High Resolution Melting (HRM) analysis in eggplant (Solanum melongena L.): A tool for microsatellite genotyping and molecular characterization of a Greek Genebank collection

The conservation and characterization of eggplant (Solanum melongena L.) genetic resources in germplasm banks has been the basis of their use in breeding projects, which has resulted in the development of new cultivars. High Resolution Melting (HRM) analysis, combined with eight microsatellite markers, has been integrated in order to facilitate the molecular identification and characterization of the eggplant germplasm, collected from the National Genebank Collection of Greece. The eight microsatellite loci used were highly informative and generated sixty three HRM profiles, which were sufficient to discriminate all eggplant landraces and cultivars studied, highlighting its potential use for cultivar genotyping. The thirty six eggplant genotypes were classified into four clusters. Hence, this assay provided a fast, cost-effective and closed-tube microsatellite genotyping method, well suited for molecular characterization of eggplant cultivars.     

国家库贮藏20年以上种子生活力与田间出苗率监测

对34种作物1.4万多份的国家库贮存种子进行了生活力监测,结果表明,贮存20年后,92.9%被监测种子的发芽率仍保持在85%以上,但有155份种子（占被监测份数的1.1%）出现了明显的下降（发芽率从80%以上降至70%以下）。发芽率显著下降的作物包括蚕豆、红小豆、黄麻、蓖麻、甜菜、西瓜、烟草、牧草。小麦等8种作物2078份种子的田间出苗率调查表明,所有种质均有出苗,但有8份种质的出苗率低于10%。出苗率与入库初始平均发芽率存在差别,平均出苗率最高的作物为普通菜豆86.2%,比该批入库初始发芽率平均值低9.3%;平均出苗率最低作物为谷子39.2%,比该批种子的入库初始发芽率平均值低51.3%。总体而言,国家库内保存的作物种子中,多数可安全保存20年以上,尤其是水稻等禾谷类作物种子,但对于蚕豆、红小豆等平均监测发芽率出现显著下降的作物种子需要增加监测频率,以确保种子的长期安全保存。     

茶梅品种资源的收集保存、鉴定评价及种质创新

综述了国内外茶梅品种的收集保存、鉴定评价及种质创新工作,其中上海市农业生物基因中心自2002年起,共收集国内外茶梅品种80余种,建立品种资源基地并对其进行了鉴定评价,筛选出3个适于上海地区的品种,并登录新品种1个。     

Molecular characterization of an international cacao collection using microsatellite markers

Plant germplasm collections invariably contain varying levels of genetic redundancy, which hinders the efficient conservation and utilization of plant germplasm. Reduction of genetic redundancies is an essential step to improve the accuracy and efficiency of genebank management. The present study targeted the assessment of genetic redundancy and genetic structure in an international cacao (Theobroma cacao L.) collection maintained in Costa Rica. A total of 688 cacao accessions maintained in this collection were genotyped with 15 simple sequence repeat (SSR) loci, using a capillary electrophoresis genotyping system. The SSR markers provided a high resolution among the accessions. Thirty-six synonymously labeled sets, involving 135 accessions were identified based on the matching of multilocus SSR profiles. After the elimination of synonymous sets, the level of redundancy caused by closely related accessions in the collection was assessed using a simulated sampling scheme that compared allelic diversity in different sample sizes. The result of the simulation suggested that a random sample of 113 accessions could capture 90% of the total allelic diversity in this collection. Principal Coordinate Analysis revealed that the Trinitario hybrids from Costa Rica shared a high similarity among groups as well as among individual accessions. The analysis of the genetic structure illustrated that the within-country/within-region difference accounted for 84.6% of the total molecular variation whereas the among-country/among-region difference accounted for 15.4%. The Brazilian germplasm contributed most to this collection in terms of total alleles and private alleles. The intercountry/interregion relationship by cluster analysis largely agreed with the geographical origin of each germplasm group and supported the hypothesis that the Upper Amazon region is the center of diversity for cacao. The results of the present study indicated that the CATIE International Cacao Collection contains a high level of genetic redundancy. It should be possible to rationalize this collection by reducing redundancy and ensuring optimal representation of the genetic diversity from distinct germplasm groups. The results also demonstrated that SSR markers, together with the statistical tools for individual identification and redundancy assessment, are technically practical and sufficiently informative to assist the management of a tropical plant germplasm collection.     

Developing an appropriate strategy to assess genetic variability in plant germplasm collections

 The value of molecular biology for monitoring the genetic status of germplasm collections is subject to practical limitations. The large number and variability of accessions held usually dictates the approach that can be employed. A quick, simple but reliable molecular protocol must be combined with an appropriate strategy for handling large sample sizes. In this study, ISSR-PCR was used to reveal genetic variability within and between accessions held in a collection of lupin germplasm. Pooling of DNA from individuals within accessions was found to be the most appropriate strategy for assessing large quantities of plant material. Band profiles generated from pools containing five individuals were fully representative of all constituent individuals used in the mix. Pools comprising 10 or 20 individuals, however, sometimes failed to contain minor bands that had been present only in the profile of one individual. Variation was observed between pools containing five different genotypes from the same accession. Routine large-scale screens are required to assess the genetic diversity and homogeneity of the lupin germplasm collection held in Reading. It is concluded that 2–3 pools of five genotypes may be sufficient to represent the genetic variability within and between accessions in the lupin and similar collections. Received: 10 August 1998 / Accepted: 13 November 1998     

Molecular studies on genetic integrity of open-pollinating species rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.) after long-term genebank maintenance

The genetic integrity of six accessions represented by 14 sub-populations of the open-pollinating species rye (Secale cereale L.) was investigated. Seeds available from a herbarium collection (first regeneration) and from the cold store (most recent regeneration) were multiplied two to fourteen times and fingerprinted using microsatellite markers. Four accessions had significantly different allele frequencies. These were multiplied seven to thirteen times. Nearly 50% of the alleles discovered in the original samples were not found in the material present in the cold store. However alleles were detected in the most recently propagated sub-populations, that were not observed in the investigated plants of the original one. The change in allele frequencies is a continuous process. Reasons for the occurrence of genetic changes and consequences for managing open pollinating species maintained in ex situ genebanks are discussed.Communicated by G. Wenzel     

The use of self-pollinated progenies as ’in-groups’ for the genetic characterization of cocoa germplasm

Theobroma cacao ) genotypes. Six primers were sufficient to distinguish all but three pairs of the 62 accessions examined. A UPGMA dendrogram was used to provide a measure of the genetic variability between genotypes. The scale was supplied by the inclusion of Theobroma grandiflora as an ’out group’ and also by the use of two contrasting progenies as ’in groups’. The ’in groups’ were obtained from the self-pollination of one plant (SPEC 54.1) known to be highly homozygous and also of a second, highly heterozygous, clone (P 19B). These reference points allowed several documentation errors to be resolved and provided a basis for identifying unwanted or low-priority material. Implications of the work for the routine maintenance of large germplasm collections are briefly discussed. Received: 20 January 1999 / Accepted: 25 May 1999     

Longevity of cryogenically stored seeds

Though cryogenic storage is presumed to provide nearly infinite longevity to cells, the actual shelf life achieved under ultra-cold temperatures has not been addressed theoretically or empirically. Here, we report measurable changes in germination of dried seeds stored under liquid nitrogen conditions for >10 years. There was considerable variability in the extent of deterioration among species and accessions within a species. Aging time courses for lettuce seeds stored at temperatures between 50 and -196 degrees C were fit to a form of the Avrami equation to determine rate coefficients and predict half-life of accessions. A reduction in the temperature dependency on aging rate, determined as a break in the Arrhenius plot, occurred at about -15 degrees C, and this resulted in faster deterioration than anticipated from extrapolation of kinetics measured at higher temperatures. The break in Arrhenius behavior occurred at temperatures in between the glass transition temperature (28 degrees C) and the Kauzmann temperature (-42 degrees C) and also coincided with a major triacylglycerol phase change (-40 to -7 degrees C). In spite of the faster than anticipated deterioration, cryogenic storage clearly prolonged shelf life of lettuce seeds with half-lives projected as approximately 500 and approximately 3400 years for fresh lettuce seeds stored in the vapor and liquid phases of liquid nitrogen, respectively. The benefit of low temperature storage (-18 or -135 degrees C) on seed longevity was progressively lost if seeds were first stored at 5 degrees C. Collectively, these results demonstrate that lowering storage temperature progressively increases longevity of seeds. However, cryogenic temperatures were not sufficient to stop deterioration, especially if initial stages of aging were allowed to progress at higher storage temperatures. This work contributes to reliable assessments of the potential benefit and cost of different genebanking strategies.