AFLP, ISSR and RAPD markers reveal high levels of genetic diversity among Lupinus spp.

The Lupinus genus includes a number of important crop species. The use of defined nucleotide sequences for the analysis of genetic diversity among these species has revealed modest levels of diversity. The aim of this study was to access AFLP, ISSR and RAPD markers to evaluate the genetic diversity among L. albus, L. angustifolius, L. cosentinii, L. hispanicus, L. luteus, L. mutabilis, L. pilosus and L. polyphyllus. Unexpectedly, low levels of genetic similarity were found (ranging from 0.205 to 0.432), regardless of the type of molecular marker used. Nevertheless, these techniques consistently showed a greater genetic similarity between L. pilosus and L. cosentinii, L. mutabilis and L. polyphyllus and among L. luteus, L. hispanicus and L. angustifolius, clearly separating the Old World from the New World species. Such low genetic similarity among Lupinus spp. is most unlikely to be due to differences in coding sequences but could be the result of a long diverging process concerning non‐coding regions, which would represent a very important proportion of these genomes.     

Genetic variation of sweet potatoes (Ipomoea batatas L.) cultivated in Chile determined by RAPDs

RAPD (randomly amplified polymorphic DNA) technology was applied to analyze the genetic variability of sweet potato germplasm existing in Chile and elsewhere. Analysis of 28 cultivars from all over the world showed polymorphic bands with all 18 primers tested. A total of 124 RAPD bands were scored with an average of 6.9 polymorphic bands per primer. These results confirm that sweet potato exhibits high genetic variation. Two groups were distinguished: one containing Peruvian cultivars, and another containing cultivars from the rest of the world. Analysis of 14 accessions from Central Chile and one from Northern Chile showed polymorphic bands with 24 of 26 primers tested, but almost all of the 140 polymorphic bands merely showed the distinctness of the Northern accession. The almost complete uniformity of the other 14 accessions shows that sweet potato germplasm collected in Central Chile has very little genetic variability and may be derived from a single cultivar. Based on these results and on historical records, some hypotheses are proposed to explain the origin of sweet potatoes cultivated in Chile. This revised version was published online in July 2006 with corrections to the Cover Date.     

梨属植物等位酶遗传多样性研究

利用超薄平板微型聚丙烯酰胺凝胶的等电聚焦电泳技术,对286份梨材料进行了等位酶遗传变异分析。在8个酶系中共检测到19个清晰位点和82个等位基因,19个位点均为多态位点,位点最大等位基因数为6,体现出梨丰富的遗传种质多样性;不同的居群具有特有等位基因;通过82个等位基因可以将286份材料完全区分开,表明等位酶基因型指纹可用作梨品种区分与鉴定的依据。     

云南籽粒苋种质资源的表型多样性分析

为了系统分析云南籽粒苋种质资源的表型遗传多样性。采用遗传多样性指数、主成分分析、相关分析和聚类分析对104份云南籽粒苋种质资源的30个表型性状进行研究。结果表明,遗传多样性指数最高的质量性状和数量性状分别是主花序形状(1.68)和主花序长度(2.07)。前10个主成分累计贡献率达到73.944%,单株鲜体重与第3、第6、第9主成分极显著正相关,单株粒重与第9主成分极显著正相关。聚类分析将104份种质划分为5类,第Ⅰ类群可为间套种亲本材料,第Ⅱ和Ⅲ类群可为大粒亲本材料,第Ⅳ类群可为优质饲用亲本材料,第Ⅴ类群可为观赏类亲本材料。云南籽粒苋种质资源表型性状具有丰富的遗传多样性,有较大的开发利用潜力。     

Genetic variability in melon based on microsatellite variation

A set of 18 simple‐sequence repeat (SSR or microsatellite) markers was used to study genetic diversity in a collection of 27 melon (Cucumis melo L.) accessions, representing a broad range of wild and cultivated melons. The materials studied were highly polymorphic for SSRs and a total of 114 alleles were detected (average of 6.3 alleles per locus). Cluster analysis suggests the division of these accessions into two major groups, largely corresponding to the division of C. melo in the two subspecies agrestis and melo. The assignment of the accession to the subspecies was generally in agreement with published reports, except for those corresponding to the ‘dudaim’ and ‘chito’ cultivar groups, which, according to the observed SSR variability, should be included in subspecies agrestis. Based on cluster analysis, five groups of accessions were defined. The two most divergent groups include mainly accessions from the Mediterranean which form one group, and accessions from China, Japan, Korea and India forming the other. Both groups shared a low level of intra‐accession variation compared with the other groups, which suggests an erosion of their genetic variability because of drift and/or inbreeding. The remaining accessions, mainly from Central Africa and India, were more variable and may be an important source of genetic variation for melon breeding.     

随机扩增多态DNA技术在天麻遗传多样性分析中的应用

【研究目的】文章以乌天麻、黄天麻和绿天麻三种变型为研究对象,通过RAPD分析研究天麻遗传多样性,从而为天麻种源的筛选与鉴定及品种选育提供科学依据。【方法】采用改良的CTAB法提取天麻样品DNA,筛选出重复性好10条随机引物进行扩增,建立RAPD分析图谱。【结果】聚类分析表明：栽培群体乌天麻是一个高度混杂的群体,虽然个体的表现型基本相同,但从遗传物质的组成来看,基因型不一致。【结论】研究表明天麻是一个杂合群体,具有丰富的遗传多样性,其中乌天麻遗传多样性最丰富,变异复杂。     

长江下游铜鱼遗传多样性的随机扩增多态DNA(RAPD)分析

用随机扩增多态DNA(RAPD)技术对长江下游铜鱼(Coreius heterodon)20个个体的遗传多样性进行了检测,从4O个随机引物中筛选出19个对每个铜鱼的DNA进行扩增。结果表明,19个引物共检测到95条清晰且重复性好的条带,分子量在100～1500 bp之间,其中多态位点为30个,占31.58%;群体的Shannon多样性值为0.1940;个体间最大遗传距离为0.0976,最小遗传距离为0.0003。通过与其他鱼类的遗传多样性的研究结果比较可初步判断,长江下游铜鱼群体的遗传多样性较低。由于没有以前的遗传多样性分析资料,因此不能评判过度捕捞和自然环境的破坏等因素对铜鱼遗传多样性的影响程度。     

RFLP variation and genetic relationships in cultivated cucumber

Summary Two sets of cucumber (Cucumis sativus L.) germplasm were used to determine the potential use of restriction fragment length polymorphisms (RFLPs) for estimating genetic relationships. Sixteen accessions [15 domesticated variety sativus and one feral variety hardwickii (PI 183967)] of diverse origin were used to assess RFLP variation in cucumber, and to determine if genetic relationships based on RFLPs were similar to those obtained by isozyme analysis. Additionally, 35 commercial lines or cultivars were surveyed to determine genetic relationships among and within common cucumber types (narrow genetic base). The 16 accessions were surveyed with 440 low copy clones from two libraries (Pst I partial genomic and cDNA) using two restriction enzymes. Data from a subset of 104 random (mapped and unmapped) and a set of 30 mapped RFLPs were used to estimate genetic relationships among the 16 cultigens. Variability was low among RFLPs (33% of all probes) and putative alleles ( 2.2 polymorphic fragments/probe). RFLP variation between sativus lines and hardwickii (21±4%) was greater than among sativus lines (12±2%). RFLPs among the 16 accessions revealed genetic relationships which agree with those obtained using isozymes. Genetic relationships estimated using mapped and unmapped RFLPs were similar. The 35 elite lines were surveyed using a set of 40 RFLPs from 3 libraries (Pst I and EcoR I partial genomic and cDNA) to evaluate the discriminatory value of RFLPs among and between commercial cucumber types. The RFLP-derived genetic relationships among this germplasm were in agreement with predictions based on fruit type and pedigree information. Thus, RFLPs are a useful addition to the morphological characters and isozyme loci currently used for taxonomic classification and plant variety protection of cucumber.     

Relationship between hybrid performance and genetic diversity based on RAPD markers in wheat, Triticum aestivum L.

Random amplified polymorphic DNA (RAPD) markers were generated from 20 wheat, Triticum aestivum lines. Fifty-four fragments generated by six primers of a 10-mer arbitrary sequence were used to study their potential power in differentiating parents with different characteristics and predicting the yield performance of hybrids produced from these parents. Experimental results showed that the 20 wheat lines were divided into four groups. Group I was characterized by more grains per spike, group II by heavy grains and group III by more spikes per unit area and short plants; group IV was similar to group III but had a much higher biomass yield and grain yield. Hybrids from parents in different groups were generally superior to most hybrids from parents in the same group. Both yield performance and heterosis of hybrids from parents between group I and group III were much better than those of other intergroup hybrids. These results suggest that, based on RAPD markers, it is possible to differentiate wheat lines with different performances and that the classification of parents from these markers is of predictive value for developing superior hybrids. However, genetic distance (GD) based on RAPD markers was not significantly correlated with hybrid performance and heterosis. It appears to be impossible to predict hybrid performance from GD itself.     

Evaluating genetic variation and relationships among two bromegrass species and their hybrid using RAPD and AFLP markers

The two most widely grown bromegrass species in North America are smooth bromegrass (Bromus inermis Leyss.), a hay type grass, and meadow bromegrass (Bromus riparius Rehmann), a pasture type grass. Recently a hybrid bromegrass population between the two species has been produced as a dual-purpose hay-pasture grass. Molecular markers have the potential to improve selection procedures to enhance bromegrass breeding. The objective of this study was to use RAPD and AFLP markers to determine genetic relationships and variations among bromegrass populations. Forty-three RAPD markers from 21 primers and 83 AFLP markers from seven primer combinations were used. Both marker types were able to group the individuals into their respective populations. The relationships among the individuals within each of the populations were not similar between the two marker types. Analysis of molecular variance (AMOVA) detected greater within-population variation than among-population variation for both marker types. The highest variation was observed in the hybrid population followed by meadow and then smooth bromegrass. The inter-population distance from both markers indicated that the highest genetic distance was between meadow and smooth bromegrass and lowest between smooth and hybrid bromegrass, which reflect the breeding history of the hybrid population. This study showed that both markers are capable of differentiating bromegrass genotypes into their respective populations, detecting genetic variation and relationships of the populations. Results of this study suggest that these two markers can be used in the future to enhance the current breeding practices in bromegrass, however, AFLP markers would be the marker of choice due to the high number of polymorphic markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic relationships of macadamia cultivars and species revealed by AFLP markers

World production of macadamia nuts is based on two species, the smooth shell Macadamia integrifolia Maiden and Betche, and the rough shell Macadamiatetraphylla L.A.S. Johnson, and their hybrids. One hundred and five AFLP markers were used to analyze 26 macadamia accessions representing four species: M. integrifolia, M. tetraphylla,M. ternifolia, and M. hildebrandii as well as a wild relative,Hicksbeachia pinnatifolia (rose nut).Each macadamia accession showed distinct AFLP fingerprints indicating a significant level of genetic variation in this macadamia germplasm collection. The fourMacadamia species included in this study were clearly separated using cluster analysis with AFLP marker data. Based on a single accession, the separation of M. ternifolia from M. integrifoliasuggested the relatively distant genetic relationship between these two species and casts doubts on the notion that the M. ternifolia may be a variant of M. integrifolia. Within the major cluster ofM. integrifolia, nine established smooth shell cultivars were separated into two sub-clusters, suggesting the heterozygous nature of the original gene pool that had contributed to macadamia variety improvement programs. M. hildebrandii and H. pinnatifoliaformed a distinct cluster and share dramatically less genetic similarity with the other Macadamia species. Additional data would be needed to clarify the phylogenetic nature and status of M. hildebrandii in the genus Macadamia. This revised version was published online in July 2006 with corrections to the Cover Date.     

甘薯和巴西牵牛18S rRNA基因的克隆和序列分析

【研究目的】为甘薯和侵染甘薯病毒的基因表达研究提供内参基因序列信息。【方法】分别以‘商薯19’、‘北京553’两甘薯品种和巴西牵牛的基因组DNA为模板,利用PCR方法克隆甘薯和巴西牵牛18S rRNA基因序列。【结果】测序结果表明,获得的供试两甘薯品种和巴西牵牛的18S rRNA基因序列长度均为1630 bp;序列比对结果表明,甘薯和巴西牵牛与裂叶牵牛、烟草等双子叶植物的18S rRNA基因相应序列的一致性均达98%以上,与单子叶植物Lilium superbum的18S rRNA基因序列也有较高的一致性。【结论】从两甘薯品种和巴西牵牛的基因组中克隆出了18S rRNA基因部分序列,研究结果不仅为利用18S rRNA基因作为内参基因分析甘薯和侵染甘薯病毒基因的表达研究提供了序列依据,而且可为甘薯和巴西牵牛的分子系统学研究提供序列参考。     

Assessment of genetic diversity among selected groundnut germplasm. I: RAPD analysis

Assessment of genetic diversity in a crop species is prerequisite to its improvement. The use of germplasm with distinct DNA profiles will help to generate genetically diversified breeding populations. The aims of the present experiment were to study molecular diversity among selected groundnut accessions and identify those with distinct DNA profiles for mapping and genetic enhancement. Twenty‐six accessions and eight primers of a 10‐mer were selected for random amplified polymorphic DNA assay. The genetic similarity (S_ij) ranged from 59.0% to 98.8%, with an average of 86.2%. Both multidimensional scaling and unweighted pair‐group method with arithmetic averages (UPGMA) dendrograms revealed the existence of five distinct clusters. However, this classification could not be related to known biological information about the accessions falling into different clusters. Some accessions with diverse DNA profiles (ICG 1448, 7101, and 1471, and ICGV 99006 and 99014) were identified for mapping and genetic enhancement in groundnut.     

Geographical distribution of genetic variation in Stylosanthes scabra revealed by RAPD analysis

A large number of S. scabra accessions have been accumulated worldwide. The majority of them were collected from Brazil and most of the others came from either Colombia or Venezuela. One hundred of these accessions, selected to represent the geographical distribution of the S. scabra collection held at the Australian Tropical Forages Genetic Resource Centre, were analysed using RAPD as markers. Seven of these accessions were found not to be S. scabra. Of the S. scabra accessions, the average dissimilarity value among Brazilian accessions (0.053) was much lower than that among Colombian (0.074) or Venezuelan (0.088) accessions, with an overall dissimilarity value of 0.059 among all the S. scabra accessions. Based on their dissimilarity values, most of these accessions could be separated into five groups. Geographical distributions for most accessions in each of these groups were well defined. Limited long distance introductions/dispersions of S. scabra between these regions were detected and they were mainly confined to Brazilian genotypes. The clustering results based on RAPD were compared with those based on morphological-agronomical characters, and the groups produced by the two different methods did not always match. Possible reasons for this discrepancy are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessment of genetic relationships among Cucumis spp. by SSR and RAPD marker analysis

The first successful production of a sterile interspecific hybrid obtained from a cross between Cucumis hystrix Chakr. (2n = 2x = 24) and Cucumis sativus var. sativus L. (2n =2x = 14), and its subsequent fertility restoration through chromosome doubling provide an effective means for investigating genetic relationships among Cucumis spp. In this study, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to investigate relationships among C. s. var. sativus L., C. s. var. hardwickii (R.) Alef., C hystrix, C. hytivus Chen & Kirkbride (the amphidiploid species from chromosome doubling of the C. sativus x C. hystrix interspecific hybrid, 2n = 38), C. melo (2n =2x = 24) and C. metuliferus Meyer and Naudin (2n =2x= 24). A total of 109 SSR bands and 398 RAPD primed sites were used to calculate Jaccard's distance coefficients for cluster analysis using a unweighted pair‐group method using an arithmetic averaging (UPGMA) algorithm. The genetic relationships identified using SSR and RAPD markers were highly concordant, such that the correlation between SSR and RAPD genetic distance (GD) estimates was r = 0.94. SSR and RAPD analysis of 22 accessions allowed for their grouping into two distinct groups designated as CS and CM. While group CS consisted of 11 C. sativus genotypes, and the C. hytivus and C. hystrix accessions, group CM included six C. melo genotypes and C. metuliferus. The GD values between C. hystrix and C. sativus ascribed by SSR and RAPD matrices were 0.59 and 0.57, respectively. These GDs were smaller than those detected between C. hystrix and C melo (0.87 and 0.70 derived from SSR and RAPD markers, respectively).     

Use of RAPD and microsatellite (SSR) variation to assess genetic relationships among populations of tetraploid alfalfa, Medicago sativa

The level of random amplified polymorphic DNA (RAPD) and microsatellite variation present in four ecotypes and two varieties of alfalfa (lucerne) from Italian and Egyptian germplasm sources was evaluated. A sample of 100 plants from 10 populations was analysed by means of 41 RAPD markers and 37 simple sequence repeat (SSR) markers. Both molecular approaches revealed a high degree of genetic diversity within each of the cultivated populations and enabled each of the plants considered to be uniquely fingerprinted. The genetic relationships among plants and populations were analysed by computing AMOVA (analysis of molecular variance) and F_ST analyses. RAPDs were able to separate the Italian populations from the Egyptian variety. SSRs allowed strong separation of the four Italian alfalfa ecotypes. It was concluded that RAPD and microsatellites could be useful and powerful tools for assessing genetic variation and genetic relationships in tetraploid alfalfa.     

澳洲坚果种质资源形态性状的遗传多样性分析

为了解引入澳洲坚果种质的遗传背景,以64份澳洲坚果种质为试材,采用41个形态性状对其进行了遗传多样性分析。41个形态性状的差异性和R型聚类分析结果表明：各性状间差异达到极显著水平;在欧氏距离（D）≈11.72处,41个性状聚为两组,组内各性状的欧氏距离较远,绝大多数性状间相对独立,对澳洲坚果种质的演化各具独立的意义,在澳洲坚果的分类中有较好的分辨能力。Q型聚类分析结果表明：64份种质的欧氏距离在3.15~12.39之间,具有较高的遗传多样性。在D≈12处可将64份种质划分为二个类群：第一类群仅包含T2 1份种质,为澳洲坚果栽培种的近缘种;第二类群包含剩下的63份种质,为澳洲坚果的栽培种。第二类群在D≈10处,可细分成3个亚类：第Ⅰ亚类包括7份种质（即695、Tet.-2、900、D4、HY、Tet.-1和广9）,属于粗壳种与光壳种的杂交种;第Ⅱ亚类包括A4和A16两个品种,属于粗壳种与光壳种的杂交种,但更偏重于光壳种;第Ⅲ亚类包括其他54份种质,都是光壳种的后代。获得的聚类结果与现有的分类体系一致;同时,获得了64份种质的形态性状聚类的亲缘关系图,其在生产种植中的品种搭配、育种中的杂交亲本选择和引种方面都具有指导意义。     

Evaluation of genetic variation in Lachenalia bulbifera (Hyacinthaceae) using RAPDs

RAPD (randomly amplified polymorphic DNA) analyses were carried out on 21 accessions of Lachenalia bulbifera (Cyrillo) Engl. Five pre-selected primers produced an average of 88% polymorphisms. Fifteen of the 21 accessions could be identified using the five primers. In a pairwise comparison genetic distance values ranging from 0.11 to 1.08 were obtained. These values reveal a high amount of variation within the species. The genetic distance values within the tetraploid and hexaploid groups on the south coast were low, but values were high between the groups on the south coast and those on the west coast. A dendogram was constructed from the RAPD banding profiles, using UPGM cluster analysis. The dendogram clusters certain accessions together. These clusters are supported by their geographical locality and chromosome data. The hexaploid group, tetraploid group and octoploid group on the south coast are respectively clustered together. It is concluded that RAPDs can be used to assess the genetic variation at an intra-specific level in Lachenalia. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assessment of genetic variation in a collection of lentil using molecular tools

A collection of lentil landraces, mostly from Italy, but including Mediterranean and foreign germplasm as reference, was evaluated using RAPDs, microsatellite-primed PCR and ISSRs. A low level of useful polymorphic bands was detected with the former two markers, whereas ISSRs revealed a higher degree of variation. The UPGMA trees, based on Jaccard similarity index matrices, obtained with RAPD and ISSR respectively, did not produce similar clusters. However, in both cases, the accession which differentiated the most from the others was from Ethiopia, a country where also other cultivated species have shown a remarkable degree of variation. On the other hand, Italian accessions showed a trend to group together. ISSR markers proved to be useful for distinguishing closely related genotypes, and possibly for substantiating the genetic peculiarity of ecotypes applying for the obtainment of origin and quality marks. This revised version was published online in July 2006 with corrections to the Cover Date.