Identification of Genetic Purity of Bitter Gourd Hybrid by ISSR Markers

Objective] This study was conducted to verify the feasibiIity of ISSR marker for identifying genetic purity of bitter gourd hybrid, and thus to provide an effective method for seed purity test in production practices. [Method] The DNA fin-gerprints of a bitter gourd cuItivar Xiuyu 1 and its parents were analyzed using IS-SR marker with 91 primers. [Result] Two primers ISSR-845 and ISSR-891 which ampIified two DNA bands of 510 and 300 bp respectiveIy from F1 generation and its parents were screened out from 91 primers. ISSR-845 couId distinguish the male parent from F1 hybrid and the female parent, whiIe ISSR-891 couId distinguish the female parent inbred Iine from Xiuyu 1. Seed purity test with the specific markers gave the same resuIt with fiIed trials based on morphoIogical identification. [Conclu-sion] ISSR marker is an accurate, simpIe and effective method for seed purity test bitter gourd hybrid, and thus can be used in production practices.

The Application of ISSR Markers to Identify the Fertility Restorer Gene Rf6 in T. timopheevii Cytoplasmic MaleSterile Wheat(Triticum aestivum)

Inter-simple sequence repeat (ISSR) analysis was carried out on a F2 population of 147 plants derived from a cross between a wheat male fertility restorer line 2114 and a male sterile line ND44A. Out of 43 primers examined, 18 primers produced distinguishable, polymorphic bands between the two parents. Linkage analysis in the mapping population showed that two markers UBC-808 and UBC-848 were closely linked with the restorer gene Rf6 of the Triticum timopheevii CMS system. The distance between the two markers and the restorer gene was 7.9 cM and 4.9 cM, respectively. Also two parents were screened with 181 pairs of SSR primers, of which, 34.3% showed polymorphisms. But no locus was found linked with the restorer gene. Compared with the SSR technique, the ISSR approach used in the experiment provided more information and proved to be a valuable method to identify alien fragments.

Genetic variation of the genus Kengyilia by ISSR markers

We investigated the genetic variation within 32 accessions distributed to 14 species and one variety by using ISSR(inter-simple sequence repeat)markers.The results showed that genetic variation was relatively higher among the accessions.A total of 593 bands were amplified by 12 ISSR primers,of which 535 bands(90.2%)were polymorphic.Eleven to 80 polymorphic bands were amplified from each prime,with an average of 44.6 bands.The interspecies GS(genetic similarity)value ranged from 0.430 to 0.866,and the average was 0.620.Cluster analysis showed that all accessions could be classified into 4 groups by ISSR markers.The different accessions in a species were clustered together,but they had genetic variation in molecular levels.There was obvious interspecies genetic variation.Species with similar morphological characteristics and from the same areas or neighboring geographical regions were clustered together and had close relationships.ISSR markers are useful in analyzing interspecies variation in Kengyilia.

Morphological and ISSR molecular markers reveal genetic diversity of wild hawthorns (Crataegus songorica K. Koch.) in Xinjiang, China

The wild hawthorn species, Crataegus songorica K. Koch., is an important wild germplasm resource in Xinjiang, China that has been endangered in recent years. The genetic diversity of C. songorica K. Koch. germplasm in five populations from Daxigou, Xinjiang, China were evaluated based on phenotypic traits and ISSR molecular markers to provide basic infor- mation on resource protection, rational utilization and genetic improvement. The F-value for the phenotypic differentiation coefficient of the 33 traits measured ranged from 0.266 to 15.128, and mean value was 13.85%. The variation among populations was found to be lower than that within population. A total of 303 loci were detected within the five populations by 12 primers. Within 298,polymorphic loci, the polymorphism was 98.35%, showing a high genetic diversity in C. songorica K. Koch. The gene diversity within population, total population genetic diversity, genetic differentiation coefficient and gene flow were 0.2779, 0.3235, 0.1408, and 3.0511, respectively. Our results showed that C. songorica K. Koch. from Xinjiang has a high level of genetic diversity at both the phenotypic and molecular levels. Significant genetic differentiation existed within population and the differentiation trend showed a regional association. And in this study, in situ and ex situ conser- vation approaches were raised for wild hawthorn protection utilization.

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.

Establishment and Optimization of ISSR-PCR Reaction System for Cymbidium ensifolium (Linn.) Sw

[Objective] This research aimed to search a best method for extracting the genomic DNA of Cymbidium ensifolium and establish the optimized ISSR-PCR reaction system.[Method] Genomic DNA was extracted from C.ensifolium leaves by modified CTAB method.ISSR-PCR reaction system for C.ensifolium was optimized.[Result] High-quality genomic DNA was obtained from C.ensifolium.The 25 μl optimized ISSR-PCR reaction system for C.ensifolium contained 2.5 μl 10× PCR buffer,2.5 mmol/L MgCl2,240 ng template DNA,160 μmol/L dNTPs,1.25 U Taq DNA polymerase,0.4 μmol/L primer and 15.78 μl ddH2O.The optimal PCR procedures were：94 ℃ pre-denaturation for 5 min and then 40 cycles,94 ℃ denaturation for 30 s,50-60 ℃ annealing for 30 s （annealing temperature according to different primers）,72 ℃ extension for 50 s and a 72 ℃ extension for 7 min.[Conclusion] An optimized ISSR-PCR reaction system for C.ensifolium was established,which provides a basis for further study on genetic diversity of C.ensifolium by using ISSR molecular marker technique.

ISSR Marker and ITS Sequence Study of Melampsora Larici-populina

To compare the differences in intertranslation space of ribosomal DNA （ITS） of Melampsora larici-populina, between the isolates from China and isolates from other countries, this study investigated ITS sequences and ITS polygenetic tree based on 11 isolates that were collected from 5 races in different parts of China. The results indicated that there was no difference among the ITS sequences of 11 isolates from China. The ITS sequence of isolates from China was more homogeneous with that of isolates from Britain compared with France, Germany, and Canada. Intersimple sequence repeat （ISSR） markers were also used to study the genetic division of Melampsora larici-populina, and the results showed that the 11 tested isolates could be divided into Western population and Northern population. Genetic diversity index of race C2 was significantly different from that of races C4, C3, and C1, and no significant differences were observed among the other races. Pathogenicity division of races must not harmonize with their genetic division, except race C2. The ITS region is conservative, and ITS sequence is not fit for studying the differences that existed among the races. ISSR marker can be used for intraspecies population study, and Melampsora larici-populina in China can be divided into two populations.

Development of a stable SCAR marker for rapid identification of Ganoderma lucidum Hunong 5 cultivar using DNA pooling method and inter-simple sequence repeat markers

The cultivar Ganoderma lucidum Hunong 5 was obtained using cross-breeding. Hunong 5 has high commercial value due to its high polysaccharide and triterpene content, This is the first report of using a DNA pooling method to develop a stable sequence characterized amplified region （SCAR） marker for rapid identification of the G. lucidum Hunong 5 cultivar. The SCAR marker was developed by first generating and sequencing a distinctive inter simple sequence repeat （ISSR） fragment （882 bp） from G. lucidum Hunong 5 cultivar. A stable SCAR primer pair GLH5F/GLH5R were obtained to identify the cultivar and the SCAR marker is a DNA fragment of 773 bp.

Study on Genetic Diversity of Turf Bamboo Based on ISSR Marker

[ Objective ] This study aimed to analyze the genetic diversity of turf bamboo species by using ISSR molecular marker technology. [ Method] Excellent turf bamboo species imported from France and domestic ornamental turf bamboo species were used as experimental materials for ISSR analysis, cluster analysis was conducted on 10 species of turf bamboo materials based on the obtained ISSR molectfiar ma^kers. [ Result] A total of 201 clear bands with good repeatability and high polymorphism were amplified with 21 ISSR primers, with a polymorphism rate of 93.1% ; similarity coefficients between different turf bamboo species ranged from 0.275 to 0.571, with an average similarity coefficient of 0. 357 ; according to the results of ISSR markers, 10 different ornamental turf bamboo species were di- vided into three categories by using UPGMA cluster analysis method. [Conclusion] Turf bamboo with different sources had relatively high genetic diversity, this study had provided theoretical and technical basis for the breeding, cultivation and vromotion of ornamental turf bamboo.

Low genetic diversity and high genetic differentiation in the critically endangered Omphalogramma souliei (Primulaceae):implications for its conservation

Omphalogramma souliei Franch. is an endangered perennial herb only distributed in alpine areas of SW China. ISSR markers were applied to determine the genetic variation and genetic structure of 60 individuals of three populations of O. souliei in NW Yunnan, China. The genetic diversity at the species level is low with P=42.5% （percentage of polymorphic bands） and Hsp=0.1762 （total genetic diversity）. However, a high level of genetic differentiation among populations was detected based on different measures （Nei＇s genetic diversity analysis： Gst=0.6038; AMOVA analysis： Fst=0.6797）. Low level of genetic diversity within populations and significant genetic differentiation among populations might be due to the mixed mating system in which xenogamy predominated and autogamy played an assistant role in O. souliei. The genetic drift due to small population size and limited current gene flow also resulted in significant genetic differentiation. The assessment of genetic variation and differentiation of the endangered species provides important information for conservation on a genetic basis. Conservation strategies for this rare endemic species are proposed.

Diversity among Isolates of the Tangerine Pathotype of Alternaria alternata

The tangerine pathotype of Alternaria alternata is the aetiological agent of Alternaria brown spot on tangerines. In the state of Paraíba, Brazil, its occurrence on “Dancy” tangerine trees is associated with genetic aspects as well as the influence of environmental conditions on reproduction and dissemination within and between populations. The aim of the present study was to evaluate the diversity of isolates of this pathogen using morphophysiological and molecular markers. For the analysis of mycelial growth and sporulation, 30 isolates from different locations were examined at 24-hour intervals until the seventh day, when the spores were quantified. The 30 isolates were characterised based on molecular markers (ISSR) and genetic similarity (Jaccard index). A factor arrangement was used: 30 isolates, four media (ODA, PDA, LEA and V8), three light regimes (continuous dark, alternating light and continuous light) and three temperatures (15°C, 25°C and 35°C), with 12 repetitions. Groups 1, 2 and 3 presented low genetic variability. Group 4 showed high genetic variability of the isolates obtained from the Massaranduba (state of Paraíba-Brazil) producing region and higher mycelial growth and sporulation of A. alternata. The continuous light regime and the temperature 25°C in PDA and V8 media were the ideal conditions for the mycelial growth and sporulation, respectively, of the isolates of A. alternata.

Genetic relationship between parasitized and non-parasitized Haloxylon ammodendron in the Alxa Desert

Haloxylon ammodendron （C. A. Mey.） Bunge is a host for the holoparasitic plant Cistanche deserticola Y. C. Ma, the original source of medicinal material known as Herba Cistanchis. The inter-simple sequence repeat marker was used to assess the genetic variations and relationships among six accessions ofH. ammodendron with a total of 120 individuals collected from three localities in the Alxa Desert, Inner Mongolia, China. At each locality, individuals both parasitized （PP） by C. deserticola and non-parasitized （NP） were sampled. The results showed that Nei＇s gene diversity and Shannon＇s index of PP accessions were higher, but were not significantly different, from those of NP accessions. An unweighted pair-group method arithmetic average dendrogram showed two clusters, one that included all PP accessions, and the other the NP accessions. Genetic differentiation therefore existed between PP and NP accessions, which might be attributed to low gene flow between the NP and PP groups （Nm〈 1）. However, the relationship between genetic distance and geographic distance within each group, although not statistically significant in this study, might be associated with high gene flow in both the NP and PP groups.

Taxonomic Status of Daduhe Loquat （Eriobotrya prinoides var. dadunensis）

The taxonomic status of Daduhe loquat （E. prinoides var. dadunensis） was studied through analyzing genetic relationships among Oakleaf loquat （E. prinoides）, Daduhe loquat and Common loquat （E. japonica） using inter simple sequence repeats （ISSR） molecular marker and morphologic marker in this paper. Based on ISSR marker research, the similarity coefficient between Oakleaf loquat and Common loquat was lower than the similarity coefficient between Oakleaf loquat and Daduhe loquat while the similarity coefficient between Daduhe loquat and Common loquat was intermediate. The highest additivity was obtained when Daduhe loquat was regarded as the undetermined hybrid （45.8%）. The specific bands of Oakleaf loquat and Common loquat were present in Daduhe loquat. Based on morphologic traits research, Daduhe loquat was also between Oakleaf loquat and Common loquat but a little leaning to Oakleaf loquat. All the results support that Daduhe loquat was hybrid of Oakleaf loquat and Common loquat.