Construction of a high-density reference linkage map of tea (Camellia sinensis)

A few linkage maps of tea have been constructed using pseudo-testcross theory based on dominant marker systems. However, dominant markers are not suitable as landmark markers across a wide range of materials. Therefore, we developed co-dominant SSR markers from genomic DNA and ESTs and constructed a reference map using these co-dominant markers as landmarks. A population of 54 F₁ clones derived from reciprocal crosses between ‘Sayamakaori’ and ‘Kana-Ck17’ was used for the linkage analysis. Maps of both parents were constructed from the F₁ population that was taken for BC₁ population. The order of most of the dominant markers in the parental maps was consistent. We constructed a core map by merging the linkage data for markers that detected polymorphisms in both parents. The core map contains 15 linkage groups, which corresponds to the basic chromosome number of tea. The total length of the core map is 1218 cM. Here, we present the reference map as a central core map sandwiched between the parental maps for each linkage group; the combined maps contain 441 SSRs, 7 CAPS, 2 STS and 674 RAPDs. This newly constructed linkage map can be used as a basic reference linkage map of tea.     

DNA分子标记在银杏中的应用

DNA分子标记广泛用于生物研究的许多方面。本文简要介绍了RFLP、RAPD、AFLP、SSR及ISSR等几种目前常用分子标记的原理,归纳总结了分子标记在银杏中的应用研究进展。(1)获得了2个雄性特有的RAPD标记和2个雌性特有的AFLP标记,为银杏的早期性别鉴定及相关基因克隆奠定了基础;(2)采用RAPD标记和ISSR标记对我国部分栽培品种进行了分子鉴别和分类的研究,编制了一些品种的DNA指纹检索表;(3)利用RAPD和ISSR标记对一些群体、个体及栽培品种或变异类型进行了遗传分化和遗传多样性研究,结果发现银杏具有较高的遗传多样性,群体间、个体间、栽培品种及类型间都存在不同程度的遗传分化;(4)利用ISSR标记对一些个体的遗传杂合性进行了研究,结果显示个体的平均杂合率为43.53%;(5)构建了包含62个RAPD标记、19个连锁群的银杏分子遗传图谱;(6)探索了银杏优先保护种群的确定。分子标记在银杏其它方面的应用还很少。今后,除了继续对上述方面进行深入系统的研究外,还应充分运用DNA分子标记技术,开展银杏的分子标记辅助选择育种、种质评价与鉴别及保育生物学等方面的研究。     

黄瓜远缘群体分子遗传连锁图谱的构建和分析

以野生黄瓜品种和普通栽培黄瓜品种作亲本获得的142个F2群体为材料,采用AFLP,SRAP,SSR等分子标记进行遗传分析,构建了包含10个连锁群,有159个标记组成的黄瓜遗传连锁图谱,其中包括112个AFLP标记,39个SRAP标记和8个SSR标记。该遗传图谱覆盖基因组长度743.11 cM,平均图距4.67 cM。     

Molecular characterisation of cultivars of apple (Malus × domestica Borkh.) using microsatellite (SSR and ISSR) markers

In this study, two microsatellite-based methodologies (SSR and ISSR) were evaluated for potential use in fingerprinting and determination of the similarity degree between 41 commercial cultivars of apple previously characterised using RAPD and AFLP markers. A total of 13 SSR primer sets was used and 84 polymorphic alleles were amplified. Seven ISSR primers yielded a total of 252 bands, of which 176 (89.1%) were polymorphic. Except for cultivars obtained from somatic mutations, all cultivars were easily distinguishable employing both methods. The similarity coefficient between cultivars ranged from 0.20 to 0.87 for SSR analysis and from 0.71 to 0.92 using the ISSR methodology. Dendrograms constructed using UPGMA cluster analysis revealed a phenetic classification that emphasises the existence of a narrow genetic base among the cultivars used, with the Portuguese cultivars revealing higher diversity. This study indicates that the results obtained based on the RAPD, AFLP, SSR and ISSR techniques are significantly correlated. The marker index, based on the effective multiplex ratio and expected heterozygosity, was calculated for both analyses (MI = 1.7 for SSR and MI = 8.4 for ISSR assays) and the results obtained were directly compared with previous RAPD and AFLP data from the same material. The SSR and ISSR markers were found to be useful for cultivar identification and assessment of phenetic relationships, revealing advantages, due to higher reproducibility, over other commonly employed PCR-based methods, namely RAPD and AFLP. This revised version was published online in August 2006 with corrections to the Cover Date.     

分子标记在木兰科植物研究中的应用

林木研究中常采用的分子标记技术主要包括RFLP、RAPD、AFLP、SSR及ISSR等。本文综述了这些分子标记技术的原理、优缺点。归纳总结了分子标记在木兰科植物中的应用研究进展:(1)利用RAPD、RFLP、cpDNA基因系列测定(psbA-trnH、atpB-rbcL、matK、ndhF)等分子标记在分子水平上对一些群体、个体进行了亲缘关系和分类研究;(2)利用RAPD、SSR和ISSR标记对一些群体、个体进行了遗传结构和遗传多样性研究;(3)采用DAF和RAPD获得了厚朴的DNA指纹图谱。分子标记在木兰科植物的其它方面的应用还很少。今后,除了继续对上述方面进行深入系统的研究外,还应充分运用分子标记技术,开展木兰科植物的分子遗传图谱、分子标记辅助选择育种、保育生物学等方面的研究。     

The first genetic maps of cashew (Anacardium occidentale L.)

Cashew (Anacardium occidentale) is a widespread tropical tree crop that is grown primarily for its nuts and has a global production of over 2 million Mt. In spite of its economic importance to many countries, however, no linkage map containing STS anchor sites has yet been produced for this species. This is largely attributable to a prolonged juvenile phase of the tree (limiting mapping to F₁ progenies) and difficulty in effecting sufficient hand-pollinations to create mapping populations of effective size. Here, we produce an F₁ mapping population of 85 individuals from a cross between CP 1001 (dwarf commercial clone) and CP 96 (giant genotype), and use it to generate two linkage genetic maps comprising of 205 genetic markers (194 AFLP and 11 SSR markers). The female map (CP 1001) contains 122 markers over 19 linkage groups and the male map (CP 96) comprises 120 markers assembled over 23 linkage groups. The total map distance of the female map is 1050.7 cM representing around 68% genome coverage, whereas the male map spans 944.7 cM (64% coverage). The average map distance between markers is 8.6 cM in the female map and 7.9 cM in the male map. Homology between the two maps was established between 13 linkage groups of the female map and 14 of the male map using 46 bridging markers that include 11 SSR markers. These maps represent a platform from which to identify loci controlling economically important traits in this crop.     

Development of SCAR and CAPS Markers for a Partially Dominant Yellow Seed Coat Gene in Brassica Napus L

Summary We have previously identified 2 RAPD and 8 AFLP markers that were associated with yellow seed coat gene in a stable and pure yellow-seeded DH line No. 2127-17, however, they were not suitable in large-scale MAS. In this paper, it reported our efforts in developing rapid and reliable SCAR and CAPS markers from the RAPD and AFLP markers. Based on the sequence information in the regions of the most closely linked 4 AFLP and 2 RAPD markers, one SCAR (SCS1130) and one CAPS (SCA1) maker were successfully developed. Linkage analysis on a 127 DH lines population derived from the cross Hui5148-2 × No. 2127-17 revealed that they were tightly flanked (3.2 cM, 3.8 cM respectively). The resulting SCS1130 and SCA1 were further evaluated in backcross breeding families and demonstrated more accurate and valuable in MAS breeding. The development of these new markers would facilitate and accelerate the yellow seed coat breeding in Brassica napus.     

Genetic mapping of AFLP markers in Japanese bunching onion (<Emphasis Type="Italic">Allium fistulosum</Emphasis>)

Summary The first genetic linkage map of Japanese bunching onion (Allium fistulosum) based primarily on AFLP markers was constructed using reciprocally backcrossed progenies. They were 120 plants each of (P₁)BC₁ and (P₂)BC₁ populations derived from a cross between single plants of two inbred lines: D1s-15s-22 (P₁) and J1s-14s-20 (P₂). Based on the (P₂)BC₁ population, a linkage map of P₁ was constructed. It comprises 164 markers – 149 amplified fragment length polymorphisms (AFLPs), 2 cleaved amplified polymorphic sequences (CAPSs), and 12 simple sequence repeats (SSRs) from Japanese bunching onion, and 1 SSR from bulb onion (A. cepa) – on 15 linkage groups covering 947 centiMorgans (cM). The linkage map of P₂ was constructed with the (P₁)BC₁ population and composed of 120 loci – 105 AFLPs, 1 CAPS, and 13 SSRs developed from Japanese bunching onion and 1 SSR from bulb onion – on 14 linkage groups covering 775 cM. Both maps were not saturated but were considered to cover the majority of the genome. Nine linkage groups in P₂ map were connected with their counterparts in P₁ map using co-dominant anchor markers, 13 SSRs and 1 CAPS.     

Mapping Co, a gene controlling the columnar phenotype of apple, with molecular markers

The columnar phenotype is a very valuable genetic resource for apple breeding because of its compact growth form determined by the dominant gene Co. Using bulked segregant analysis combined with several DNA molecular marker techniques to screen the F₁ progeny of Spur Fuji × Telamon (heterozygous for Co), 9 new DNA markers (6 RAPD, 1 AFLP and 2 SSRs) linked to the Co gene were identified. A total of 500 10-mer random primers, 56 pairs of selective AFLP primers and 8 SSR primer pairs were screened. One RAPD marker S1142₆₈₂, and the AFLP marker, E-ACT/M-CTA₃₄₆, were converted into SCAR markers designated SCAR₆₈₂ and SCAR₂₁₆, respectively. These markers will enable early selection in progenies where Co is difficult to identify. The Co gene was located between the SSR markers CH03d11 and COL on linkage group 10 of the apple genetic linkage map. Finally, a local genetic map of the region around the Co gene was constructed by linkage analysis of the nine new markers and three markers developed earlier.     

A consensus genetic map of chickpea (Cicer arietinum L.) based on 10 mapping populations

A consensus genetic map of chickpea (Cicer arietinum L.) was constructed by merging linkage maps from 10 different populations, using STMS (Sequence-tagged Microsatellite Sites) as bridging markers. These populations derived from five wide crosses (C. arietinum × Cicer reticulatum) and five narrow crosses (Desi × Kabuli types) were previously used for mapping genes for several agronomic traits such as ascochyta blight, fusarium wilt, rust resistance, seed weight, flowering time and days to flower. The integrated map obtained from wide crosses consists of 555 loci including, among other markers, 135 STMSs and 33 cross-genome markers distributed on eight linkage groups and covers 652.67 cM. The map obtained from narrow crosses comprises 99 STMSs, 3 SCARs, 1 ASAP, fusarium resistance gene, 5 morphological traits as well as RAPD and ISSR markers distributed on eight linkage groups covering 426.99 cM. Comparison between maps from wide and narrow crosses reflects a general coincidence, although some discrepancies are discussed. Medicago truncatula cross-genome markers were BLASTed against the M. truncatula pseudogenome permitting assignments of chickpea linkage groups LGI, II, III, IV, V and VI on Medicago chromosomes 2, 5, 7, 1, 3 and 4, respectively. A marker detectable on Medicago chromosome 4 were also located on LGVIII, This consensus map is an important progress to assist breeders for selecting suitable markers to be used in marker-assisted selection (MAS).     

绿豆遗传连锁图谱的整合

利用绿豆及其近缘种的701对SSR引物,对现有绿豆遗传连锁图谱进行补充,结果在高感豆象绿豆栽培种Berken和高抗豆象绿豆野生种ACC41两亲本间筛选到多态性SSR引物104对。群体分析后,结合其他分子数据,使用作图软件Mapmaker/Exp 3.0b,获得一张含有179个遗传标记和12个连锁群,总长1831.8cM、平均图距10.2cM的新遗传连锁图谱,包括97个SSR标记,91个来自绿豆近缘种;RFLP标记76个;RAPD标记4个;STS标记2个。对32个绿豆、小豆共用SSR标记在遗传连锁图谱的分布分析发现,二个基因组间有一定程度的同源性,共用标记在连锁群上的排列顺序基本上一致,只有部分标记显示绿豆和小豆基因组在进化过程中发生了染色体重排;利用新图谱对ACC41的抗绿豆象主效基因重新定位,仍定位于I(9)连锁群,与其相邻分子标记的距离均小于8cM,其中与右翼SSR标记C220的距离约2.7cM。与原图谱比较,新定位的抗性基因与其相邻标记的连锁更加紧密。     

Male and female genetic linkage map of hops, Humulus lupulus

A male and female linkage map of hop has been constructed using 224 DNA polymorphisms (106 amplified fragment length polymorphisms (AFLPs), three random amplified polymorphic DNAs (RAPDs), one RAPD‐sequence‐tagged‐site (STS), and three microsatellite (STSs) segregating in an F₁ population of the English cultivar ‘Wye Target’‐the German male breeding line ‘85/54/15’. Linkage between these loci was estimated using JOINMAP Version 2.0. The final map for the female parent consisted of 110 loci assigned to eight linkage groups covering a distance of 346.7 cM. For the male map, 57 loci could be mapped on nine linkage groups spanning over 227.4 cM. One of these male linkage groups (Gr09‐M) presumably represents the Y chromosome, since all markers assigned (10 AFLPs, three RAPDs and one STS) were closely linked to the male sex (M). Because of their sex‐specific segregation, 10 doubly heterozygous AFLPs spanning a distance of 18.7 cM could be identified as markers describing the X chromosome, which is part of the male and female map. Three STMSs, which had already proved useful in hop genotyping, could be integrated as codominant locus‐specific markers and thus allowed to produce reliable allelic bridges between the female and male counterparts.     

Genotyping safflower (Carthamus tinctorius) cultivars by DNA fingerprints

Summary Carthamus tinctorius (2n = 2x = 24) (family Asteraceae), commonly known as safflower, is widely cultivated in agricultural production systems of Asia, Europe, Australia and the Americas as a source of high-quality vegetable and industrial oil. India ranks first in the production of safflower oil. Fourteen cultivars, widely cultivated in various agro-climatic regions of India, have been fingerprinted by RAPD, ISSR, and AFLP markers utilizing 36, 21 primers, and 4 primer combinations, respectively. On an individual assay basis, AFLP has proven to be the best marker system as compared with the other two markers applied as assessed by high discriminating power (0.98), assay efficiency index (33.2), marker index (18.2), resolving power (40.62), and genotype index (0.856). Thirty-six RAPD and 21 SSR primers could differentiate a maximum of eight and four cultivars, respectively, whereas, two AFLP primer combinations could fingerprint all the 14 cultivars. To understand genetic relationships among these cultivars, Jaccard's similarity coefficient and UPGMA clustering algorithm were applied to the three marker data sets. Mean genetic similarities ranged from 0.689 (AFLP) to 0.952 (ISSR). Correlation coefficient comparisons between similarity matrices and co-phenetic matrices obtained with the three markers revealed that AFLP displayed no congruence vis-a-vis RAPD and ISSR data. However, strong correlation was observed between RAPD and ISSR marker systems. This paper reports the start of molecular biology programme targeting nuclear genome of safflower, a major world oilseed crop about whose genetics very little is known.     

A new citrus linkage map based on SRAP,SSR, ISSR,POGP, RGA and RAPD markers

Sequence-related amplified polymorphism (SRAP), simple sequence repeats (SSR), inter-simple sequence repeat (ISSR), peroxidase gene polymorphism (POGP), resistant gene analog (RGA), randomly amplified polymorphic DNA (RAPD), and a morphological marker, Alternaria brown spot resistance gene of citrus named as Cabsr caused by (Alternaria alternata f. sp. Citri) were used to establish genetic linkage map of citrus using a population of 164 F₁ individuals derived between ‘Clementine’ mandarin (Citrus reticulata Blanco ‘Clementine) and ‘Orlando’ tangelo’ (C. paradisi Macf. ‘Duncan’ × C. reticulata Blanco ‘Dancy’). A total of 609 markers, including 385 SRAP, 97 RAPD, 95 SSR, 18 ISSR, 12 POGP, and 2 RGA markers were used in linkage analysis. The ‘Clementine’ linkage map has 215 markers, comprising 144 testcross and 71 intercross markers placed in nine linkage groups. The ‘Clementine’ linkage map covered 858 cM with and average map distance of 3.5 cM between adjacent markers. The ‘Orlando’ linkage map has 189 markers, comprising 126 testcross and 61 intercross markers placed in nine linkage groups. The ‘Orlando’ linkage map covered 886 cM with an average map distance of 3.9 cM between adjacent markers. Segregation ratios for Cabsr were not significantly different from 1:1, suggesting that this trait is controlled by a single locus. This locus was placed in ‘Orlando’ linkage group 1. The new map has an improved distribution of markers along the linkage groups with fewer gaps. Combining different marker systems in linkage mapping studies may give better genome coverage due to their chromosomal target site differences, therefore fewer gaps in linkage groups.     

DNA分子标记在野生大豆遗传多样性研究中的应用进展

为了研究陕西省不同居群野生大豆的遗传多样性,本研究归纳了几种常见DNA分子标记技术（RFLP、RAPD、AFLP、SSR、ISSR、SNP）的原理及特点,综述了不同DNA分子标记在野生大豆遗传多样性研究中的应用现状。分析了不同DNA分子标记在野生大豆遗传多样性研究上的优缺点,提出了SSR标记的发展前景,以及在研究野生大豆遗传多样性上存在的优势。     

A genetic map of an interspecific diploid pseudo testcross population of coffee

Coffee is globally one of the most important export crops and is a prominent part of the economy in more than 50 countries in Latin America, Africa and Asia. In Colombia, it has been the leading export commodity for more than a century. However, genetic research on coffee has been rather sparse and mainly focused on the two major cultivated species, Coffea arabica L. and C. canephora P., leaving unexplored the genetic potential in other species. In this study, an interspecific mapping population consisting of 101 F₁ hybrid plants from a cross between the diploid species C. liberica and C. eugenioides was evaluated for genetic segregation at 618 molecular marker loci. Of these, 168 SSRs and two ESTs exhibited polymorphic patterns that allowed segregation analysis and genetic linkage estimations. A genetic map consisting of 146 co-dominant loci and 11 predicted linkage groups was constructed using the mapping software JoinMap 3.0. The conjoined maternal/paternal map length is 798.68 cM, has an average saturation density of 6.01 cM/interval, and covers an estimated 66–86 % of the diploid coffee genome. Approximately 24 % of loci had null alleles, and 23.5 % exhibited segregation distortion. Knowledge derived from this study has important applications for quantitative trait locus analysis and marker-assisted selection in Colombian coffee breeding programs.     

Analysis of molecular variance and population structure in southern Indian finger millet genotypes using three different molecular markers

The genetic relationship among 42 genotypes of finger millet collected from different geographical regions of southern India was investigated using random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), and simple sequence repeats (SSR) markers. Ten RAPD primers produced 111 polymorphic bands. Five ISSR primers produced a total of 61 bands. Of these, 23 bands were polymorphic. The RAPD and ISSR fingerprints revealed 71.3 and 37.4% polymorphic banding patterns, respectively. Thirty-six SSR primers yielded 83 scorable alleles in which 62 were found to be polymorphic. Out of 36 SSR primers used, 14 primers (46.6%) produced polymorphic bands. The SSR primer UGEP7 produced a maximum number of six alleles. Mean polymorphic information content (PIC) of RAPD, ISSR and SSR were 0.44, 0.28, and 0.14, respectively. Molecular variances among the population were 2, 11, and 1% for RAPD, ISSR, and SSR markers, respectively. SSR produced 99% molecular variance within individuals. RAPD and ISSR markers produced a low level of molecular variance within individuals. The STRUCTURE (model-based program) analysis revealed that the 42 finger millet genotypes could be divided into a maximum of four subpopulations. Based on the Bayesian statistics, each RAPD and SSR marker produced three subpopulations (K=3), while ISSR marker showed four subpopulations (K=4). This study revealed that RAPD and SSR markers could narrow down the analysis of population structure and it may form the basis for finger millet breeding and improvement programs in the future.     

甘蓝型油菜遗传图谱的构建及芥酸含量的QTL分析

一个由甘蓝型油菜品种Quantum (黄花、低芥酸)和人工合成的甘蓝型油菜品系No.2127-17（白花、高芥酸）为亲本材料建立的DH群体中芥酸呈现单基因的遗传模式。为了发展与芥酸紧密连锁的分子标记对其实行有效的控制,随机选择121个结实正常的DH系为作图群体,利用SSR和RAPD标记构建了一张甘蓝型油菜的遗传连锁图谱。在亲本间检测     

用RAPD、ISSR和AFLP标记分析系谱关系明确的甘薯品种的亲缘关系

用RAPD、ISSR和AFLP标记对系谱关系明确的7个甘薯品种进行了亲缘关系分析。24个RAPD引物、14个ISSR引物和9对AFLP引物分别扩增出173、174和168条多态性带。3种分子标记在检测甘薯品种间遗传差异上相关程度高，其中RAPD与ISSR之间的相关系数最大为0.9328。用ISSR标记估计的品种间遗传距离为0.1286~1.0932，平均0.4883，大于其余2个标记的估计值。3种分子标记皆可揭示甘薯品种的亲缘关系，其中ISSR标记产生的聚类图与系谱图最吻合，认为ISSR标记更适于分析甘薯品种的亲缘关系。