空间搭载水稻种子后代基因组多态性及其与空间重离子辐射关系的探讨

神舟三号飞船搭载带核径迹辐射探测器的水稻种子装置,回收后应用随机扩增多态性DNA(randomamplified polymorphic DNA,RAPD)技术,分析了201粒升空种子长出植株的基因组多态性。在检测的189个基因座位范围内,30.2%的植株中发现与地面对照不同的扩增带,单株的多态性座位数为1￣25。特异扩增带的测序及单核苷酸多态性(single-nucleotide polymorphism,SNP)分析进一步证明了空间搭载水稻种子确实可导致当代植株基因组发生变异。同一技术分析个别种子连续世代的基因组多态性,结果显示,当代的部分多态性可遗传至后代。7粒受空间高原子序数、高能粒子轰击的种子,在当代植株均显示不同程度的基因组多态性,从胚受粒子击中的3粒种子后代中,筛选出农艺性状明显变异的突变株系,初步暗示了空间高能重离子辐射对诱导基因组的多态性,乃至遗传性表型变异的有效性。     

Genomic polymorphism in consecutive generation rice plants from seeds on board a spaceship and their relationship with space HZE particles

Dry rice seeds (Oryza sativa, subspecies indica) were sandwiched between nuclear track detectors, aboard the Chinese spaceship Shenzhou-3 for seven days. The seeds were recovered and the genomic polymorphism in 201 plants developed from these seeds was studied using random amplified polymorphism DNA analysis. When compared with plants from ground-based control seeds, the genomic polymorphic bands were amplified in 30.2% of the plants from the seeds exposed in space. The results for sequencing and SNP analyses of the polymorphic bands verified the single nucleotide variations in these plants. Genomic polymorphisms in the consecutive generations of individual plants of the seeds from space were also discovered. Seven seeds receiving hits of HZE (high atomic number and high energy) particles from space were selected for further analyses and variable genomic polymorphisms were detected in all plants that developed from these seeds. Among them, the embryos of three seeds were hit at least once, and mutants with significant changes in agronomic traits were only found in later generations of these seeds. This result implies that the HZE particles of space are effective in inducing the changes of plant genome of inherited phenotypes. Translated from Acta Biophysica Sinica, 2006, 22(2): 131–137 [译自: 生物物理学报]     

Genetic variability of Old Portuguese bread wheat cultivars assayed by IRAP and REMAP markers

Retrotransposons (RTNs) constitute informative molecular markers for plant species as a result of their ability of integrating into a multitude of loci throughout the genome and thereby generating insertional polymorphisms between individuals. Inter-retrotransposon amplified polymorphisms (IRAPs) and the retrotransposon-microsatellite amplified polymorphisms (REMAPs) are marker systems based on long terminal repeats (LTRs) RTNs, developed for plants, that have been widely used for evolution, genetic diversity, DNA fingerprinting of cultivars and varieties, genetic mapping linkage and for detection of genetic rearrangements induced by polyploidisation. In the present study, we aimed to analyse the genetic variability among 48 Old Portuguese bread wheat cultivars using both IRAP and REMAP markers. Five IRAP and six REMAP primer combinations were used. IRAP produced 103 polymorphic fragments in a total of 113 bands. On average, 22.6 bands were amplified per IRAP primer combination. The bands ranged in size from 250 to 5000 bp. The REMAP primer combinations allowed the amplification of 53 bands, 51 of them polymorphic. An average of 8.8 REMAP bands was scored per primer combination. The REMAP bands ranged from 250 to 3000 bp. Both marker systems presented high percentages of polymorphism. However, IRAP markers were suitable for detecting genetic variability at the individual level and did not differentiate higher taxa. The REMAP maker system allowed the clustering by botanical variety and identified most of the homonym bread wheat cultivars.     

空间搭载诱导水稻种子突变的分子标记多态性分析

以卫星空间搭载广东水稻品种特籼占13干种子,返地种植后经5代选择、培育,获得一批形态及育性变异的突变体及品系（种）,如株高变矮,稻穗变大,雄性不育等。为了探索空间诱变的本质,对选出的6个突变体及2个优良品系,选用了130个10－mer随机扩增多态性DNA（RAPD经物和17对扩增片段长度多态性（AFLP）引物组合,分别对其基因组DNA进行多态性位点扫描分析,两种方法的结果均显示：不同的突变体与原种DNA之间存在不同程度的多态性差异,且由两法得到的结果较接近,为6％－12％。此结果从分子水平上进一步证明了空间环境确实对植物种子存在诱变作用。     

Activation of transposable elements and insertional polymorphism in Opuntia offspring as assessed by inter-retrotransposon amplified polymorphism markers

Activation of retrotransposon is a pivotal factor in the genesis of genetic polymorphism. Retrotransposon-based molecular markers are excellent tools for detecting genetic diversity and genomic changes associated with their activity. The objective of this study was to use IRAP markers to detect integration events of retrotransposon in Opuntia, and to compare IRAP and ISSR polymorphisms. To achieve these aims, five IRAP and five ISSR markers were analyzed on three varieties and their progenies. All IRAP primers showed an increase in the percentage of polymorphism, number of total bands, and polymorphic bands in the seedlings compared to their mother plants; that is, the offspring showed 13, 24 and 27 more bands than the mother plants from Tobarito, Montesa and Copena varieties, respectively. Conversely, sexual reproduction did not proportionally affect the variation in the number of ISSR bands, and neither did polymorphisms between mother plants and their offspring. Cluster and multivariate analyses based on IRAP and ISSR data revealed a clear separation between varieties, and there was no overlapping between seedlings of different varieties. The activation of retrotransposons in seedlings of opuntia with variable frequencies was evidenced. The presence of insertion and active retrotransposons may help to undertake studies on genetic diversity and evolution of Opuntia.     

SRAP在检测黄瓜基因组多态性中的特征

将SRAP (Sequence-Related Amplified Polymorphism)应用于黄瓜(Cucumis sativus L.)遗传图谱和耐高温QTL的定位过程中, 发现SRAP在检测黄瓜亲本基因组多态性中呈现出一些特征。对于每个正向引物, 在与12个不同的反向引物组合时, 产生多态性条带的引物组合数均在5~8个之间; 而对于每个反向引物, 在与11个不同的正向引物组合时, 产生多态性的引物组合数则在2~11个之间, 差异较大。反向引物SA4或EM6与研究的所有正向引物组合时产生的多态性条带分子量完全相同, 这些条带可能是由反向单引物扩增而来的。引物组合OD3ME11扩增出的多态性条带存在共分离现象。同时对利用SRAP的这些特征指导我们的研究进行了讨论。     

应用SRAP标记对莲藕资源的聚类分析

利用一种新的分子标记SRAP技术对17个莲藕品种进行了DNA多态性分析。选取7对引物扩增基因组DNA,共获得168条带,其中159条为多态性条带,每对引物平均提供24个标记信息。由UPMGA方法得到的聚类分析结果表明了17个品种间的遗传关系:(1)亚洲莲与美洲莲之间有明显的差异;(2)在亚洲莲内部,藕莲和花莲有明显的遗传分化,在花莲内部亚洲莲与美洲莲的杂交后代和其它花莲品种之间存在明显的差异;(3)SRAP标记是作分子图谱的好标记,但很难区分遗传关系较近的品种。     

Molecular Diversity in some Ghanaian Cowpea [<Emphasis Type="Italic">Vigna unguiculata</Emphasis> L. (Walp)] Accessions

Cowpea [Vigna unguiculata L. (Walp)] is grown mainly for its protein-rich grains and is consumed in various forms in sub-Saharan Africa. Average grain yield in farmers’ fields is generally low due to a number of biotic and abiotic stresses. One hundred and six cowpea accessions from Ghana, which had previously been evaluated for seedling drought tolerance, were used for this study. This paper attempts to use three multi-locus PCR-based molecular markers; simple sequence repeats (SSR), inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphisms (REMAP), to analyse genetic diversity in the cowpea accessions. Analysis of the polymorphic bands data indicated that 101 alleles were amplified among 121 cowpea genotypes (83.4%) from 16 SSR primer pairs out of a total of 30 SSR primer pairs. Likewisely, a total of 66 (54.5%) polymorphic bands were obtained from IRAP and a total of 114 (94.2%) highly polymorphic bands obtained from REMAP analysis. The outcome indicated the highly polymorphic nature of the DNA markers, as small groups of these molecular markers were found to be able to identify each of the accessions used. Microsatellite markers (SSRs) and retrotransposon-based markers, like IRAP and REMAP, were found to be highly polymorphic and informative, suggesting that genomic fingerprinting has a major role in characterizing populations.     

IRAP and REMAP for retrotransposon-based genotyping and fingerprinting

Retrotransposons can be used as markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, retrotransposon-microsatellite amplified polymorphism (REMAP) analysis and inter-retrotransposon amplified polymorphism (IRAP) analysis, that require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and for agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in 1-2 d.     

Start codon targeted polymorphism for evaluation of functional genetic variation and relationships in cultivated peanut (<Emphasis Type="Italic">Arachis</Emphasis><Emphasis Type="Italic">hypogaea</Emphasis> L.) genotypes

Cultivated peanut possesses an extremely narrow genetic basis. Polymorphism is considerably difficult to identify with the use of conventional biochemical and molecular tools. For the purpose of obtaining considerable DNA polymorphisms and fingerprinting cultivated peanut genotypes in a convenient manner, start codon targeted polymorphism technique was used to study genetic diversity and relatedness among 20 accessions of four major botanical varieties of peanut. Of 36 primers screened, 18 primers could produce unambiguous and reproducible bands. All 18 primers generated a total of 157 fragments, with a mean of 8.72 ranging from 4 to 17 per primer. Of 157 bands, 60 (38.22%) were polymorphic. One to seven polymorphic bands were amplified per primer, with 3.33 polymorphic bands on average. Polymorphism per primer ranged from 14.29 to 66.67%, with an average of 36.76%. The results revealed that not all accessions of the same variety were grouped together and high genetic similarity was detected among the tested genotypes based on cluster analysis and genetic distance analysis, respectively. Further, accession-specific markers were observed in several accessions. All these results demonstrated the following: (1) start codon targeted polymorphism technique can be utilized to identify DNA polymorphisms and fingerprint cultivars in domesticated peanut, and (2) it possesses considerable potential for studying genetic diversity and relationships among peanut accessions.     

Changes in AFLP and SSR DNA polymorphisms induced by short-term space flight of rice seeds

Differences of both amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) polymorphisms were compared between the 60-d-old rice (Oryza sativa L. cv. DH7) and F3 rice plants (SP3) derived from seed, which endured a 7-d-space flight in March 2002. Total leaf AFLP DNA bands amplified from 22 primer pairs were 537 in DH7, whereas 562 in SP3. From the total 267 SSR DNA bands generated by 267 primer pairs, 39 were polymorphic with 22 larger (56 %) or 17 smaller (44 %) fragment size bands. The greatest numbers of AFLP DNA bands were amplified by primer E1M1 in DH7 (33) and E3M1 in SP3 (35), whilst the least by E4M3 in DH7 (14) and E5M2 in SP3 (16).     

Genetic stability of cryopreserved shoot tips of <Emphasis Type="Italic">Rubus</Emphasis> germplasm

Questions often arise concerning the genetic stability of plant materials stored in liquid nitrogen for long time periods. This study examined the genetic stability of cryopreserved shoot tips of Rubus germplasm that were stored in liquid nitrogen for more than 12 yr, then rewarmed and regrown. We analyzed the genetic stability of Rubus grabowskii, two blackberry cultivars (“Hillemeyer” and ‘Silvan’), and one raspberry cultivar (“Mandarin”) as in vitro shoots and as field-grown plants. No morphological differences were observed in greenhouse-grown cryopreserved plants when compared to the control mother plants. In the field, cryopreserved plants appeared similar but were more vigorous than mother plants, with larger leaves, fruit, and seeds. Single sequence repeats (SSR) and amplified fragment length polymorphism (AFLP) analyses were performed on shoots immediately after recovery from cryopreservation and on shoots subcultured for 7 mo before analysis. Ten SSR primers developed from “Marion” and “Meeker” microsatellite-enriched libraries amplified one to 15 alleles per locus, with an average of seven alleles and a total of 70 alleles in the four genotypes tested. No SSR polymorphisms were observed between cryopreserved shoots and the corresponding mother plants regardless of subculture. Although no polymorphisms were detected in shoots analyzed immediately after recovery from cryopreservation, AFLP polymorphisms were detected in three of the four Rubus genotypes after they were subcultured for 7 mo. Field-grown plants from the polymorphic shoot tips of R. grabowskii and ‘Silvan’ displayed the same AFLP fingerprints as their corresponding mother plants. Only long-cultured in vitro shoot tips displayed polymorphisms in vitro, and they were no longer detected when the plants were grown ex vitro. The transitory nature of these polymorphisms should be carefully considered when monitoring for genetic stability.     

Genetic diversity of Hibiscus tiliaceus (Malvaceae) in China assessed using AFLP markers

Amplified fragment length polymorphism (AFLP) markers were used to investigate the genetic variations within and among nine natural populations of Hibiscus tiliaceus in China. DNA from 145 individuals was amplified with eight primer pairs. No polymorphisms were found among the 20 samples of a marginal population of recent origin probably due to a founder effect. Across the other 125 individuals, 501 of 566 bands (88.5%) were polymorphic, and 125 unique AFLP phenotypes were observed. Estimates of genetic diversity agreed with life history traits of H. tiliaceus and geographical distribution. AMOVA analysis revealed that most genetic diversity resided within populations (84.8%), which corresponded to results reported for outcrossing plants. The indirect estimate of gene flow based on phiST was moderate (Nm=1.395). Long-distance dispersal of floating seeds and local environments may play an important role in shaping the genetic diversity of the population and the genetic structure of this species.     

Inheritance pattern of RAPD markers in Melipona quadrifasciata (Hymenoptera: Apidae, Meliponinae).

Melipona quadrifasciata is an important pollinator agent in several regions of Brazil. Data concerning the genetics of this species are scarce in the literature. In this work we used the random amplified polymorphic DNA (RAPD) technique to determine the degree of polymorphism and the inheritance pattern of these molecular markers in this species. Our ultimate goal is to establish tools to be used in the study of the genomic organization of M. quadrifasciata. Genomic DNA from progenies F(1) and BC(1) were assayed with 79 different primers, yielding an average of 6.67 bands and 1.68 polymorphisms per primer. Three types of polymorphisms were detected: band presence/absence, band intensity, and fragment-length polymorphisms. Most of the observed polymorphisms were band presence/absence, typical of RAPD-dominant markers. The number of observed polymorphisms and their segregation in accordance with a Mendelian proportion confirm the importance of this technique for genome analysis of species like M. quadrifasciata that are poorly studied at the genetic level.     

莲品种资源的SRAP遗传多样性分析

利用分子标记SRAP技术对国内广泛栽培以及新近育成的39个莲品种进行了DNA多态性分析。选取5对引物扩增基因组DNA,共获得101条带,其中88条为多态性条带,每对引物平均提供20个标记信息。由UPMGA方法得到的聚类分析结果表明了39个品种间的遗传关系,结果表明:花莲、籽莲和藕莲三大类群有明显的界限,花莲和籽莲的遗传距离较近,藕莲与它们的遗传距离较远。分子方差分析结果表明:莲品种间和品种内均存在遗传变异,藕莲品种内的遗传变异略低于品种间的遗传变异,而诱变籽莲、诱变花莲和常规籽莲品种内遗传变异均大于品种间的遗传变异,尤其是诱变籽莲、诱变花莲品种内遗传变异占总变异的分量分别超过了70%和60%,这种品种内的遗传变异,一方面对于莲的高产稳产具有重要的意义,另一方面也说明了从这些国内广泛栽培的品种以及新近育成的莲品种中可以直接进一步选育出更优良的新品种。     

A comparative analysis of genetic diversity in blackgram genotypes using RAPD and ISSR markers

Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to study the DNA polymorphism in elite blackgram genotypes. A total of 25 random and 16 ISSR primers were used. Amplification of genomic DNA of the 18 genotypes, using RAPD analysis, yielded 104 fragments that could be scored, of which 44 were polymorphic, with an average of 1.8 polymorphic fragments per primer. Number of amplified fragments with random primers ranged from two (OPA-13) to nine (OPK-4) and varied in size from 200 bp to 2,500 bp. Percentage polymorphism ranged from 16.6% (OPK-7) to a maximum of 66.6% (OPE-5, OPH-2, and OPK-8), with an average of 42.7%. The 16 ISSR primers used in the study produced 101 bands across 18 genotypes, of which 55 were polymorphic. The number of amplified bands varied from two (ISSR 858) to ten (ISSR 810), with a size range of 200–2,200 bp. The average numbers of bands per primer and polymorphic bands per primer were 6.3 and 3.4, respectively. Percentage polymorphism ranged from 25% (ISSR 885) to 100% (ISSR 858), with an average percentage polymorphism of 57.5% across all the genotypes. The 3-anchored primers based on poly(GA) and poly(AG) motifs produced high average polymorphisms of 54.98% and 58.32%, respectively. ISSR markers were more efficient than the RAPD assay, as they detected 57.4% polymorphic DNA markers in Vigna mungo as compared to 42.7% for RAPD markers. The Mantel test between the two Jaccards similarity matrices gave r =0.32, showing low correlation between RAPD- and ISSR-based similarities. Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared, whereas the pattern of clustering of the genotypes remained more or less the same in ISSR and combined data of RAPD and ISSR.     

Random amplified polymorphic DNA (RAPD) markers detect a single phenotype in lysimachia minoricensis J.J. Rodr. (Primulaceae), A wild extinct plant

Lysimachia minoricensis is a Mediterranean (Balearic Islands) endemic that is extinct in the wild but extant in botanical gardens. Previously, no variation at 22 isozyme loci was revealed in more than 150 analysed plants. Random amplified polymorphic DNA (RAPD) analysis was used to examine genetic variation among five individuals from each of eight botanical garden accessions (40 plants). No polymorphisms were detected at 201 amplified bands. This is the first report of RAPD monomorphism in a nonapomictic vascular plant. The lack of detectable genetic variation suggests that an extremely reduced gene pool was recovered in the field before its extinction. Although the screening of other genomic markers is feasible, it is suggested that the knowledge of biological and autoecological features should be prioritized before new re-introductions are attempted.     

Evidence for genomic changes in transgenic rice (Oryza sativa L.) recovered from protoplasts

The occurrence of genomic modifications in transgenic rice plants recovered from protoplasts and their transmission to the self-pollination progeny has been verfied with the random amplified polymorphic DNA (RAPD) approach. The plant was the Indica-type rice (Oryza sativa L.) cultivar Chinsurah Boro II. The analysed material was: (1) microspore-derived embryogenic rice cells grown in suspension culture, (2) transgenic plants recovered from protoplasts produced from the cultured cells and (3) the self-pollination progeny (two successive generations) of the transgenic plants. DNA purified from samples of these materials was PCR-amplified with different random oligonucleotide primers and the amplification products were analysed by agarose gel electrophoresis. Band polymorphism was scored and used in band-sharing analyses to produce a similarity matrix. Relationships among the analysed genomes were expressed in a dendrogram.The extensive DNA changes evidenced in cultured cells demonstrate the occurrence of somaclonal variation in the material used to produce protoplasts for gene transfer. Quantitatively reduced DNA changes were also found in the resulting transgenic plants and i their self-pollination progenies.While confirming the stability of the foreign gene in transgenic plants, this work gives molecular evidence for the occurrence of stable genomic changes in transgenic plants and points toin vitro cell culture as the causative agent. RAPDs are shown to be a convenient tool to detect and estimate the phenomenon at the molecular level. The methodology is also proposed as a fast tool to select those transgenic individuals that retain the most balanced genomic structure and to control the result of back-crosses planned to restore the original genome.     

Randomly amplified polymorphic DNA-polymerase chain reaction analysis of two different populations of cultured Korean catfishSilurus asotus

Genetic similarity and diversity of cultured catfishSilurus asotus populations collected from two areas in western Korea were examined using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Out of 20 random primers tested, 5 produced 1344 RAPD bands ranging from 8.2 to 13.6 polymorphic bands per primer. The polymorphic bands in these populations ranged from 56.4% to 59.6%. Polymorphic bands per lane within populations ranged from 4.9% to 5.3%. The similarity within the Kunsan population varied from 0.39 to 0.82 with a mean (± SD) of 0.56 ± 0.08. The level of bandsharing values was 0.59 ± 007 within the catfish population from Yesan. The genetic similarity in cultured catfish populations may have been caused because individuals from two populations were reared in the same environmental conditions or by inbreeding during several generations. However, in view of bandsharing values, polymorphic bands and also the specific major bands that were inter-population-specific, significant genetic differentiation between these populations were present even if bandsharing (BS) values were somewhat numerically different. Therefore, the number of RAPD polymorphisms identified in this study may be sufficient to permit estimating genetic similarity and diversity. However, in future, additional populations, sampling sites and individuals will be necessary to make up for these weak points.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.