Genetic Mapping of Laminaria japonica and L. Iongissima Using Amplified Fragment Length Polymorphism Markers in a ＂Two-Way Pseudo- Testcross＂ Strategy

With a ＂two-way pseudo-testcross＂ mapping strategy, we applied the amplified fragment length polymorphism （AFLP） markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F1 cross family （Laminaria iongissima Aresch. × L. Japonica Miyabe） with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1：1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3：1 Mendelian segregation ratio. Among the loci with 1：1 segregating ratios, 79 loci were ordered in 14 linkage groups （648.6 cM） of the paternal map, and 72 loci were ordered in 14 linkage groups （601.9 cM） of the maternal map. The average density of loci was approximately 1 per 8 cM. To Investigate the homologies between two parental maps, we used 58 loci segregated 3：1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.

Development of Sequence Characterized Amplified Region (SCAR) Primers for the Detection of Resistance to Sporisorium reiliana in Maize

Head smut of maize （Zea mays L.）, which was caused by Sporisorium reiliana, occurred in most of the maize growing areas of the world. The purpose of this study was to develop SCAR markers for map-based cloning of resistance genes and MAS. Two sets of BC3 progenies, one （BC3Q） derived from the cross Qi319 （resistance）×Huangzao 4 （susceptible）, the other （BC3M） from Mol7 （resistance）× Huangzao 4 （susceptible）, were generated. Huangzao 4 was the recurrent parent in both progenies. A combination of BSA （bulked segregant analysis） with AFLP （amplified fragment length polymorphism） method was applied to map the genes involving the resistance to S. reiliana, and corresponding resistant and susceptible bulks and their parental lines were used for screening polymorphic AFLP primer pairs. One fragment of PI3M61-152 was converted into SCAR （sequence charactered amplified fragment） marker S130. The marker was mapped at chromosome bin 2.09, the interval of a major QTL region previously reported to contribute to S. reiliana resistance. Furthermore, S130 was highly and facilitate map-based cloni associated with resistance to S. reiliana, and could be useful for marker-assisted selection ng of resistance genes.

Isolation and characterization of the AGAMOUS homologous gene NTAG in Chinese narcissus (Narcissus tazetta var. chinensis Roem)

Amaryllidaceae, a monocot plant family, consists of many important ornamental bulb flower species. Chinese narcissus （Narcissus tazetta var. chinensis Roem）, its flowers developed at high temperatures and bloomed at lower temperatures during the Chinese Spring Festival, is a traditional Chinese flower with high economic and ornamental value. To study its flower development, a full length cDNA containing MADS box domain from narcissus was isolated by a reverse transcription polymerase chain reaction （RT-PCR） with degenerate oligo-nucleotide primers derived from a conserved MADS- and K-box domain sequence. The 5＇ and the 3＇ regions of the gene were amplified using the PCR protocol for the rapid amplification of cDNA ends （RACE）. The 690 bp open reading frame encodes 230 amino acid residues. A comparison of the deduced amino acid sequence of NTAG with the sequence of other MADS box proteins showed 91.3% amino acid identities with HAG （Hyacinthus orientalis）. Sequence analysis and alignment showed significant similarity with other AG homologues. RNA blot analysis indicated that the narcissus NTAG gene was expressed only in reproductive organs, especially in stamens and carpels. These results indicated that the NTAG gene was an AG homologue and that the AG genes appeared to be structurally and functionally conserved between dicots and monocots.

Genetic Relationships among Prunus mume var. pendula Using AFLP Markers

Genetic relationships among Prunus mume var. pendula were studied by using AFLP markers. 18 accessions representing 14 cultivars of Prunus mume var. pendula were selected from the germplasm collection at the Research Center of China Mei Flower. Seven Mse I-EcoR I AFLP primer combinations revealed 450 legible bands, and 269 of which were polymorphic markers. A similarity matrix was prepared using the simple matching coefficient of similarity and Neis (72) distance coefficient. A UPGMA dendrogram demonstrated the genetic relationships of the cultivars. The information given by AFLP markers was basically consistent with the morphological classification and the evolutionary history of the morphotypes, and roughly supported the new revised classification system for Chinese Mei Cultivars. But there were still several exceptions: 1) the Guhong Chuizhi inserted between the Tiaoxue Chuizhi and the Danfen Chuizhi; 2) the Wufu Chuizhi kept off the Pink Pendant Form, and the Moshan Chuizhi was removed from Viridiflora Pendant Form; 3) the Danbi Chuizhi and the Shuangbi Chuizhi of Viridiflora Pendant Form got together well but fell within the Pink Pendant Form.

Impact of Crop Rotation on Pathotype and Genetic Structure of Phythophthora sojae in Fields

To estimate the impact of crop rotation on the pathotype and genetic structure of Phythophthora sojae in fields, 372 isolates of P. sojae were obtained from long-term localisation experimental fields in Heilongjiang Province of China. The hypocotyl inoculation method was used to characterize the virulence of P. sojae on 13 differential cultivars, and the amplified fragment length polymorphism（AFLP） technique was used to analyze difference in the genetic structure of P. sojae. The results indicated that an abundant diversity of genetic structures and pathotypes of P. sojae, a more uniform distribution of pathotypes and less dominance of pathotypes occurred in corn-soybean and wheat-soybean rotation fields than in a continuous soybean mono-cropping field. These findings suggested that P. sojae did not easily become the dominant race in rotation fields, which maintain disease resistance in soybean varieties. Therefore, the results of this study suggested that Phytophthora stem and root rot of soybeans could be effectively controlled by rotating soybeans with non-host crops of corn and wheat.

Molecular Study of the Genetic Variability of Pumpkins Landraces from Brazilian Amazon

The Cucurbita maxima Duchesne is a vegetable crop plant cultivated and maintained by traditional Amazon communities, Brazil. The situation is worsened by the possibility of disappearance of local populations and genetic variability of this specie, taking into account the today changes promoted in family farming. The aim of this study was to estimate the current levels of genetic variability of local cultivars through the use of molecular markers (Amplified Fragment Length Polymorphism—AFLP). We chose to collect in two distinct micro regions in order to identify possible influences of geographic isolation and different levels of market requirements in the conservation of the genetic variability of the C. maxima. For the molecular analysis, bulk samples of fresh leaves of 15 plants/half-sibling family were collected in paper bags. There were 34 samples from the half-sib families. The analysis of the results half-sib obtained by methods of estimation of genetic variation by molecular markers shows that the forms of cultivation and management adopted by family farmers maintain the identities of the local/landraces (native cultivars) and, at the same time, the levels of diversity for the assurance of adaptability macro-environmental.

Range-wide genetic diversity in natural populations of Larix principis-rupprechtii Mayr.

Prince Rupprecht’s larch(Larix principis-rupprechtii Mayr.),a deciduous conifer,widely grows in middle and high elevations of Northern China.Its natural distribution has sharply decreased and has become fragmented,which may have resulted in the loss of genetic variation.In this study,ten natural populations across the entire range of this species were analyzed using amplifi ed fragment length polymorphism markers.A total of 309 loci were detected from 225 individuals of these populations,of which 261(84.5%)were polymorphic.At the species level,the genetic diversity was high(average of the Nei’s genetic diversity H e=0.2602,and Shannon’s information index I=0.3967).The results of molecular variance analysis showed that 90.71%of the genetic diversity occurred within populations.The genetic diff erentiation among populations was moderate as a whole(F ST=0.0929,G ST=0.1510),which is consistent with the moderate level of gene fl ow among populations(N m=2.8116).Based on the unweighted pair group method with arithmetic mean and STRU CTU RE analysis,these populations were grouped into three genetically distinct clusters.The degree of inter-population diff erentiation(G ST=0.1338)for the south group was larger than that for the north group(G ST=0.0915).There was a signifi cant correlation between genetic distance and geographic distance across the species range(r=0.316,P<0.05).Genetic diversity was signifi-cantly associated with longitude but not elevation or climatic factors.The populations with high genetic diversity from each cluster are therefore recommended for future conservation and management of this species.

Loss of Genetic Diversity of Domesticated Panax notoginseng F H Chen as Evidenced by ITS Sequence and AFLP Polymorphism： A Comparative Study with P. stipuleanatus H T Tsai et K M Feng

In the present study, we evaluated the genetic diversity of Panax notoginseng F H Chen, a domesticated species, and P. stipuleanatus H T Tsai et K M Feng, an endangered wild species in southeastern Yunnan and adjacent areas in Vietnam, using sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA and amplified fragment length polymorphism (AFLP) markers. Twenty-four accessions from three plantations of P. notoginseng and 51 samples from eight populations of P. stipuleanatus were assayed. A total of 694 bp of partial sequences of 18S, ITS 1, 5.8S, ITS2, and partial sequences of 26S were obtained. No sequence variation was detected within P. notoginseng and nine sites (1.30%) were variable in P. stipuleanatus. Two-thirds of the variable sites were found between Langqiao and other populations. In P. notoginseng, four pairs of AFLP primer combinations generated 312 bands, of which 240 (76.9%) were polymorphic and 60.15% of the polymorphisms were harbored within plantations. Approximately 41.0% and 66.9% of bands were polymorphic in population D7 and 5589, respectively. In P.stipuleanatus, the same four primer combinations produced 346 bands, of which 334 (96.5%) were polymorphic and approximately 62.14% of polymorphisms were maintained within populations. Considerable variations were observed. The percentage of polymorphic bands ranged from 50.2% to 84.9% and the average over populations was 70.9%. Cluster analysis did not show correlation of genetic differentiation with the distinctive leaf morphology of P. stipuleanatus (i.e. one form with bipinnatifid leaflets and the other with undivided leaflets). Because over 40% of genetic variations were maintained among populations and because of the very restricted distribution of P. stipuleanatus, all natural populations of this species should be conserved in situ. Considering that there are variations in P. notoginseng within and among plantations, we suggest establishing a genetic resource conservation garden or reintroducing P. notoginseng into its native habitats in southwestern China. Such reintroduction should be carefully executed after large-scale screening of genetic variation within the species.

Drug-resistant gene based genotyping for Acinetobacter baumannii in tracing epidemiological events and for clinical treatment within nosocomial settings

Background Acinetobacter baumannfi has emerged as an important pathogen related to serious infections and nosocomial outbreaks around the world. However, of the frequently used methods, pulsed-field gel electrophoresis （PFGE） and amplified fragment length polymorphism （AFLP） in Acinetobacter baumannfi genotyping lack the direct molecular proof of drug resistance. This study was conducted to establish a typing method based on drug resistant gene identification in contrast to traditional PFGE and AFLP in the period of nosocomial epidemic or outbreak. Methods From January 2005 to October 2005, twenty-seven strains of Acinetobacter species from Intensive Care Units, the Second Affiliated Hospital in Ningbo were isolated, including both epidemic and sporadic events. Susceptibility test, PFGE, AFLP and drug resistance gene typing （DRGT） were carried out to confirm the drug resistance and analyze the genotyping, respectively. PFGE was used as a reference to evaluate the typeability of DRGT and AFLP. Results Twenty-seven strains of Acinetobacter displayed multiple antibiotic resistance and drug resistant genes, and β-1actamase genes were detected in 85.2% strains. The result of DRGT was comparable to PFGE in Acinetobacter strains with different drug resistance though a little difference existed, and even suggested a molecular evolution course of different drug-resistant strains. AFLP showed great polymorphism between strains and had weak ability in distinguishing the drug resistance. Conclusion Compared to AFLP and PFGE, DRGT is useful to analyze localized molecular epidemiology of nosocomial infections and outbreaks, which would benefit clinical diagnosis and therapy.

Use of the Metabolomics Approach to Characterize Chinese Medicinal Material Huangqi

Integration of the genetic and metabolic fingerprinting can provide a new approach to differentiate similar Traditional Chinese Medical （TCM） materials. Two leguminous plants, Mojia Huangqi and Menggu Huangqi, are important medical herbs and share great similarities in morphology, chemical constituent, and genomic DNA sequence. The taxonomy of Mojia Huangqi and Menggu Huangqi has been debated for more than 50 years and discrimination of TCM materials directly affects the pharmacological and clinical effects. AFLP based genetic fingerprinting and GC-TOF/MS-based meta- bolic fingerprinting were used to successfully discriminate the two species. The results of AFLP supported the opinion that Menggu Huangqi was a variant of Mojia Huangqi. The metabolic fingerprinting showed growth locations have greater impacts on the metabolite composition and quantity than the genotypes （cultivated versus wild） in Menggu Huangqi. The difference of some soluble sugars, fatty acids, proline, and polyamine reflected plant adaptation to different growth environments. Using multivariate and univariate statistical analysis, three AFLP markers and eight metabolites were identified as candidate DNA and metabolic markers to distinguish the two herb materials. The correlation network between AFLP markers and metabolites revealed a complex correlation network, which indicated the special metabolic pathways and the regulation networks of Huangqi.

Cloning and Characterization of the Microspore Development-Related Gene BcMF2 in Chinese Cabbage Pak-Choi （Brassica campestris L. ssp. chinensis Makino）

For the sake of providing some important information relevant to the study of the molecular mechanism of genic male sterility in plants, gene differential expression in flower buds at different developmental stages, as well as in rosette leaves, florescence leaves, and scapes was analyzed using cDNA amplified fragment length polymorphism (cDNA-AFLP) in the genic male sterile A and fertile B line of Chinese cabbage pak-choi. Following amplification of 125 pairs of primer combinations, 11 differential fragments were obtained, of which eight were from the B line and the other three were from the A line. Of 11 differential fragments, four were verified by Northern hybridization that were expressed preferentially in fertile flower buds. Results of GenBank BLAST showed that one fragment was with unknown function, whereas the other fragments have strong nucleotide sequence similarities with the polygalacturonase (PG) gene, the pectinesterase (PE) gene, and the polygalacturonase inhibitory protein (PGIP4) gene. Only full-length cDNA from the differential fragment BcMF-A18T16-1 was amplified by rapid amplification of cDNA ends (RACE) and Northern analysis showed that this fragment was expressed only in medium and large-sized flower buds of the B line. The full-length cDNA, designated as BcMF2 (Brassica campestris Male Fertile 2), was 1 485 bp long and was composed of a 1 263-bp open reading frame, which had 83% nucleotide similarity to a PG gene from Arabidopsis encoding polygalacturonase. Analysis of the basic structure of the protein revealed that it had one polygalacturonase active site (RVTCGPGHGLSVGS) at 256th site of amino acids and was classified as being a member of family 28 of the glycosyl hydrolases. The role of the BcMF2 gene on microspore development is discussed in the present paper.

A High-throughput Genomic Tool： Diversity Array Technology Complementary for Rice Genotyping

Diversity array technology （DART^TM） was a genotyping tool characterized gel-independent and high throughput. The main purpose of present study is to validate DArT for rice （Oryza sativa L.）genotyping in a high throughput manner. Technically, the main objective was to generate a rice general purpose gene pool, and optimize this genomic tool in order to evaluate rice germplasm genetic diversity. To achieve this, firstly, a generalpurpose DArT array was developed. Ten representatives from 24 varieties were hybridized with the general-purpose array to determine the informativeness of the clones printed on the array. The informative 1 152 clones were re-arrayed on a slide and used to fingerprint 17 of 24 germplasms. Hybridizing targets prepared from the germplasm to be assayed to the DNA array gave DNA fingerprints of germplasms. Raw data were normalized and transformed into binary data, which were then analyzed by using NTSYSpc （Numerical taxonomy system for cluster and ordination analysis, v. 2.02j） software package. The graphically displayed dendrogram derived from the array experimental data was matched with simple sequence repeats genotyping outline and varieties＇ pedigree deviation of the different varieties. Considering DArT is a sequence-independent genotyping approach, it will be applied in studies of the genetic diversity and the gene mapping of diverse of organisms, especially for those crops with less-developed molecular markers.

Analysis of genetic diversity in banana cultivars(Musa cvs.) from the South of Oman using AFLP markers and classification by phylogenetic,hierarchical clustering and principal component analyses

Banana is an important crop grown in Oman and there is a dearth of information on its genetic diversity to assist in crop breeding and improvement programs.This study employed amplified fragment length polymorphism(AFLP) to investigate the genetic variation in local banana cultivars from the southern region of Oman.Using 12 primer combinations,a total of 1094 bands were scored,of which 1012 were polymorphic.Eighty-two unique markers were identified,which revealed the distinct separation of the seven cultivars.The results obtained show that AFLP can be used to differentiate the banana cultivars.Further classification by phylogenetic,hierarchical clustering and principal component analyses showed significant differences between the clusters found with molecular markers and those clusters created by previous studies using morphological analysis.Based on the analytical results,a consensus dendrogram of the banana cultivars is presented.

Assessment of Genetic Stability Among In Vitro Plants of Arachis retusa Using RAPD and AFLP Markers for Germplasm Preservation

Arachis retusa Krapov. et W. C. Gregory et Valls is endemic in the West-central region of Brazil, occurring In areas endangered by human actions. The establishment of in vitro preservation methods for wild species of Arachis is an alternative to seed banks for germplasm storage, multiplication and distribution. The risk of genetic changes Induced by tissue culture and the monitoring of the genetic stability of the biological material before, during and after storage must be considered In the context of conservation. Random amplified polymorphlc ONA （RAPO） and amplified fragment length polymorphism （AFLP） fingerprinting were used to evaluate the genetic stability of in vitro plants originated from cotyledons and embryo axes of A. retusa. Cotyledons originated shoots through direct organogenesls and embryo axes displayed muItishoot formation Induced by 110 mmol/L and 8.8 mmol/L BAP, respectively. Ninety genomlc regions （loci） generated from RAPO and 372 from AFLP analyses were evaluated. All amplified fragments detected by both techniques in plants derived from the two explant types were monomorphic. The results Indicate that the recovered shoots are genetically stable at the assessed genomic regions.

Effects of Logging on the Genetic Diversity of Quercus tiaoloshanica Chun et Ko in a Tropical Montane Forest of Hainan Island, Southern China

Quercus tiaoloshanica Chun et Ko, which has a narrow range of distribution, is one of the important endemic species of the tropical montane rain forest on Hainan Island, southern China. Long-term logging and habitat destruction have resulted in population decline and distribution retreat of Q. tiaoloshanica. To determine the impact of logging on the genetic diversity of Q. tiaoloshanica, the authors investigated the genetic structures using amplified fragment length polymorphism markers in four regenerated stands after logging and in one unlogged stand. Compared with the unlogged stand, the effective number of alleles per locus dropped by 1.0% in selective logging stands and by 2.0% in clear logging stands, corresponding to reductions of 3.8% and 5.2%, respectively, in mean Nei＇s gene diversity and 2.9% and 3.5%, respectively, in mean Shannon diversity index. No substantial genetic erosion was detected in any of the regenerated stands owing to the high tree density and high heterogeneity of the Q. tiaoloshanica stands investigated. Meanwhile, there was no natural regeneration of the species observed in a Dacrydium pierrei Hickel plantation 700 m away from the regenerated stands, suggesting the limited ability of seed dispersal of Q. tiaoloshanica. Clear logging should be undertaken cautiously because the total number of plant species dropped by 15.2% in the clear-logged stands compared with the unlogged stand. To conserve the genetic diversity of this species, as well as the plant biodiversity of tropical forests, the habitats of Q. tiaoloshanica should be protected against exploitation in terms of agricultural or other forms of land use, and some mature trees should be preserved as seed sources to maintain an adequate regeneration base for this species in the management of logging.

AFLP analysis of genetic diversity and relationship among some Chinese domestic ducks and wild ducks

The amplified fragment length polymorphic(AFLP)technique was used to analyze the genome DNA polymorphism among 8 breeds of domestic ducks and 2 species of wild ducks.Nine of the 17 selected primers pairs gave reproducible polymorphic DNA amplification bands.The amplified bands ranged from 44 to 83 per primer pair.Of the 513 AFLP markers obtained,498 were polymorphic.The proportion of polymorphic loci was 97.1%.The genetic distance(D)and similarity coefficients(GS)were calcu-lated based on the polymorphic data.Between domestic ducks D ranged from 0.331 to 0.589,while between domes-tic ducks and the wild ducks,it ranged from 0.298 to 0.520(vs.Anas Platyrhynchos)and from 0.316 to 0.522(vs.A.Poecilorhyncha),respectively.The variance analysis showed no significant difference between the two groups of data,which indicated that both mallard and spot-billed ducks made contributions to domestic duck evolution.A dendrogram was constructed according to the D value.