Mapping genetic diversity of cherimoya (Annona cherimola Mill.): application of spatial analysis for conservation and use of plant genetic resources

There is a growing call for inventories that evaluate geographic patterns in diversity of plant genetic resources maintained on farm and in species' natural populations in order to enhance their use and conservation. Such evaluations are relevant for useful tropical and subtropical tree species, as many of these species are still undomesticated, or in incipient stages of domestication and local populations can offer yet-unknown traits of high value to further domestication. For many outcrossing species, such as most trees, inbreeding depression can be an issue, and genetic diversity is important to sustain local production. Diversity is also crucial for species to adapt to environmental changes. This paper explores the possibilities of incorporating molecular marker data into Geographic Information Systems (GIS) to allow visualization and better understanding of spatial patterns of genetic diversity as a key input to optimize conservation and use of plant genetic resources, based on a case study of cherimoya (Annona cherimola Mill.), a Neotropical fruit tree species. We present spatial analyses to (1) improve the understanding of spatial distribution of genetic diversity of cherimoya natural stands and cultivated trees in Ecuador, Bolivia and Peru based on microsatellite molecular markers (SSRs); and (2) formulate optimal conservation strategies by revealing priority areas for in situ conservation, and identifying existing diversity gaps in ex situ collections. We found high levels of allelic richness, locally common alleles and expected heterozygosity in cherimoya's putative centre of origin, southern Ecuador and northern Peru, whereas levels of diversity in southern Peru and especially in Bolivia were significantly lower. The application of GIS on a large microsatellite dataset allows a more detailed prioritization of areas for in situ conservation and targeted collection across the Andean distribution range of cherimoya than previous studies could do, i.e. at province and department level in Ecuador and Peru, respectively.     

Interpretation of triose phosphate isomerase isozymes in the cherimoya (Annona cherimola Mill.)

Detailed interpretation of triose phosphate isomerase (TPI) isozymes in seed plants has been restricted to only a few species. Three sets of TPI bands are regularly observed in the cherimoya(Annona cherimola), a primitive angiosperm. The slowest, set I, is expressed as one or three bands; the second-slowest set II, as one or two bands; and the fastest, set IV, as one or three bands. A faint set III, just cathodal to set IV, is detected rarely with overstaining. Set IV bands are expressed in macerated extracted pollen but not in pollen leachate. Dissociation-reassociation experiments reveal that the set II bands are heterodimers involving, in part, the enzymes involved in the set I bands. These data combined with those from full-sib progeny analysis lead us to propose a three-locus model to explain the TPI isozyme banding patterns in cherimoya. Sets I and IV consist of the allelic products of individual, single loci. Sets I and II occur in the cytoplasm. Set IV occurs in organelles. Set II isozymes are the intergenic heterodimers of the locus coding for set I and the locus coding for set III. Our results reported here are contrasted with the TPI isozyme patterns known for other vascular plants and suggest that the locus coding for set III may be a duplication of very ancient origin.This work was supported, in part, by funding from the Elvenia J. Slosson Endowment Fund.     

Genetic diversity and conservation and utilization of plant genetic resources

Biodiversity refers to variation within the living world, while genetic diversity represents the heritable variation within and between populations of organisms, and in the context of this paper, among plant species. This pool of genetic variation within an inter-mating population is the basis for selection as well as for plant improvement. Thus, conservation of this plant genetic diversity is essential for present and future human well-being. During recent years, there has been increasing awareness of the importance of adopting a holistic view of biodiversity, including agricultural biodiversity, conservation for sustainable utilization and development. These principles have been enshrined in the Convention on Biological Diversity and the Global Plan of Action of the Food and Agriculture Organization of the United Nations. The emphasis is now to understand the distribution and extent of genetic diversity available to humans in plant species, so that the genetic diversity can be safely conserved and efficiently used. It is generally recognized that plant genetic diversity changes in time and space. The extent and distribution of genetic diversity in a plant species depends on its evolution and breeding system, ecological and geographical factors, past bottlenecks, and often by many human factors. Much of the large amount of diversity of a species may be found within individual populations, or partitioned among a number of different populations.A better understanding of genetic diversity and its distribution is essential for its conservation and use. It will help us in determining what to conserve as well as where to conserve, and will improve our understanding of the taxonomy and origin and evolution of plant species of interest. Knowledge of both these topics is essential for collecting and use of any plant species and its wild relatives. In order to mange conserved germplasm better, there is also a need to understand the genetic diversity that is present in collections. This will help us to rationalize collections and develop and adopt better protocols for regeneration of germplasm seed. Through improved characterization and development of core collections based on genetic diversity information, it will be possible to exploit the available resources in more valuable ways.     

Genetic structure of Araucaria angustifolia (Araucariaceae) populations in Brazil: implications for the in situ conservation of genetic resources

The distribution of the genetic variation within and among natural populations of A. ANGUSTIFOLIA growing in different regions in Brazil was assessed at microsatellite and AFLP markers. Both markers revealed high gene diversity ( H = 0.65; AR = 9.1 for microsatellites and H = 0.27; P = 77.8 % for AFLPs), moderate overall differentiation ( RST = 0.13 for microsatellites and FST = 0.10 for AFLPs), but high divergence of the northernmost, geographically isolated population. In a Bayesian analysis, microsatellite data suggested population structure at two levels: at K = 2 and at K = 3 in agreement to the geographical distribution of populations. This result was confirmed by the UPGMA dendrogram based on microsatellite data (bootstrap support > 95 %). Non-hierarchical AMOVA revealed high variation among populations from different A POSTERIORI defined geographical groups. The genetic distance between sample locations increased with geographical distance for microsatellites ( R = 0.62; P = 0.003) and AFLPs ( R = 0.32; P = 0.09). This pattern of population differentiation may be correlated with population history such as geographical isolation and postglacial colonization of highlands. Implications of the population genetic structure for the conservation of genetic resources are discussed.     

PERMANENT GENETIC RESOURCES: Development of 52 new polymorphic SSR markers from cherimoya (Annona cherimola Mill.): transferability to related taxa and selection of a reduced set for DNA fingerprinting and diversity studies

Fifty-two single locus polymorphic microsatellites were developed using two genomic libraries digested with HaeIII and RsaI of cherimoya cv. Fino de Jete enriched in CT/AG repeats. A total of 222 alleles were detected with the selected simple sequence repeats (SSRs). The observed and expected heterozygosities ranged from 0.08 to 0.73 and from 0.20 to 0.84, respectively. Most of the SSRs were transferable to other species in the Annonaceae. A set of 20 microsatellites was selected to facilitate the exchange of data among laboratories.     

Importance of genetic characterization and conservation of plant genetic resources: The breeding system and genetic diversity of wild soybean (Glycine soja)

Abstract Plant genetic resources play an important role in the improvement of cultivated plants. To characterize and evaluate the ecological and reproductive features of wild soybean ( Glycine soja Sieb. et Zucc.), which is the most probable ancestor of cultivated soybean ( G. max (L) Merr.), the breeding system and genetic diversity of G. soja were investigated. The extent of natural cross-pollination of G. soja was estimated in four populations along the Omono River in Akita Prefecture, Japan by examining allozyme variation. Although it has been previously believed that G. soja is autogamous, as is cultivated soybean, the mean multilocus outcrossing rate ( t m) estimate was 13%. These values are much higher than the outcrossing rate previously reported for both G. soja and G. max . Frequent visits by honeybees and carpenter bees to flowers were also observed, which supported this conjecture. Furthermore, to evaluate the genetic variation of G. soja as a genetic resource, the genetic structure of 447 populations over Japan were analyzed. Wild soybean populations had a higher degree of variation of isozyme loci. The G ST coefficient of gene differentian values among the sites within the district were particularly high, revealing that the isozyme genotype was greatly different among site populations and homogeneous within the sites. The genetic differentiation among nine districts was observed in the allele frequencies of a few loci, indicating that geographic isolation in the wild soybean population was effectively created through the distance between the districts. The difference in the allele frequency among the districts may be produced under genetic drift. Finally, the importance of the preservation of natural plant populations and the habitats of wild progenitors (i.e. the in situ conservation of plant genetic resources) was emphasized.     

Mapping the geographical distribution of genetic variation in the genus Lens for the enhanced conservation of plant genetic diversity

Plant genetic resource conservation strategies, informed by an understanding of the geographical distribution of genetic variation within species, are likely to result in a wider representation of conserved diversity in ex situ gene banks and in situ genetic reserves. The main objective of this study was to map the geographical distribution of genetic variation, as revealed by randomly amplified polymorphic DNA (RAPDs), in four wild relatives of the cultivated lentil, namely Lens culinaris ssp. orientalis, L. odemensis, L. ervoides and L. nigricans . Areas of high diversity and unique diversity were located for each taxon, and regions where further germplasm collection was most likely to yield novel genetic variation were identified. There were centres of diversity for L. culinaris ssp. orientalis in southeast Turkey and northwest Syria, and in south Syria and Jordan. A centre of diversity was found to exist in Sweida province, south Syria, for L. odemensis , and for L. ervoides along the coastal border region between Syria and Turkey stretching down along the Syrian coast. There was a centre of diversity for L. nigricans in west Turkey. Analytical techniques previously used at the species level were found to be useful at the genotypic level to objectively target areas for future collection missions, to increase diversity in ex situ collections and to target areas for in situ conservation.     

On the verge of extinction: genetics of the critically endangered Iberian plant species, Borderea chouardii (Dioscoreaceae) and implications for conservation management

Borderea chouardii is a relictual and dioecious, strictly sexually reproducing, long-living geophyte of the Dioscoreaceae family. Previous biological and demographic studies have indicated the existence of a uniformly distributed panmictic population of this taxon at the southernmost Spanish pre-Pyrenean mountain ranges where it occurs in rather inaccessible crevices of a single limestone cliff. However, individuals of B. chouardii are spatially subdivided into two subpopulations located, respectively, on the upper and lower parts of the cliff, and vertically separated 150 m. Because of its extreme rarity, B. chouardii was the first Iberian taxon to have a specific conservation plan and has been included in several red lists under the category of critically endangered (CR). However, no previous attempts have been conducted to analyse the fine scale evolutionary mechanisms involved in its present microspatial distribution. Genetic diversity and population structure have been investigated through the analysis of neutral hypervariable markers such as simple sequence repeats (SSRs) and randomly amplified polymorphic DNAs (RAPDs) to unravel the impact of life history traits in the differentiation of the two subpopulations. Both types of molecular markers were unequivocal in distinguishing two genetically distinct groups of individuals corresponding to their spatial separation. However, SSRs detected a higher level of subpopulation differentiation (F(ST) = 0.35, R(ST) = 0.32) than RAPDs (F(ST) = 0.21). SSR data indicated significant deviation from random dispersal of genes and genotypes between the two groups, suggesting that mating occurs mainly among individuals within subpopulations, thus, favouring the divergence between the two groups. This microevolutionary differentiation scenario might have been caused by a coupled effect of past genetic drift and reproductive isolation, as a result of strong glacial age bottlenecks and inefficient dispersal system of pollen and seeds, respectively. The identification of such genetic structure in this narrow endemic prompts a modification of the management strategies of its single extant population.     

Interpopulation genetic divergence in European grayling (Thymallus thymallus, Salmonidae) at a microgeographic scale: implications for conservation

In order to provide guidelines for conservationand management of the severely declining LakeSaimaa (eastern Finland) European grayling(Thymallus thymallus, Salmonidae), weinvestigated the microgeographic geneticstructure of three populations from the watersystem using 17 microsatellite loci andmitochondrial DNA polymerase chainreaction-restriction fragment lengthpolymorphism analysis. Microsatellites revealedlow levels of intrapopulation genetic diversityand substantial divergence between populationssampled from spawning sites separated by aslittle as 55 kilometers. Mitochondrial analysisindicated the occurrence of two compositehaplotypes within Lake Saimaa. The nucleotidesubstitution estimates between the haplotypessuggested their separation to markedly predatethe late Pleistocene period. The populationsexhibited marked frequency differences for thetwo mitochondrial haplotypes, reinforcing theview of limited interpopulation gene flowwithin Lake Saimaa. An individual basedmicrosatellite Neighbor-Joining dendrogramdemonstrated clustering of all the specimensaccording to their sampling origin. Individualassignment tests revealed 100% assignmentsuccess of individuals into their population oforigin and 100% exclusion (p <0.05) from all alternative referencepopulations. These findings exemplify that T. thymallus populations may be genetically highly structured over small geographical scales and provide a good starting point for the development of appropriate conservation strategies for Lake Saimaa grayling.     

生境片断化对植物种群遗传结构的影响及植物遗传多样性保护

生境片断化是指大而连续的生境变成空间隔离的小种群的现象。生境片断化对植物种群遗传效应包括生境片断化过程中的取样效应及其后的小种群效应（遗传漂变、近交等）。理论研究表明,生境片断化后,植物种群的遗传变异程度将降低,而残留的小种群间的遗传分化程度将升高。然而对一些植物的研究表明,生境片断化对植物种群的遗传效应要受其他一些因素的影响,如世代长度、片断化时间、片断种群的大小、基因流的改变等。最后,针对生境     

Geographic pattern of genetic variation in Pinus resinosa: area of greatest diversity is not the origin of postglacial populations

Genetic diversity is low in natural populations of red pine, Pinus resinosa, a species that has a vast range across north-eastern North America. In this study, we examined 10 chloroplast microsatellite or simple sequence repeats (cpSSR) loci in 136 individuals from 10 widespread populations. Substantial variation for the cpSSR loci was observed in the study populations. The contrast with red pine's lack of variation for other types of loci is likely to be due to the higher mutation rates typical of SSR loci. The amount of variation is lower than that generally found for cpSSR loci in other pine species. In addition, the variation exhibits a striking geographical pattern. Most of the genetic diversity is among populations, with little within populations, indicating substantial isolation of and genetic drift within many populations in the southern half of the species distribution. The greatest diversity now occurs in the north-eastern part of New England, which is especially intriguing because this entire area was glaciated. Thus the centre of diversity cannot be the origin of postglacial populations, rather it is likely caused by admixture, most probably because of influences from two separate refugia. Furthermore, the pattern indicates that the spread of red pine since the last glaciation is rather more complex than usually described, and it likely includes more than one refugia, complex migration routes, and postglacial-retreat isolation and genetic drift among shrinking populations in regions of the present southern range.     

Microsatellite genetic variation in the Chinese endemic Eucommia ulmoides (Eucommiaceae): implications for conservation

Eucommia ulmoides, a traditional Chinese medicinal plant, is endangered as a consequence of long‐term and widespread harvest in the late 20th century. It has been widely cultivated as a source of herbal medicine and for use in the organic chemical industry in China. In this study, eight microsatellite markers were applied to investigate genetic diversity in E. ulmoides. Three hundred individuals from one semi‐wild population and nine cultivated populations across its main production area were collected. A high level of genetic diversity at population levels (H_E = 0.716) was observed. The highly outcrossed mating system, high longevity of E. ulmoides and seed admixture may be responsible for high genetic variation within populations. A genetic bottleneck was observed in one population. Populations were only slightly differentiated from one another (F_ST = 0.063); this was also supported by AMOVA, which revealed that 94.05% of the total variation resided within populations. This is probably attributable to long‐distance gene flow mediated by the exchange of seeds by local farmers. Implications of these results for the conservation of genetic resources of E. ulmoides are discussed. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 775–785.     

Microsatellite analysis of the inbreeding grass weed Barren Brome (Anisantha sterilis) reveals genetic diversity at the within- and between-farm scales

Nine microsatellites were used to screen 131 samples of Barren Brome (Anisantha sterilis: synonym Bromus sterilis) collected from within the fields of three English farms [from Oxfordshire (Oxon), Leicestershire (Leics) and Wiltshire (Wilts)] and eight seeds taken from samples of each of 10 farms across England, UK. Most individuals (approximately 97%) were homozygous. Polymorphism occurred at all nine loci in all three farms sampled at the field scale, and at most loci for nine of the other 10 farm samples. Between three and 11 alleles were found per locus. Gene diversity (D = 1 - summation operator p(i)2) ranged from 0.088 to 0.760. Polymorphism occurred among individuals within and among fields, and farms. Some alleles were found in only one farm. On the basis of the alleles at all nine loci in the 211 sampled plants, a total of 92 (44%) different genotypes was identified. Clustering analysis using the unweighted pair group method with arithmetic averages (UPGMA) for the combined Oxon, Wilts and Leics samples did not cluster them into their respective farms. Similarly, a phenogram of samples from all 10 farms showed considerable mixing of individuals with respect to farm origins. Identification of genotypes on field plans showed evidence of both spatial localization and mixing. Previous reports have suggested that A. sterilis is strictly inbreeding with little intrapopulation variation at the genetic level. Our data reveal that A. sterilis exists as numerous separate and genetically different lines, which are maintained by inbreeding but which very occasionally outcross. Possible explanations for this pattern of high genetic diversity are discussed.     

Low genetic diversity and high genetic differentiation in the critically endangered Omphalogramma souliei (Primulaceae):implications for its conservation

Omphalogramma souliei Franch. Is an endangered perennial herb only distributed in alpine areas of SW China. ISSR markers were applied to determine the genetic variation and genetic structure of 60 individuals of three populations of O. Souliei in NW Yunnan, China. The genetic diversity at the species level is low with P= 42.5% (percentage of polymorphic bands) and Hsp=0.1762 (total genetic diversity). However, a high level of genetic differentiation among populations was detected based on different measures (Nei's genetic diversity analysis: Gst=0.6038; AMOVA analysis: Fst=0.6797). Low level of genetic diversity within populations and significant genetic differentiation among populations might be due to the mixed mating system in which xenog-amy predominated and autogamy played an assistant role in O. Souliei. The genetic drift due to small population size and limited current gene flow also resulted in significant genetic differentiation. The assessment of genetic variation and differentiation of the endangered species provides important information for conservation on a genetic basis. Conservation strategies for this rare endemic species are proposed.     

岛屿植物舟山新木姜子居群遗传多样性的RAPD分析

基于随机扩增多态 DNA(RAPD)方法分析了舟山群岛濒危植物舟山新木姜子 (N eolitsea sericea) 6个居群的遗传多样性及分化程度。 10条随机引物扩增出 84个可分析位点 ,多态位点百分比 (PPL)为 3 8.10 %。经 POPOGENE分析发现 ,舟山新木姜子居群平均水平的多态位点百分比 (PPL )为 2 3 .18% ,Nei' s基因多样度 (HE)为 0 .0 793 ,Shannon信息指数 (H )为 0 .12 0 1,与其它岛屿植物比较具有中等偏低水平的遗传多样性 ;岛屿各居群间遗传分化程度较高 (Gst=0 .3 646) ;地理距离与遗传距离之间具有显著相关性 (r=0 .7697,P=96.62 % ) ,岛屿隔离效应是导致居群间遗传分化的重要因素。结合居群遗传多样性及UPGMA聚类分析 ,推测普陀山岛舟山新木姜子部分个体可能为大猫岛迁入的后裔 ,而朱家尖岛舟山新木姜子则由人为移植自普陀山岛。基于舟山新木姜子的物种保护及资源利用 ,建议加强现有自然居群的就地保护 ,促进居群自然更新 ;建立种质资源库 ,收集不同岛屿的种源进行混合繁殖 ,促进基因交流 ;选育优良品系用于海岛植被恢复及园林观赏     

Low genetic diversity and high genetic differentiation in the critically endangered Omphalogramma souliei (Primulaceae): implications for its conservation

Omphalogramma souliei Franch. is an endangered perennial herb only distributed in alpine areas of SW China. ISSR markers were applied to determine the genetic variation and genetic structure of 60 individuals of three populations of O. souliei in NW Yunnan, China. The genetic diversity at the species level is low with P=42.5% (percentage of polymorphic bands) and Hsp=0.1762 (total genetic diversity). However, a high level of genetic differentiation among populations was detected based on different measures (Nei's genetic diversity analysis: Gst=0.6038; AMOVA analysis: Fst=0.6797). Low level of genetic diversity within populations and significant genetic differentiation among populations might be due to the mixed mating system in which xenogamy predominated and autogamy played an assistant role in O. souliei. The genetic drift due to small population size and limited current gene flow also resulted in significant genetic differentiation. The assessment of genetic variation and differentiation of the endangered species provides important information for conservation on a genetic basis. Conservation strategies for this rare endemic species are proposed.     

A multilocus population genetic survey of the greater sage-grouse across their range

The distribution and abundance of the greater sage-grouse (Centrocercus urophasianus) have declined dramatically, and as a result the species has become the focus of conservation efforts. We conducted a range-wide genetic survey of the species which included 46 populations and over 1000 individuals using both mitochondrial sequence data and data from seven nuclear microsatellites. Nested clade and structure analyses revealed that, in general, the greater sage-grouse populations follow an isolation-by-distance model of restricted gene flow. This suggests that movements of the greater sage-grouse are typically among neighbouring populations and not across the species, range. This may have important implications if management is considering translocations as they should involve neighbouring rather than distant populations to preserve any effects of local adaptation. We identified two populations in Washington with low levels of genetic variation that reflect severe habitat loss and dramatic population decline. Managers of these populations may consider augmentation from geographically close populations. One population (Lyon/Mono) on the southwestern edge of the species' range appears to have been isolated from all other greater sage-grouse populations. This population is sufficiently genetically distinct that it warrants protection and management as a separate unit. The genetic data presented here, in conjunction with large-scale demographic and habitat data, will provide an integrated approach to conservation efforts for the greater sage-grouse.     

应用微卫星标记分析不同桔小实蝇种群的遗传多样性

为探究不同桔小实蝇Bactrocera dorsalis (Hendel)地理种群的遗传变异、入侵来源和扩散情况,利用13对引物对中国南方10省区、泰国、夏威夷、菲律宾和老挝的30个桔小实蝇种群共180个个体的遗传多样性水平进行了研究。 Popgene32和NTSYS-pc2.10e软件分析结果表明：30个不同桔小实蝇种群的遗传相似度在0.3599~0.9153范围内。种群的Nei氏基因多样性指数平均为0.6464±0.1026,Shannon信息指数I平均为1.2845±0.2632, 提示桔小实蝇种群具有较丰富的遗传多样性。UPGMA聚类分析显示,福建地区和海南地区分别独立一支,广东地区和台湾地区种群聚成一支,而广西、泰国、湖南、云南、老挝、四川、 重庆和贵州地区聚为一大支系。据此提出泰国种群和老挝种群是最早入侵我国的种群,云南地区是最早的入侵地,广西地区可能为又一较早入侵地。