AFLP标记在小香羊遗传多态性检测中的应用

研究了AFLP标记在研究小香羊遗传多态性方面应用的可行性和该山羊个体基因组DNA的AFLP扩增结果。实验应用10条AFLP引物,用PstI酶切,对15只小香羊基因组DNA进行AFLP反应,共获得113个AFLP标记,单引物获得的标记数在2～19之间,小香羊群体相似系数AFLP研究结果为0．913(0．814～0．980)。该研究为评价小香羊的遗传稳定性提供了相关的参数,准确评价尚待和其它品种对比研究后确定。     

Multilocus population structure of Tapesia yallundae in Washington State

Population genetic structure of the fungal wheat pathogen Tapesia yallundae in Washington State was determined using genetically characterized amplified fragment length polymorphic (AFLP) markers and mating-type (MAT1-1 or MAT1-2). Segregation and linkage relationships among 164 AFLP markers and MAT were analysed using 59 progeny derived from an in vitro cross. Alleles at 158 AFLP loci and the mating-type locus segregated in a 1:1 ratio. Ten unlinked markers were chosen to determine genetic and genotypic diversity and to test the hypothesis of random mating and population differentiation among five subpopulations of T. yallundae representative of the geographical distribution of wheat production in eastern Washington. Among 228 isolates collected, overall gene diversity was high (h = 0.425) and a total of 91 unique multilocus genotypes (MLG) were identified, with 32 MLG occurring at least twice. The overall population genetic structure was consistent with random mating based on the segregation of mating-type, index of association (IA), parsimony tree length permutation test (PTLPT) and genotypic diversity analyses. However, clonal genotypes were found within each subpopulation and were also distributed among the five subpopulations. No significant differences in allele frequencies were found among the five subpopulations for all 10 loci based on contingency table analysis (G2) and Wier & Cockerham's population differentiation statistic theta (theta = -0.008, P = 0.722). T. yallundae appears to consist of a large homogeneous population throughout eastern Washington with both sexual and asexual reproduction contributing to the observed population genetic structure despite no report of sexual fruiting bodies of T. yallundae occurring under natural field conditions.     

Population genetics of the yellow fever mosquito in Trinidad: comparisons of amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers

Recent development of DNA markers provides powerful tools for population genetic analyses. Amplified fragment length polymorphism (AFLP) markers result from a polymerase chain reaction (PCR)-based DNA fingerprinting technique that can detect multiple restriction fragments in a single polyacrylamide gel, and thus are potentially useful for population genetic studies. Because AFLP markers have to be analysed as dominant loci in order to estimate population genetic diversity and genetic structure parameters, one must assume that dominant (amplified) alleles are identical in state, recessive (unamplified) alleles are identical in state, AFLP fragments segregate according to Mendelian expectations and that the genotypes of an AFLP locus are in Hardy-Weinberg equilibrium (HWE). The HWE assumption is untestable for natural populations using dominant markers. Restriction fragment length polymorphism (RFLP) markers segregate as codominant alleles, and can therefore be used to test the HWE assumption that is critical for analysing AFLP data. This study examined whether the dominant AFLP markers could provide accurate estimates of genetic variability for the Aedes aegypti mosquito populations of Trinidad, West Indies, by comparing genetic structure parameters using AFLP and RFLP markers. For AFLP markers, we tested a total of five primer combinations and scored 137 putative loci. For RFLP, we examined a total of eight mapped markers that provide a broad coverage of mosquito genome. The estimated average heterozygosity with AFLP markers was similar among the populations (0.39), and the observed average heterozygosity with RFLP markers varied from 0.44 to 0.58. The average FST (standardized among-population genetic variance) estimates were 0.033 for AFLP and 0.063 for RFLP markers. The genotypes at several RFLP loci were not in HWE, suggesting that the assumption critical for analysing AFLP data was invalid for some loci of the mosquito populations in Trinidad. Therefore, the results suggest that, compared with dominant molecular markers, codominant DNA markers provide better estimates of population genetic variability, and offer more statistical power for detecting population genetic structure.     

Genetic variation and population structure of oriental migratory locust, Locusta migratoria manilensis, in China by allozyme, SSRP-PCR, and AFLP markers

Allozyme analysis, microsatellite primer PCR (SSRP-PCR), and amplified fragment length polymorphism (AFLP) techniques were used to assess genetic diversity and population structure of the Chinese oriental migratory locust, Locusta migratoria manilensis. A total of 299 PCR markers (67 SSRPs and 232 AFLPs) were detected in eight populations, of which 98.7% were polymorphic markers. The proportion of polymorphic loci (95.5-98.8%) by SSRP+AFLP markers indicated no significant differences between populations, and all populations exhibited a similar level of variability; results of the allozyme analysis demonstrated that 19 loci gave rise to a lower level of polymorphism (55.6-66.7%). The genetic distances between the populations were relatively low. Shannon's index and Nei's gene diversity showed low differentiation among the populations. Allozyme analysis, however, reflected greater similarity and smaller differentiation between the populations than those shown by SSRP and AFLP markers. Neighbor-joining dendrograms derived from both the allozyme and SSRP+AFLP markers showed that the genetic distances among Chinese oriental migratory locust populations were not greatly influenced by geographic distance and breeding habitats.     

Genetic differentiation of Helicoverpa armigera (Hübner) and H. assulta (Guenée) (Lepidoptera: Noctuidae) based on AFLP markers

Abstract Here we use amplified fragment length polymorphism (AFLP) to assess genetic differentiation of Helicoverpa armigera and H. assulta . The results indicated that both species-specific fingerprints and cluster analysis showed the ability of AFLP technique to discriminate the two sibling species; among a total 1963 AFLP markers amplified from nine primer combinations: 777 (39.6%) were H. armigera -specific, 602 (30.7%) were H. assulta -specific, and 584 (29.7%) were common bands. The mean number of H. armigera -specific bands was significantly more than that of H. assulta -specific bands for nine primer combinations ( P < 0.05); the intraspecific distance of H. armigera and H. assulta was 0.123 0 and 0.110 7 respectively, and the interspecific distance was 0.178 3. In addition, the percentage of polymorphic loci and estimated average heterozygosity were used to estimate genetic diversity of the two species. This study therefore demonstrates that AFLP analysis is a sensitive and reliable technique to study genetic differentiation and genetic relationships between species and provides sufficient molecular markers for future linkage map construction, location and eventual cloning of genes involved in traits differentiation.     

Genetic diversity analysis using lowly polymorphic dominant markers: the example of AFLP in pigs

DNA markers are commonly used for large-scale evaluation of genetic diversity in farm animals, as a component of the management of animal genetic resources. AFLP markers are useful for such studies as they can be generated relatively simply; however, challenges in analysis arise from their dominant scoring and the low level of polymorphism of some markers. This paper describes the results obtained with a set of AFLP markers in a study of 59 pig breeds. AFLP fingerprints were generated using four primer combinations (PC), yielding a total of 148 marker loci, and average harmonic mean of breed sample size was 37.3. The average proportion of monomorphic populations was 63% (range across loci: 3%-98%). The moment-based method of Hill and Weir (2004, Mol Ecol 13:895-908) was applied to estimate gene frequencies, gene diversity (F(ST)), and Reynolds genetic distances. A highly significant average F(ST) of 0.11 was estimated, together with highly significant PC effects on gene diversity. The variance of F(ST) across loci also significantly exceeded the variance expected under the hypothesis of AFLP neutrality, strongly suggesting the sensitivity of AFLP to selection or other forces. Moment estimates were compared to estimates derived from the square root estimation of gene frequency, as currently applied for dominant markers, and the biases incurred in the latter method were evaluated. The paper discusses the hypotheses underlying the moment estimations and various issues relating to the biallelic, dominant, and lowly polymorphic nature of this set of AFLP markers and to their use as compared to microsatellites for measuring genetic diversity.     

The inheritance and chromosomal localization of AFLP markers in a non-inbred potato offspring

AFLP^TM is a new technique to generate large numbers of molecular markers for genetic mapping. The method involves the selective amplification of a limited number of DNA restriction fragments out of complex plant genomic DNA digests using PCR. With six primer combinations 264 segregating AFLP amplification products were identified in a diploid backcross population from non-inbred potato parents. The identity of an AFLP marker was specified by the primer combination of the amplification product and its size estimated in bases. The segregating AFLP amplification products were mapped by using a mapping population with 217 already known RFLP, isozyme and morphological trait loci. In general, the AFLP markers were randomly distributed over the genome, although a few clusters were observed. No indications were found that AFLP markers are present in other parts of the genome than those already covered by RFLP markers. Locus specificity of AFLP markers was demonstrated because equally sized amplification products segregating from both parental clones generally mapped to indistinguishable maternal and paternal map positions. Locus specificity of AFLP amplification products will allow to establish the chromosomal identity of linkage groups in future mapping studies.Since AFLP technology is a multi-locus detection system, it was not possible to identify the AFLP alleles which belong to a single AFLP locus. The consequences of a genetic analysis based on single alleles, rather than on loci with two or more alleles on mapping studies using progenies of non-inbred parents are discussed.     

Genetic Variation and Population Structure of Oriental Migratory Locust, Locusta migratoria manilensis, in China by Allozyme, SSRP-PCR, and AFLP Markers

Allozyme analysis, microsatellite primer PCR (SSRP-PCR), and amplified fragment length polymorphism (AFLP) techniques were used to assess genetic diversity and population structure of the Chinese oriental migratory locust, Locusta migratoria manilensis. A total of 299 PCR markers (67 SSRPs and 232 AFLPs) were detected in eight populations, of which 98.7% were polymorphic markers. The proportion of polymorphic loci (95.5–98.8%) by SSRP+AFLP markers indicated no significant differences between populations, and all populations exhibited a similar level of variability; results of the allozyme analysis demonstrated that 19 loci gave rise to a lower level of polymorphism (55.6–66.7%). The genetic distances between the populations were relatively low. Shannon’s index and Nei’s gene diversity showed low differentiation among the populations. Allozyme analysis, however, reflected greater similarity and smaller differentiation between the populations than those shown by SSRP and AFLP markers. Neighbor-joining dendrograms derived from both the allozyme and SSRP+AFLP markers showed that the genetic distances among Chinese oriental migratory locust populations were not greatly influenced by geographic distance and breeding habitats.     

Molecular mapping of the rf1 gene for pollen fertility restoration in sorghum (Sorghum bicolor L.)

We report the molecular mapping of a gene for pollen fertility in A1 (milo) type cytoplasm of sorghum using AFLP and SSR marker analysis. DNA from an F₂ population comprised of 84 individuals was screened with AFLP genetic markers to detect polymorphic DNAs linked to fertility restoration. Fifteen AFLP markers were linked to fertility restoration from the initial screening with 49 unique AFLP primer combinations (+3/+3 selective bases). As many of these AFLP markers had been previously mapped to a high-density genetic map of sorghum, the target gene (rf1) could be mapped to linkage group H. Confirmation of the map location of rf1 was obtained by demonstrating that additional linkage group-H markers (SSR, STS, AFLP) were linked to fertility restoration. The closest marker, AFLP Xtxa2582, mapped within 2.4 cM of the target loci while two SSRs, Xtxp18 and Xtxp250, flanked the rf1 locus at 12 cM and 10.8 cM, respectively. The availability of molecular markers will facilitate the selection of pollen fertility restoration in sorghum inbred-line development and provide the foundation for map-based gene isolation. Received: 22 August 2000 / Accepted: 18 October 2000     

Geographic variation of the major histocompatibility complex in Eastern Atlantic grey seals (Halichoerus grypus)

Pathogen-driven balancing selection maintains high genetic diversity in many vertebrates, particularly in the major histocompatibility complex (MHC) immune system gene family, which is often associated with disease susceptibility. In large natural populations where subpopulations face different pathogen pressures, the MHC should show greater genetic differentiation within a species than neutral markers. We examined genetic diversity at the MHC-DQB locus and nine putatively neutral microsatellite markers in grey seals (Halichoerus grypus) from eight United Kingdom (UK) colonies, the Faeroe Islands and Sable Island, Canada. Five DQB alleles were identified in grey seals, which varied in prevalence across the grey seal range. Among the seal colonies, significant differences in DQB allele and haplotype frequencies and in average DQB heterozygosity were observed. Additionally, the DQB gene exhibited greater differentiation among colonies compared with neutral markers, yet a weaker pattern of isolation by distance (IBD). After correcting for the underlying IBD pattern, subpopulations breeding in similar habitats were more similar to one another in DQB allele frequencies than populations breeding in different habitats, but the same did not hold true for microsatellites, suggesting that habitat-specific pathogen pressure influences MHC evolution. Overall, the data are consistent with selection at MHC-DQB loci in grey seals with both varying selective pressures and geographic population structure appearing to influence the DQB genetic composition of breeding colonies.     

Amplified fragment length polymorphism (AFLP) in soybean: species diversity, inheritance, and near-isogenic line analysis

Amplified fragment length polymorphism (AFLP) analysis is a PCR-based technique capable of detecting more than 50 independent loci in a single PCR reaction. The objectives of the present study were to: (1) assess the extent of AFLP variation in cultivated (Gycine max L. Merr.) and wild soybean (G. soja Siebold & Zucc.), (2) determine genetic relationships among soybean accessions using AFLP data, and (3) evaluate the usefulness of AFLPs as genetic markers. Fifteen AFLP primer pairs detected a total of 759 AFLP fragments in a sample of 23 accessions of wild and cultivated soybean, with an average of 51 fragments produced per primer pair per accession. Two-hundred and seventy four fragments (36% of the total observed) were polymorphic, among which 127 (17%) were polymorphic in G. max and 237 (31%) were polymorphic in G. soja. F₂ segregation analysis of six AFLP fragments indicated that they segregate as stable Mendelian loci. The number of polymorphic loci detected per AFLP primer pair in a sample of 23 accessions ranged from 9 to 27. The AFLP phenotypic diversity values were greater in wild than in cultivated soybean. Cluster and principal component analyses using AFLP data clearly separated G. max and G. soja accessions. Within the G. max group, adapted soybean cultivars were tightly clustered, illustrating the relatively low genetic diversity present in cultivated soybean. AFLP analysis of four soybean near-isogenic lines (NILs) identified three AFLP markers putatively linked to a virus resistance gene from two sources. The capacity of AFLP analysis to detect thousands of independent genetic loci with minimal cost and time requirements makes them an ideal marker for a wide array of genetic investigations.     

花椰菜遗传图谱的构建及NBS-LRR类抗性同源基因在图谱中的定位

利用AFLP和NBS profiling技术, 以花椰菜自交系“AD白花”与高代自交不亲和系“C-8”杂交得到的F1代自交产生的F2代分离群体为材料, 构建了第一个花椰菜遗传连锁图谱。该图谱由234个AFLP标记和21个NBS标记构成了9个连锁群, 总图距为668.4 cM, 标记间平均距离为2.9 cM。每个连锁群包含的位点数从12到47个, 相邻两标记之间的距离范围是0~14.9 cM。NBS标记分布在8个连锁群中, 这些标记大部分聚在一起。本研究为今后的基因定位及重要农艺性状的分析提供框架图。此外, 研究NBS profiling 方法在花椰菜中的稳定性和有效性以及NBS-LRR类RGA在花椰菜基因组中的分布和特点。     

Analysis of genetic variation in selected stocks of hatchery flounder,Paralichthys olivaceus,using AFLP markers

Amplified fragment length polymorphism (AFLP) analysis was performed in order to evaluate genetic characteristics of one common population and two selective hatchery populations of flounder Paralichthys olivaceus. A group of 60 genotypes belonging to three populations was screened using 10 different AFLP primer combinations. A total of 491 loci were produced in the three studied populations. The loci of 65.78%, 61.47% and 60.92% were polymorphic over all the genotypes tested in common, susceptible and resistant populations, respectively. The number of polymorphic loci detected by single primer combination ranged from 21 to 43. The average heterozygosity of common, susceptible and resistant populations was 0.1656, 0.1609 and 0.1586, respectively, which showed no significant difference. Compared with the common population, the two selective hatchery populations, susceptible and resistant, showed significant genetic differences including a smaller (P < 0.05) number of total loci, a smaller (P < 0.05) number of total polymorphic loci and a smaller (P < 0.05) percentage of low frequency (0–0.2) polymorphic loci. AFLP banding pattern was transformed into binary data and matrices were processed with POPGENE and TFPGA software. Similarity relationships were described graphically by a dendrogram, which clustered the three populations. The AFLP fingerprinting technique was confirmed to be a reproducible and sensitive tool for the study of population genetics of flounder. The present study confirmed that it was important to detect the genetic variability of the selective hatchery populations for the conservation of natural flounder resources.     

Genetic diversity among oat varieties of worldwide origin and associations of AFLP markers with quantitative traits

One hundred and fourteen oat (Avena sativa L.) varieties of worldwide origin were evaluated for genetic diversity based on 77 molecular polymorphisms produced by eight selective AFLP primer combinations. Genetic similarity, calculated using the DICE coefficient, was used for cluster analysis and principal component analysis was applied. In addition population structure was explored to identify discrete subpopulations based on allele frequency. Although clustering and population structure showed relationships with region and country of origin, there was no obvious relationship to hull presence or hull colour. Oat varieties originating from European breeding programs showed less diversity than varieties originating from North and South America. Associations between AFLP markers and agronomic traits (grain yield, groat yield, panicle emergence, plant height, and lodging) as well as kernel quality traits (kernel weight, test weight, screening percent and groat percent) were also investigated. Marker-trait associations were tested using a naïve simple regression model and five additional models that account for population structure. Significant associations were found for 23 AFLP markers, with many of these affecting multiple traits. This study demonstrates that diversity can be significantly enhanced using a global collection, and provides evidence for marker-trait associations that can be validated in segregating populations and exploited through marker-assisted selection.     

Inheritance and usefulness of AFLP markers in channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and their F1, F2, and backcross hybrids

Eight primer combinations were used to investigate the application of amplified fragment length polymorphism (AFLP) markers in catfish for genetic analysis. Intraspecific polymorphism was low among channel catfish or blue catfish strains. Interspecific AFLP polymorphism was high between the channel catfish and blue catfish. Each primer combination generated from 70 to more than 200 bands, of which 38.6–75.7% were polymorphic between channel catfish and blue catfish. On average, more than 20 polymorphic bands per primer combination were produced as quality markers suitable for genetic analysis. All AFLP markers were transmitted into channel catfish?×?blue catfish F1 hybrids, except rare markers that were heterozygous in the parents and therefore were segregating in F1 hybrids. The two reciprocal channel catfish?×?blue catfish F1 hybrids (channel catfish female?×?blue catfish male; blue catfish female?×?channel catfish male) produced identical AFLP profiles. The AFLP markers were inherited and segregated in expected Mendelian ratios. At two loci, E8-b9 and E8-b2, markers were found at significantly lower frequencies than expected with F2 and backcross hybrids which had been selected for increased growth rates. The reproducibility of AFLP was excellent. These characteristics of the catfish AFLP markers make them highly useful for genetic analysis of catfish, especially for construction of genetic linkage and quantitative trait loci maps, and for marker-assisted selection.     

Molecular genetic linkage maps for allotetraploid Leymus wildryes (Gramineae: Triticeae).

Molecular genetic maps were constructed for two full-sib populations, TTC1 and TTC2, derived from two Leymus triticoides x Leymus cinereus hybrids and one common Leymus triticoides tester. Informative DNA markers were detected using 21 EcoRI-MseI and 17 PstI-MseI AFLP primer combinations, 36 anchored SSR or STS primer pairs, and 9 anchored RFLP probes. The 164-sib TTC1 map includes 1069 AFLP markers and 38 anchor loci in 14 linkage groups spanning 2001 cM. The 170-sib TTC2 map contains 1002 AFLP markers and 36 anchor loci in 14 linkage groups spanning 2066 cM. Some 488 homologous AFLP loci and 24 anchor markers detected in both populations showed similar map order. Thus, 1583 AFLP markers and 50 anchor loci were mapped into 14 linkage groups, which evidently correspond to the 14 chromosomes of allotetraploid Leymus (2n = 4x = 28). Synteny of two or more anchor markers from each of the seven homoeologous wheat and barley chromosomes was detected for 12 of the 14 Leymus linkage groups. Moreover, two distinct sets of genome-specific STS markers were identified in these allotetraploid Leymus species. These Leymus genetic maps and populations will provide a useful system to evaluate the inheritance of functionally important traits of two divergent perennial grass species.     

Conversion of AFLP markers associated with FHB resistance in wheat into STS markers with an extension-AFLP method.

Amplified fragment length polymorphism (AFLP) has proven a powerful tool for tagging genes or quantitative trait loci (QTLs) of interest in plants. However, conversion of AFLP markers into sequence-tagged site (STS) markers is technically challenging in wheat owing to the complicated nature of its genome. In this study, we developed an "extension-AFLP" method to convert AFLP markers associated with Fusarium head blight (FHB) resistance into STS markers. When an AFLP marker of interest was detected with an EcoRI+3-MseI+4-selective primer combination, the PCR product was used as a template for an additional selective amplification with four primer pairs, in which one additional selective base (either A, C, G, or T) was added to the 3' end of one of the two primers. The extended primer pair that produced the targeted band was further extended by adding each of the four selective nucleotide bases for the next round of selective amplification. Extension selective amplification was performed until the target bands became clear enough for subsequent cloning and sequencing. By using the extension-AFLP method, we successfully converted two AFLP markers located on chromosome 3BS and associated with FHB resistance into STS markers. Our results indicated that the extension-AFLP method is an efficient approach for converting AFLP markers into STS markers in wheat. The developed STS markers might be used for marker-assisted selection (MAS) for FHB resistance in wheat breeding programs.     

Genetic diversity and structure in fragmented populations of the endangered species Ranunculus cabrerensis (Ranunculaceae): implications for conservation

Ranunculus cabrerensis is an endemic and endangered species of the Northwestern Iberian Peninsula. The molecular markers AFLP and ISSR were used to investigate the genetic diversity and population structure of four populations across its known distribution. Fifteen selective primer combinations of AFLP and seventeen ISSR primer combinations produced a total of 2830 and 103 unambiguously repeatable fragments respectively, of which 97.57 and 81.38% were polymorphic for both markers. The genetic diversity of R. cabrerensis at species level was high (H _E = 0.294 by ISSR and H _E = 0.191 by AFLP) and differentiation between sampled locations was also relatively high (G _ST = 0.316 and 0.158 by ISSR and AFLP analysis respectively) compared to other studies of endangered and rare species using the same techniques. The analysis of molecular variance (AMOVA) indicated that the main genetic variation was within sampled locations (73% by AFLP; 52% by ISSR), even though the variation among locations was also significant. Principal Coordinates, NeighborNet and Bayesian analyses revealed a weak but significant relationship between the genetic structures of different populations in R. cabrerensis, with gene flow acting as a homogenizing force that prevents stronger differentiation of populations. Finally, suggestions for conservation strategies to preserve the genetic resources of this species are outlined.     

Comparative study of the discriminating capacity of AFLP and ISTR markers for genetic analysis of<Emphasis Type="Italic">Agave fourcroydes</Emphasis>

Two DNA-based marker systems, amplified fragment legnth polymorphism (AFLP) and inverse sequence-tagged repeat (ISTR), were evaluated with respect to their discriminating capacity and efficiency in genetically analyzing henequen (Agave fourcroydes). Samples were taken from a mother plant, sucker-derived daughter plants, and bulbils. ISTR analysis produced more polymorphic markers than AFLP and had a better capacity for quantifying genetic diversity through an average number of alleles per locus, expected heterozygosis of polymorphic loci, expected heterozygosity, effective number of alleles per locus, and total number of effective alleles. ISTR also showed superior discriminatory capacity.     

黑石顶南亚热带常绿阔叶林优势种群黄果厚壳桂的AFLP分析

以扩增片段长度多态性（AFLP）分析了黑石顶南亚热带常绿阔叶林优势种群黄果厚壳桂（Cryptocarya concinna)在三个不同群落类型中的遗传多态性及遗传结构,并比较了该3个种群间的遗传分,以4对AFLP选择引物组合产生了大量扩增表型带,用AMOVA、PHYLIP等软件分析计算了种群的遗传结构。结果表明黑石顶黄果厚0壳桂种群具有高的种群内遗传多态性、低的种群间遗传分析。并讨论了种群遗传变化与生境的关系。