and populations. These data indicate the following on cassava's origin: (1) genetic variation in the crop is a subset of that found in the wild M. esculenta subspecies, suggesting that cassava is derived solely from its conspecific wild relative. (2) Phenetic analyses group cassava with wild populations from the southern border of the Amazon basin, indicating this region as the likely site of domestication. (3) Manihot pruinosa, while closely related to M. esculenta (and possibly hybridizing with it where sympatric), is probably not a progenitor of the crop. Genetic differentiation among the wild populations is moderately high (F:(ST) = 0.42, rho(ST) = 0.54). This differentiation has probably arisen primarily through random genetic drift (rather than mutation) following recent population divergence.     

Population genetics of Manihot esculenta ssp. flabellifolia gives insight into past distribution of xeric vegetation in a postulated forest refugium area in northern Amazonia

The Guianas have often been proposed as a forest refugium; however, this view has received little testing. Studies of population genetics of forest taxa suggest that the central part of French Guiana remained forested, while the southern part (currently forested) may have harboured more open vegetation. Insights into the population structure of species restricted to non-forested habitats can help test this hypothesis. Using six microsatellite loci, we investigated the population genetics of French Guianan accessions of Manihot esculenta ssp. flabellifolia , a taxon restricted to coastal savannas and to rocky outcrops in the densely forested inland. Coastal populations were highly differentiated from one another, and our data suggest a recent colonization of these savannas by M. esculenta ssp. flabellifolia in a west-to-east process. Coastal populations were strongly differentiated from inselberg populations, consistent with an ancient separation of these two groups, with no or low subsequent gene flow. This supports the hypothesis that the central part of the region may have remained forested since the Last Glacial Maximum, impeding the establishment of Manihot . Contrary to coastal populations, inselberg Manihot populations were strikingly homogeneous at a broad spatial scale. This suggests they were connected until recently, either by a large continuous savanna area or by smaller, temporary disturbed areas shifting in space.     

Development of microsatellite primers for Jatropha curcas (Euphorbiaceae) and transferability to congeners

? Premise of the study: Microsatellite primers were developed for Jatropha curcas (Euphorbiaceae), a tree species with large potential for biofuel production, to investigate its natural genetic diversity and mating system to facilitate the establishment of tree improvement and conservation programs. ? Methods and Results: Using a protocol for genomic library enrichment, 104 clones containing 195 repeat motifs were identified. Primer pairs were developed for 40 microsatellite loci and validated in 41 accessions of J. curcas from six provenances. Nine loci were polymorphic revealing from two to eight alleles per locus, and six primers were able to amplify alleles in the congeners J. podagrica, J. pohliana, and J. gossypifolia, but not in other Euphorbiaceae species, such as Hevea brasiliensis, Manihot esculenta, or Ricinus communis. ? Conclusions: The primers developed here revealed polymorphic loci that are suitable for genetic diversity and structure, mating system, and gene flow studies in J. curcas, and some congeners.     

Population history of Manihot esculenta (Euphorbiaceae) inferred from nuclear DNA sequences

The nature of gene flow in plants -- including the propensity for interspecific introgression -- makes them interesting candidates for phylogeographical analysis. Plant phylogeography studies have been limited, however, by the availability of suitable intraspecific variation. In this study, DNA sequence variation from a nuclear gene [Glyceraldehyde 3-phosphate dehydrogenase; (G3pdh)] was used to examine the population history of Manihot esculenta ssp. flabellifolia and a potentially hybridizing species, M. pruinosa. These species occur in the rainforest-savanna ecotone adjoining the Amazon basin, a region believed to have undergone major habitat shifts since the Pleistocene. Geographical distributions of the G3pdh haplotypes indicate genetic isolation-by-distance across the range of M. esculenta ssp. flabellifolia. However, there is greater genetic similarity between northeastern and western populations than would be expected given the present species distribution. A nested clade analysis suggests that northeastern and western populations were connected by gene flow until relatively recently, when they became fragmented. This inferred fragmentation event is consistent with post-Pleistocene habitat shifts proposed for the Amazon basin. At the interspecific level, haplotype sharing with M. pruinosa may reflect either recent interspecific introgression or incomplete lineage sorting between these closely related species.     

Evolution under domestication: contrasting functional morphology of seedlings in domesticated cassava and its closest wild relatives

Although cassava (Manihot esculenta ssp. esculenta) is asexually propagated, farmers incorporate plants from seedlings into planting stocks. These products of sex are exposed to selection, which in agricultural environments should favour rapid growth. To examine whether seedling morphology has evolved under domestication, we compared domesticated cassava, its wild progenitor (M. esculenta ssp. flabellifolia) and their sister species (M. pruinosa) under controlled conditions. Field observations complemented laboratory study. In both wild taxa, the hypocotyl did not elongate (hypogeal germination) and cotyledons remained enclosed in the testa. In domesticated cassava, the hypocotyl elongated (epigeal germination), and cotyledons emerged and became foliaceous. The difference in hypocotyl elongation was fixed, whereas cotyledon morphology varied with environmental conditions in M. pruinosa. Comparative analysis suggests that epigeal germination is primitive in Manihot, that the lineage including wild ancestors of cassava evolved hypogeal germination--which confers greater tolerance to risks in their savanna environment--and that with domestication, there was a reversion to epigeal germination and photosynthetic cotyledons, traits conferring high initial growth rates in agricultural habitats.     

Genetic diversity of traditional South American Landraces of Cassava (Manihot esculenta Crantz): an analysis using microsatellites

The extent and structure of the genetic variability of traditional varieties of cassava (Manihot esculenta Crantz) have been little documented, despite considerable evidence for this crop? great varietal diversity in traditional agroecosystems. We used microsatellite markers to assess the genetic structure of traditional landraces of sweet and bitter cassava collected from five South American sites. As reference, we used a sample of 38 accessions from a world collection of cultivated cassava. For a total of 10 loci examined, we found 15 alleles that were not represented in this sample. Ten of these had been previously detected in wild Manihot species. The geographical structure of genetic variability was weak, but the genetic differentiation between bitter and sweet landraces was significant, suggesting that each form had evolved separately after domestication. Our results showed that traditional landraces form an important source of genetic diversity and merit more attention from managers of crop genetic resources.     

Microsatellite markers for <Emphasis Type="Italic">Juglans cinerea</Emphasis> L. and their utility in other Juglandaceae species

Ten polymorphic microsatellite markers were found to amplify in butternut (Juglans cinerea; Juglandaceae). These microsatellite loci were found to amplify across most of nine other species and five hybrids examined. Loci were highly polymorphic, with 18 to 32 alleles per locus across species. These nuclear microsatellite markers will be useful in examining genetic diversity within and among populations of butternut, and in distinguishing butternut from interspecific hybrids.     

Characterization of polymorphic microsatellite loci in Yucca filamentosa

Yuccas and their pollinator moths are a textbook example of mutualism, yet we lack sufficiently variable markers to properly study the population genetics of the plants. We characterized 13 polymorphic microsatellite loci for Yucca filamentosa by screening primers derived from an expressed sequence tag database. We found four to 13 alleles per locus and the observed heterozygosity ranged from 0.31 to 1. These markers will be useful in future ecological studies of Y. filamentosa.     

Characterization of six microsatellite loci in Myrica faya (Myricaceae) and cross amplification in the endangered endemic M. rivas-martinezii in Canary Islands, Spain

Six novel polymorphic microsatellite markers were isolated from enriched libraries in Myrica faya Ait., recently renamed Morella faya, (fayatree, firetree, or firebush) in order to examine the genetic diversity in natural populations. Also, test cross-specific amplification and genetic diversity in Myrica rivas-martinezii, which is endemic on the Canary islands. Microsatellite loci were screened in 225 individuals of both species from different islands of the Canarian archipelago. All markers were successfully amplified from both Myrica species, with an average number of 6.5 and 9.3 alleles per locus in M. rivas-martinezii and M. faya, respectively. There was no evidence for linkage disequilibrium between loci, and the probability of null alleles ranged from 0.01 to 0.17.     

Development of microsatellite markers for the small yellow croaker Larimichthys polyactis (Sciaenidae) by cross-species amplification

The small yellow croaker (Larimichthys polyactis) is a highly valued fish for human consumption found in the Western Pacific that was considered endangered until recently because of overfishing. We selected microsatellite markers for this species from markers developed for Miichthys miiuy, also of the family Sciaenidae. Among 43 markers polymorphic for M. miiuy, 11 were found to be polymorphic for L. polyactis. Characterization of these 11 loci was made based on 30 L. polyactis individuals collected by trawling in the Zhoushan Fishing Ground, Zhejiang Province, China. Total genomic DNA was isolated from fin clips. The number of alleles per locus ranged from 4 to 10, with a mean of 5.82, while the effective number of alleles ranged from 1.64 to 10.00, with a mean of 3.22. Observed and expected heterozygosities ranged from 0.17 to 0.72 and from 0.39 to 0.81, respectively. Significant deviation from Hardy-Weinberg equilibrium was found at four loci, after applying Bonferroni's correction. There was no significant association between any of the pairs of microsatellite loci, hence allelic variation at these loci was considered independent. These 11 polymorphic microsatellite loci will be useful for genetic diversity analysis and molecular-assisted breeding for L. polyactis.     

青鱼微卫星标记的开发与特性分析

青鱼(Mylopharyngodon piceus)是中国最为重要的淡水养殖鱼类。开发青鱼的微卫星标记能为青鱼的遗传多样性分析提供更多工具。本研究使用磁珠富集法,利用生物素探针(CA)10和(GACA)6,富集得到青鱼基因组微卫星片段,进一步通过设计微卫星引物检验其在青鱼原种群体中的有效性和多态性水平。结果显示,所构建文库中849个克隆含有微卫星序列,通过利用PCR技术在吴江原种青鱼36个个体中进行多态性筛选,获得了25个多态性微卫星位点。其平均等位基因数(Na)和有效等位基因数(Ne)分别为7.08和3.526,平均观测杂合度(Ho)和期望杂合度(He)分别为0.602和0.619,平均多态信息含量(PIC)为0.568。其中,Mp23、Mp27和Mp35这3个位点极显著偏离哈迪-温伯格平衡(P 0.01)。本研究开发的微卫星标记能为青鱼种质资源的评价和保护等研究提供工具。     

Characterization of polymorphic microsatellite DNA markers for Acacia saligna (Labill.) H.L.Wendl. (Mimosaceae)

A genomic library was constructed and 10 novel polymorphic microsatellite markers developed for Acacia saligna, a species complex being developed for agroforestry across southern Australia. Polymorphism was investigated for these markers in 30 individuals from two subspecies. Alleles per locus averaged 4.0 for ssp. saligna and 5.2 for ssp. lindleyi. Population specific alleles were detected for all loci. The microsatellite markers will be used for diagnostic assessment of the four main subspecies of the species complex.     

SNPs,SSRs and inferences on cassava’s origin

Despite its importance as a staple food throughout the tropics, the root crop cassava (it Manihot esculenta ssp. esculenta) has traditionally not been a major focus of research. One basic question about cassava that remained unresolved until recently concerns the crop’s origin. This paper describes analyses of SNPs (single nucleotide polymorphisms) and SSR (simple sequence repeat) variation as a means of tracing cassava’s evolutionary and geographical origins. Genetic diversity was examined in a sample of 20 cassava varieties that are representative of germplasm diversity within the crop, and in 212 individuals collected from wild populations of two closely related Manihot species. SNP and indel variation was examined in portions of two low copy nuclear genes, BglA and Hnl. Inferences from these genes were compared to those from previously examined loci, including the low copy nuclear gene G3pdh and 5 SSR loci. For all genes examined, SNPs and SSR alleles are shared between domesticated cassava and a specific geographical subset of wild Manihot populations, which suggests the following: (1) Cassava was likely domesticated from a single wild Manihot species, M. esculenta ssp. flabellifolia, rather than from multiple hybridizing species, as traditionally believed; and (2) the crop most likely originated in the southern Amazon basin.     

Isolation and characterization of 19 novel microsatellite loci in Rhododendron aureum and Rhododendron brachycarpum (Ericaceae)

Nineteen polymorphic microsatellite loci are described for Rhododendron aureum Georgi (Ericaceae), an endangered species in Korea, and the closely related Rhododendron brachycarpum. The sequences containing repeat motifs were identified using low-depth next generation sequencing and 148 microsatellite loci were examined for amplification success and the detection of polymorphisms. All 19 loci were polymorphic and the number of alleles for these markers varied from 4 to 25, with an average of 15 alleles per locus. Three R. aureum loci showed departure from Hardy–Weinberg equilibrium (HWE) and one R. brachycarpum locus deviated significantly from HWE. These microsatellite loci will be useful for investigating the genetic diversity and population structure of both species.     

Development of microsatellite markers in a large perennial herb, Veratrum album ssp. oxysepalum

We developed 11 polymorphic microsatellite [simple sequence repeat (SSR)] loci from genomic DNA of Veratrum album ssp. oxysepalum using a dual-suppression polymerase chain reaction technique and an improved method. These markers, with four to 17 alleles per locus, identified 47 genotypes in 48 samples collected from a population in Hokkaido, Japan. The observed and expected heterozygosities ranged from 0.042 to 0.938 and from 0.448 to 0.909, respectively. These SSR markers will be available to identify genets and evaluate genetic diversity of V. album ssp. oxysepalum.     

Development and Characterization of RAPD and Microsatellite Markers for Genetic Variation Analysis in the Critically Endangered Yellow Catfish <Emphasis Type="Italic">Horabagrus nigricollaris</Emphasis> (Teleostei: Horabagridae)

Random-amplified polymorphic DNA (RAPD) and microsatellite markers were developed and used for the analysis of genetic variability in the critically endangered yellow catfish Horabagrus nigricollaris, sampled from the Chalakkudy River, Kerala, India. Eight RAPD and five microsatellite markers were detected to genotype the species. In RAPD, the 73 fragments were 20.55% polymorphic, whereas 4 polymorphic loci (80%) were obtained in microsatellites. In microsatellites, the number of alleles across the 5 loci was 1-5, and the range of heterozygosity was 0.25-0.5. The mean observed number of alleles was 2.4, and the effective number was 1.775 per locus. The average heterozygosity across all investigated samples was 0.29, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods report a low degree of gene diversity and lack of genetic heterogeneity in the population of H. nigricollaris, emphasizing the need for fishery management, conservation, and rehabilitation of this species.     

Isolation and characterization of 10 SSR markers of Momordica charantia (Cucurbitaceae)

? Premise of the study: Microsatellite markers were isolated and characterized from the genome of Momordica charantia (bitter melon) to be applied in studies of genetic diversity and population structure. ? Methods and Results: Twenty-five microsatellite loci were isolated from the genome of bitter melon using the Fast Isolation by AFLP of Sequences COntaining Repeats (FIASCO) method. Ten loci were polymorphic, and the number of alleles per locus ranged from three to seven, with the observed heterozygosity ranging from 0.46 to 0.65. The markers also amplified successfully in the related species M. cochinchinensis and Cucurbita pepo. ? Conclusions: These markers will have potential utility for applications in genetic diversity evaluation, molecular fingerprinting, identification, comparative genomics analysis, and genetic mapping in Momordica species, as well as in C. pepo.