Optimization of SSR-PCR Reaction System and Screening of Polymorphic Primers for RIL Parents

[Objective] This study aimed to establish a high-efficiency and stable SSR amplification system and screen polymorphic primers in order to further compare the tri-group probability analysis method and traditional QTL mapping method. [Method] In this study, we preliminarily screened the 605 pairs of primers evenly distributed on the 12 chromosomes through investigating their polymorphism performance in amplification, and established an optimized SSR-PCR reaction system. [Result] A 10μL SSR-PCR reaction system suitable for rice was set up as fol ows： 2 μl of 10 × Buffer, 2.0 mmol/L Mg2＋ （final concentration）, 0.5 mmol/L dNTPs, 1.0 μmol/L primers （final concentration）, 1 μl of DNA template, 0.15 U Taq DNA polymerase. Among the SSR primers distributed over the genome, 142 pairs that were polymorphic upon the parents were screened. [Conclusion] This study lays a good foundation for sub-sequent QTL mapping studies.

Screening of Primers for DNA Barcoding of Sedum Plants

[ Objective] This study aimed to screen primers for DNA barcoding of Sedum plants. [ Method] Sedum lineare Thunb., S. spectabile Boreau, S. ta- tarinowii Maxim, S. aizoon L. and S. sarmentosum Bunge were used as experimental materials. Nuclear DNA and chloroplast DNA were extracted from these five Sedum plants with CTAB method for primer screening by PCR amplification. PCR products were detected by agarose gel electrophoresis. [ Result] Four sequences suitable for DNA barcoding of Sedum plants were preliminarily screened from six candidate sequences （psbA-trnH, ropB, rbcL, rpoC1, ITS 2, marK）, including psbA-trnH, ropB, rpoC1 and ITS 2. The amplification rate of these four sequences reached 100%. [ Conclusion] This study provided basis for DNA barcoding of Sedum plants.

DNA Extraction and ISSR Primer Screening of Camellia chekiangoleosa

[Objective] The aim was to screen out simple methods of DNA extraction and effective ISSR primers suitable to all germplasm materials of Camellia chekiangoleosa.[Method] The modified CTAB method was used in the extraction of the genomic DNA,50 ISSR primers from other Camellia plants were used in the ISSR-PCR amplification for 20 germplasm materials from 10 populations in Fujian,Zhejiang,Jiangxi and Anhui Province.[Result] Pure DNA could be obtained rapidly by using the improved CTAB method,and the 20 selected effective primers had rich polymorphism,clear bands and good repeatability.337 DNA bands were obtained,of which 281 bands were polymorphic,accounting for 83.4% of the total amplified bands.And 16.85 bands could be amplified with a primer,averagely.[Conclusion] The selected 20 primers could be effectively applied to ISSR analysis of the germplasm resources of C.chekiangoleosa in Zhejiang.

Primer Screening on Germplasm Resources of Mallotus oblongiolus by ISSR Molecular Marker

[ Objective] To provide effective primers for the rapid and accurate ISSR analysis of the germplasm materials of Mallotus oblongiolus （Miq.） Muello-Arg.. [Method] The modified CTAB method was used in the extraction of the genomic DNA. 99 ISSR primers were used in the ISSR-PCR amplification for 20 germplasm materials from 10 populations in Hainan Island, so that some primers, which were suitable to all gerplasm materials of M. oblongiolu, could be selected. [ Result] 15 effective primers with characteristics of rich polymorphism, clear bands, and good repeatability were selected from 99 test primers. The 15 primers selected were used in the ISSR-PCR amplification for 66 germplasm materials of M. oblongiolus. From all of which the abundant and distinct DNA fingerprintings could be obtained. 286 DNA bands were obtained, and of which 231 bands were polymorphic, which amounted to 80.77% of the total bands amplified. And 19.1 bands could be obtained with each primer, averagely. [ Conclusion] The 15 primers selected could be effectively applied to ISSR analysis of the germplasm resources of M. oblongiolus.

Sequence-related amplified polymorphism primer screening on Chinese fir(Cunninghamia lanceolata(Lamb.) Hook)

Chinese fir（Cunninghamia lanceolata（Lamb.）Hook） is one of the most important coniferous tree species used for timber production in China. Here, we conducted a sequence-related amplified polymorphism（SRAP） primer screening assay with a total of 594 primer combinations,using 22 forward and 27 reverse primers on four representative Chinese fir genotypes. The obtained results indicated that Chinese fir genomic DNA has a notable amplification bias on the employed forward or reverse primer nucleotides（30selection bases）. Out of the tested primer sets, 35 primer combinations with clearly distinguished bands, stable amplification, and rich polymorphism were selected and identified as optimal primer sets. These optimal primer pairs gave a total of 379 scorable bands,including 265 polymorphic bands, with an average of 10.8bands and 7.6 polymorphic bands per primer combination.The produced band number for each optimal primer set ranged from 7 to 14 with a percentage of polymorphic bands spanning from 33.3 to 100.0 %. These primer combinations could facilitate the next SRAP analysis assays in Chinese fir.

Evaluation of SRAP markers efficiency in genetic diversity of Aspergillus flavus from peanut-cropped soils in China

In order to evaluation the efficiency of SRAP markers on genetic diversity of Aspergillus flavus,we screened out 17sets of primer pairs which could produce clear and reproducible SRAP bands from 150 SRAP primer pairs.The size of SRAP fragments ranged from 120 to 2100 bp.Primer pair Me10/Em9 produced the maximum number of polymorphic bands(12 bands),while Me8/Em13 produced the fewest number of polymorphic bands(only 1).Through analysis genetic diversity ability of different sets of primer pairs,the set of 12 primer pairs was selected for SRAP genetic marker of A.flavus.Cluster analysis was performed based on the genetic similarity coefficients,which ranged from 0.53 to 0.89.A dendrogram assembled using the unweighted pair-group method with arithmetic averages grouped A.flavus samples into 5 main clusters.The results suggested that SRAP marker is a useful molecular technology for the diversity of A.flavus from peanut soils in China.