A preliminary study for identification of candidate AFLP markers under artificial selection for shell color in pearl oyster Pinctada fucata

Pearl oyster Pinctada fucata is widely cultured to produce seawater pearl in South China, and the quality of pearl is significantly affected by its shell color. Thus the Pearl Oyster Selective Breeding Program (POSBP) was carried out for the shell color and growth traits. The black (B), gold (G), red (R) and white (W) shell strains with fast growth trait were achieved after five successive generation selection. In this study, AFLP technique was used to scan genome of four strains with different shell colors to identify the candidate markers under artificial selection. Eight AFLP primer combinations were screened and yielded 688 loci, 676 (98.26%) of which were polymorphic. In black, gold, red and white strains, the percentage of polymorphic loci was 90.41%, 87.79%, 93.60% and 93.31%, respectively, Nei's gene diversity was 0.3225, 0.2829, 0.3221 and 0.3292, Shannon's information index was 0.4801, 0.4271, 0.4825 and 0.4923, and the value of F_ST was 0.1805. These results suggested that the four different shell color strains had high genetic diversity and great genetic differentiation among strains, which had been subjected to the continuous selective pressures during the artificial selective breeding. Furthermore, six outlier loci were considered as the candidate markers under artificial selection for shell color. This study provides a molecular evidence for the inheritance of shell color of P. fucata.     

Conservation genetics of endangeredBrasenia schreberi based on RAPD and AFLP markers

Brasenia schreberi J.F. Gmelin is a declared endangered species found in the lakes and ponds of South Korea. For planning its conservation strategy, we examined the genetic diversity within and among six populations, using randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). Polymorphisms were more frequently detected per loci with AFLP (69.3%) than RAPD (36.8%). High genetic diversity was recognized within populations: polymorphic loci (PPL) values ranged from 36.3% in the CJM population to 74.5% in the GGT population, with a mean value of 47.8% based on AFLP markers. Great genetic differentiation (θ^B) was detected among the six populations (0.670 on RAPD and 0.196 on AFLP), and we calculated a low rate of gene flow (N_em), i.e., 0.116 on RAPD and 0.977 on AFLP. Furthermore, a Mantel test revealed that no correlation existed between genetic distances and geographical distances among the six local populations, based on RAPD or AFLP markers. These results are attributed to a number of factors, including an insufficient length of time for genetic diversity to be reduced following a natural decline in population size and isolation, adaptation of the genetic system to small population conditions, and a restricted gene flow rate. Based on both its genetic diversity and population structure, we suggest that a strategy for conserving and restoringB. schreberi must focus on maintaining historical processes, such as high levels of outbreeding, while monitoring increased gene flow among populations. This is because a reduction in genetic diversity as a result of genetic drift is undesirable.     

Genetic diversity in wild populations of Paulownia fortunei

The genetic diversities of 16 Paulownia fortunei populations involving 143 individuals collected from 6 provinces in China were analyzed using amplified fragment length polymorphism (AFLP). A total of 9 primer pairs with 1169 polymorphic loci were screened out, and each pair possessed 132 bands on average. The percentage of polymorphic bands (98.57%), the effective number of alleles (1.2138–1.2726), Nei’s genetic diversity (0.1566–0.1887), and Shannon’s information index (0.2692–0.3117) indicated a plentiful genetic diversity and different among Paulownia fortune populations. The genetic differentiation coefficient between populations was 0.2386, while the gene flow was 1.0954, and the low gene exchange promoted genetic differentiation. Analysis of variance indicated that genetic variation mainly occurred within populations (81.62% of total variation) rather than among populations (18.38%). The 16 populations were divided by unweighted pair-group method with arithmetic means (UPGMA) into 4 groups with obvious regionalism, in which the populations with close geographical locations (latitude) were clustered together.     

Identification and Mapping of Amplified Fragment Length Polymorphism Markers Linked to Shell Color in Bay Scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F₁ families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.     

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.     

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 diversity and structure of western white pine (Pinus monticola) in North America: a baseline study for conservation, restoration, and addressing impacts of climate change

Western white pine (Pinus monticola) is an economically and ecologically important species in western North America that has declined in prominence over the past several decades, mainly due to the introduction of Cronartium ribicola (cause of white pine blister rust) and reduced opportunities for regeneration. Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity and structure among populations at 15 sites (e.g., provenances) across the native range of western white pine. The level of genetic diversity was different among 15 populations tested using 66 polymorphic AFLP loci. Nei’s gene diversity (H _E) at the population level ranged from 0.187 to 0.316. Genetic differentiation (G _ST) indicated that 20.1% of detected genetic variation was explained by differences among populations. In general, populations below 45^oN latitude exhibited a higher level of genetic diversity than higher latitude populations. Genetic distance analysis revealed two major clades between northern and southern populations, but other well-supported relationships are also apparent within each of the two clades. The complex relationships among populations are likely derived from multiple factors including migration, adaptation, and multiple glacial refugia, especially in higher latitudes. Genetic diversity and structure revealed by this study will aid recognition and selection of western white pine populations for species management and conservation programs, especially in consideration of current and future climate changes.     

Genetic diversity and biogeography of the traditional Chinese medicine, Gardenia jasminoides, based on AFLP markers

Gardenia jasminoides Ellis is used in traditional Chinese medicine (TCM) in China. Levels of genetic variation and patterns of population structure within and among eight wild or cultivated populations of G. jasminoides Ellis in China were investigated using amplified fragment length polymorphism (AFLP) markers. Of the 11 primers screened, four produced highly reproducible AFLP bands. Using these primers, 244 discernible DNA fragments were generated with 165 bands (67.6%), were polymorphic, indicating considerable genetic variation at the species level. In contrast, there were relatively low levels of polymorphism at the population level with the percentage of polymorphic bands (PPB) ranging from 36.89% to 59.43%. Genetic diversity within populations ranged from 0.2086 to 0.3108, averaging 0.2392 at the species level. A high level of genetic differentiation among populations was detected based on Nei's genetic diversity analysis (76.59%), Shannon's index analysis (64.8%) and AMOVA analysis (72.75%). No significant statistical differences (analysis of molecular variance [AMOVA], p = 0.0639) in AFLP variation were found between regions. However, the variance among populations and within populations differed significantly (p < 0.001). An indirect estimate of historical levels of gene flow (N_m = 1.7448) was consistent with the high mean genetic identity (mean I = 0.9263) found among populations. There is an association between geographic and genetic distances between populations. Presently gene change exists between populations.     

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.     

AFLP and RAPD analyses of genetic diversity of wild and/or weedy Guizotia (Asteraceae) from Ethiopia

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) markers were used to provide estimates of the comparative genetic variation within and among populations of various Guizotia taxa with the goal of conserving and utilizing their genetic diversity. The percentage of polymorphic loci (P(S)) ranged from 28.5%-90% (AFLP) and 85.6%-99.6% (RAPD). The overall gene diversity estimate () has shown slight variation among taxa ranging from 0.32-0.37 (AFLP) and from 0.22 to 0.28 (RAPD). The within population diversity of "Chelelu" and "Ketcha" was found to be unexpectedly high. Both parameters used to estimate population differentiation (G(ST) and F(ST)) revealed the highest population differentiation G. zavattarii in followed by G. arborescens. Genetic variation among populations within a taxon was highly significant for all the five taxa as revealed by AMOVA (P<0.0001). The need for immediate conservation activities for G. arborescens and G. zavattarii, and factors that contribute to the existing genetic variability and population genetic structures are discussed.     

Analysis of genetic variability and population structure of the endemic medicinal Limonium sinense using molecular markers

Limonium sinense is an endemic medicinal herb used to treat fever, hemorrhage and other disorders. In the present study, population genetic diversity was elucidated using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) primers. Percentage of polymorphic bands, Nei's gene diversity and Shannon's information index revealed a high level of genetic diversity at species level. The analysis of molecular variance revealed that 69.88% (RAPD), 71.19% (ISSR) and 70.97% (AFLP) of variability were partitioned among individuals within populations, which indicated the coherent trend by Gst (0.3849/0.3577/0.3670). Gene flow number (Nm) was 0.581/0.618/0.612, which indicated that there was a limited gene exchange between populations. The UPGMA clustering results showed that the genetic distance had no significant correlation with geographic distance. These results indicate that these markers were reliable tools for the differentiation and determination of the genetic diversity among the populations of L. sinense and the conservation of existing natural population is necessary.     

Evaluation of genetic diversity of Panicum turgidum Forssk from Saudi Arabia

The genetic diversity of 177 accessions of Panicum turgidum Forssk, representing ten populations collected from four geographical regions in Saudi Arabia, was analyzed using amplified fragment length polymorphism (AFLP) markers. A set of four primer-pairs with two/three selective nucleotides scored 836 AFLP amplified fragments (putative loci/genome landmarks), all of which were polymorphic. Populations collected from the southern region of the country showed the highest genetic diversity parameters, whereas those collected from the central regions showed the lowest values. Analysis of molecular variance (AMOVA) revealed that 78% of the genetic variability was attributable to differences within populations. Pairwise values for population differentiation and genetic structure were statistically significant for all variances. The UPGMA dendrogram, validated by principal coordinate analysis-grouped accessions, corresponded to the geographical origin of the accessions. Mantel’s test showed that there was a significant correlation between the genetic and geographical distances (r = 0.35, P < 0.04). In summary, the AFLP assay demonstrated the existence of substantial genetic variation in P. turgidum. The relationship between the genetic diversity and geographical source of P. turgidum populations of Saudi Arabia, as revealed through this comprehensive study, will enable effective resource management and restoration of new areas without compromising adaptation and genetic diversity.     

Genetic diversity of Chinese alligator (Alligator sinensis) revealed by AFLP analysis: an implication on the management of captive conservation

Chinese alligator (Alligator sinensis) is one of the most critically endangered species among 23 extant crocodiles in the world. To prevent the extinction of the species, a captive propagation started at early 1980s, and the total number of alligator was brought up to 10 thousands from dozens of founder in 2000. But several genetic investigations showed those alligators were under an extremely low genetic diversity status with few detectible polymorphic loci. To get more insight into its genetic diversity for the management of captive Chinese alligator, AFLP was adopted to characterize variations in the population. Total of 347 bands were generated from 47 individuals using 3 primer combinations, of which 203 (58.50%) were polymorphic, and 35 AFLP phenotypes were revealed from those individuals. Comparing the results between RAPD and AFLP analysis on almost same sample set clearly indicated that AFLP is more efficient in revealing polymorphic loci, especially in those populations with extremely low genetic diversity. In present three assays, electrophoresis profile also displayed 3 individuals possessing very highly polymorphic AFLP phenotypes that were never been found by RAPD and mtDNA D-loop sequencing, implicating that we should offer these individuals more breeding opportunities to maintain the genetic diversity in the population and restrict those carrying few polymorphic loci from reproduction.     

Assessing genetic diversity of populations of topmouth culter (Culter alburnus) in China using AFLP markers

Genetic diversity of five wild populations and a cultured population of topmouth culter (Culter alburnus) was investigated using amplified fragment length polymorphism (AFLP). A total of 373 reproducible bands amplified with seven AFLP primer combinations were obtained from 163 fish. The percentage of polymorphic loci ranged widely from 37.0% to 69.2% within distinct populations. The cultured population appeared to have a lower level of polymorphism (37.0%), gene diversity (0.121 ± 0.188) and Shannon's Information index (0.183 ± 0.263) than the wild populations. Analysis of molecular variance (AMOVA) revealed that average F_ST value overall loci was 0.2671, and the percentage of variation within population (73.29%) was larger than among populations (26.71%) (P < 0.01). The six populations were clustered into two major clades with UPGMA. The results from analysis of population pairwise gene flow indicated moderate gene flow among populations. Our study indicated that the genetic diversity of the cultured population was reduced compared with the wild populations. Geographic isolation, habitat, and artificial selection all may have played important roles in population differentiation. The information may be beneficial to future broodstock selection and defining conservation management for the different populations of topmouth culter.     

Genetic variability, population size and reproduction potential in Ligularia sibirica (L.) populations in Estonia

Ligularia sibirica (L.) Cass. (Asteraceae) is a EU Habitats Directive Annex II plant species that has suffered a lot from human-caused major changes in quality and availability of habitats in Estonia. The aim of this study was to find out if the observed decline in population size is reflected in the amount of genetic variation and fertility in remnant populations of this species. AFLP technique was used for that purpose. Genetic diversity within populations was assessed as the percentage of polymorphic loci in a given population and average gene diversity over loci. The degree of genetic differentiation among populations and genetic differentiation between pairs of populations was estimated. The amount of viable seeds per flower stem was compared among populations and between years (2007 and 2008). Average genetic diversity over loci and proportion of polymorphic loci in L. sibirica populations were significantly correlated with population size, suggesting the action of genetic drift and/or inbreeding. No correlation was found between genetic and geographic distances. Natural barriers like forests may have been efficiently preventing seed migration even between geographically closer populations. Results of this study suggest that genetic erosion could be partially responsible for the lower fitness in smaller populations of this species.     

Assessing genetic diversity of wild populations of Prenantȁ9s schizothoracin, Schizothorax prenanti, using AFLP markers

Prenantȁ9s schizothoracin, Schizothorax prenanti, an endemic fish to China, has undergone a dramatic decline in numbers due to human impacts. We studied its genetic diversity in three tributaries of the Yangtze River: the Qingyi River, which has many hydropower dams, and the Dadu River and Muli River where many hydropower dams are being proposed. Using amplified fragment length polymorphism (AFLP), 621 loci were amplified with seven AFLP primer combinations in 45 individuals. The loci were highly polymorphic and heterozygous (87% polymorphism, 30% heterozygosity). The genetic distances within populations were large. The analysis of molecular variance demonstrated that most variation occurred within populations. The estimated fixation index (Φ_st) value averaged over all polymorphic loci across the three rivers was 0.0837, indicating a moderate genetic differentiation. The differentiations between populations were significant, and population structure was strong. The results suggested that China had wild populations of Prenantȁ9s schizothoracin with considerable genetic diversity in the Muli, Dadu and Qingyi rivers. The proposals to dam these rivers should take into account the importance of conserving their genetic quality.     

Amplified fragment length polymorphism analysis to identify the genetic structure of the Gymnocypris przewalskii (Kessler, 1876) population from the Qinghai Basin, China

Amplified fragment length polymorphism (AFLP) was used to analyse the genetic structure of 45 individuals of Gymnocypris przewalskii (Kessler, 1876), an endangered and state‐protected rare fish species, from three areas [the Heima (HM), Buha (BH) and Shaliu rivers (SL), all draining into Qinghai Lake]. A total of 563 polymorphic loci were detected. The HM, BH and SL populations have 435, 433 and 391 loci, respectively ( Zhu and Wu, 1975 ), which account for 77.26%, 76.91% and 69.45% of the total number of polymorphic loci of each population, respectively. The Nei indices of genetic diversities (H) of the three populations were calculated to be 0.2869 (HM), 0.2884 (BH) and 0.2663 (SL), respectively. Their Shannon informative indices are 0.4244, 0.4251 and 0.3915, respectively. Research results show that the mean genetic distance between HM and BH is the smallest (0.0511), between BH and SL is the second shortest (0.0608), and between HM and SL is the largest (0.0713), with the mean genetic distance among the three populations being over 0.05. Data mentioned above indicate that the three populations have a certain genetic differentiation. The total genetic diversity (H_t = 0.3045) and the mean value of genetic diversity within the population (H_s = 0.2786) indicate that the variations have mainly come from within the population.     

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 diversity of the expansive grass Brachypodium pinnatum in a changing landscape: Effect of habitat age

Perennial grasses constitute a major group of species showing a dramatic decline of biodiversity in successional plant communities. Using AFLP markers, we examined 12 populations of the expansive grass Brachypodium pinnatum differing in habitat age (30–50, ca. 100 and >300 years old) in order to determine whether clonal diversity of populations, genetic variation, and the relative importance of clonal propagation versus sexual reproduction change with grassland age. Five AFLP primer combinations gave a total of 517 bands, 79% of which were polymorphic. 314 different multilocus lineages were distinguished among the 453 samples analyzed. The number of genotypes (G) and clonal richness (R) decreased with habitat age, while the distribution of the frequency of genets changed from many clones of similar size to dominance by one or a few large clones. We consider these results to give evidence of significant role of sexual reproduction in the early phases of colonization and prevalence of clonal growth and competitive exclusion of less adapted genotypes in the later ones. However, habitat age had only marginal effect on genetic diversity, as percentage of polymorphic loci (PPL) within all the populations analyzed was similar, viz. 38.6–43.5%.     

Molecular identification of AFLP fragments associated with elytra color variation of the Asian ladybird beetle,Harmonia axyridis (Coleoptera: Coccinellidae)

The Asian ladybird beetle, Harmonia axyridis shows polymorphism in elytra color patterns. However, it is uncertain whether these color patterns are regulated by genetic factors. This investigation used amplified fragment length polymorphism (AFLP) analysis to determine any genetic causes of the variability of color patterns. Using four individuals of each group, AFLP analysis produced 37 polymorphic bands. Among several polymorphic bands, six AFLP markers were associated with elytra color patterns after further analysis using six additional individuals of each group. These polymorphic sites were sequenced but did not match DNA sequence data deposited in GenBank. Based on the color-associated AFLP markers, SCAR primers were designed for PCR amplification of genomic DNA. These primers (SCAR 12 and SCAR 44) were used to analyze color-associated loci and/or alleles of H. axyridis DNA. SCAR 12 primers designed from a Spectabilis type-specific fragment (AFLP 12) amplified a specific band of 530 bp in four Spectabilis individuals, but not in the insects with other color patterns.