Chromosomal location of AFLP markers in common wheat utilizing nulli-tetrasomic stocks.

Amplified fragment length polymorphism (AFLP) markers with a total of 256 EcoRI + ANN - MseI + CNN primer combinations were investigated employing the common wheat cultivar Triticum aestivum 'Chinese Spring.' On average, 103 fragments per primer combination were amplified, ranging from a maximum of 226 fragments to a minimum of 18 fragments. The primer combinations E + AAA - M + CNN and E + ATT - M + CNN produced very few distinct fragments. By using 15 randomly chosen EcoRI + ANN - MseI + CNN primer combinations, 928 AFLP markers were allocated to wheat chromosomes, of which 131 were assigned to specific chromosome arms. These AFLP markers were locus-specific and randomly distributed on the different chromosomes. In addition, 6 and 41 AFLP markers were simultaneously absent in two nulli-tetrasomics (NTs) of both homoeologous and non-homoeologous groups, respectively, whereas additional fragments were detected in N1BT1A, N5AT5D, and N6BT6A lines.     

Isolation and Characterization of Male-Specific DNA Markers in the Rock Bream Oplegnathus fasciatus

Sex-specific DNA markers applicable were very useful for elucidating the sex-determination mechanism and sex control in fishes. In the present study, amplified fragment-length polymorphism (AFLP) approach with 144 primer combinations was employed to identify sex-specific markers in the rock bream. Four male-specific AFLP fragments were identified which were designated as Opl286, Opl237, Opl422, and Opl228. Further sequence analysis of the sex markers’ genomic region revealed subtle differences between the males and females. We identified four male-specific single-nucleotide polymorphisms (SNPs) and a deletion of 8 bp in marker Opl286, six male-specific SNPs in marker Opl237, three male-specific SNPs in marker Opl422, and eight male-specific SNPs and 1 bp inversions in marker Opl228. Specific primers were designed based on the nucleotide variation in the sequences to develop a simple polymerase chain reaction method for identifying the genetic sex of rock bream. As a result, three out of the four male-specific markers were converted into SNP markers. The male-specific AFLP markers and AFLP-derived SNP markers were tested in 100 individuals collected from three locations around the coast of Zhoushan, yielding reproducible sex identification. These male-specific DNA markers are a useful tool for the identification of the sex-determining locus in rock bream and for guiding artificial breeding programs.     

Identification of a male-specific AFLP marker in a functionally dioecious fig,<Emphasis Type="Italic"> Ficus fulva</Emphasis> Reinw. ex Bl. (Moraceae)

A male-specific amplified fragment length polymorphism (AFLP) marker was identified in the functionally dioecious fig species, Ficus fulva. A total of 89 polymorphic fragments from three primer combinations were produced, of which one (246 bp) was present in all males (n=23) and absent in all females (n=24) of two populations. This strong association suggests a tight chromosomal linkage between the AFLP marker and the sex-controlling locus. Further analysis indicated that the marker segregated in open-pollinated progenies from natural populations in a 1:1 ratio (n=156), implying that males are the heterogametic sex. Chromosome preparations showed no evidence for morphologically distinct sex chromosomes. The low frequencies of associated markers argue against a morphologically cryptic non-recombining sex chromosome. The sex-locus is therefore likely to be autosomal. The male-specific AFLP marker was sequenced and converted into a sequence characterised amplified region (SCAR) marker. This SCAR marker produced a fragment of equal size in males and females, suggesting that sequence divergence between male- and female-specific chromosomal regions is low.Publication 3311 NIOO-KNAW Netherlands Institute of Ecology     

High-density linkage maps and sex-linked markers for the black tiger shrimp (Penaeus monodon)

We report on the construction of sex-specific high-density linkage maps and identification of sex-linked markers for the black tiger shrimp (Penaeus monodon). Overall, we identified 44 male and 43 female linkage groups (2n = 88) from the analysis of 2,306 AFLP markers segregating in three full-sib families, covering 2,378 and 2,362 cM, respectively. Twenty-one putatively homologous linkage groups, including the sex-linkage groups, were identified between the female and male linkage maps. Six sex-linked AFLP marker alleles were inherited from female parents in the three families, suggesting that the P. monodon adopts a WZ-ZZ sex-determining system. Two sex-linked AFLP markers, one of which we converted into an allele-specific assay, confirmed their association with sex in a panel of 52 genetically unrelated animals.     

SRAP技术研究烟粉虱遗传多样性

采用AFLP、SRAP2种标记方法分别对2个烟粉虱Bemisia tabaci Gennadius种群(一品红、甘蓝)的多态性进行分析。结果表明,(1)2种方法平均每对引物组合产生的条带数分别为29.4和21.8。(2)AFLP法每对引物组合产生10～23条多态性带,平均17.20条,多态性带的比例平均为57.93%。SRAP法每对引物组合产生5～18条多态性带,平均13.3条,多态性带的比例平均为60.59%。(3)前者的基因多样性范围为0.1503～0.2838,平均为0.2297;后者的基因多样性范围为0.0977～0.2911,平均为0.2332。证明利用SRAP技术和AFLP技术研究烟粉虱的遗传多样性是有效的。     

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.     

Conversion of AFLP markers to sequence-specific markers for closely related lines in rice by use of the rice genome sequence

DNA polymorphism between two major japonica rice cultivars, Nipponbare and Koshihikari, was identified by AFLP. Eighty-four polymorphic AFLP markers were obtained by analysis with 360 combinations of primer pairs. Nucleotide sequences of 73 markers, 29 from Nipponbare and 44 from Koshihikari, were determined, and 46 AFLP markers could be assigned to rice chromosomes based on sequence homology to the rice genome sequence. Specific primers were designed for amplification of the regions covering the AFLP markers and the flanking sequences. Out of the 46 primer pairs, 44 amplified single DNA fragments, six of which showed different sizes between Nipponbare and Koshihikari, yielding codominant SCAR markers. Eight primer pairs amplified only Nipponbare sequences, providing dominant SCAR markers. DNA fragments amplified by 13 primer pairs showed polymorphism by CAPS, and polymorphism of those amplified by 13 other primer pairs were detected by PCR-RF-SSCP (PRS). Nucleotide sequences of the other four DNA fragments were determined in Koshihikari, but no difference was found between Koshihikari and Nipponbare. In total, 40 sequence-specific markers for the combination of Nipponbare and Koshihikari were produced. All the SNPs identified by AFLP were detectable by CAPS and PRS. The same method was applicable to a combination of Kokoromachi and Tohoku 168, and 23 polymorphic markers were identified using these two rice cultivars. The procedure of conversion of AFLP-markers to the sequence-specific markers used in this study enables efficient sequence-specific marker production for closely related cultivars.     

Development of S-SAP markers based on an LTR-like sequence from Medicago sativa L.

The Sequence-Specific Amplification Polymorphism (S-SAP) method, recently derived from the Amplified Fragment Length Polymorphism (AFLP) technique, produces amplified fragments containing a retrotransposon LTR sequence at one end and a host restriction site at the other. We report the application of this procedure to the LTR of the Tms1 element from Medicago sativa L. Genomic dot-blot analysis indicated that Tms1 LTRs represent about 0.056% of the M. sativa genome, corresponding to 16 x 10(3) copies per haploid genome. An average of 66 markers were amplified for each primer combination. Overall 49 polymorphic fragments were reliably scored and mapped in a F(1) population obtained by crossing diploid M. falcata with M. coerulea. The utility of the LTR S-SAP markers was higher than that of AFLP or SAMPL (Selective Amplification of Microsatellite Polymorphic Loci) markers. The efficiency index of the LTR S-SAP assay was 28.3, whereas the corresponding values for AFLP and SAMPL markers were 21.1 and 16.7, respectively. The marker index for S-SAP was 13.1, compared to 8.8 for AFLP and 9.5 for SAMPL. Application of the Tms1 LTR-based S-SAP to double-stranded cDNA resulted in a complex banding pattern, demonstrating the presence of Tms1 LTRs within exons. As the technique was successfully applied to other species of the genus Medicago, it should prove suitable for studying genetic diversity within, and relatedness between, alfalfa species.     

Species identification of five penaeid shrimps using PCR-RFLP and SSCP analyses of 16S ribosomal DNA

DNA-based molecular markers for differentiation of five penaeid shrimps (Penaeus monodon, P. semisulcatus, Feneropenaeus merguiensis, Litopenaeus vannamei and Marsupenaeus japonicus) were developed based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and single-stranded conformation polymorphism (SSCP) of 16S ribosomal (r) DNA. Differentiation of P. monodon, P. semisulcatus and L. vannamei can be unambiguously carried out by PCRRFLP of 16S rDNA(560) whereas P. semisulcatus and M. japonicus shared a BABB mitotype. These shrimps were successfully discriminated by SSCP analysis of 16S rDNA(560). Nevertheless, the amplification success for L. vannamei and F. merguiensis was not consistent when tested against larger sample sizes. As a result, 16S rDNA(560) of an individual representing the most common mitotype of each species was cloned and sequenced. The new primer pair was designed and tested against the large sample sizes (312 bp product, N = 185). The amplification success was consistent across all species. PCR-RFLP of 16S rDNA(312) was as effective as that of 16S rDNA(560). Differentiation of all shrimp species were successfully carried out by SSCP analysis.     

Inheritance of amplified fragment length polymorphism markers and their utility in population genetic analysis of Plecoglossus altivelis

The reproducibility, mode of inheritance and polymorphism of amplified fragment length polymorphism (AFLP) markers were examined in ayu Plecoglossus altivelis (Salmoniformes: Plecoglossidae). The AFLP markers were highly reproducible, their inheritance following Mendelian expectations. The number of fragments amplified (34–134), polymorphic ratio (0·15–0·78) and average heterozygosity (0·02–0·25) of the AFLP markers showed significant variation among six primer pairs and among ayu populations, including a landlocked Lake‐Biwa population, two amphidromous populations ( P. a. altivelis ) and two Ryukyu‐ayu populations ( P. a. ryukyuensis ). Although AFLP analysis provided similar results in intra‐population diversity and relationships among populations to those found by analyses of allozymes, microsatellites and mitochondrial DNA sequences, AFLPs showed higher polymorphisms and hence greater distinction between genetically close populations.     

High-resolution DNA fingerprinting of parthenogenetic root-knot nematodes using AFLP analysis

Amplified fragment length polymorphism (AFLP) analysis has been used to characterize 15 root-knot nematode populations belonging to the three parthenogenetic species Meloidogyne arenaria , M. incognita and M. javanica. Sixteen primer combinations were used to generate AFLP patterns, with a total number of amplified fragments ranging from 872 to 1087, depending on the population tested. Two kinds of polymorphic DNA fragments could be distinguished: bands amplified in a single genotype, and bands polymorphic between genotypes (i.e. amplified in not all but at least two genotypes). Based on presence/absence of amplified bands and pairwise similarity values, all the populations tested were clustered according to their specific status. Significant intraspecific variation was revealed by AFLP, with DNA fragments polymorphic among populations within each of the three species tested. M. arenaria appeared as the most variable species, while M. javanica was the least polymorphic. Within each specific cluster, no general correlation could be found between genomic similarity and geographical origin of the populations. The results reported here showed the ability of the AFLP procedure to generate markers useful for genetic analysis in root-knot nematodes.     

AFLP Analysis Confirms Exclusive Maternal Genomic Contribution of Meiogynogenetic Sea Bass (Dicentrarchus labrax L.)

Meiogynogenesis was induced in the European sea bass Dicentrarchus labrax L. by fertilizing eggs with UV-irradiated sperm followed by inhibition of the second meiotic division by a cold shock. Putative gynogenetic progeny derived from three groups of breeders were analyzed for maternal inheritance using amplified fragment length polymorphism (AFLP) markers. Discrimination of fingerprints was based on male-specific bands, which were absent in females. Four of 64 MseI/EcoRI primer pairs used to analyze parental polymerase chain reaction products were selected to screen progeny for paternal AFLP markers in each group. Four to 11 diagnostic bands per fish confirmed the gynogenetic origin of the progeny. AFLP analysis determined that 89.5%, 100%, and 100% of the sea bass from groups 1, 2, and 3, respectively, were gynogenetic. Our results show that AFLP analysis is suitable for verification of gynogenesis in fish.     

糙皮侧耳(Pleurotus ostreatus)的AFLP指纹图谱分析

在对糙皮侧耳(Pleurotus ostreatus)的AFLP分析条件进行优化的基础上,利用该技术建立了14株产自不同地区的糙皮侧耳：DNA指纹图谱,并进行了数据分析。结果表明,在合成的14条引物的不同组合中,引物对E-3／M-3可以产生较多的DNA多态片段,E-AGC／M-CAT引物对的扩增效果最好,共获得184条DNA扩增带,其中多态性条带i101条,占54．89％。利用UPGMA法对所获数据进行聚类分析,计算得到糙皮侧耳菌株之间的遗传距离,发现不同品种间遗传距离差异较大,从0．192到0．754,说明糙皮侧耳的遗传多样性比较丰富。绘制的指纹分析树状图表明,14个糙皮侧耳菌株被分为6个组群,其中P17和杂3的相似性系数最高,达到了81．2％,而侧5与其他菌株的亲缘关系相对最远。     

Characterization of Iberian pig genotypes using AFLP markers

The use of the AFLP (amplified fragment length polymorphism) technique for the characterization of highly inbred Iberian pig breed genotypes and the detection of strain-specific polymorphisms is demonstrated. Twelve different primer combinations were used on individual DNA samples from animals belonging to two black hairless Iberian pig strains, Guadyerbas and Coronado. These amplification reactions allowed the detection of more than 1700 amplification products of which 26 were identified as strain-specific markers, present in all individuals of one strain and absent in the other. Comparison of male and female amplification products within one strain also allowed the identification of 8 male-specific amplified bands. AFLP showed a great power of marker detection due to a high multiplex ratio and high reproducibility. Comparison of similarity and co-ancestry coefficient matrices also showed the usefulness of AFLP markers to estimate genetic relationships between individuals pigs.     

Genetic relationships of Aglaonema species and cultivars inferred from AFLP markers

BACKGROUND AND AIMS: Aglaonema is an important ornamental foliage plant genus, but genetic relationships among its species and cultivars have not been reported. This study analysed genetic relatedness of 54 cultivars derived from nine species using amplified fragment length polymorphism (AFLP) markers. METHODS: Initially, 48 EcoRI + 2/MseI + 3 primer set combinations were screened, from which six primer sets that showed clear scoreable and highly polymorphic fragments were selected and used for AFLP reactions. AFLP fragments were scored and entered into a binary data matrix as discrete variables. Jaccard's coefficient of similarity was calculated for all pair-wise comparisons among the 54 cultivars, and a dendrogram was constructed by the unweighted pair-group method using the arithmetic average (UPGMA). KEY RESULTS: The number of AFLP fragments generated per primer set ranged from 59 to 112 with fragment sizes varying from 50 to 565 bp. A total of 449 AFLP fragments was detected, of which 314 were polymorphic (70 %). All cultivars were clearly differentiated by their AFLP fingerprints. The 54 cultivars were divided into seven clusters; cultivars within each cluster generally share similar morphological characteristics. Cluster I contains 35 cultivars, most of them are interspecific hybrids developed mainly from A. commutatum, A. crispum or A. nitidum. However, Jaccard's similarity coefficients among these hybrids are 0.84 or higher, suggesting that these popular hybrid cultivars are genetically much closer than previously thought. This genetic similarity may imply that A. nitidum and A. crispum are likely progenitors of A. commutatum. CONCLUSIONS: Results of this study demonstrate the efficiency and ease of using AFLP markers for investigating genetic relationships of ornamental foliage plants, a group usually propagated vegetatively. The AFLP markers developed will help future Aglaonema cultivar identification, germplasm conservation and new cultivar development.     

Conversion of AFLP markers to sequence-specific PCR markers in barley and wheat

 Conversion of amplified fragment length polymorphisms (AFLPs) to sequence-specific PCR primers would be useful for many genetic-linkage applications. We examined 21 wheat nullitetrasomic stocks and five wheat-barley addition lines using 12 and 14 AFLP primer combinations, respectively. On average, 36.8% of the scored AFLP fragments in the wheat nullitetrasomic stocks and 22.3% in the wheat-barley addition lines could be mapped to specific chromosomes, providing approximately 461 chromosome-specific AFLP markers in the wheat nullitetrasomic stocks and 174 in the wheat-barley addition lines. Ten AFLP fragments specific to barley chromosomes and 16 AFLP fragments specific to wheat 3BS and 4BS chromosome arms were isolated from the polyacrylamide gels, re-amplified, cloned and sequenced. Primer sets were designed from these sequences. Amplification of wheat and barley genomic DNA using the barley derived primers revealed that three primer sets amplified DNA from the expected chromosome, five amplified fragments from all barley chromosomes but not from wheat, one amplified a similar-sized fragment from multiple barley chromosomes and from wheat, and one gave no amplification. Amplification of wheat genomic DNA using the wheat-derived primer sets revealed that three primer sets amplified a fragment from the expected chromosome, 11 primer sets amplified a similar-sized fragment from multiple chromosomes, and two gave no amplification. These experiments indicate that polymorphisms identified by AFLP are often not transferable to more sequence-specific PCR applications. Received: 30 June 1998 / Accepted: 26 October 1998     

魔芋种质资源的AFLP分析

利用AFLP技术对13份魔芋种质资源进行了DNA多态性分析。从10对引物中筛选出2对引物扩增基因组DNA,共获得112条带,其中64条为多态性标记,每个引物平均提供56个标记信息。UPMGA法聚类分析结果表明:魔芋资源的遗传多态性比较丰富,西盟魔芋和珠芽魔芋的关系比较近,白魔芋和花魔芋的关系比较近。