Development of single nucleotide polymorphism markers in <Emphasis Type="Italic">Theobroma cacao</Emphasis> and comparison to simple sequence repeat markers for genotyping of Cameroon clones

Single nucleotide polymorphism (SNP) markers are increasingly being used in crop breeding programs, slowly replacing simple sequence repeats (SSR) and other markers. SNPs provide many benefits over SSRs, including ease of analysis and unambiguous results across various platforms. We have identified and mapped SNP markers in the tropical tree crop Theobroma cacao, and here we compare SNPs to SSRs for the purpose of determining off-types in clonal collections. Clones are used as parents in breeding programs and the presence of mislabeled clones (off-types) can lead to the propagation of undesired traits and limit genetic gain from selection. Screening was performed on 186 trees representing 19 Theobroma cacao clones from the Institute of Agricultural Research for Development (IRAD) breeding program in Cameroon. Our objectives were to determine the correct clone genotypes and off-types using both SSR and SNP markers. SSR markers that amplify 11 highly polymorphic loci from six linkage groups and 13 SNP markers that amplify eight loci from seven linkage groups were used to genotype the 186 trees and the results from the two different marker types were compared. The SNP assay identified 98% of the off-types found via SSR screening. SNP markers spread across multiple linkage groups may serve as a more cost-effective and reliable method for off-type identification, especially in cacao-producing countries where the equipment necessary for SSR analysis may not be available.     

Comparative Assessment of SSR and AFLP Markers for Evaluation of Genetic Diversity and Conservation of Fig, <Emphasis Type="Italic">Ficus carica</Emphasis> L., Genetic Resources in Tunisia

This study characterises the genetic variability of fig, Ficus carica L., using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. It compares the efficiency and utility of the two techniques in detecting variation and establishing genetic relationships among Tunisian fig cultivars. Our results show that using both marker systems, the Tunisian fig germ plasm is characterised by having a large genetic diversity at the deoxyribonucleic acid level, as most of AFLP bands were detected and all SSR markers were polymorphic. In fact, 351 (342 polymorphic) and 57 (57 polymorphic) bands were detected using AFLP and SSR primers, respectively. SSR markers were the most polymorphic with an average polymorphic information content value of 0.94, while AFLP markers showed the highest effective multiplex ratio (56.9) and marker index (45.2). The effective marker index was recorded highest (4.19) for AFLP markers and lowest (0.70) for the SSR ones. Our results demonstrate that (1) independent as well as combined analyses of cluster analyses of SSR and AFLP fragments showed that cultivars are clustered independently from their geographical origin, horticultural classifications and tree sex; (2) the analysis of molecular variance allowed the partitioning of genetic variation within and among fig groups and showed greater variation within groups and (3) AFLP and SSR markers datasets showed positive correlation. This study suggests the SSR and AFLP markers are suitable for diversity analysis and cultivars fingerprinting. An understanding of the genetic diversity and population structure of F. carica in Tunisia can also provide insight into the conservation and management of this species.     

QTL mapping ofFusarium moniliforme ear rot resistance in maize. 1. Map construction with microsatellite and AFLP markers

To map the QTLsof Fusarium moniliforme ear rot resistance inZea mays L., a total of 230 F₂ individuals, derived from a single cross between inbred maize lines R15 (resistant) and Ye478 (susceptible), were genotyped for genetic map construction using simple sequence repeat (SSR) markers and amplified fragment length polymorphism (AFLP) markers. We used 778 pairs of SSR primers and 63 combinations of AFLP primers to detect the polymorphisms between parents, R15 and Ye478. From the polymorphic 30 AFLP primer combinations and 159 SSR primers, we scored 260 loci in the F₂ population, among which 8 SSR and 13 AFLP loci could not be assigned to any of the linkage groups. An integrated molecular genetic linkage map was constructed by the remaining 151 SSR and 88 AFLP markers, which distributed throughout the 10 linkage groups of maize and spanned the genome of about 3463.5 cM with an average of 14.5 cM between two markers. On 4 chromosomes, we detected 5 putative segregation distortion regions (SDR_s), including 2 new ones (SDR₂ and SDR₇). The other 3 SDR_s were located near the regions where gametophyte genes were mapped, indicating that segregation distortion could be partially caused by gametophytic factors.     

Assessment and comparison of AFLP and SSR based molecular genetic diversity in Indian isolates of Ascochyta rabiei, a causal agent of Ascochyta blight in chickpea (Cicer arietinum L.)

Ascochyta blight (AB), caused by Ascochyta rabiei (Pass.) Labr. (anamorph), is the most damaging disease of chickpea (Cicer arietinum L.) and is a serious biotic stress constraint for chickpea production. To understand the molecular diversity in A. rabiei populations of India, a total of 64 isolates collected from AB-infected chickpea plants from different agroclimatic regions in the North Western Plain Zone (NWPZ) of India were analyzed with 11 AFLP (amplified fragment length polymorphism) and 20 SSR (simple sequence repeat) markers. A total of 9 polymorphic AFLP primer pairs provided a total of 317 fragments, of which 130 were polymorphic and showed an average PIC value 0.28. Of the SSR markers, 12 showed polymorphism and provided a total of 29 alleles with an average PIC value 0.35. To the best of our knowledge, this is the first report on a comparison of AFLP and SSR diversity estimates in A. rabiei populations. The dendrogram developed based on AFLP and SSR data separately, as well as on the combined marker dataset, grouped the majority of AB isolates as per geographic regions. Model based population structure analysis revealed four distinct populations with varying levels of ancestral admixtures among 64 isolates studied. Interestingly, several AFLP primer combinations and SSR markers showed the locus/allele specific to AB isolates of certain regions, e.g., Hisar, Sriganganagar, Gurdaspur, and Sundarnagar. Genetic variability present in AB isolates of the NWPZ of India suggests the continuous monitoring of changes in A. rabiei population to anticipate the breakdown of AB resistance in chickpea cultivars grown in India.     

Using morphological diagnosis and molecular markers to assess the clonal fidelity of micropropagated Echinacea purpurea regenerants

Both morphological characteristics and amplified fragment length polymorphism (AFLP) markers were used to validate the genetic fidelity of 1 080 field-grown Echinacea purpurea plants regenerated from leaf explants of donor T5-9. Morphological diagnosis revealed that 1 067 out of 1 080 regenerants were normal, while 13 regenerants were aberrant. AFLP analysis was further performed to assess DNA variations among donor, 43 sampled normal regenerants and all 13 aberrant regenerants. Seven primer combinations generated 471 fragments among donor and normal regenerants, of which 9 fragments were polymorphic. The same primer pairs generated 484 fragments for aberrant regenerants, of which 417 fragments were polymorphic. UPGMA clustering indicated that 42 normal regenerants and donor fell into same cluster at similarity scale of > 0.99, while all 13 aberrant regenerants and one morphologically normal regenerant comprised the other clusters. AFLP analysis indicated that these 14 regenerants are off-types.     

Characterization of fusarium wilt-resistant and fusarium wilt-susceptible somaclones of banana cultivar rastali (Musa AAB) by random amplified polymorphic DNA and retrotransposon markers

Two DNA fingerprinting techniques, random amplified polymorphic DNA (RAPD) and inter-retrotransposon amplified polymorphism (IRAP), were used to characterize somaclonal variants of banana. IRAP primers were designed on the basis of repetitive and genome-wide dispersed long terminal repeat (LTR) retrotransposon families for assessing the somaclonal variation in 2Musa clones resistant and susceptible toFusarium oxysporum f. sp.cubense race 4. RAPD markers successfully detected genetic variation within and between individuals of the clones. IRAP makers amplified either by a single primer or a combination of primers based on LTR orientation successfully amplified different retrotransposons dispersed in theMusa genome and detected new events of insertions. RAPD markers proved more polymorphic than IRAP markers. Somaclonal variation seems to be the result of numerous indels occurring genome-wide accompanied by the activation of retroelements, as a result of stress caused by micropropagation. It is concluded that characterization of the somaclonal variants requires more than one DNA marker system to detect variation in diverse components of the genome.     

Molecular characterization of intra-population variability of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. using DNA based molecular markers

Jatropha curcas L. (Euphorbiaceae) has acquired a great importance as a renewable source of energy with a number of environmental benefits. Very few attempts were made to understand the extent of genetic diversity of J. curcas germplasm. In the present study, efforts were made to analyze the genetic diversity among the elite germplasms of J. curcas, selected on the basis of their performance in field using random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR). The plants were selected on the basis of height, canopy circumference, number of seeds per fruit, weight of 100 seeds, seed yield in grams per plant and oil content. Out of 250 RAPD (with 26 primers), 822 AFLP (with 17 primers) and 19 SSR band classes, 141, 346 and 7 were found to be polymorphic, respectively. The percentage polymorphism among the selected germplasms using RAPD, AFLP and SSR was found to be 56.43, 57.9, and 36.84, respectively. The Jaccard’s similarity coefficient was found 0.91, 0.90 and 0.91 through RAPD, AFLP and SSR marker systems, respectively. Principle component analysis (PCA) and dendrogarm analysis of genetic relationship among the germplasm using RAPD, AFLP and SSR data showed a good correlation for individual markers. The germplasm JCC-11, 12, 13, 14 and 15 whose yield found to be high were clustered together in dendrogram and PCA analysis though JCC11 is geographically distinct from others. In overall analysis JCC6 (in RAPD), JCC8 (in AFLP) and JCC 6 and JCC10 (in SSR) were found genetically diverse. Characterization of geographically distinct and genetically diverse germplasms with varied yield characters is an important step in marker assisted selection (MAS) and it can be useful for breeding programs and QTL mapping.     

Characterization and fine mapping of <Emphasis Type="Italic">RppQ</Emphasis>, a resistance gene to southern corn rust in maize

Southern corn rust (SCR) is a fungal disease caused by Puccinia polysora Underw, which can infect maize and may result in substantial yield losses in maize production. The maize inbred line Qi319 carries the SCR resistance gene RppQ. In order to identify molecular markers linked to the RppQ gene, several techniques were utilized including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP). In addition, sequence characterized amplified region (SCAR) techniques combined with bulked segregant analysis (BSA) were used. Seven RAPD markers, eight SSR markers, and sixty-three AFLP primer combinations amplified polymorphisms between two parents and two bulk populations. A large F₂ population was used for genetic analysis and for fine mapping of the RppQ gene region. One AFLP polymorphic band, M-CAA/E-AGC₃₂₄, was converted to a SCAR marker, MA7, which was mapped to a position 0.46 cM from RppQ. Finally, the RppQ gene was mapped between the SCAR marker MA7 and the AFLP marker M-CCG/E-AGA₁₅₇ with distances of 0.46 and 1.71 cM, respectively.     

Molecular characterization and identification of markers for toxic and non-toxic varieties of <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. using RAPD,AFLP and SSR markers

Jatropha curcas L., a multipurpose shrub has acquired significant economic importance for its seed oil which can be converted to biodiesel, is emerging as an alternative to petro-diesel. The deoiled seed cake remains after oil extraction is toxic and cannot be used as a feed despite having best nutritional contents. No quantitative and qualitative differences were observed between toxic and non-toxic varieties of J. curcas except for phorbol esters content. Development of molecular marker will enable to differentiate non-toxic from toxic variety in a mixed population and also help in improvement of the species through marker assisted breeding programs. The present investigation was undertaken to characterize the toxic and non-toxic varieties at molecular level and to develop PCR based molecular markers for distinguishing non-toxic from toxic or vice versa. The polymorphic markers were successfully identified specific to non-toxic and toxic variety using RAPD and AFLP techniques. Totally 371 RAPD, 1,442 AFLP markers were analyzed and 56 (15.09%) RAPD, 238 (16.49%) AFLP markers were found specific to either of the varieties. Genetic similarity between non-toxic and toxic verity was found to be 0.92 by RAPD and 0.90 by AFLP fingerprinting. In the present study out of 12 microsatellite markers analyzed, seven markers were found polymorphic. Among these seven, jcms21 showed homozygous allele in the toxic variety. The study demonstrated that both RAPD and AFLP techniques were equally competitive in identifying polymorphic markers and differentiating both the varieties of J. curcas. Polymorphism of SSR markers prevailed between the varieties of J. curcas. These RAPD and AFLP identified markers will help in selective cultivation of specific variety and along with SSRs these markers can be exploited for further improvement of the species through breeding and Marker Assisted Selection (MAS).     

Development of microsatellite markers in canary seed (<Emphasis Type="Italic">Phalaris canariensis</Emphasis> L.)

Annual canarygrass, commonly known as canary seed (Phalaris canariensis L.), is a self-pollinated diploid cereal (2n = 12) with a genome size of 3,800 Mbp. Canary seed is presently used for bird-feed with a potential to develop it for human consumption. Marker-assisted selection can be used to accelerate breeding of new canary seed cultivars. Microsatellites or simple sequence repeat (SSR) markers generally show a high degree of polymorphism in different plant genera. FIASCO (Fast Isolation by AFLP of Sequences COntaining repeats) was used to generate microsatellite markers specific for canary seed. An enriched SSR (AG)₁₇ library derived from DNA isolated from a canary seed cultivar (CDC Togo) was produced. Analysis and DNA sequencing of the library resulted in 744 clones from which 132 primer pairs were designed. Seventy-eight functional markers amplified unique products from canary seed DNA. These SSR markers revealed the biodiversity among a panel of 48 canary seed accessions. Polymorphic information content (PIC) values of 37 polymorphic microsatellites ranged from 0.08 to 0.73 with an average of 0.36.     

Limited variability of CTG/CAG repeats in <Emphasis Type="Italic">Lycopersicon</Emphasis> nuclear DNA

Seven clones containing (CTG)_n/(CAG)_n repeats (n ≥ 4) were isolated by screening Lycopersicon esculentum genomic DNA. Four of the clones contained more than one simple sequence repeat (SSR). The SSRs were analyzed in several L. esculentum cultivars after polymerase chain reaction (PCR) amplification. No length variations were observed, suggesting considerable locus stability. Five clones are from transcribed regions, which might explain the lack of cultivar variations. However the conservation of CTG repeats was limited as differences in some transcribed loci were registered between L. pennellii and other Lycopersicon species. It is noted that in Lycopersicon trinucleotide repeat variation might be used for species identification.     

Genetic relationships and clonal identity in a collection of commercially relevant poplar cultivars assessed by AFLP and SSR

A collection of 66 poplar commercial clones widely cultivated in Italy, China and in other countries of southern Europe and belonging to various poplar species and hybrids, have been fingerprinted using both amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) techniques. Three AFLP primer combinations and six SSRs unambiguously genotyped the analysed poplar collection, with the exception of three groups of six, four and two individuals, which turned out to be indistinguishable even if they met the standards currently applied for distinctness, uniformity and stability (DUS) testing when registered. High levels of variation were detected with both molecular techniques; a total of 201 AFLP bands were amplified of which 96% turned out to be polymorphic and up to 15 SSR alleles were identified at a single locus, with a mean of 9.3 alleles per locus in the case of Populus × canadensis. The probability of matching fortuitously any two genotypes at all the SSR loci in the case of P. × canadensis was less then 7.5×10^–9. The AFLP-derived dendrogram and principal coordinate analysis (PCOORDA) clustered the clones with respect to their taxonomic classification, and allowed their genetic interrelationships to be established. Correct identification of poplar varieties is essential for ensuring the effective correspondence between the real and the declared identity of a clone, to avoid commercial frauds, and to establish breeding programmes. Molecular markers may play a major role to satisfy all these needs.     

Genetic structure of island populations of <Emphasis Type="Italic">Prunus lannesiana</Emphasis> var. <Emphasis Type="Italic">speciosa</Emphasis> revealed by chloroplast DNA,AFLP and nuclear SSR loci analyses

The wild flowering cherry Prunus lannesiana var. speciosa is highly geographically restricted, being confined to the Izu Islands and neighboring peninsulas in Japan. In an attempt to elucidate how populations of this species have established we investigated the genetic diversity and differentiation in seven populations (sampling 408 individuals in total), using three kinds of genetic markers: chloroplast DNA (cpDNA), amplified fragment length polymorphisms (AFLPs), and 11 nuclear SSR polymorphic loci. Eight haplotypes were identified based on the cpDNA sequence variations, 64 polymorphic fragments were scored for the AFLP markers, and a total of 154 alleles were detected at the 11 nuclear SSR loci. Analysis of molecular variance showed that among-population variation accounted for 16.55, 15.04 and 7.45% of the total detected variation at the cpDNA, AFLPs, and SSR loci, respectively. Thus, variation within populations accounted for most of the genetic variance for all types of markers, although the genetic differentiation among populations was also highly significant. For cpDNA variation, no clear structure was found among the populations, except that of the most distant island, although an “isolation by distance” pattern was found for each marker. Both neighbor-joining trees and structure analysis indicate that the genetic relationships between populations reflect geological variations between the peninsula and the islands and among the islands. Furthermore, hybridization with related species may have affected the genetic structure, and some genetic introgression is likely to have occurred.     

Molecular mapping of the Oregon Wolfe Barleys: a phenotypically polymorphic doubled-haploid population

A phenotypically polymorphic barley (Hordeum vulgare L.) mapping population was developed using morphological marker stocks as parents. Ninety-four doubled-haploid lines were derived for genetic mapping from an F₁ using the Hordeum bulbosum system. A linkage map was constructed using 12 morphological markers, 87 restriction fragment length polymorphism (RFLP), five random amplified polymorphic DNA (RAPD), one sequence-tagged site (STS), one intron fragment length polymorphism (IFLP), 33 simple sequence repeat (SSR), and 586 amplified fragment length polymorphism (AFLP) markers. The genetic map spanned 1,387 cM with an average density of one marker every 1.9 cM. AFLP markers tended to cluster on centromeric regions and were more abundant on chromosome 1 (7H). RAPD markers showed a high level of segregation distortion, 54% compared with the 26% observed for AFLP markers, 27% for SSR markers, and 18% for RFLP markers. Three major regions of segregation distortion, based on RFLP and morphological markers, were located on chromosomes 2 (2H), 3 (3H), and 7 (5H). Segregation distortion may indicate that preferential gametic selection occurred during the development of the doubled-haploid lines. This may be due to the extreme phenotypes determined by alleles at morphological trait loci of the dominant and recessive parental stocks. Several molecular markers were found to be closely linked to morphological loci. The linkage map reported herein will be useful in integrating data on quantitative traits with morphological variants and should aid in map-based cloning of genes controlling morphological traits. Received: 23 August 2000 / Accepted: 15 December 2000     

Association of AFLP and SSR markers with agronomic and fibre quality traits in <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.

Molecular markers linked to QTL contributing to agronomic and fibre quality traits would be useful for cotton improvement. We have attempted to tag yield and fibre quality traits with AFLP and SSR markers using F₂ and F₃ populations of a cross between two Gossypium hirsutum varieties, PS56-4 and RS2013. Out of 50 AFLP primer combinations and 177 SSR primer pairs tested, 32 AFLP and four SSR primers were chosen for genotyping F₂ individuals. Marker-trait associations were studied for eight agronomic and five fibre quality traits through simple and multiple regression analysis (MRA) using a set of 92 AFLP polymorphic loci and four SSR markers. Simple linear regression analysis (SLRA) identified 23 markers for eight different traits whereas multiple regression analysis identified 30 markers for at least one of the 13 traits. SSR marker BNL 3502 was consistently identified to be associated with fibre strength. While all the markers identified in SLRA were also detected in MRA, as many as 16 of the 30 markers were identified to be associated with respective traits in both F₂ and F₃ generations. The markers explained up to 41 per cent of phenotypic variation for individual traits. A number of markers were found to be associated with multiple traits suggesting clustering of QTLs for fibre quality traits in cotton.     

An SSR-based linkage map of Capsicum annuum

There are five cultivated species of pepper, of which Capsicum annuum is the most widely cultivated as a vegetable or spice and the main experimental material of most pepper breeding programs. However, the number of simple sequence-repeat (SSR) markers known for C. annuum is limited. To develop SSR markers for Capsicum species, we constructed four SSR-enriched libraries from the genomic DNA of C.␣annuum, sequenced 1873 clones, and isolated 626 unique SSR clones. A higher percentage of these SSR markers were taken from dinucleotide motif libraries than from trinucleotide motif libraries. Primer pairs for the 626 SSR clones were synthesized and tested for polymorphisms; 594 amplified products were detected with the expected size. However, only 153 products were polymorphic between the parents of our mapping population. Using 106 highly reproducible pairs from the primer pairs, we constructed a linkage map of C. annuum in an intraspecific doubled haploid population (n=117) that contains nine previously reported SSRs as well as AFLP, CAPS, and RAPD markers and the trait of fruit pungency. The map contains 374 markers, including 106 new SSR markers distributed across all 13 linkage groups, and covers 1042 cM. The polymorphism information content (PIC) of these new SSR markers was calculated using 14 lines of Capsicum species. The average number of alleles per locus was 2.9 and the average PIC value was 0.46, even within C. annuum. The SSR markers developed in this study will be useful for mapping and marker-assisted selection in pepper breeding, and the linkage map provides a reference genetic map for Capsicum species.     

Localization of BAC clones on mitotic chromosomes of <Emphasis Type="Italic">Musa acuminata</Emphasis> using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization

A bacterial artificial chromosome (BAC) library of banana (Musa acuminata) was used to select BAC clones that carry low amounts of repetitive DNA sequences and could be suitable as probes for fluorescence in situ hybridization (FISH) on mitotic metaphase chromosomes. Out of eighty randomly selected BAC clones, only one clone gave a single-locus signal on chromosomes of M. acuminata cv. Calcutta 4. The clone localized on a chromosome pair that carries a cluster of 5S rRNA genes. The remaining BAC clones gave dispersed FISH signals throughout the genome and/or failed to produce any signal. In order to avoid the excessive hybridization of repetitive DNA sequences, we subcloned nineteen BAC clones and selected their ‘low-copy’ subclones. Out of them, one subclone gave specific signal in secondary constriction on one chromosome pair; three subclones were localized into centromeric and peri-centromeric regions of all chromosomes. Other subclones were either localized throughout the banana genome or their use did not result in visible FISH signals. The nucleotide sequence analysis revealed that subclones, which localized on different regions of all chromosomes, contained short fragments of various repetitive DNA sequences. The chromosome-specific BAC clone identified in this work increases the number of useful cytogenetic markers for Musa.     

Integration genetic linkage map construction and several potential QTLs mapping of Chinese shrimp (<Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis>) based on three types of molecular markers

In this study, totally 54 selected polymorphic SSR loci of Chinese shrimp (Fenneropenaeus chinensis), in addition with the previous linkage map of AFLP and RAPD markers, were used in consolidated linkage maps that composed of SSR, AFLP and RAPD markers of female and male construction, respectively. The female linkage map contained 236 segregating markers, which were linked in 44 linkage groups, and the genome coverage was 63.98%. The male linkage map contained 255 segregating markers, which were linked in 50 linkage groups, covering 63.40% of F. chinensis genome. There were nine economically important traits and phenotype characters of F. chinensis were involved in QTL mapping using multiple-QTL mapping strategy. Five potential QTLs associated with standard length (q-standardl-01), with cephalothorax length (q-cephal-01), with cephaloghorax width (q-cephaw-01), with the first segment length (q-firsel-01) and with anti-WSSV (q-antiWSSV-01) were detected on female LG1 and male LG44 respectively with LOD > 2.5. The QTL q-firsel-01 was at 73.603 cM of female LG1. Q-antiWSSV-01 was at 0 cM of male LG44. The variance explained of these five QTLs was from 19.7–33.5% and additive value was from −15.9175 to 7.3675. The closest markers to these QTL were all SSR, which suggested SSR marker was superior to AFLP and RAPD in the QTL mapping.     

Characterization and pharmacological properties of in vitro propagated clones of <Emphasis Type="Italic">Echinacea tennesseensis</Emphasis> (Beadle) Small

Tissue culture techniques have been used to establish and maintain a repository of medicinal Echinacea. In vitro clones obtained from hypocotyls of germinated seeds, varied macroscopically, microscopically and exhibited variation in immune enhancing activity. Two in vitro produced clones of Echinacea tennesseensis (Beadle) Small (ETN 03 and ETN 11) were identified as high and low activity based on the activation of human monocytes. Phenotypic analyses of ETN 03 and ETN 11 clones were done using AFLP (Amplified Fragment Length Polymorphism) assay. Results of the AFLP assay revealed that no mutation has occurred during in vitro multiplication, storage, and acclimatization into soil. Plants of ETN 03, ETN 11 clones were cultivated for two growing seasons. Extracts of their dry leaves and roots exhibited immune enhancing activity; however, the variation in activity noticed between clones during micropropagation diminished and was no longer statistically relevant.     

Genetic mapping of the pear scab resistance gene Vnk of Japanese pear cultivar Kinchaku

Pear scab (caused by Venturia nashicola) is one of the most harmful diseases of pears, especially Japanese and Chinese pear species. The molecular identification and early selection of resistant plants could greatly improve pear breeding. We have identified the position of the scab resistance gene, designated Vnk in an indigenous Japanese pear cultivar Kinchaku, within the pear genome by using simple sequence repeat (SSR) markers derived from pear and apple. The position of Vnk was identified in the central region of linkage group 1 of Kinchaku. Several amplified fragment length polymorphism (AFLP) markers linked to Vnk were obtained by bulked segregant analysis. Among them, the AFLP marker closest to Vnk was converted into a sequence tagged site (STS) marker. Four random amplified polymorphic DNA (RAPD) markers previously found to be loosely associated with Vnk (Iketani et al. 2001) were successfully converted into STS markers. Six markers (one SSR Hi02c07 and five STSs converted from AFLP and RAPD) showed tight linkages to Vnk, being mapped with distances ranging from 2.4 to 12.4 cM. The SSR CH-Vf2, which was isolated from a BAC clone of the contig containing the apple scab gene Vf, was mapped at the bottom of linkage group 1 in Kinchaku, suggesting that the Vnk and Vf loci are located in different genomic regions of the same homologous linkage group.