蓖麻生物工程研究进展

蓖麻是一种高蓄能植物和工业原料植物,具有很大的开发利用价值。本文从组织培养和遗传转化两个方面并结合本实验室的工作综述了蓖麻生物工程研究的最新进展。在蓖麻组织培养方面,不同的外植体中以成熟种子的胚轴最适宜,而在不同激素中以TDZ诱导丛生芽的效率最高,并以此为基础建立了蓖麻离体再生体系。在遗传转化方面,不同的转化方法中以农杆菌介导法最适合蓖麻转化。蓖麻胚轴对卡那霉素不敏感,潮霉素是蓖麻转化的适宜筛选剂。文中指出了蓖麻生物工程研究中存在的问题,并对应用生物技术培育蓖麻新品种和促进蓖麻生产的可能性进行了讨论。     

Genetic Diversity Among Jatropha and Jatropha-Related Species Based on ISSR Markers

Jatropha curcas (jatropha) is a potential biodiesel crop. A major limitation in production is that jatropha remains wild with low genetic variation. Related species/genera in the Euphorbiaceae can potentially be used for its genetic improvement. In this study, we employed inter-simple sequence repeats (ISSRs) to assess genetic variation among 30 accessions of jatropha, two accessions of bellyache bush (Jatropha gossypifolia), two accessions of spicy jatropha (Jatropha integerrima), two accessions of bottleplant shrub (Jatropha podagrica), and three accessions of castor bean hybrids. Genetic relationships were evaluated using 27 of 86 ISSR markers, yielding 307 polymorphic bands with polymorphism contents ranging from 0.76 to 0.95 for IMPN 1 and UBC 807 markers, respectively. Dice’s genetic similarity coefficient ranged from 0.39 to 0.99, which clearly separated the plant samples into seven groups at the coefficient of 0.48. The first group comprised J. curcas from Mexico, the second group comprised J. curcas from China and Vietnam, the third group comprised J. curcas from Thailand, the fourth group was J. integerrima, the fifth group was J. gossypifolia, the sixth group was J. podagrica, and the last and most distinct group was Ricinus communis. Analysis of molecular variance revealed that 63% of the variability was attributable to variation among groups, while 37% was due to variation within groups. Based on Nei’s genetic distance, the population from G2 (J. curcas from China) and G4 (J. curcas from Vietnam) had the least ISSR variability (0.0668), whereas G8 (R. communis) and Jatropha spp. displayed the highest distance (0.6005–0.7211).     

Castor bean organelle genome sequencing and worldwide genetic diversity analysis

Castor bean is an important oil-producing plant in the Euphorbiaceae family. Its high-quality oil contains up to 90% of the unusual fatty acid ricinoleate, which has many industrial and medical applications. Castor bean seeds also contain ricin, a highly toxic Type 2 ribosome-inactivating protein, which has gained relevance in recent years due to biosafety concerns. In order to gain knowledge on global genetic diversity in castor bean and to ultimately help the development of breeding and forensic tools, we carried out an extensive chloroplast sequence diversity analysis. Taking advantage of the recently published genome sequence of castor bean, we assembled the chloroplast and mitochondrion genomes extracting selected reads from the available whole genome shotgun reads. Using the chloroplast reference genome we used the methylation filtration technique to readily obtain draft genome sequences of 7 geographically and genetically diverse castor bean accessions. These sequence data were used to identify single nucleotide polymorphism markers and phylogenetic analysis resulted in the identification of two major clades that were not apparent in previous population genetic studies using genetic markers derived from nuclear DNA. Two distinct sub-clades could be defined within each major clade and large-scale genotyping of castor bean populations worldwide confirmed previously observed low levels of genetic diversity and showed a broad geographic distribution of each sub-clade.     

Joint analysis of phenotypic and molecular diversity provides new insights on the genetic variability of the Brazilian physic nut germplasm bank

The genetic variability of the Brazilian physic nut (Jatropha curcas) germplasm bank (117 accessions) was assessed using a combination of phenotypic and molecular data. The joint dissimilarity matrix showed moderate correlation with the original matrices of phenotypic and molecular data. However, the correlation between the phenotypic dissimilarity matrix and the genotypic dissimilarity matrix was low. This finding indicated that molecular markers (RAPD and SSR) did not adequately sample the genomic regions that were relevant for phenotypic differentiation of the accessions. The dissimilarity values of the joint dissimilarity matrix were used to measure phenotypic + molecular diversity. This diversity varied from 0 to 1.29 among the 117 accessions, with an average dissimilarity among genotypes of 0.51. Joint analysis of phenotypic and molecular diversity indicated that the genetic diversity of the physic nut germplasm was 156% and 64% higher than the diversity estimated from phenotypic and molecular data, respectively. These results show that Jatropha genetic variability in Brazil is not as limited as previously thought.     

Isolation and characterization of nine microsatellite markers from Coffea arabica L., showing wide cross‐species amplifications

Genetic improvement of coffee (Coffea arabica L.) is constrained by low genetic diversity and lack of genetic markers, suitable screening tools, information on the genetic make‐up of available gene pool and long generation time. In this context, use of DNA markers such as microsatellites that provide high genetic‐resolution becomes highly desirable. Here, we report the development of nine new microsatellite markers from partial genomic library of an elite variety of Coffea arabica. The developed microsatellites revealed robust cross‐species amplifications in 17 related species of coffee, and their Polymorphic Information Content varied from 0 to 0.6, 0 to 0.78 and 0.67 to 0.90 for the arabica, robusta genotypes and species representatives, respectively. The data thus suggest their potential use as genetic markers for assessment of germplasm diversity and linkage analysis of coffee.     

Tissue culture-mediated biotechnological intervention in pomegranate: a review

The past 30 years have witnessed a series of systematic biotechnological advances made in pomegranate. These encompass optimization and establishment of in vitro culture techniques including micropropagation, somatic embryogenesis, synthetic seed production, plant regeneration via callus-mediated shoot organogenesis, adventitious shoot regeneration, anther culture, tetraploid induction and genetic transformation. This review attempts to provide a comprehensive account on the tissue culture-mediated biotechnological interventions made in pomegranate aimed at complementing conventional programmes for improvement of this nutraceutically important fruit crop.     

Assessing the genetic diversity of tobacco germplasm using intersimple sequence repeat and inter-retrotransposon amplification polymorphism markers

The genetic diversity of 118 tobacco accessions, including flue‐cured tobacco, sun‐/air‐cured tobacco, burley tobacco, oriental tobacco and wild tobacco, was characterised using intersimple sequence repeat (ISSR) and inter‐retrotransposon amplification polymorphism (IRAP) markers. ISSR and IRAP banding patterns and genetic distance (GD) values showed the low level of genetic diversity within and among cultivated tobacco types. There was higher GD and average heterozygosity among wild tobacco types than those among cultivated tobacco. Genetic diversity of tobacco germplasm was low, with a high level of genetic identity (>0.77) between the different types. However, neighbour‐joining cluster analysis of marker‐based GDs showed that the accessions from the same tobacco type, as classified by manufacturing quality traits, were nearly clustered into the same group. These results will help in the formulation of appropriate strategies for variety improvement in tobacco, and ISSR and IRAP markers of the genetic diversity will contribute to further study and improvement of tobacco.     

Isolation of novel microsatellites using FIASCO by dual probe enrichment from Jatropha curcas L. and study on genetic equilibrium and diversity of Indian population revealed by isolated microsatellites

Jatropha curcas L. belongs to family Euphorbiaceae, native to South America attained significant importance for its seed oil which can be converted to biodiesel, a renewable energy source alternative to conventional petrodiesel. Very few attempts were made to isolate novel microsatellite markers and assessment of the extent of genetic equilibrium and diversity that exists in J. curcas. Therefore, the present investigation was undertaken to isolate the novel microsatellites and access genetic equilibrium, diversity that exists among 44 diverse germplasm collected from distinct geographical areas in India using isolated microsatellites. The overall efficiency of the enrichment of microsatellite by dual probe in the present study found to be 54% and among the sequences obtained the percentage of sequences having suitable flanking regions for the primer designing was found to be 89.58%. The mean co-efficient of genetic similarity (CGS) was found to be 0.97. The overall diversity obtained by microsatellites was found to be low in comparison with the diversity reported by multilocus markers systems observed in earlier studies; however, the good allele polymorphism was observed. The overall dendrogram of microsatellite analysis resulted in random clustering of germplasm and not in accordance to geographical area of collection. The present study, diversity analysis using microsatellite markers concludes the low genetic diversity and genetic disequlibrium of J. curcas in India and will provide pavement for further intra-population studies on narrow geographical areas to understand the population genetic structure, phylogeography and molecular ecological studies. The germplasm characterized, and the microsatellite markers isolated and characterized in the present study can be employed efficiently in breeding programs for genetic improvement of the species through marker assisted selection and QTL analysis, for further genetic resource management and help in making the J. curcas as potential crop with superior agronomical traits.     

Genetic variability and population structure in a collection of inbred lines derived from a core germplasm of castor

Castor (Ricinus communis L.) is an industrially important oilseed crop and is the only source of an unusual fatty acid, ricinoleic acid in plant species. The castor oil and its products have numerous industrial uses including biofuel; hence, the demand for castor oil is ever increasing globally. Current productivity levels in castor are inadequate to meet the requirement, which underscores the need for breeding high yielding cultivars with better adaptability by exploiting diverse genetic resources. This study reports development and characterization of a set of inbred lines derived from a core germplasm collection of castor. The panel of 144 inbreds exhibited an excellent phenotypic diversity for morpho-agronomic traits related to plant architecture and yield components. However, SSR allelic diversity appears to be only moderate. The average number of alleles per SSR locus in the genotype panel was 3.0 and mean gene diversity was 0.38. Nevertheless, a majority of the inbred pairs (77 %) had very less estimated kinship coefficients (<0.05) suggesting that they were not related by pedigree. A very low level of genetic relatedness among the genotypes and absence of population structure suggest that this genotype panel consists of ideal set of materials for association mapping studies aiming at molecular breeding of key traits in castor. To our knowledge, this is the first report on the development and characterization of a large inbred collection representing the bulk of genetic diversity in castor, which can be further exploited for genetic, physiological and molecular studies towards achieving higher productivity.     

RAPD fingerprinting of blackcurrant (Ribes nigrum L.) cultivars

Ribes nigrum germplasm was screened for random amplified polymorphic DNA (RAPD) markers. Fiftyfour markers were identified which generated individual fingerprints for each of 21 cultivars. Genetic variation within R. nigrum germplasm, as detected by RAPDs, demonstrated that the genetic basis for improvement of blackcurrant is narrower than would be expected by the analysis of parentage.     

小麦地方品种繁殖更新过程中的遗传多样性变化分析

采用分别保存于长期库及中期库的3个小麦地方品种的6份材料,进行了9项农艺性状及35个与农艺性状相关的微卫星标记检测,每份材料选取30个单株进行遗传多样性与遗传结构分析。结果表明:(1)更新前,3个小麦地方品种均为遗传异质性群体,在SSR位点上的异质度分别为57.14%、48.57%和5.71%。(2)在农艺性状表现上,只有温泉小麦3在株高和穗粒数上更新后比更新前显著增加,其他材料无显著差异。(3)在SSR位点多态性表现上,3个品种在更新后均发生了遗传多样性变化,8个与粒重、产量、生育期性状相关位点存在等位位点丢失现象,其中2个与粒重、生育期相关位点频率变化显著。(4)综合农艺性状调查与SSR分子标记检测结果发现,3个品种更新前后在多样性指数上无显著差异,遗传分化系数Gst分别为0.0269、0.0324和0.0380,即更新前后遗传差异分别为2.69%、3.24%和3.80%。上述结果建议,经繁殖更新的小麦种质资源能够比较完好地保持其遗传多样性和遗传结构。对于遗传异质性小麦地方品种在繁殖更新后存在遗传多样性丢失的危险,为了保证更新前后的遗传完整性,建议在繁殖更新过程中每个品种至少应保持300个单株的群体。     

Assessment of genetic diversity in Indian rice germplasm (Oryza sativa L.): use of random versus trait-linked microsatellite markers

Assessment of genetic diversity in a crop germplasm is a vital part of plant breeding. DNA markers such as microsatellite or simple sequence repeat markers have been widely used to estimate the genetic diversity in rice. The present study was carried out to decipher the pattern of genetic diversity in terms of both phenotypic and genotypic variability, and to assess the efficiency of random vis-à-vis QTL linked/gene based simple sequence repeat markers in diversity estimation. A set of 88 rice accessions that included landraces, farmer’s varieties and popular Basmati lines were evaluated for agronomic traits and molecular diversity. The random set of SSR markers included 50 diversity panel markers developed under IRRI’s Generation Challenge Programme (GCP) and the trait-linked/gene based markers comprised of 50 SSR markers reportedly linked to yield and related components. For agronomic traits, significant variability was observed, ranging between the maximum for grains/panicle and the minimum for panicle length. The molecular diversity based grouping indicated that varieties from a common centre were genetically similar, with few exceptions. The trait-linked markers gave an average genetic dissimilarity of 0.45 as against that of 0.37 by random markers, along with an average polymorphic information constant value of 0.48 and 0.41 respectively. The correlation between the kinship matrix generated by trait-linked markers and the phenotype based distance matrix (0.29) was higher than that of random markers (0.19). This establishes the robustness of trait-linked markers over random markers in estimating genetic diversity of rice germplasm.     

Transference of natural diversity from the apomictic germplasm of Paspalum notatum to a sexual synthetic population

Genetic improvement in apomictic forage species has been restricted because of the absence of genetic variability in sexual germplasm with the same ploidy level. Following a new breeding scheme, a sexual synthetic tetraploid population (SSTP) of Paspalum notatum has been generated. The objectives of this work were: (a) to evaluate the genetic variability in SSTP by means of molecular markers, morphologic and agronomic traits, and seed fertility and quality traits and (b) to assess the transference of genetic variability from the apomictic germplasm to the sexual one. Molecular markers revealed a twofold higher level of variability in the SSTP in comparison with the sexual germplasm utilised for its generation, and similar levels with the apomictic ones; moreover, markers showed that most of the variability was inherited from the apomictic germplasm. Morphologic and agronomic traits and seed fertility and quality traits showed high levels of variation in the three groups of genotypes indicating that the new breeding scheme was effective in transferring variability from the apomictic germplasm to the SSTP. This new population will be useful in breeding of P. notatum, and the breeding scheme used for its generation may be used in other apomictic species.     

Utilization of in silico EST–SSR markers for diversity studies in castor (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.)

Castor (Ricinus communis L.) a chief non-edible oilseed crop has numerous industrial applications. Systematic genetic diversity analysis utilizing DNA based markers has been quick and reliable method that ensures selection of diverse parents for exploitation of higher levels of heterosis in breeding programs. From NCBI database, 63,852 EST sequences of castor were mined. One thousand one hundred and five (1105) EST–SSRs and 1652 repeat motifs sequences were identified from 20,495 non-redundant unigene sequences. Repeat motifs consisted of 29.7 % mono nucleotide repeats, 24.8 % di nucleotide repeats, 27.27 % tri nucleotide repeats and 3.94 % tetra nucleotide repeats. Twenty eight primer pairs were chosen from SSR-containing ESTs to determine genetic diversity among 27 castor accessions. Twelve EST–SSRs showed polymorphism. Number of alleles detected were 2–3 with an average of 2.33 per locus. 150–400 bp was the size of an allele. Dendrogram analysis grouped the 27 accessions into two separate clusters. Genetic similarity coefficient of dendrogram ranged from 0.24 to 0.83. The polymorphic information content value of 0.28–0.49 revealed medium level of diversity in castor. Results of present study indicated that EST–SSRs to be efficient markers for genetic diversity studies. Knowledge on level of diversity existing in castor genotypes would be useful for breeders to plan efficient hybrid breeding programme.     

Does epigenetic polymorphism contribute to phenotypic variances in <Emphasis Type="Italic">Jatropha curcas</Emphasis> L.?

Background  

There is a growing interest in Jatropha curcas L. (jatropha) as a biodiesel feedstock plant. Variations in its morphology and seed productivity have been well documented. However, there is the lack of systematic comparative evaluation of distinct collections under same climate and agronomic practices. With the several reports on low genetic diversity in jatropha collections, there is uncertainty on genetic contribution to jatropha morphology.     

Biotechnological advances in mango (Mangifera indica L.) and their future implication in crop improvement: a review

Biotechnology can complement conventional breeding and expedite the mango improvement programmes. Studies involving in vitro culture and selection, micropropagation, embryo rescue, genetic transformation, marker-assisted characterization and DNA fingerprinting, etc. are underway at different centers worldwide. In vitro culture and somatic embryogenesis of several different genotypes have been achieved. The nucellus excised from immature fruitlets is the appropriate explant for induction of embryogenic cultures. High frequency somatic embryogenesis has been achieved in some genotypes; however, some abnormalities can occur during somatic embryo germination. Embryo rescue from young and dropped fruitlets can improve the hybridization success in a limited flowering season. Protocols for protoplast culture and regeneration have also been developed. In vitro selections for antibiotic tolerance and fungal toxin resistance have been very promising for germplasm screening. Genetic transformation using Agrobacterium tumefaciens has been reported. Genes that are involved with fruit ripening have been cloned and there have been attempts to deliver these genes into plants. DNA fingerprinting and studies on genetic diversity of mango cultivars and Mangifera species are also being conducted at several research stations. The purpose of this review is to focus upon contemporary information on biotechnological advances made in mango. It also describes some ways of overcoming the problems encountered during in vitro propagation of mango.     

Genetic diversity and structure among natural populations of Sindora glabra in Hainan Island,China as revealed by ISSR markers

Sindora glabra, one of the second-class national protective plants in China, has important ecological and commercial values. To understand the genetic diversity and structure of S. glabra, eleven natural populations from four areas of Hainan Island in China were analyzed by inter-simple sequence repeat (ISSR) markers. Thirteen primers were screened out and used to amplify 157 DNA samples. A total of 122 bands were obtained, among which 114 (93.4%) bands were polymorphic. Genetic parameters including average number of effective alleles (1.547), Nei's gene diversity (0.321), Shannon's information index (0.482), and gene differentiation coefficient (0.1944) revealed a high level of genetic diversity maintained in S. glabra populations. The variation within populations accounted for 85.6% of total variation based on analysis of molecular variance. Genetic distance analysis showed that 11 populations could be divided into three groups and populations from the same areas were classified as one group, suggesting that high genetic diversity of S. glabra was attributable to geographic isolation. Together, introduction of germplasm from distant natural distribution areas would be a sound strategy for germplasm resource conservation of S. glabra and selection of elite individuals from populations of far relationship for hybridization is of great importance for breeding improvement programs in future.     

Genetic diversity analysis and transferability of cereal EST-SSR markers to orchardgrass (Dactylis glomerata L.)

The diversity and genetic relationships among 74 orchardgrass accessions were analyzed using cereal EST-SSRs and orchardgrass SSR markers in order to estimate genetic variability and compare the level of diversity. In total, 190 polymorphic bands were detected with an average of 6.3 alleles per SSR loci. The average polymorphic rate (P) for the species was 84.63%, suggesting a high degree of genetic diversity. The molecular variance analysis (AMOVA) showed that the proportion of variance explained by within- and among-geographical groups diversity was74.87% and 25.13%, respectively. The distinct geographical divergence of orchardgrass was revealed between Americas and Oceania. The ecogeographical conditions such as climate and soil, genetic drift and mating system could be the crucial factors for genetic divergence. Furthermore, the study also indicated that northern Africa, Europe and temperate Asia might be the diversity differentiation center of orchardgrass. The result will facilitate the breeding program and germplasm collection and conservation.