Restriction fragment length polymorphism diversity in soybean

Summary Fifty-eight soybean accessions from the genus Glycine, subgenus Soja, were surveyed with 17 restriction fragment length polymorphism (RFLP) genetic markers to assess the level of molecular diversity and to evaluate the usefulness of previously identified RFLP markers. In general, only low levels of molecular diversity were observed: 2 of the 17 markers exhibited three alleles per locus, whereas all others had only two alleles. Thirty-five percent of the markers had rare alleles present in only 1 or 2 of the 58 accessions. Molecular diversity was least among cultivated soybeans and greatest between accessions of different soybean species such as Glycine max (L.) Merr. and G. soja Sieb. and Zucc. Principal component analysis was useful in reducing the multidimensional genotype data set and identifying genetic relationships.     

Assessment of genetic diversity among and within Achillea species using amplified fragment length polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity of 57 Achillea accessions belonging to five species, A. millefolium, A. filipendulina, A. tenuifolia, A. santolina and A. biebersteinii. Nine AFLP primer combinations were used, which produced 301 polymorphic bands. In most species, a high level of genetic variation was detected among the genotypes. The Jaccard's similarity indices (J), based on AFLP profiles, were subjected to UPGMA cluster analysis. Application of Mantel's test for cophenetic correlation to the cluster analysis indicated the high fitness of the accessions to a group (r = 0.918). The dendrogram generated revealed five major groups corresponding to five species. The principle coordinate analysis (PCoA) data confirmed the results of the clustering. Among the species, A. teunifolia and A. santolina showed the greatest and the least genetic diversity, respectively. A. filipendulina accessions were acquired primarily from the same ecological regions of western Iran. Accessions belonging to A. biebersteinii originated from the Isfahan province and were separated from other species at the root of the dendrogram. The results of the clustering method, based on AFLP markers, corresponded closely with the geographical origins of the genotypes. The results of the present study could contribute to a better understanding and management of conservation and exploitation of the Achillea germplasm.     

Population structure and genetic diversity of Huperzia serrata (Huperziaceae) based on amplified fragment length polymorphism (AFLP) markers

The levels and pattern of the genetic variation within and among natural populations of Huperzia serrata were investigated using amplified fragment length polymorphism markers. Seven primer combinations used in the study amplified 615 discernible bands with 532 (86.5%) being polymorphic, indicating a considerable high level of genetic diversity at the species level. AMOVA analysis revealed a low level of genetic differentiation among the ten populations. The UPGMA cluster of all samples showed that individuals from the same population occasionally failed to cluster in one distinct group. A Mantel test showed no significant correlation between genetic distance and geographical distance (r = 0.278, P = 0.891), suggesting that the gene flow was not restricted geographically. A number of factors that might affect the genetic profiles of H. serrata included clonal growth, selective effect of niche and outcrossing, as well as the effective wind-dispersal of spores.     

Genetic diversity of Aegiceras corniculatum (Myrsinaceae) revealed by amplified fragment length polymorphism (AFLP)

Aegiceras corniculatum is a cryptoviviparous mangrove tree distributed in the Indo-West Pacific. The genetic structure of 13 populations of A. corniculatum from South China, Malay Peninsula, Sri Lanka, and North Australia, was assessed by amplified fragment length polymorphism (AFLP) markers. Our results showed a relatively high level of genetic variation at the species level (P = 92%, H_E = 0.294 and H_s = 0.331 ± 0.001). The value of G_ST was 0.698, suggesting significant genetic differentiation among populations. At the population level, however, genetic diversity was low (P = 24%, H_E = 0.086 and H_s = 0.127 ± 0.001). When populations were grouped according to geographic regions, i.e., South China, Malay Peninsula and Sri Lanka, it was inferred from analysis of molecular variance (AMOVA) that about half the total variation (49%) was accounted for differentiation between regions. A UPGMA dendrogram based on genetic distance also revealed five major clades corresponding to geographical regions within the distribution of A. corniculatum, although the precise relationships among the clades were not fully concordant with expected geographical delineations and need further study.     

Genetic diversity and structure of natural Pinus tabulaeformis populations in North China using amplified fragment length polymorphism (AFLP)

Chinese pine (Pinus tabulaeformis carr.), endemic to China, is a conifer species with extensive and fragmented distribution in North China. In this study, the genetic diversity and structure of 20 natural populations of this species were investigated using amplified fragment length polymorphism (AFLP) markers. A total of 445 fragments were revealed with 8 pairs of primers, 379 (85.17%) of which were polymorphic. A moderate level of genetic diversity was detected at the species level (Shannon's information index I = 0.356, Nei's gene diversity H_E = 0.271) and at the population level (I = 0.219, H_E = 0.206). Most of genetic variation was within populations while a considerable level of genetic differentiation was detected (G_ST = 0.352, Ф_ST = 0.304). The high differentiation could be attributed to the complex and fragmented habitats, and a limited gene flow among populations (N_m = 0.572). The Mantel test indicated that there was significant correlation (r = 0.455, P < 0.001) between Nei's genetic distance and geographical distance among all the populations. The results suggested that proper countermeasures should be taken to prevent the habitat further deterioration and maintain the genetic diversity of this species.     

Amplified fragment length polymorphism analysis of the population structure and genetic diversity of Phoebe zhennan (Lauraceae), a native species to China

Phoebe zhennan S. Lee et F. N. Wei (Lauraceae), is the main source of Gold Phoebe, a rare and extremely valuable wood in China. However it has undergone a dramatic decline. In this study, we used 12 amplified fragment length polymorphism primer combinations to assay 92 accessions, which were highly representative of the entire P. zhennan germplasm. It revealed that P. zhennan consisted of three genetic populations, named as SCZ (central Sichuan), CQH (eastern Sichuan, Chongqin, Hubei and Hunan) and YG (Yunnan and Guizhou), probably owing to natural selection caused by topography differences. The CQH population further diverged into two geographical sub-populations: CD-CQ (SCD and west region of Chongqin) and HB-HN (eastern side of Chongqin, Hubei and Hunan). The loci were moderately polymorphic (40.4%). The genetic distance between SCZ and YG was the highest, between CD-CQ and HB-HN the lowest. Pairwise fixation indices (Ф_PT) between any inferred populations were significant. This rare species exhibited low genetic diversity; therefore, the results provided significant data related to the conservation and management of P. zhennan. That is, with this genetic information, land managers are equipped with better tools allowing them to more effectively protect this species and its limited genetic diversity.     

Cleaved AFLP (cAFLP), a modified amplified fragment length polymorphism analysis for cotton

In certain plant species including cotton (Gossypium hirsutum L. or Gossypium barbadense L.), the level of amplified fragment length polymorphism (AFLP) is relatively low, limiting its utilization in the development of genome-wide linkage maps. We propose the use of frequent restriction enzymes in combination with AFLP to cleave the AFLP fragments, called cleaved AFLP analysis (cAFLP). Using four Upland cotton genotypes (G. hirsutum) and three Pima cotton (G. barbadense), we demonstrated that cAFLP generated 67% and 132% more polymorphic markers than AFLP in Upland and Pima cotton, respectively. This resulted in 15.5 and 25.5 polymorphic cAFLP markers per AFLP primer combination, as compared to 9.1 and 11.0 polymorphic AFLP. The cAFLP-based genetic similarity (GS) is generally lower than the AFLP-based GS, even though both marker systems are overall congruent. In some cases, cAFLP can better resolve genetic relationships between genotypes, rendering a higher discriminatory power. Given the high-resolution power of capillary-based DNA sequencing system, we further propose that AFLP and cAFLP amplicons from the same primer combination can be pooled as one sample before electrophoresis. The combination produced an average of 18.5 and 31.0 polymorphic markers per primer pair in Upland and Pima cotton, respectively. Using several restriction enzyme combinations before pre-selective amplification in combination with various frequent 4 bp-cutters or 6 bp-cutters after selective amplification, the pooled AFLP and cAFLP will provide unlimited number of polymorphic markers for genome-wide mapping and fingerprinting.     

Genetic characteristics of two genes for resistance to soybean mosaic virus in PI486355 soybean

Soybean [Glycine max (L.) Merr.] PI486355 is resistant to all the identified strains of soybean mosaic virus (SMV) and possesses two independently inherited resistance genes. To characterize the two genes, PI486355 was crossed with the susceptible cultivars Lee 68 and Essex and with cultivars Ogden and Marshall, which are resistant to SMV-G1 but systemically necrotic to SMV-G7. The F₂ populations and F₂₃ progenies from these crosses were inoculated with SMV-G7 in the greenhouse. The two resistance genes were separated in two F₃₄ lines, LR1 and LR2, derived from Essex x PI486355. F₁ individuals from the crosses of LR1 and LR2 with Lee 68, Ogden, and York were tested with SMV-G7 in the greenhouse; the F₂ populations were tested with SMV-G1 and G7. The results revealed that expression of the gene in LR1 is gene-dosage dependent, with the homozygotes conferring resistance but the heterozygotes showing systemic necrosis to SMV-G7. This gene was shown to be an allele of the Rsv1 locus and was designated as Rsv1-s. It is the only allele identified so far at the Rsv1 locus which confers resistance to SMV-G7. Rsv1-s also confers resistance to SMV-G1 through G4, but results in systemic necrosis with SMV-G5 and G6. The gene in LR2 confers resistance to strains SMV-G1 through G7 and exhibits complete dominance. It appears to be epistatic to genes at the Rsv1 locus, inhibiting the expression of the systemic necrosis conditioned by the Rsv1 alleles. SMV-G7 induced a pin-point necrotic reaction on the inoculated primary leaves in LR1 but not in LR2. The unique genetic features of the two resistance genes from PI486355 will facilitate their proper use and identification in breeding and contribute to a better understanding of the interaction of SMV strains with soybean resistance genes.     

Amplified fragment length polymorphism (AFLP): a review of the procedure and its applications

Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter and restriction site sequence, with additional nucleotides at the 3′-end, are used as selective agents to amplify a subset of ligated fragments. Polymorphisms are identified by the presence or absence of DNA fragments following analysis on polyacrylamide gels. This technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for the positional cloning of genes of interest. However, its application is rapidly expanding in bacteria and higher eukaryotes for determining genetic relationships and for epidemiological typing. This review describes the AFLP procedure, and recent, novel applications in the molecular fingerprinting of DNA from both eukaryotic and prokaryotic organisms. Received 19 December 1997/ Accepted in revised form 3 June 1998     

Evaluation of soybean RFLP marker diversity in adapted germ plasm

Summary Soybean RFLP markers have been primarily developed and genetically mapped using wide crosses between exotic and adapted genotypes. We have screened 38 soybean lines at 128 RFLP marker loci primarily to characterize germ plasm structure but also to evaluate the utility of RFLP markers identified in unadapted populations. Of these DNA probes 70% detected RFLPs in this set of soybean lines with an average polymorphism index of 0.30. This means that only 1 out of 5 marker loci was informative between any particular pair of adapted soybean lines. The variance associated with the estimation of RFLP genetic distance (GD_R) was determined, and the value obtained suggested that the use of more than 65–90 marker loci for germ plasm surveys will add little precision. Cluster analysis and principal coordinate analysis of the GD_R matrix revealed the relative lack of diversity in adapted germ plasm. Within the cultivated lines, several lines adapted to Southern US maturity zones also appeared as a separate group. GD_R data was compared to the genetic distance estimates obtained from pedigree analysis (GD_P). These two measures were correlated with r = 0.54 for all 38 lines, but the correlation increased to r = 0.73 when only adapted lines were analyzed.     

RFLP mapping using near-isogenic lines in the soybean [Glycine max (L.) Merr.]

Summary A molecular marker analysis of a near-isogenic line (NIL), its donor parent (DP), and its recurrent parent (RP) can provide information about linkages between molecular markers and a conventional marker introgressed into the NIL. If the DP and RP possess different alleles for a given molecular marker, and if the NIL possesses the same allele as the DP, then it is reasonable to presume a linkage between that molecular marker and the introgressed marker. In this study, we examined the utility of RFLPs as molecular markers for the NIL genemapping approach. The allelic status of fifteen RFLP loci was determined in 116 soybean RP/NIL/DP line sets; 66 of the Clark RP type and 50 of the Harosoy RP type. Of the 1740 possible allelic comparisons (116 NILs x 15 RFLP loci), 1638 were tested and 462 (33.9%) of those were informative (i.e., the RP and DP had different RFLP alleles). In 15 (3.2%) of these 462 cases the NIL possessed the DP-derived RFLP allele, leading to a presumption of linkage between the RFLP locus and the introgressed conventional marker locus. Two presumptive linkages, pK-3 — and pK-472 — Lf _i, were subsequently confirmed by cosegregation linkage analysis. Although not yet confirmed, two other associations, pk-7 ab and pK-229 — y ₉ seemed to be plausible linkages, primarily because the pk-7 — ab association was detected in two independently derived NILs and both markers of the pK-229 — y ₉ association were known to be linked to Pb. The data obtained in this investigation indicated that RFLP loci were useful molecular markers for the NIL gene-mapping technique.Published as Paper no. 9101, Journal Series, Nebraska Agric. Res. Div. Project no. 12-091. Research partially funded by a grant from the Nebraska Soybean Development, Utilization, and Marketing Board     

扩增片段长度多态性实验技术及在动物遗传多样性研究中的应用

扩增片段长度多态性(AFLP)是一种有效的分子遗传标记方法,具有经济、简便、模板需要量少、重复性高、结果可靠等优点。目前AFLP在动物方面的应用还不是很多,处于初级阶段,主要用于鉴定分类关系、种群遗传多样性分析、遗传连锁图谱构建等方面。     

Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.     

Restriction fragment length polymorphism evaluation of six peanut species within the Avachis section

Summary Restriction fragment length polymorphisms (RFLP) were assessed among accessions within six peanut species of the Arachis section: tetraploid cultivated species, A. hypogaea; tetraploid wild species, A. monticola; and four diploid wild species, A. batizocoi,A. cardenasii, A. duranensis and A. glandulifera. While the two tetraploid species did not show polymorphism with 16 PstI-generated random genomic probes, two of seven seed cDNA probes detected polymorphisms. The RFLP variation detected by two seed cDNA probes appeared to be related to structural changes occurring within tetraploid species. The botanical var. fastigiata (Valencia market type) of A. hypogaea subspecies fastigiata was shown to be the most variable. Arachis monticola was found to be more closely related to A. hypogaea subspecies hypogaea than to subspecies fastigiata. Diploid species A. cardenasii, A. duranensis, and A. glandulifera showed considerable intraspecific genetic diversity, but A. batizocoi showed little polymorphism. The genetic distance between the cultivated peanut and wild diploid species was found to be closest for A. duranensis.Florida Agricultural Experiment Station, Journal Series No. R-01493     

Genetic diversity and population structure of spottedtail goby (Synechogobius ommaturus) based on AFLP analysis

Larval dispersal may have an important impact on genetic structure of benthic fishes. To examine population genetic structure of spottedtail goby Synechogobius ommaturus, samples from five different locations of China and Kunsan population in Korea were analyzed by using amplified fragment length polymorphism (AFLP) technology. A total of 253 bands were identified from 91 individuals by 5 primer combinations and the percentage of polymorphic bands was 43.87%. The average gene diversity was 0.0794 ± 0.1470 and Shannon’s information index was 0.1279 ± 0.2138. The pairwise F_st values ranged from 0.022 to 0.201. The results of AMOVA analysis indicated that 90.54% of the genetic variation contained within populations and 9.46% occurred among populations. The gene flow estimates (Nm) demonstrated that different gene flow existed among populations from 0.994 to 11.114. No significant genealogical branches or clusters were recognized on the UPGMA tree. The results support the hypothesis that larval dispersal ability can be responsible for the genetic diversity and population structuring.     

Geographical diversity and genetic relationships among Cedrus species estimated by AFLP

Genetic diversity was described in 17 cedar populations covering the geographical range of the four species of the genus Cedrus. The study was conducted using amplified fragment length polymorphism (AFLP) on haploid tissues (megagametophytes). Eleven selective AFLP primer pairs generated a total of 107 polymorphic amplification products. Correspondence and genetic distance analyses indicated that Cedrus deodara constitutes a separate gene pool from the Mediterranean cedars. Within Mediterranean cedars, we distinguished two groups: the first one is made of Cedrus atlantica, while the second one is made of Cedrus libani and Cedrus brevifolia, these latter two species being genetically similar despite important divergence previously observed for morphological and physiological traits. The lowest intrapopulation variability was found in the two C. deodara populations analyzed. Surprisingly, C. brevifolia, the endemic taxon from the island of Cyprus that is found in small and fragmented populations, showed one of the highest levels of diversity. This unexpected pattern of diversity and differentiation observed for C. brevifolia suggests a recent divergence rather than a relictual, declining population. Patterns of diversity within- and among-populations were used to test divergence and fragmentation hypotheses and to draw conclusions for the conservation of Cedrus gene pools.     

AFLP标记的特点及其在昆虫学研究中的应用

扩增片段长度多态性（AFLP）是一种新兴的很有效的分子遗传标记方法, 它通过对基因组DNA限制性内切酶酶切片段进行选择性扩增而揭示多态性,具有快速、经济简便、不需要预先知道模板DNA的信息、模板需要量少、重复性高、结果可靠及具有很高的信息含量等优点。AFLP也具有缺点,主要是标记是显性的,同其他显性标记一样,不能区分杂合体和纯合体,因而不能更好地估算种群遗传的变异,对种群遗传结构的分析不能提供更多的统计信息;AFLP技术较复杂,而且经常使用放射性同位素,对模板DNA质量要求也较高。为了克服AFLP的这些缺点,人们又在其基础上发展了其他相关技术,例如AFRP、SAMPL、DALP和TE-AFLP等。目前AFLP在昆虫方面的应用还不是很多,处于初级阶段,主要应用在生态型鉴定、种群遗传分析、连锁图谱构建等方面,相信随着其技术的发展完善,必将会越来越多地应用于昆虫学的研究中。     

Genetic analysis of field germination in soybean (Glycine max. (L) merill.)

Summary Genetic analysis for germination percentage was carried out in the F₃ and F₄ generations of a diallel cross involving six promising genotypes of soybean. Results indicated a high amount of genetic variability and a moderately high heritability together with genetic advance, suggesting a possible improvement for this character through hybridization and selection. Correlations at different levels revealed a strong negative association of germination with only one seed character: seed weight. This observation was further confirmed from path coefficient analysis. These findings strongly suggest that to base selection on seed weight which may not influence the seed quality of soybean.     

Amplified fragment length polymorphism (AFLP) as suitable markers to study orobanche cumana genetic diversity

Orobanche cumana Wallr., an obligate root parasite of sunflower can cause severe damage to this crop. The genetic diversity obtained with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) on two Orobanche populations were compared. Nei and Li distance matrices obtained with both methods among the two populations were correlated significantly according to Mantel's test and could partition the populations. The sampling variance of genetic distances within and among populations estimated using bootstrap procedure were not significantly different between the two techniques. The principal difference between the two techniques is that AFLP markers gave a higher degree of resolution for discriminating closely related germplasm than RAPD.     

Amplified fragment length polymorphism (AFLP) analysis of genetic variation in Moringa oleifera Lam

Moringa oleifera is an important multipurpose tree introduced to Africa from India at the turn of this century. Despite limited knowledge of the levels of genetic diversity and relatedness of introduced populations, their utilization as a source of seed for planting is widespread. In order to facilitate reasoned scientific decisions on its management and conservation and prepare for a selective breeding programme, genetic analysis of seven populations was performed using amplified fragment length polymorphism (AFLP) markers. The four pairs of AFLP primers ( Pst I/ Mse I) generated a total of 236 amplification products of which 157 (66.5%) were polymorphic between or within populations. Analysis of molecular variance ( AMOVA ) revealed significant differences between regions and populations, even though outcrossing perennial plants are expected to maintain most variation within populations. A phenetic tree illustrating relationships between populations suggested at least two sources of germplasm introductions to Kenya. The high levels of population differentiation detected suggest that provenance source is an important factor in the conservation and exploitation of M. oleifera genetic resources.