Conservation genetics of Heritiera littoralis (Sterculiaceae), a threatened mangrove in China,based on AFLP and ISSR markers

AFLP and ISSR markers were used to determine the genetic variations in eight mangrove and non-mangrove populations of Heritiera littoralis (Sterculiaceae), a threatened species in China. Our results showed a moderate to high level of genetic variation in this species (P = 63.69%, H_T = 0.20 for AFLP; P = 76.07%, H_T = 0.22 for ISSR), and a relatively high level of genetic differentiation among populations (G_ST = 0.24 for AFLP and 0.27 for ISSR). Life history traits, long-distance dispersal of floating seeds, and local environments may play important roles in shaping the genetic diversity and genetic structure of this species. Investigation of the plant’s reproductive capacity showed that the natural seed production of H. littoralis was low, as was the germination rate and the transformation rate from juvenile to adult. H. littoralis is seriously threatened and is in urgent need of conservation in China.     

Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon

Three different types of molecular markers, RAPD, AFLP and RFLP were used to measure genetic diversity among six genotypes of Cucumis melo L. Each line represented a different melon genotype: Piel de Sapo, Ogen, PI161375, PI414723, Agrestis and C105. A number of polymorphic RAPD, AFLP and RFLP bands were scored on all materials and the genetic similarity measured. Clustering analysis performed with the three types of markers separated the genotypes into two main groups: (1) the sweet type, cultivated melons and (2) the exotic type, not cultivated melons. While the data obtained suggest that all three types of markers are equally informative, AFLPs showed the highest efficiency in detecting polymorphism. Received: 30 December 1999 / Accepted: 24 January 2000     

Genetic diversity analysis and conservation of the endangered Chinese endemic herb Dendrobium officinale Kimura et Migo (Orchidaceae) based on AFLP

Dendrobium officinale is a critically endangered perennial herb endemic to China. Determining the levels of genetic diversity and patterns of population genetic structure of this species would assist in its conservation and management. Data of 12 populations were used to assess its genetic diversity and population structure, employing the method of amplified fragment length polymorphism (AFLP). A high level of genetic diversity was detected (H (E) = 0.269) with POPGENE. As revealed by AMOVA analysis, there was moderate variation between pairs of populations with Phi(ST) values ranging from 0.047 to 0.578 and on average 26.97% of the genetic variation occurred among populations. Three main clusters were shown in UPGMA dendrogram using TFPGA, which is consistent with the result of principal coordinate ananlysis (PCO) using NTSYS. Keeping a stable environment is critical for the in situ conservation and management of this rare and endangered plant, and for ex situ conservation it is important to design an integrated germplasm bank.     

Application of AFLP, RAPD and ISSR markers to genetic mapping of European and Japanese larch

Genetic linkage maps have been increasingly developed for a wide variety of plants, using segregating populations such as F₂s or backcrosses between inbred lines. These pedigrees are rarely available in outbred species like forest trees which have long generation times. Thus genetic mapping studies have to use peculiar pedigrees and markers in appropriate configurations. We constructed single-tree genetic linkage maps of European larch (Larix decidua Mill.) and Japanese larch [Larix kaempferi (Lamb.) Carr.] using segregation data from 112 progeny individuals of an hybrid family. A total of 266 markers (114 AFLP, 149 RAPD and 3 ISSR loci) showing a testcross configuration, i.e.heterozygous in one parent and null in the other parent, were grouped at LOD 4.0, θ=0.3. The maternal parent map (L. decidua)consisted of 117 markers partitioned within 17 linkage groups (1152 cM) and the paternal parent map (L. kaempferi) had 125 markers assembled into 21 linkage groups (1206 cM). The map distance covered by markers was determined by adding a 34.7-cM independence distance at the end of each group and unlinked marker. It reached 2537 cM and 2997 cM respectively for European larch and Japanese larch, and represented respectively a 79.6% and 80.8% coverage of the overall genome. A few 3:1 segregating markers were used to identify homologous linkage groups between the European larch and the Japanese larch genetic maps. The PCR-based molecular markers allowed the construction of genetic maps, thus ensuring a good coverage of the larch genome for further QTL detection and mapping studies. Received: 15 March 1999 / Accepted: 29 March 1999     

硬核资源遗传多样性的AFLP分析（英文）

为了解云南省硬核[Scleropyrum wallichianum (Wight et Arn.) Arn.]的遗传多样性,采用AFLP标记分析了7个居群84份种质材料的遗传变异。结果表明,从64对引物组合中挑选出多态性较好的引物8对,共扩增出1 728条带,其中多态性条带1 388条,多态性百分率为80.14%。硬核在物种水平的多样性指数分别为Na=1.416, Ne=1.179, H=0.137, I=0.225,在居群水平上分别为H=0.111,I=0.175;在遗传相似性系数为0.52时,这些种质材料可分为3组,其中易武居群具有丰富的遗传变异,大部分的遗传变异存在于居群内,而在0.05置信区间内居群间遗传变异仅为11.5%;居群间的遗传距离和地理距离无显著相关性(r=0.0323, P=0.5820)。因此,硬核资源可采用就地和迁地保护策略,以增加其遗传多样性。     

Analysis of genetic diversity in Aconitum kongboense L. revealed by AFLP markers

The systematic evaluation of the molecular diversity encompassed in Aconitum kongboense L. inbreds or parental lines offers an efficient means of exploiting the heterosis in A. kongboense as well as for management of biodiversity. An excellent and novel DNA-based molecular, amplified fragment length polymorphisms (AFLPs) markers, was firstly used to analysis the genetic diversity in A. kongboense genotypes. Out of 256 primers screened, a total of ten combinations successfully produced scorable, clear, reproducible and relatively high polymorphism bands, 64.12% of which were polymorphic. The values of number of polymorphic loci (NPL), percentage of polymorphic loci (PPB), observed number of alleles (Na) and effective number of alleles (Ne) were highest in population 3 (NPL = 77, PPB = 68.75%, Na = 1.688 ± 0.466, Ne = 1.412 ± 0.397) and lowest in population 2 (NPL = 57, PPB = 50.89%, Na = 1.509 ± 0.502, Ne = 1.273 ± 0.340). In addition, Jaccard's similarity coefficients among different populations varied from 0.45 to 1.00 with an average of 0.55. The data collected will contribute to identification, rational exploitation and conservation of germplasms of A. kongboense, and potentially useful to aid its breeding.     

用RAPD和AFLP的方法对中国卤虫(Artemia)种及亲缘关系的研究

利用ＲＡＰＤ（随机扩增多态ＤＮＡ）和ＡＦＬＰ（扩增片段长度多态性）技术对不同种及种群卤虫的关系进行分析。 １０１个随机引物对４种卤虫Ａｆｒａｎｃｉｓｃａｎａ、Ａ ｕｒｍｉａｎａ、Ａ ｓｉｎｉｃａ和Ａ．ｐａｒｔｈｅｎｏｇｅｎｅｌｉｃａ基因组ＤＮＡ进行扩增,平均每个种获得７５１条带,其中４５８条带为多态性标记,每个引物提供平均７４个标记信息,聚类结果表明Ａ．ｓｉｎｉｃａ是不同于其他旧大陆两性生殖卤虫的一个独立的种。对来自 １５个种及品系的卤虫的 ＡＦＬＰ分析显示了非常好的遗传多态性,采用 １２对引物检测到 ５９４条带,其中 ４８０个为多态性标记。聚类结果表明来自西藏的两性生殖卤虫为不同于中国内陆两性生殖卤虫的新种。而孤雌生殖卤虫在进化过程中可能是多源的,中国内陆和沿海的孤雌生殖卤虫是沿着不同的途径进化的,内陆和沿海的孤雌生殖卤虫可能为不同的种。     

Linkage map of Japanese black pine based on AFLP and RAPD markers including markers linked to resistance against the pine needle gall midge

Macrogametophytes derived from the seeds of a tree resistant to pine needle gall midge (PGM) were analyzed using amplified fragment length polymorphism (AFLP). A total of 244 segregating loci were detected among 71 macrogametophytes. Combining the AFLP results with previously reported segregation data for 127 random amplified polymorphic DNA (RAPD) markers, 157 AFLP and 50 RAPD markers with confirmed map positions were assigned to 20 linkage groups and three pairs covering 2085.5 cM with an average distance of 10.1 cM. The total map distance covers about 77.1–78.4% of the total genome, estimated to be approximately 2665–2719 cM in length. Thus, using AFLP markers, the previous RAPD map of this tree was improved in terms of the average distance between markers, the total map distance, and coverage of the genome. Three RAPD markers linked to a gene associated with resistance to PGM were also located on this map. Rceived: 14 April 2000 / Accepted: 21 August 2000     

重金属离子含量对利川莼菜生长的影响

莼菜原产于湖北省利川市,因其富含果胶质而具有极高的营养价值,是中国传统药食两用植物,也是国家一级珍稀濒危水生植物。为更好地保护莼菜这一珍稀植物资源,本研究以利川市高、中、低产莼菜田为研究对象,初步分析田中水和土壤的重金属离子含量对莼菜生长的影响,并了解其生长所需要的部分环境条件。结果显示:三类田的水质都达到了一类水质标准,其中锌离子含量只在高-低产田间表现出显著差异,在高、中产田间,中、低产田间未见显著差异;土壤中重金属离子含量总体处于三级水平,说明累积量较少,土质尚好。同时三类田中水为中性而土壤为偏酸性,且未表现出显著性差异。相关性分析表明水土中重金属离子并未对莼菜生长和产量产生实质影响,研究结果为下一步深入分析重金属离子对莼菜生长的影响机制以及制定资源保护策略奠定了基础。     

Comparative analysis of genetic variation among <Emphasis Type="Italic">Xanthomonas axonopodis pv manihotis</Emphasis> isolated from the western states of Nigeria using RAPD and AFLP

Xanthomonas axonopodis pv manihotis is the causal agent of cassava bacterial blight (CBB) worldwide. CBB disease is a major constraint to cassava cultivation, and losses can be extremely severe in regions where highly susceptible cultivars are grown. To develop an efficient disease management policy, the genetic diversity of the pathogens population must be known. There is dearth of information on the genetic diversity of X. axonopodis pv manihotis population in Nigeria. We used RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism), a PCR-based technique, to characterize the X. axonopodis pv manihotis isolates from the western States of Nigeria. Thirteen strains Xam and 2 reference strains were tested with eight primers combination of AFLP and 4 RAPD primers. RAPD amplified DNA fragment data showed four major clusters at 80 % similarity coefficient level and two strains were not clustered by this analysis. Strains Kwa76A and Ond48A were also separated in the principal component analysis of the same data. Numerical analysis differentiated the AFLP patterns into four distinct clusters and grouped two strains separately at 66 % similarity. PCA assembly grouped the bacterial strains into 4 and one of the strains was singled out from the others. The two DNA analyses techniques seem to be complimentary to one another and informative on the genomic structure of Xam population in Western Nigeria. The genetic analysis presented here contributes to understanding of the Xam population structure in Western Nigeria.     

极度濒危植物五针白皮松的保护遗传学研究

用随机扩增多态(RAPD)方法对极度濒危植物五针白皮松(Pinus squamata X.W.Li)的遗传多样性和居群遗传结构进行了研究。14个随机引物共获得93条RAPD谱带,其中6条为多态带,多态位点百分率仅为6．45％,遗传多样性极低。Shannon指数I和Nei指数h在种内也只有0．020和0．030,两个亚居群间(半阴坡亚居群与半阳坡亚居群)遗传分化程度不高,遗传分化系数Gst只有0．110,与大多数松科植物近似,居群每代迁移数为4．032。五针白皮松极低的遗传多样性可能是由于它在演化过程中遭受过严重的灾害,造成严重的瓶颈效应,丧失其大部分遗传变异。在随后的演化过程中由于遗传漂变、自交衰退等小种群现象,导致遗传多样性的进一步丧失。另外,阔叶树种对其生存的挤压和人类的干扰也是导致五针白皮松遗传多样性降低的因素之一。本文最后对该结果的保护生物学意义进行了评价。     

Assessment of genetic diversity in Azadirachta indica using AFLP markers

 Genetic diversity was estimated in 37 neem accessions from different eco-geographic regions of India and four exotic lines from Thailand using AFLP markers. Seven AFLP selective primer combinations generated a total of 422 amplification products. The average number of scorable fragments was 60 per experiment, and a high degree (69.8%) of polymorphism was obtained per assay with values ranging from 58% to 83.8%. Several rare and accession-specific bands were identified which could be effectively used to distinguish the different genotypes. Genetic relationships within the accessions were evaluated by generating a similarity matrix based on the Jaccard index. The phenetic dendrogram generated by UPGMA as well as principal correspondence analysis separated the 37 Indian genotypes from the four Thai lines. The cluster analysis indicated that neem germplasm within India constitutes a broad genetic base with the values of genetic similarity coefficient ranging from 0.74 to 0.93. Also, the Indian genotypes were more dispersed on the principal correspondence plot, indicating a wide genetic base. The four lines from Thailand, on the other hand, formed a narrow genetic base with similarity coefficients ranging from 0.88 to 0.92. The lowest genetic similarity coefficient value (0.47) was observed between an Indian and an exotic genotype. The level of genetic variation detected within the neem accessions with AFLP analysis suggests that it is an efficient marker technology for delineating genetic relationships amongst genotypes and estimating genetic diversity, thereby enabling the formulation of appropriate strategies for conservation and tree improvement programs. Received: 20 October 1998 / Accepted: 28 November 1998     

A genetic map of Asparagus officinalis based on integrated RFLP, RAPD and AFLP molecular markers

 An integrated genetic map of the dioecious species Asparagus officinalis L. has been constructed on the basis of RFLP, RAPD, AFLP and isoenzyme markers. The segregation analysis of the polymorphic markers was carried out on the progeny of five different crosses between male and female doubled-haploid clones generated by anther culture. A total of 274 markers have been organized to ten linkage groups spanning 721.4 cM. Since the haploid chromosome number of asparagus is ten, the established linkage groups probably represent the different chromosomes; however, the only group associated with a specific chromosome is the one which includes sex, whose determinant genes have been located on chromosome 5. A total of 33 molecular markers (13 RFLPs, 18 AFLPs, 2 RAPDs and 1 isoenzyme) have been located on this chromosome. The closest marker to the sex determinant is the AFLP SV marker at 3.2 cM. Received: 26 March 1998 / Accepted: 30 April 1998     

Genetic diversity and population structure of endangered Isoetes coreana in South Korea based on RAPD analysis

Isoetes coreana Chung and Choi is on the list of critically endangered species in South Korea. Using randomly amplified polymorphic DNA (RAPD) markers, we investigated the genetic diversity within and between seven local populations of I. coreana. Ten RAPD primers produced a total of 94 bands, of which 59 (62.8%) were polymorphic. A low level of genetic diversity was recognized within the populations of I. coreana: polymorphic loci (P), with values ranging from 3.4% to 33.9%, and a mean value of 15.5% being observed. Analysis of molecular variance (AMOVA) showed that genetic diversity was greater among populations (81.6%) than within populations (19.7%) or among the three regions included in this study (Han River, Youngsan River, and Nakdong River). In addition, a high degree of genetic differentiation (θ^B = 0.742) was detected among the populations. These results indicate that genetic differentiation has occurred very rapidly. However, the rate of gene flow between populations was found to be as low as 0.059, irrespective of the genetic and geographical distances between the populations, which indicates that genetic drift must have played an important role in forming the present populations of I. coreana. Because a reduction of genetic diversity as a result of genetic drift is undesirable, increasing the gene flow between populations of Korean quillwort I. coreana should be considered as a conservation strategy.     

我国部分金莲花种质资源遗传多样性的RAPD研究

用RAPD标记对来自不同产地的24份金莲花种质从分子水平开展遗传多样性研究,为综舍评价我国金莲花资源现状及植物资源的合理保护提供参考。结果显示,30条RAPD引物共扩增获得194条清晰可辨条带,其中多态性条带108条,多态性位点百分率为55．67％,金莲花总多样性指数为0．1460,居群内遗传多样性为0．0591,Shannon’s多样性指数为0．2248;通过UPGMA进行聚类分析,将24份金莲花种质划分为3个大类,根据总遗传多样性和居群内遗传多样性计算不同居群间遗传分化系数为0．6902,基因流估计平均值为0．2244。研究结果表明,目前我国金莲花种质资源的遗传多样性指教偏低,应该尽快采取相应措施,对不同金莲花资源进行合理保护。     

Estimation of outcrossing rate in a breeding population of Eucalyptus urophylla with dominant RAPD and AFLP markers

 Eucalyptus breeding is typically conducted by selection in open-pollinated progenies. As mating is controlled only on the female side of the cross, knowledge of outcrossing versus selfing rates is essential for maintaining adequate levels of genetic variability for continuous gains. Outcrossing rate in an open-pollinated breeding population of Eucalyptus urophylla was estimated by two PCR-based dominant marker technologies, RAPD and AFLP, using 11 open-pollinated progeny arrays of 24 individuals. Estimated outcrossing rates indicate predominant outcrossing and suggest maintenance of adequate genetic variability within families. The multilcous outcrossing rate (t_m) estimated from RAPD markers (0.93±0.027), although in the same range, was higher (α>0.01) than the estimate based on AFLP (0.89±0.033). Both estimates were of similar magnitude to those estimated for natural populations using isozymes. The estimated Wright’s fixation index was lower than expected based on t_m possibly resulting from selection against selfed seedlings when sampling plants for the study. An empirical analysis suggests that 18 is the minimum number of dominant marker loci necessary to achieve robust estimates of t_m. This study demonstrates the usefulness of dominant markers, both RAPD and AFLP, for estimating the outcrossing rate in breeding and natural populations of forest trees. We anticipate an increasing use of such PCR-based technologies in mating-system studies, in view of their high throughput and universality of the reagents, particularly for species where isozyme systems have not yet been optimized. Received: 25 March 1997 / Accepted: 13 May 1997     

Assessment of genetic diversity through RAPD,ISSR and AFLP markers in Podophyllum hexandrum: a medicinal herb from the Northwestern Himalayan region

Total synthesis of podophyllotoxin is an expensive process and availability of the compound from the natural resources is an important issue for pharmaceutical companies that manufacture anticancer drugs. In order to facilitate reasoned scientific decisions on its management and conservation for selective breeding programme, genetic analysis of 28 populations was done with 19 random primers, 11 ISSR primers and 13 AFLP primer pairs. A total of 92.37 %, 83.82 % and 84.40 % genetic polymorphism among the populations of Podophyllum were detected using RAPD, ISSR and AFLP makers, respectively. Similarly the mean coefficient of gene differentiation (Gst) were 0.69, 0.63 and 0.51, indicating that 33.77 %, 29.44 % and 26 % of the genetic diversity resided within the population. Analysis of molecular variance (AMOVA) indicated that 53 %, 62 % and 64 % of the genetic diversity among the studied populations was attributed to geographical location while 47 %, 38 % and 36 % was attributed to differences in their habitats using RAPD, ISSR and AFLP markers. An overall value of mean estimated number of gene flow (Nm) were 0.110, 0.147 and 0.24 from RAPD, ISSR and AFLP markers indicating that there was limited gene flow among the sampled populations.     

Genetic analyses of micropropagated and regenerated plantlets of banana as assessed by RAPD and ISSR markers

A cultivar of dessert banana, namely, Nanjanagudu Rasabale (NR), classified under group “silk” (of genotype AAB), is seriously under the threat of extinction due to its susceptibility to bacterial wilt and bunchy-top virus disease. A regeneration protocol using tissue culture method was developed (Venkatachalam et al. 2006), where a large number of plantlets were regenerated from leaf base explants. Simultaneously, a micropropagation protocol was also developed where high levels of up to 53.28 μM of benzylamino purine (BAP) and 55.80 μM of kinetin (Kn) were used. The progressive increase of cytokinins levels resulted in concomitant increase in shoot number, with a maximum of 80 shoot buds per segment in BAP (31.08 μM). The plantlets were analyzed for their genetic stability using randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers. A total of 50 RAPD and 12 ISSR primers resulted in 625 distinct and reproducible bands showing homogeneous RAPD and ISSR patterns. Band intensity histogram of each gel confirmed their monomorphic nature with no genetic variation among the plantlets analyzed. The present study has established for the first time that the regeneration and rapid micropropagation protocol developed through the present study will be of great use in conserving the endangered cultivar – NR – without risk of genetic instability.     

Genetic relationships among Mediterranean Pistacia species evaluated by RAPD and AFLP markers

Polymorphisms among Mediterranean basin Pistacia species and accessions within species were assessed by random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses. Twenty-eight Pistacia accessions representing six species from geographically diverse locations in the Mediterranean area were analyzed. With RAPD, a total of 259 DNA fragments were amplified by 27 pre-selected primers, 254 were polymorphic fragments. AFLP analysis with 15 primer sets, produced 954 (93%) polymorphic bands out of a total of 1026. A Mantel test revealed an extremely high correlation (r=0.99) between similarity matrices generated from RAPD and AFLP data sets, indicating that similar results were obtained by the two techniques. Dendrograms constructed from the similarity matrices showed that Pistacia species could be clustered into two groups, one group containing all the #E5/E5#. lentiscus and the second group containing all other accessions. The latter group was divided into two subgroups, one consisting of #E5/E5#. palaestina and #E5/E5#. terebinthus; the other consisting of #E5/E5#. atlantica, #E5/E5#. khinjuk and #E5/E5#. vera. P. vera and P. khinjuk were highly similar, as were P. palaestina and P. terebinthus.     

Reproducibility testing of RAPD,AFLP and SSR markers in plants by a network of European laboratories

A number of PCR-based techniques can be used to detect polymorphisms in plants. For their wide-scale usage in germplasm characterisation and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn requires that they can be standardised to yield reproducible results, so that direct collation and comparison of the data are possible. This article describes a network experiment involving several European laboratories, in which the reproducibility of three popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), amplified fragment length polymorphism (AFLP) and sequence-tagged microsatellites (SSR). For each technique, an optimal system was chosen, which had been standardised and routinely used by one laboratory. This system (genetic screening package) was distributed to different participating laboratories in the network and the results obtained compared with those of the original sender. Different experiences were gained in this exchange experiment with the different techniques. RAPDs proved difficult to reproduce. For AFLPs, a single-band difference was observed in one track, whilst SSR alleles were amplified by all laboratories, but small differences in their sizing were obtained.