Variation of cytosine methylation in 57 sweet orange cultivars

Sweet orange is an important group of citrus cultivars, which includes a number of bud sport cultivars. Little is known about the CpG methylation status of the CCGG sequences in the orange genome. In this study, methylation-sensitive amplification polymorphism (MSAP), based on the application of isoschizomers (Hpa II and Msp I), was first used to analyze cytosine methylation patterns in 57 orange cultivars that were not fully differentiated by regular DNA molecular markers. Three types of bands were generated from ten primer pairs. Type I bands were present following restriction with Eco RI + Hpa II and Eco RI + Msp I; type II or type III were present only following restriction with either Eco RI + Hpa II or with Eco RI + Msp I. The total number of these three types of bands was 802, 72, and 157, respectively. Among these, the number of polymorphic bands were 244 (30.2%), 23 (31.9%), and 32 (20.4%), in type I, II and III, respectively. The methylation patterns of these 57 cultivars are discussed and assessed by dendrograms derived from the analysis of polymorphic MSAP bands. The distribution of polymorphic bands of the above three types demonstrate the methylation patterns and frequency at the cytosine loci. We suggest that methylation events could be more frequent than demethylation events, and that the methylation patterns maybe associated with phenotypic traits.     

Monitoring of cultivar identity in tissue culture-derived date palms using RAPD and AFLP analysis

In the present study, two polymerase chain reaction (PCR)-based methods namely, randomly amplified polymophic DNA (RAPD) and amplification fragment length polymorphism (AFLP) were employed to assess genetic variations, which may appeared, in tissue culture-derived date palm (Phoenix dactylifera) offshoots. Analysis of RAPD banding patterns generated by PCR amplification using 37 random primers gave no evidences for somaclonal variations and the percentage of polymorphic bands in a total of 259 scored bands was zero. Meanwhile, analysis of AFLP banding patterns generated using 13 primer combinations pointed to minor genetic variations in the AFLP banding patterns. The percentage of genetic variations (polymorphism) in tissue culture-derived date palm offshoots belonging to cultivars Sakkoty, Gandila and Bertamoda was 2.6, 0.79 and 1 %, respectively, as revealed by AFLP analysis. The low percentage of genetic variations confirms the genetic stability of tissue culture-derived dry date palm cultivars.     

Anther culture and Hordeum bulbosum-derived barley doubled haploids mutations and methylation

Anther culture and Hordeum bulbosum-derived doubled haploid (DH) lines of barley (Hordeum vulgare L.) were analyzed for RFLP and RAPD polymorphisms. Polymorphisms were not detected in the anther culture-or H. bulbosum-derived DH lines among 273 RFLP and 89 polymerase chain reaction (PCR)-amplified DNA fragments assayed. It was calculated that base substitution or small deletion/insertion mutations had not been induced among 401 640 by screened. Large deletion/insertion mutations were not observed among 33 Mb screened. Polymorphisms were observed when DNA was digested with the methylation-sensitive restriction enzymes HpaII and MspI: these RFLPs originated primarily from the anther culture-derived doubled haploids. The data indicate that heritable DNA methylation changes had occurred during DH production, particularly with the anther culture method.     

DNA methylation profiles differ between field- andin vitro-grown leaves of apple

A reliable method has been previously developed to detect cytosine methylation at the 5′-CCGG-3′ sequence using isoschizomers (Msp I and Hpa II) and a modified amplified fragment length polymorphism (AFLP) technique. With this method, DNA methylation profiles were investigated in leaf tissues of apple (Malus × domestica cv. Gala) grown under two different growth conditions, field and tissue culture. A total of 1,622 AFLP bands were detected using 32 pairs of primers, and these banding patterns were assembled into three groups. Type I AFLP bands were present in both EcoR I/Hpa II and EcoR I/Msp I lanes. Type II bands were present in the EcoR I/Msp I lanes, but not in EcoR I/Hpa II lanes. Type III bands were present in EcoR I/Hpa II lanes, but not in the EcoR I/Msp I lanes. For leaf tissues of field- and in vitro-grown apples, the ratios of types I, II, and III to the total number of amplified fragments were 70 %, 24 %, and 6 %, and 71 %, 23 %, and 6 %, respectively. Although the ratios of the three types of banding patterns were similar in both leaf tissues, a few bands specific to either field-grown trees or in vitro-grown shoots were observed. This study provided evidence that changes in methylation occurred in apple leaf tissues subjected to tissue culture growth conditions.     

Methylation imprinting was observed of mouse mo-2 macrosatellite on the pseudoautosomal region but not on chromosome 9

Mouse mo-2 macrosatellites consisting of 31-bp tandem repeat units are mainly located at two loci in the C57BL/6 genome, one being at the centromere-distal telomeric region of chromosome 9 and the other at the pseudoautosomal (PA) region of chromosomes X and Y. The two clustes constitute approximately 300 kb and 150 kb, respectively. Southern analysis of a methylation-sensitive enzyme, HpaII-digested DNA showed that the mo-2 macrosatellites are detected as more than 30 polymorphic bands. Comparison of those bands between reciprocally crossed F₁ mice revealed that approximately 20% of the allele-specific fragments exhibit different band intensities depending on the sex of the parent of origin. The differential methylation is observed in the mo-2 macrosatellite on the PA region but not in that on chromosome 9. Several fragments including the 3.4-kb fragment without internal HpaII site are more clearly detected when paternally derived, suggesting that the male-derived macrosatellite is undermethylated. Interestingly the difference is much more remarkable in inter-subspecific F₁ mice between C57BL/6 and MSM than F₁ between C57BL/6 and C3H/He. This suggests the presence of a modifier(s) that affect(s) the methylation of mo-2 in the MSM genome.     

Evaluation of Genetic and Epigenetic Modification in Rapeseed （Brassica napus） Induced by Salt Stress

Salinity is an important limiting environmental factor for rapeseed production worldwide. In this study, we assessed the extent and pattern of DNA damages caused by salt stress in rapeseed plants. Amplified fragment length polymorphism （AFLP） analysis revealed dose-related increases in sequence alterations in plantlets exposed to 10-1000 mmol/L sodium chloride. In addition, individual plantlets exposed to the same salt concentration showed different AFLP and selected region amplified polymorphism banding patterns. These observations suggested that DNA mutation in response to salt stress was random in the genome and the effect was dose-dependant. DNA methylation changes in response to salt stress were also evaluated by methylation sensitive amplified polymorphism （MSAP）. Three types of MSAP bands were recovered. Type Ⅰ bands were observed with both isoschizomers Hpa Ⅱ and Msp Ⅰ, while type Ⅱ and type Ⅲ bands were observed only with Hpa Ⅱ and Msp Ⅰ, respectively. Extensive changes in types of MSAP bands after NaCI treatments were observed, including appearance and disappearance of type Ⅰ, Ⅱ and Ⅲ bands, as well as exchanges between either type Ⅰand type Ⅱ or type Ⅰ and type Ⅲ bands. An increase of 0.2-17.6% cytosine methylated CCGG sites were detected in plantlets exposed to 10- 200 mmol/L salt compared to the control, and these changes included both de novo methylation and demethylation events. Nine methylation related fragments were also recovered and sequenced, and one sharing a high sequence homology with the ethylene responsive element binding factor was identified. These results demonstrated clear DNA genetic and epigenetic alterations in planUets as a response to salt stress, and these changes may suggest a mechanism for plants adaptation under salt stress.     

Genome and species relationships in genus<Emphasis Type="Italic"> Avena</Emphasis> based on RAPD and AFLP molecular markers

Species and genome relationships among 11 diploid (A and C genomes), five tetraploid (AB and AC genomes) and two hexaploid (ACD genome) Avena taxa were investigated using amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPD) markers. The two primer pairs used for the AFLP reactions produced a total of 354 polymorphic bands, while 187 reproducible bands were generated using ten RAPD primers. Genetic similarities amongst the entries were estimated using the Jaccard and Dice algorithms, and cluster analyses were performed using UPGMA and neighbor joining methods. Principle coordinate analysis was also applied. The highest cophenetic correlation coefficient was obtained for the Jaccard algorithm and UPGMA clustering method (r=0.99 for AFLP and r=0.94 for RAPD). No major clustering differences were present between phenograms produced with AFLPs and RAPDs. Furthermore, data produced with AFLPs and RAPDs were highly correlated (r=0.92), indicating the reliability of our results. All A genome diploid taxa are clustered together according to their karyotype. The AB genome tetraploids were found to form a subcluster within the A_s genome diploids (AFLPs), indicating their near-autoploid origin. The AC genome tetraploids are clustered to the ACD genome hexaploids. Finally, the C genome diploids form an outer branch, indicating the major genomic divergence between the A and C genomes in Avena.Communicated by J.S. Heslop-Harrison     

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.     

AFLP-Based detection of DNA methylation

By using the isoschizomersHpa II andMsp I which display differential sensitivity to cytosine methylation, a modified amplified fragment length polymorphism (AFLP) technique has been developed to investigate DNA methylation profiles in eukaryotic organims. Genomic DNA was digested with a mixture ofEcoR I and one of the isoschizomers, and ligated to oligonucleotide adapters. After two rounds of selective PCR amplification, followed by DNA separation on a Long Ranger gel electrophoresis, a subset of restriction fragments can be displayed on an X-ray film. Comparison of AFLP banding patterns betweenHpa II andMsp I revealed the extent of DNA methylation. The technique has been successfully applied in this study to investigate DNA methylation profiles of apple (Malus domestica cv. Gala) genomic DNA extracted from leaves of field-grown adult trees andin vitro-grown shoot cultures. The results showed that up to 25 percent of AFLP bands were derived from methylated sequences, and among those, a few bands unique to either adult trees orin vitro shoots were observed. These results demonstrated that this protocol is effective in identifying methylated DNA profiles. Both first authors have contributed equally to this work.     

Citrus somatic hybrid: an alternative system to study rapid structural and epigenetic reorganization in allotetraploid genomes

Citrus somatic hybrids produced in the past years provide a novel opportunity to study the immediate effects of allopolyploidization on genome structure and methylation. Here, we present a first attempt to investigate the alterations in genome structure and methylation in three sets of citrus somatic allotetraploids and their diploid parents using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques. Our results indicate that all the allotetraploids mainly have the AFLP and MSAP banding patterns containing specific bands from both parents plus some alterations. The incidences of the AFLP polymorphic bands in allotetraploids show a range from 4.61 to 7.88 %, while from 12.50 to 15.67 % of the sites are methylated. In addition, the proportions of callus-parent-specific DNA structure and methylation alterations are much greater than those of leaf-parent-specific alterations in the somatic hybrids. Furthermore, we find that the somatic hybrids take on a greater divergence from the callus parent and a closer relationship to leaf parent in all groups of plants by dendrogram analysis based on AFLP or MSAP data. Taken together, our results suggest that somatic hybrids are very useful in elucidating the immediate changes that occur in newly synthesized allotetraploid.     

Newly developed MSAP analysis reveals the different polymorphism patterns in transgenic tobacco plants with the dsRNA MET1 gene

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we isolated NtMET1 from Nicotiana tabacum cv. Havana (SR1) and obtain transgenic plants that reduced MET1 expression level with the double-strand RNA (dsRNA) MET1 gene. Transgenic tobacco plants showed dwarf and abnormal flower development when compared with the wild type. Using methylation-sensitive amplified polymorphism (MSAP) analysis, the patterns of cytosine methylation in transformed plants and the wild type were compared. MseI/HpaII selection primers showed an interesting polymorphism, and 153 DNA bands of interest were detected. Among these, 30 selective fragments were sequenced and analyzed with a BLAST search by successful MSAP modifications. The homology search showed that the transposons and tandem repeated sequences were related to the phenotypes. These results suggested that the decreased degree of methylation by dsRNA strategy caused abnormal growth and development in N. tabacum.     

Search for methylation-sensitive amplification polymorphisms associated with the mantled variant phenotype in oil palm (Elaeis guineensis Jacq).

The methylation-sensitive amplification polymorphism (MSAP) technique has been employed on somatic embryo-derived oil palms (Elaeis guineensis Jacq.) to identify methylation polymorphisms correlated with the "mantled" somaclonal variation. The variant phenotype displays an unstable feminization of male organs in both male and female flowers. Using MSAP, the methylation status of CCGG sites was compared in three normal versus three mantled regenerants sampled in clonal populations obtained through somatic embryogenesis from four genotypically distinct mother palms. Overall, 64 selective primer combinations were used and they have amplified 23 markers exhibiting a differential methylation pattern between the two phenotypes. Our results indicate that CCGG sites are poorly affected by the considerable decrease in global DNA methylation that has been previously associated with the mantled phenotype. Each of the 23 markers isolated in the present study could discriminate between the two phenotypes only when they were from the same genetic origin. This result hampers at the moment the direct use of MSAP markers for the early detection of variants, even though valuable information on putative target sequences will be obtained from a further characterization of these polymorphic markers.     

Anther culture and Hordeum bulbosum-derived barley doubled haploids mutations and methylation

Anther culture and Hordeum bulbosum-derived doubled haploid (DH) lines of barley (Hordeum vulgare L.) were analyzed for RFLP and RAPD polymorphisms. Polymorphisms were not detected in the anther culture-or H. bulbosum-derived DH lines among 273 RFLP and 89 polymerase chain reaction (PCR)-amplified DNA fragments assayed. It was calculated that base substitution or small deletion/insertion mutations had not been induced among 401 640 by screened. Large deletion/insertion mutations were not observed among 33 Mb screened. Polymorphisms were observed when DNA was digested with the methylation-sensitive restriction enzymes HpaII and MspI: these RFLPs originated primarily from the anther culture-derived doubled haploids. The data indicate that heritable DNA methylation changes had occurred during DH production, particularly with the anther culture method.     

A population genetic study of the endangered plant species Limonium dufourii (Plumbaginaceae) based on amplified fragment length polymorphism (AFLP)

Limonium dufourii ( Plumbaginaceae ) is a triploid species with obligate apomictic reproduction and is endemic to the East Mediterranean coast of Spain, where it is present in only six populations, most of which have a very low number of individuals. Genetic variation and population structure in this species was studied using amplified fragment length polymorphisms (AFLPs) as markers, using the same individuals as in a previous study with random amplified polymorphic DNA (RAPD). Three different primers provided 252 bands of which 51 were polymorphic among the 152 individuals analysed. Those polymorphic bands were able to define 65 different phenotypes, of which all but two were present in only one population. The comparative analyses of data from AFLPs with those from RAPDs show a high degree of concordance. Additionally, and given the nature of these markers, we propose the estimation of nucleotide divergences from AFLP patterns. Relationships among the different AFLP patterns and the estimates of population genetic parameters obtained with this evolutionary distance are in good agreement with previous results. These analyses show that substantial genetic variability and differentiation exist within and among populations of L. dufourii . Their higher reproducibility and the possibility of obtaining estimates of nucleotide divergence make AFLPs a much better DNA fingerprinting technique.     

Vernalization-induced changes of the DNA methylation pattern in winter wheat.

Vernalization is a cold treatment that induces or accelerates flowering and insures that temperate-zone plants will not flower until after winter. There is evidence that vernalization results in DNA demethylation that induces flowering. Differences in DNA methylation can be determined using methylation-sensitive amplified fragment length polymorphisms (AFLPs). Methylation-sensitive AFLPs utilize restriction enzyme isoschizomers that are differentially sensitive to methylation, producing polymorphisms related to methylation differences as opposed to sequence differences. Near-isogenic lines (NILs) have been developed for spring vs. winter habit in wheat (Triticum aestivum) and allow for the study of a single vernalization locus. In this study, differences in the methylation pattern were determined for spring and winter NILs, as well as for unvernalized and vernalized individuals. Winter wheat was more highly methylated than spring wheat and methylation-related AFLPs were produced between winter and spring wheat. Changes in the methylation pattern were observed at the end of vernalization, one week after the end of vernalization, and four weeks after the end of vernalization of winter wheat. However, the most methylation differences were observed one week after removal of winter wheat from cold treatment. Our data suggest that there is not only a vernalization-induced demethylation related to flower induction, but there is also a more general and non-specific demethylation of sequences unrelated to flowering. Two methylation-related AFLPs induced by vernalization were shared among all of the winter NILs.     

DNA-methylation alterations and exchanges during in vitro cellular differentiation in rose (<Emphasis Type="Italic">Rosa hybrida</Emphasis> L.)

DNA-methylation profiles of leaf tissues of Rosa hybrida cv. Carefree Beauty collected from in vivo-grown greenhouse plants, in vitro-grown proliferating shoots at different passages, regenerants of embryogenic callus, regenerants of organogenic callus, as well as calli from undifferentiated callus (UC), embryogenic callus, and organogenic callus were investigated using an amplified fragment-length polymorphism (AFLP)-based detection technique. Three types of AFLP bands were recovered. Type I bands were observed with both isoschizomers Msp and HpaII, while type II and type III bands were observed only with MspI and HpaII, respectively. Sequence analysis of the three types of AFLP bands revealed that a nonmethylated MspI/HpaII-recognition site 5-CCGG-3 resulted in a type I band, while an inner 5-methylcytosine generated most type II and type III bands. About 40% of inner and 20% of outer cytosines in 5-CCGG-3 sequences were fully methylated, and only a few hemimethylated outer cytosines were observed. Changes in types of AFLP bands among different tissues were frequently observed, including appearance and disappearance of type I, II, and III AFLP bands, as well as exchanges between either type I and type II or type I and type III AFLP bands. Methylation alterations of outer cytosines in 5-CCGG-3 sequences triggered appearance and disappearance of type I and II AFLP bands. Methylation changes of both outer and inner cytosines resulted in either removal or generation of type III AFLP bands. Methylation alteration of an inner cytosine was responsible for exchange between type I and type II, while hemimethylation of an outer cytosine accounted for exchange between type I and type III AFLP bands. During UC induction, a significant DNA-methylation alteration was detected in both inner and outer cytosines. Variations in methylation profiles significantly differed between somatic embryogenesis and in vitro organogenesis. Demethylation of outer cytosines occurred at a high frequency during somatic embryogenesis, and most altered AFLP bands in embryogenic callus were passed on to its regenerants. However, most methylation-altered AFLP bands during organogenesis were recovered in shoot regenerants derived via organogenic callus. Seven tissue-specific bands were isolated, cloned, and sequenced. Blast search revealed that two of these might be derived from functional genes.Mingliang Xu and Xiangqian Li contributed equally to this paper     

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs

Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different- colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were sampled. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating independently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once. Received: 3 October 2000 / Accepted: 19 February 2001     

Epigenetic variation within <Emphasis Type="Italic">Phragmites australis</Emphasis> among lineages,genotypes, and ramets

Epigenetics is likely an important factor in morphological and physiological acclimation, phenotypic plasticity, and potentially ecological dynamics such as invasiveness. We propose that Phragmites australis is an ideal model species for studies of epigenetics as a factor in plant invasions and ecology due to natural clonal replication (controlling for genetic variation) and the co-occurrence of subspecies with distinct life history strategies such as differences in invasiveness. In earlier work, genotypes and constituent clonal ramets were identified using microsatellite markers. In this pilot study, we screened the same ramets for epigenetic variation with Methylation-Sensitive AFLPs (MS-AFLPs), a modified type of AFLP dependent on differentially methylation-sensitive restriction enzymes. We found a significant difference in epigenetic signatures between introduced and native subspecies, and found that introduced P. australis demonstrated more epigenetic variation than their native counterparts. In both subspecies we observed moderate variation between genotypes relative to the higher degree of epigenetic variation found within genotypes (among ramets), suggesting that epigenotype may be more closely aligned with microhabitat than within-subspecies genotype. Finally, we observed potential epigenetic variation by site. This is the first study to investigate natural variation in DNA methylation patterns of P. australis and establishes the baseline in our understanding of the ecological relevance of epigenetics in this species.     

Genetic and epigenetic instabilities induced by tissue culture in wild barley (<Emphasis Type="Italic">Hordeum brevisubulatum</Emphasis> (Trin.) Link)

A simple tissue culture protocol was developed for efficient plant regeneration from young inflorescence-derived calli in wild barley, Hordeum brevisubulatum (Trin.) Link, an important pasturage grass. Genetic and epigenetic instabilities in the regenerated plants (regenerants) were assessed by three molecular markers AFLP, S-SAP and MSAP. Two pools of calli derived from young inflorescences of a single donor plant and 44 randomly chosen regenerants were subjected to AFLP analysis. Results showed that 74 out of 793 scored bands were polymorphic among the studied samples, giving rise to a genetic variation frequency of 9.3%. The number of variant bands as compared to the donor plant varied greatly among the regenerants, with a small number of regenerants accumulated a large number of variant bands (maximum 55), while the majority of regenerants showed only 2–3 variant bands. A subset of regenerants together with the two pools of calli were selected for S-SAP and MSAP analysis to detect possible retrotranspositional activity of a prominent retroelement family, BARE-1, in the genomes of Hordem species, and possible alterations in cytosine methylation. S-SAP analysis showed that of the 768 scored bands, 151 were polymorphic among the analyzed samples, giving rise to a genetic variation frequency of 19.7%, albeit no evidence for retrotranspositional event was obtained based on locus-specific PCR amplifications. MSAP analysis revealed that tissue culture has caused cytosine methylation alterations in both level and pattern compared with the donor plant. Sequencing of selected variant bands indicated that both protein-coding genes and transposon/retrotransposons were underlying the genetic and epigenetic variations. Correlation analysis of the genetic and epigenetic instabilities indicated that there existed a significant correlation between MSAP and S-SAP (r = 0.8118, 1,000 permutations, P < 0.05), whereas the correlation between MSAP and AFLP (r = 0.1048) is not statistically significant. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Xiaoling Li and Xiaoming Yu contributed equally to this work.     

玉米相对饱和遗传连锁图谱构建与一种新的AFLPs共显性分析方法探讨

利用一个F2作图群体(X178×B73),首先构建了一个含有130个SSRs的玉米连锁框架图,然后用119个AFLPs位点增加图谱密度,得到一个全长1659·3cM,标记间平均间距6·66cM的玉米相对饱和连锁图。同时,对SSRs和AFLPs的一些遗传特性进行了分析,探讨了AFLP标记进行共显性分析的一种新方法。分析表明SSRs和AFLPs分子标记具有多态性和可靠性高等特点,是构建高密度分子标记遗传连锁图的有效技术。加密的玉米遗传连锁图谱为比较基因组研究、数量性状位点(quantitativetraitloci,QTLs)克隆、杂种优势机理研究以及标记辅助选择等提供了技术基础。