MSAP Marker Based Epigenetic Regulation in Symptomatic and Asymptomatic Floral Malady of Soybean (Glycine max)

Abstract: It has learned that soybean is being affected by a floral disorder known as floral malady where plants fail to develop pod and do notattendfull maturity. For this floral disorder, we present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics of non-methylation, hyper-methylation, hemi-methylation, and full methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied a technique to analyze alterations in the cytosine methylation a popular Indian soybean (Glycine max L.) genotype, JS-335.The result revealed that in the symptomatic plant, out of 392 MSAP sites, 281 (71.68%), 33 (8.41%),38 (9.69%), and 40(10.20%) found to beun-methylated, hemi-methylated, fully methylated and hyper-methylated, respectively. Whereas, the MSAP profile of asymptomatic plants revealedout of 402MSAP sites, 330 (81.28%) was un-methylated, 22(5.41%) hemi-methylated,29(7.14%) fully methylatedand 25 (6.15%) hypermethylated. In comparison with asymptomatic(18.71%) plant, approximately 10% increased methylation was noted in symptomatic(28.31%) plantprofiles. The increased levels of methylation was recorded in the symptomatic plants about 28.31%and18.71% in asymptomatic. The study showed a higher epigenetic influence on JS-335 genotype of floral malady symptomatic than same genotype of asymptomatic plant. No pod formation in symptomatic plant induce genome wide changes either in promoter or coding region of gene(s) and DNA fragments showing polymorphism related to differences in pattern and extent of methylation associated with floral malady.

MSAP analysis of DNA methylation between pogoston-type and patchouliol-type of Pogostemon cablin / 中草药

Objective: To analyze the DNA methylations between two chemical types of Pogostemon cablin using MSAP technique. Methods: Methylation sensitive amplification polymorphism (MSAP) technique was used to detect the DNA methylation degree of two chemical type and a total of 197 P. cablin samples. Results: The highest or higher values of the Shannon polymorphism index of the four types of MSAP locus information were produced in the five areas of Yangchun, Deqing, Gaozhou Datong, Lubu, and Guangning. It formed two branches with P. cablin Shipaiensis (Pogostone-type) and the rest (Patchouliol-type). The percentage of variation among the populations was 60.66%, which was far greater than that within the population. A total of 10 differential fragments were screened out, sequenced and analyzed, one of those belongs to the serine/threonine protein kinase family. Conclusion: The MSAP technique can be used to identify the different origins of P. cablin. The formation of different chemical types of P. cablinis closely related to its DNA methylation level, and further research is needed.

Analysis of genomic DNA methylation level in foxtail millet by Methylation Sensitive Amplified Polymorphism / 生物工程学报

DNA methylation is an important type of epigenetic modification in eukaryotes. In order to research genome-wide methylation levels and patterns in foxtail millet (Setaria italica), the Methylation Sensitive Amplified Polymorphism (MSAP) analysis (employing double digestion with EcoR I and Hpa II/Msp I) was established and applied in two foxtail millet cultivars (Chaogu 58 and Yugu 1). The results showed that 32 pairs of MSAP primers were selected from 100 MSAP primers, and 1 615 and 1 482 clearly distinguishable and reproducible bands were amplified from Chaogu 58 and Yugu 1 respectively, including 3 types of methylation patterns. Cytosine methylation levels of CCGG context in Chaogu 58 and Yugu 1 were characterized as 6.93% and 8.77% respectively. Such different genomic DNA methylation levels between two foxtail millet varieties may provide a preliminary reference for the cultivation of this crop from a novel epigenetic viewpoint.

Comparison of the phenolic contents and epigenetic and genetic variability of wild and cultivated watercress (Rorippa nasturtium var. aquaticum L.)

Background: Epigenetic modifications are key factors modulating the expression of genes involved in the synthesis of phytochemicals. The knowledge of plant epigenetic and genetic variations can contribute to enhance the production of bioactive compounds. These issues have been little explored thus far in Rorippa nasturtium var. aquaticum L. (watercress), an edible and medicinal plant. The aim of the current study was to determine and compare the phenolic composition and epigenetic and genetic variations between wild and cultivated watercress. Results: Significant differences were found in the quantitative phenolic composition between wild and cultivated watercress. The eight primer combinations used in the methylation-sensitive amplification polymorphism (MSAP) method revealed different epigenetic status for each watercress type, the cultivated one being the most epigenetically variable. The genetic variability revealed by the EcoRI/MspI amplification profile and also by eight inter-simple sequence repeat (ISSR) primers was different between the two types of watercress. The results of the Mantel test showed that the correlation between genetic and epigenetic variations has diminished in the cultivated type. Cluster analyses showed that the epigenetic and genetic characterizations clearly discriminated between wild and cultivated watercress. Conclusions: Relevant chemical, epigenetic, and genetic differences have emerged between wild and cultivated watercress. These differences can contribute to fingerprint and develop quality control tools for the integral and safety use and the commercialization of watercress. The richness of epialleles could support the development of tools to manipulate the watercress epigenome to develop high bioproduct­producing cultivars

In vitro autotetraploid induction and analysis on DNA methylation diversity of Platycodon grandiflorum / 中国中药杂志

In order to investigate the epigenetic variations between diploid and autotetraploid of Platycodon grandiflorus. The diploid buds of P. grandiflorus were soaked in the mixture of different concentration colchicines and 0.002 g•mL ⁻¹ dimethyl sulphoxide (DMSO)．The identification of autotetraploid plants were based on morphological characteristics, chromosome number and flow cytometry. And then the level and pattern of DNA methylation explored by using the technology of methylation sensitive amplified polymorphism (MSAP)．The result demonstrated that the buds soaked in 0.2% colchicines and 0.002 g•mL ⁻¹ DMSO solution for 12 h was ideal conditions to induce autotetraploid of P. grandiflorus, with induction rate of 32.0%.The diploid and tetraploid plants existed distinctly differences in morphological indexes.Totally,1 586 bands were amplified by 20 pairs of selective primers, of which 764 and 822 bands were detected in diploid and autotetraploid respectively. The total methylation ratio,full methylation ratio and hemimethylated ratio were 91.25%,61.25% and 30.65% in diploid of P. grandiflorus,respectively.However,the total methylation ratio,full methylation ratio and hemimethylated ratio of autotetraploid of P. grandiflorus were 86.13%,54.38% and 31.75%, respectively. Compared with diploid, the genomic DNA total methylate ratio and full methylation ratio of autotetration plants decreased by 6.02% and 7.14%.But the hemimethylated ratio of autotetraploid was higher than that of diploid, which more than 1.6%. All this results indicated that DNA methylation patterns have adjusted during the polyploidy process．.

Effect of nickel chloride on Arabidopsis genomic DNA and methylation of 18S rDNA

Background In recent years, nickel (Ni) has been widely applied in industrial and agricultural production and has become a kind of environmental pollution. In this study, the effect of nickel chloride (NiCl2) with different concentrations on Arabidopsis genomic stability and DNA methylation has been demonstrated. The nucleolus variation and 18S rDNA methylation after NiCl2 treatment have been analyzed. Results The results are as follows: (1) The NiCl2 could result in heritable genomic methylation variations. The genomic DNA methylation variations have been detected by methylation-sensitive amplified polymorphism (MSAP) molecular markers, and the result showed that after NiCl2 treatment, there was methylation variation in T0 generation seedlings, and partial site changes maintained in T1 generation, which suggested that the effects of NiCl2 on DNA methylation could be heritable in offspring. (2) NiCl2 brought deformity and damage to nucleolar structure in Arabidopsis root tip cells, and the damage was positively correlated with the NiCl2 concentration. 3. In the nucleolus, there was an increased cytosine methylation in 18S rDNA. The plant nucleolus variation and 18S rDNA methylation may be used as an examination indicator for Ni pollution in soil or plant. Conclusions NiCl2 application caused variation of DNA methylation of the Arabidopsis genomic and offspring's. NiCl2 also resulted in nucleolar injury and deformity of root tip cells. The methylation rate of 18S rDNA also changed by adding NiCl2.

Optimization of MSAP Reaction System forCyathula officinalisKuan and Primers Screening / 世界科学技术-中医药现代化

This study was aimed to establish and optimize the reaction system of MSAP for analysis on DNA methylation inCyathula officinalis Kuan of Sichuanmedicinal. The tender leaves ofC. officinalisKuan were used as materials. Orthogonal method was used in the study of main factors which affected the system quality of MSAP. The results showed that the optimal reaction systems of MSAP included: enzyme digestion (20μL), 300 ng DNA, 1.5μL EcoRI, 1.5μL HpaII or 1.5μL MspI (HpaII); ligation (25μL): 15μL digestion products, 1μL EcoR I adaptor, 1μL Hpa II/Msp I adaptor, 0.2μL T4 ligase; pre-amplication mixture (25μL): ligation products 1μL, rTaq polymerase 1 U, each primer 1.5μL, dNTP 2μL; selective amplification mixture (25μL): pre-amplification product was diluted 50 times, rTaq polymerase 1 U, each primer 1.5μL, dNTP 3μL. The optimal MSAP reaction system was used to screen for 6 pairs of effective primers from 256 pairs of primers of MSAP. It was concluded that the optimized system ensured the stable and clear bands and the screened 6 pairs of primers were with good specificity. It provided useful references for further studies of epigenetic onC. officinalisKuan DNA methylation and MSAP analysis for other medicinal plants.

Analysis of genetic and epigenetic variation in in vitro propagated potato somatic hybrid by AFLP and MSAP marker

Background: Genetic and epigenetic changes (DNA methylation) were examined in the tissue-culture propagated interspecific potato somatic hybrids between dihaploid Solanum tuberosum and S. pinnatisectum. Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) were applied to detect the genetic and epigenetic changes, respectively in the somatic hybrids mother plants (1st cycle) and their regenerants (30th cycles sub-cultured). Results: To detect genetic changes, eight AFLP primer combinations yielded a total of 329 scorable bands of which 49 bands were polymorphic in both mother plants and regenerants. None of the scorable bands were observed in term of loss of original band of mother plant or gain of novel band in their regenerants. AFLP profiles and their cluster analysis based on the Jaccard’s similarity coefficient revealed 100% genetic similarity among the mother plant and their regenerants. On the other hand, to analyze epigenetic changes, eight MSAP primer pair combinations detected a few DNA methylation patterns in the mother plants (0 to 3.4%) and their regenerants (3.2 to 8.5%). Out of total 2320 MSAP sites in the mother plants, 2287 (98.6%) unmethylated, 21 (0.9%) fully methylated and 12 (0.5%) hemi-methylated, and out of total 2494 MSAP sites in their regenerants, 2357 (94.5%) unmethylated, 79 (3.1%) fully methylated and 58 (2.3%) hemi-methylated sites were amplified. Conclusion: The study concluded that no genetic variations were observed among the somatic hybrids mother plants and their regenerants by eight AFLP markers. However, minimum epigenetic variations among the samples were detected ranged from 0 to 3.4% (mother plants) and 3.2 to 8.5% (regenerants) during the tissue culture process.

Reconstruction of the skin and soft defects of hand and foot by free medial sural artery perforator flap / 中华显微外科杂志

ObjectiveTo evaluate the curative effect of reconstruction of the hand and foot defects with bones and tendons exposure using free medial sural artery perforator flap(MSAP). MethodsRadiographs of 2 cadavers injected with a modified lead oxide-gelatin mixture were digitally analyzed. Between April 2007 and December 2010, thirty-four patients with soft tissue defects in the distal limb were treated with the free MSAP flap transplantation. The sizes of the defect ranged 6 cm × 4 cm-13 cm × 8 cm, and the flaps ranged 7 cm× 5 cm-14 cm × 9 cm. These clinical cases included 25 hands and 9 feet, all of them with bones and tendons exposure.In these defects,twenty-two were clean,twelve got infections.In our cases, twenty-three flaps were nourished with single perforator vessel and else 11 with two;perforator vessel fifteen flaps were dissected one superficial vein to anastomose with that of the recipient sites in addition to accompanying vein anastomosis;The sensation of 9 flaps recovered the hands were reconstructed with cutaneous nerve anastomosis. ResultsA partition of the calf skin blood vessels,and three-dimensional reconstruction image of the sural artery were obtained.All flaps survived,five of them appeared partially violet and bubbles. Followed up 6-21 months, the cosmetic results were satisfactory and without apparent bulkiness.The flap colors were similar to recipient sites. The flap senses reconstructed with neural anastomosis recover to S2-S3. ConclusionThe new flap is very suitable to repair the soft tissue defect in the distal limbs,because the fairly constant perforator vessel,the reliable blood supply and the cosmetic shape of the MSAP flap are all advantages of it in addition to no damage to low leg chief artery and gastrocnemius.

DNA Methylation of polyploid complex of Pinellia ternata by MSAP analysis / 中草药

Objective To analyze the DNA methylation status of polyploid complex of Pinellia ternata. Methods Methylation sensitive amplified polymorphism(MSAP) technique was carried out to analyze the methylation status of polyploid complex of P.ternata. Thirty-four selective primer amplifications were used to check the status of cytosine methylation DNA samples. Results A total of 7 708 bands were obtained.Among them 5 636 bands,each representing a recognition site cleaved by one or both of the isoschizomers(Hpa Ⅱ and Msp Ⅰ),were amplified.Furthermore,methylation patterns varied among the four polyploids:heptaploid,octoploid,nonuploid,and decaploid.Total and full methylation levels in P.ternata were 54%-58% and 24.1%-24.3%.All types of MSAP patterns detected in the study belonged to two classes,type Ⅰ and Ⅱ. 52.5% of detected bands belonged to Type Ⅰ; Another 47.5% were type Ⅱ,which showed the methylation differences among the four polyploids. Conclusion The results demonstrate DNA methylation events occur in P.ternata and the general methylation levels are higher.