In vitro versus in vivo genetic divergence in potato

 The objective of this study was to compare the genetic divergence pattern in potato (Solanum tuberosum L.) under in vitro and in vivo conditions. Twenty two potato genotypes were evaluated for ten morphological characters under four in vitro conditions, and for 20 characters under four in vivo seasons. Mahalanobis’ generalized intra- and inter-group genetic distances, and the distribution of genotypes into different clusters, led to the same conclusions under both in vitro and in vivo conditions: (1) genetic diversity was not related to geographic diversity, (2) genetic distances were higher between Tuberosum and Andigena than within Tuberosum and Andigena, and (3) present-day Indian varieties have more resemblance to Tuberosum than to the Andigena group. The in vitro approach was more effective than the in vivo approach for differentiating the genotypes per se, although its effectiveness for cross prediction is known to be low. Received: 15 September 1997 / Accepted: 15 July 1998     

Molecular characterisation of inter and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA (RAPD) markers

Summary Protoplast fusion allows the transfer of both mono- and polygenic traits between species that are sexually incompatible. This approach has particular relevance for potato, and somatic hybridisation has been used to introduce a range of disease resistance genes from sexually incompatible wild species into the cultivated potato gene pool. In addition, protoplast fusion allows the resynthesis of tetraploid genotypes from pre-selected diploid or dihaploid donor parents. A limiting factor for the efficient exploitation of this technology in potato breeding is the difficulty of unequivocally identifying nuclear hybrids (heterokaryons). In order to facilitate the identification of hybrids at an early stage following fusion, Randomly Amplified Polymorphic DNA markers (RAPDs) have been used to characterise molecularly both inter- and intra-specific somatic hybrids of potato. RAPD markers detect naturally occurring polymorphism in the donor genotypes and utilise short oligonucleotide primers of arbitrary nucleotide sequence in combination with the polymerase chain reaction (PCR). The exploitation of RAPDs in the characterisation of both somatic and sexual hybrids is discussed.     

Rapid in vitro propagation,conservation and analysis of genetic stability of Viola pilosa

A protocol for in vitro propagation was developed for Viola pilosa, a plant of immense medicinal value. To start with in vitro propagation, the sterilized explants (buds) were cultured on MS basal medium supplemented with various concentrations of growth regulators. One of the medium compositions MS basal + 0.5 mg/l BA + 0.5 mg/l TDZ + 0.5 mg/l GA₃ gave best results for in vitro shoot bud establishment. Although the problem of shoot vitrification occurred on this medium but this was overcome by transferring the vitrified shoots on MS medium supplemented with 1 mg/l BA and 0.25 mg/l Kn. The same medium was found to be the best medium for further in vitro shoot multiplication. 100 % root induction from in vitro grown shoots was obtained on half strength MS medium supplemented with 1 mg/l IBA. In vitro formed plantlets were hardened and transferred to soil with 83 % survival. Additionally, conservation of in vitro multiplying shoots was also attempted using two different approaches namely slowing down the growth at low temperature and cryopreservation following vitrification. At low temperature retrieval rate was better at 10 °C than at 4 °C after conservation of in vitro multiplying shoots. In cryopreservation–vitrification studies, the vitrified shoot buds gave maximum retrieval of 41.66 % when they were precooled at 4 °C, while only 16.66 % vitrified shoots were retrieved from those precooled at 10 °C. Genetic stability of the in vitro grown plants was analysed by RAPD and ISSR markers which indicated no somaclonal variation among in vitro grown plants demonstrating the feasibility of using the protocol without any adverse genetical effects.     

Determination of genetic stability using isozymes and RFLPs in beet plants regenerated in vitro

Summary Sugar, fodder and garden beet (Beta vulgaris L.) plants have been regenerated in culture from a range of expiant material. Of the regenerants 764 were subjected to isozyme analysis using eight enzyme-specific stains, and 60 were subjected to RFLP analysis using three cDNA probes. Both molecular techniques allowed the identification of somaclonal variant plants. Assessment of the numbers of variant isozymes and restriction fragments has allowed the calculation of the approximate percentage of variant alleles occurring in any one somaclonal regenerant, namely between 0.05% and 0.1%.     

Determination of genetic stability in long-term micropropagated shoots of Pinus thunbergii Parl. using RAPD markers

Random amplified polymorphic DNA (RAPD) markers were used to determine the genetic stability of long-term (more than 10 years) micropropagated shoots of Japanese black pine (Pinus thunbergii Parl.). Thirty-six shoots consisting of three morphotypes (short, medium, and long needles) were randomly chosen from about 4,000 micropropagated shoots regenerated from the explants of a single nematode-resistant mother plant. Out of 126 primers screened, 30 gave 134 clear reproducible bands. A total of 4,824 bands obtained from these studies exhibited no aberration in RAPD banding patterns among the tested shoots. Our results show that regenerants from our plant micropropagation system are genetically stable. Received: 5 December 1997 / Revision received: 17 May 1998 / Accepted: 1 June 1998     

Construction of a genetic linkage map for roses using RAPD and AFLP markers

A segregating population of diploid rose hybrids (2n = 2x = 14) was used to construct the first linkage maps of the rose genome. A total of 305 RAPD and AFLP markers were analysed in a population of 60 F₁ plants based on a so-called ”double-pseudotestcross” design. Of these markers 278 could be located on the 14 linkage groups of the two maps, covering total map lengths of 326 and 370 cM, respectively. The average distances between markers in the maps for 93/1–117 and 93/1–119 is 2.4 and 2.6 cM, respectively. In addition to the molecular markers, genes controlling two phenotypic characters, petal number (double versus single flowers) and flower colour (pink versus white), were mapped on linkage groups 3 and 2, respectively. The markers closest to the gene for double flowers, Blfo, and to the gene for pink flower colour, Blfa, cosegregated without recombinants. The maps provide a tool for further genetic analyses of horticulturally important genes as, for example, resistance genes and a starting point for marker-assisted breeding in roses. Received: 22 September 1998 / Accepted: 12 March 1999     

High output genetic mapping of polyploids using PCR-generated markers

Summary The polymerase chain reaction (PCR) with arbitrarily selected primers has been established as an efficient method to generate fingerprints that are useful in genetic mapping and genomic fingerprinting. To further increase the productivity of mapping and fingerprinting efforts, we have altered existing protocols to include the use of the Stoffel fragment, which is derived from genetically engineered Taq polymerase. We also optimized the thermal profile of the reaction to increase the number of useful primers. In mapping of the genome of Saccharum spontaneum SES 208, a polyploid wild relative of sugarcane, these modifications allowed for an increase of 30% in the number of loci screened per primer, and an 80% increase in the number of polymorphisms per primer. Furthermore, the enzyme cost per reaction was decreased approximately 1.6-fold. Finally, there was an increase from about 70% to about 97% in the number of primers that were useful (i.e., gave a reproducible fingerprint) using our protocol. We have placed some of these markers into linkage groups.     

我国枣生物技术研究进展

生物技术的发展为枣种质资源的研究、创新及新品种培育提供了更多的途径。本文综述了近年来我国枣生物技术的研究进展,主要包括离体培养、分子标记、基因工程3个方面的内容。目前,生物技术是枣传统育种的有效补充,已成为枣遗传改良、种质资源创新和科学研究的重要技术。     

Extension of the linkage map in Citrus using random amplified polymorphic DNA (RAPD) markers and RFLP mapping of cold-acclimation-responsive loci

Genetic mapping with RAPD markers has been initiated in Citrus. Reproducible polymorphism of amplified DNA fragments was obtained with approximately half of the 140 random primers tested, revealing 266 segregating loci. These were tested for linkage using 60 BC₁ progeny from an intergeneric cross of Citrus grandis (L.) Osb. x [Citrus grandis (L.) Osb. x Poncirus trifoliata (L.) Raf.]. A core linkage map was constructed that consists of nine linkage groups containing 109 RAPD markers and 51 previously-mapped RFLP and isozyme markers. A further 79 markers that could not be ordered unambiguously because of their allelic constitution were associated with individual linkage groups and are shown in relation to the core map. The core map has a total length of 1192 cM with an average distance of 7.5 cM between loci and is estimated to cover 70–80% of the genome. Loci with distorted segregation patterns clustered on several linkage groups. Individual clusters of loci were skewed in allelic composition toward one or the other parent, usually C. grandis. This relatively-saturated linkage map will eventually be used to identify quantitative trait loci for cold and salt-tolerance in Citrus. As a beginning we have mapped three loci detected by a cold-acclimation-responsive cDNA.     

Development and applications of a set of chromosome-specific cytogenetic DNA markers in potato

Reliable and easy to use techniques for chromosome identification are critical for many aspects of cytogenetic research. Unfortunately, such techniques are not available in many plant species, especially those with a large number of small chromosomes. Here we demonstrate that fluorescence in situ hybridization (FISH) signals derived from bacterial artificial chromosomes (BACs) can be used as chromosome-specific cytogenetic DNA markers for chromosome identification in potato. We screened a potato BAC library using genetically mapped restriction fragment length polymorphism markers as probes. The identified BAC clones were then labeled as probes for FISH analysis. A set of 12 chromosome-specific BAC clones were isolated and the FISH signals derived from these BAC clones serve as convenient and reliable cytological markers for potato chromosome identification. We mapped the 5S rRNA genes, the 45S rRNA genes, and a potato late blight resistance gene to three specific potato chromosomes using the chromosome-specific BAC clones. Received: 19 January 2000 / Accepted: 27 March 2000     

Minimal growth conservation of potato microplants: silver thiosulfate reduces ethylene-induced growth abnormalities during prolonged storage in vitro

 Efficacy of silver thiosulfate (STS) in reducing ethylene-induced culture abnormalities during minimal growth conservation of microplants was studied in seven potato (Solanum tuberosum L.) genotypes. Different concentrations of STS (0, 1.5, 3.0, 4.5, 6.0, 7.5 and 9.0 μg ml^–1) were tested in minimal growth medium based on MS medium supplemented with 20 g l^–1 mannitol and 40 g l^–1 sucrose. STS improved the microplant growth and reduced the culture abnormalities during prolonged maintenance of potato shoot cultures in vitro. The beneficial effect of STS was most prominent for number of green leaves per microplant and leaf senescence. After 16 months of storage, desirable microplant growth was observed in cultures conserved in medium containing 6.0–9.0 μg ml^–1 STS. The profile of the peroxidase isozymes of conserved cultures did not show any apparent genetic variation due to the presence of STS in the conservation medium. Received: 2 September 1998 / Revision received: 20 November 1998 / Accepted: 12 December 1998     

Maximum-likelihood models for mapping genetic markers showing segregation distortion. 1. Backcross populations

A maximum-likelihood approach is used in order to estimate recombination fractions between markers showing segregation distortion in backcross populations. It is assumed that the distortions are induced by viability differences between gametes or zygotes due to one or more selected genes. We show that Bailey's (1949) estimate stays consistent and efficient under more general assumptions than those defined by its author. This estimate should therefore be used instead of the classical maximum-likelihood estimate. The question of detection of linkage is also discussed. We show that the order of markers on linkage groups may be affected by segregation distortion.     

EST-derived single nucleotide polymorphism markers for assembling genetic and physical maps of the barley genome

In a panel of seven genotypes, 437 expressed sequence tag (EST)-derived DNA fragments were sequenced. Single nucleotide polymorphisms (SNPs) that were polymorphic between the parents of three mapping populations were mapped by heteroduplex analysis and a genome-wide consensus map comprising 216 EST-derived SNPs and 4 InDel (insertion/deletion) markers was constructed. The average frequency of SNPs amounted to 1/130 bp and 1/107.8 bp for a set of randomly selected and a set of mapped ESTs, respectively. The calculated nucleotide diversities (π) ranged from 0 to 40.0 × 10⁻³ (average 3.1 × 10⁻³) and 0.52 × 10⁻³ to 39.51 × 10^–3 (average 4.37 × 10⁻³) for random and mapped ESTs, respectively. The polymorphism information content value for mapped SNPs ranged from 0.24 to 0.50 with an average of 0.34. As expected, combination of SNPs present in an amplicon (haplotype) exhibited a higher information content ranging from 0.24 to 0.85 with an average of 0.50. Cleaved amplified polymorphic sequence assays (including InDels) were designed for a total of 87 (39.5%) SNP markers. The high abundance of SNPs in the barley genome provides avenues for the systematic development of saturated genetic maps and their integration with physical maps. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Both R. Kota and R.K. Varshney contributed equally to this work.     

Pea Ty1-copia group retrotransposons: transpositional activity and use as markers to study genetic diversity in Pisum

The variation in transposition history of different Ty1-copia group LTR retrotransposons in the species lineages of the Pisum genus has been investigated. A heterogeneous population of Ty1-copia elements was isolated by degenerate PCR and two of these (Tps12 and Tps19) were selected on the basis of their copy number and sequence conservation between closely related species for further in-depth study of their transpositional history in Pisum species. The insertional polymorphism of these elements and the previously characterised PDR1 element was studied by sequence-specific amplification polymorphism (SSAP). Each of these elements reveals a unique transpositional history within 55 diverse Pisum accessions. Phylogenetic trees based on the SSAP data show that SSAP markers for individual elements are able to resolve different species lineages within the Pisum genus. Finally, the SSAP data from all of these retrotransposon markers were combined to reveal a detailed picture of the intra and inter-species relationships within Pisum. Received: 23 January 2000 / Accepted: 24 March 2000     

我国葫芦科植物离体培养研究进展

葫芦科植物包括多种瓜类蔬菜,对其进行离体培养研究具有重要的理论和实践意义.综述了国内在葫芦科植物器官培养、体细胞胚胎发生、花药培养、原生质体培养和体细胞杂交及离体遗传转化等方面取得的研究进展,并对葫芦科植物离体培养、遗传转化与育种的前景作了展望.     

Blood markers and genetic evolution in Cercopithecinae

Serum proteins and RBC enzymes were surveyed in 16 species (183 animals) of African guenons (tribe Cercopithecini) in order to determine their genetic polymorphism and to establish dendrograms on the basis of their allele frequencies. The molecular data obtained were compared with those of mangabeys (16 animals tested) and discussed in the light of our results inPapio andMacaca. The species surveyed wereCercopithecus neglectus, C. hamlyni, C. l'hoesti (C. l'h. l'hoesti, C. l'h. preussi, andC. l'h. solatus), C. nictitans, C. mitis (C. m. kolbi, C. m. albotorquatus, C. m. stuhlmanni, andC. m. albogularis), C. cephus, C. ascanius, C. erythrotis, C. petaurista, C. mona, C. pogonias, C. wolfi, andC. aethiops, Miopithecus talapoin, Allenopithecus nigroviridis andErythrocebus patas, Lophocebus albigena, andCercocebus torquatus. Eleven loci (ten systems) were studied in red blood cell enzymes and the Gc, Gm, Km, and Bm systems in DBP and immunoglobulin serum proteins. Most of the loci were polymorphic. Similar and different polymorphisms occur in closely related species or subspecies, particularly inCercopithecus. Guenons have phenotypes clearly distinct from mangabeys.     

Determination of genetic diversity among Saccharina germplasm using ISSR and RAPD markers

Various species of genus Saccharina are economically important brown macroalgae cultivated in China. The genetic background of the conserved Saccharina germplasm was not clear. In this report, DNA-based molecular markers such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity and phylogenetic relationships among 48 Saccharina germplasms. A total of 50 ISSR and 50 RAPD primers were tested, of which only 33 polymorphic primers (17 ISSR and 16 RAPD) had an amplified clear and reproducible profile, and could be used. Seventeen ISSR primers yielded a total of 262 bands, of which 256 were polymorphic, and 15.06 polymorphic bands per primer were amplified from 48 kelp gametophytes. Sixteen RAPD primers produced 355 bands, of which 352 were polymorphic, and 22 polymorphic bands per primer were observed across 48 individuals. The simple matching coefficient of ISSR, RAPD and pooled ISSR and RAPD dendrograms ranged from 0.568 to 0.885, 0.670 to 0.873, and 0.667 to 0.862, revealing high genetic diversity. Based on the unweighted pair group method with the arithmetic averaging algorithm (UPGMA) cluster analysis and the principal components analysis (PCA) of ISSR data, the 48 gametophytes were divided into three main groups. The Mantel test revealed a similar polymorphism distribution pattern between ISSR and RAPD markers, the correlation coefficient r was 0.62, and the results indicated that both ISSR and RAPD markers were effective to assess the selected gametophytes, while matrix correlation of the ISSR marker system (r = 0.78) was better than that of the RAPD marker system (r = 0.64). Genetic analysis data from this study were helpful in understanding the genetic relationships among the selected 17 kelp varieties (or lines) and provided guidance for molecular-assisted selection for parental gametophytes of hybrid kelp breeding.     

Simple sequence repeat marker diversity in cassava landraces: genetic diversity and differentiation in an asexually propagated crop

Cassava (Manihot esculenta) is an allogamous, vegetatively propagated, Neotropical crop that is also widely grown in tropical Africa and Southeast Asia. To elucidate genetic diversity and differentiation in the crop's primary and secondary centers of diversity, and the forces shaping them, SSR marker variation was assessed at 67 loci in 283 accessions of cassava landraces from Africa (Tanzania and Nigeria) and the Neotropics (Brazil, Colombia, Peru, Venezuela, Guatemala, Mexico and Argentina). Average gene diversity (i.e., genetic diversity) was high in all countries, with an average heterozygosity of 0.5358 ± 0.1184. Although the highest was found in Brazilian and Colombian accessions, genetic diversity in Neotropical and African materials is comparable. Despite the low level of differentiation [F_st(theta) = 0.091 ± 0.005] found among country samples, sufficient genetic distance (1-proportion of shared alleles) existed between individual genotypes to separate African from Neotropical accessions and to reveal a more pronounced substructure in the African landraces. Forces shaping differences in allele frequency at SSR loci and possibly counterbalancing successive founder effects involve probably spontaneous recombination, as assessed by parent-offspring relationships, and farmer-selection for adaptation.Communicated by H.C. Becker     

Deciphering genetic diversity analysis of saffron (Crocus sativus L.) using RAPD and ISSR markers

The existence of genetic diversity in Crocus sativus has globally remained a mystery till date. The study investigated PCR based DNA amplification profile of saffron using ISSR and RAPD based primers. A total of 38 amplicons were generated by ISSR primers in the range from 7 to 12 with an average of 9.50 bands per primer. 20 bands were found to be polymorphic and 18 were monomorphic with an average percentage of polymorphism as 52.48%. RAPD based amplification revealed a total 161 amplicons, 107 as polymorphic and 54 as monomorphic with an average percentage of polymorphism as 66.44%. Cumulative results of RAPD and ISSR demonstrated that Nei-Li’s similarity index ranged between 0.70 and 0.97. The results of AMOVA has revealed 9% of variance among populations and 91% of variance within populations, Φ PT was found as 0.089, which indicates existence of genetic differences though limited. In conclusion, the results indicate that saffron accessions are minimally genetically differentiated, which could be capitalized in future breeding programmes to ameliorate this precious crop.     

Analysis of arthropod bloodmeals using molecular genetic markers

Little is known about the transmission dynamics of human malaria and other vector-borne diseases, partly because of the limited availability and distribution of appropriate tools for quantifying human-mosquito contact rates. Recent developments in molecular biology have allowed a significant increase in the efficacy and reliability of bloodmeal identification, and DNA-based molecular markers are now being harnessed for typing arthropod bloodmeals. The extent to which these markers have been used for analysis of mosquito bloodmeals and the potential they might have for the future is discussed, and the contributions that the advent of PCR has made are examined here.