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.     

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. Received: 10 September 1997 / Accepted: 10 December 1997     

Evaluation of inter-simple sequence repeat analysis for mapping in Citrus and extension of the genetic linkage map

Inter-simple sequence repeat (ISSR) analysis was evaluated for its usefulness in generating markers to extend the genetic linkage map of Citrus using a backcross population previously mapped with restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and isozyme markers. ISSR markers were obtained through the simple technique of PCR followed by analysis on agarose gels, using simple sequence repeat (SSR) primers. Optimization of reaction conditions was achieved for 50% of the SSR primers screened, and the primers amplified reproducible polymorphic bands in the parents and progeny of the backcross population. Mendelian segregation of the polymorphic bands was demonstrated, with an insignificant number of skewed loci. Most of the SSR primers produced dominant loci; however co-dominance was observed with loci derived from three primers. A new genetic map was produced by combining the segregation data for the ISSR markers and data for the RFLP, RAPD and isozyme markers from the previous map and creating genetic linkages among all the markers using JoinMap 2.0 mapping software. The new map has an improved distribution of markers along the linkage groups with fewer gaps, and marker order showed partial or complete conservation in the linkage groups. The incorporation of ISSR markers into the genetic linkage map demonstrates that ISSR markers are suitable for genetic mapping in Citrus. Received: 3 February 2000 / Accepted: 12 May 2000     

An AFLP-based genetic linkage map of channel catfish (Ictalurus punctatus) constructed by using an interspecific hybrid resource family

Catfish is the major aquaculture species in the United States. The hybrid catfish produced by crossing channel catfish females with blue catfish males exhibit a number of desirable production traits, but their mass production has been difficult. To introduce desirable genes from blue catfish into channel catfish through introgression, a genetic linkage map is helpful. In this project, a genetic linkage map was constructed using amplified fragment length polymorphism (AFLP). A total of 607 AFLP markers were analyzed using 65 primer combinations and an interspecific backcross resource family. A total of 418 AFLP markers were assigned to 44 linkage groups. Among the remaining 189 markers, 101 were not used because of significant segregation distortion, 29 were unlinked, and 59 were eliminated because they span very large distances. The 418 AFLP markers covered 1593 cM Kosambi. The AFLP markers showed a high level of clustering that appears to be related to certain primer combinations. This linkage map will serve as the basis for mapping a greater number of markers to provide a map with high enough resolution for it to be useful for selective breeding programs using introgression.     

Random amplified polymorphic DNA (RAPD) markers in hop, Humulus lupulus: level of genetic variability and segregation in F1 progeny

The level of genetic variation in 24 hop genotypes was studied using the recently developed technique for producing random amplified polymorphic DNAs (RAPDs). Of the 60 primers screened, eight produced polymorphic RAPD bands, 38 produced bands that were monomorphic for all genotypes and 19 did not produce any amplification product. It appeared that the level of polymorphism among the genotypes was generally low. Three of the primers, A11, A17 and C9, were used to determine the stability and segregation of RAPD markers in five families with a total of 182 F₁ progeny. The segregation ratios of these markers in the f₁ progeny suggested that they were inherited in a Mendelian manner. RAPD markers were stable and may be useful for the construction of linkage maps in hop.     

High‐density interspecific genetic linkage mapping provides insights into genomic incompatibility between channel catfish and blue catfish

Catfish is the leading aquaculture species in the United States. The interspecific hybrid catfish produced by mating female channel catfish with male blue catfish outperform both of their parent species in a number of traits. However, mass production of the hybrids has been difficult because of reproductive isolation. Investigations of genome structure and organization of the hybrids provide insights into the genetic basis for maintenance of species divergence in the face of gene flow, thereby helping develop strategies for introgression and efficient production of the hybrids for aquaculture. In this study, we constructed a high‐density genetic linkage map using the hybrid catfish system with the catfish 250K SNP array. A total of 26 238 SNPs were mapped to 29 linkage groups, with 12 776 unique marker positions. The linkage map spans approximately 3240 cM with an average intermarker distance of 0.25 cM. A fraction of markers (986 of 12 776) exhibited significant deviation from the expected Mendelian ratio of segregation, and they were clustered in major genomic blocks across 15 LGs, most notably LG9 and LG15. The distorted markers exhibited significant bias for maternal alleles among the backcross progenies, suggesting strong selection against the blue catfish alleles. The clustering of distorted markers within genomic blocks should lend insights into speciation as marked by incompatibilities between the two species. Such findings should also have profound implications for understanding the genomic evolution of closely related species as well as the introgression of hybrid production programs in aquaculture.     

冬枣×宁梨巨枣的子代SRAP鉴定及遗传多样性分析

应用SRAP分子标记方法对冬枣×宁梨巨枣的子代进行了分子鉴定及遗传多样性分析。采用构建基因池的方法对SRAP分子标记引物进行筛选,从88对引物中筛选出15对多态性好、主带清晰的引物,并对子代进行了真实性鉴定及多态性分析。结果表明:(1)15对引物共产生95个多态性条带,平均每对引物产生6.3个多态性条带,显示了较高的多态性比率。(2)80个子代中44个具有父本特征带,鉴定为真杂种。子代遗传多样性及UPGMA聚类分析表明,子代个体与亲本间的遗传相似系数在0.55~0.98之间,个体差异明显。该研究结果为枣树杂交育种提供了重要的分子证据。     

Comparative Assessment of Genetic Variability in the Populations of Endemic and Endangered Yellow Catfish, Horabagrus brachysoma (Teleostei: Horabagridae), Based on Allozyme, RAPD, and Microsatellite Markers

The comparative assessment of genetic diversity using allozymes, random amplified polymorphic DNA (RAPD), and microsatellite markers was conducted in endemic and endangered yellow catfish (Horabagrus brachysoma) sampled from three locations in Western Ghats river systems of India. Among the three markers, microsatellites show more polymorphism, having 100% polymorphic loci, whereas allozymes show the least (56%). In RAPD, 60.5% of fragments were polymorphic. Observed heterozygosity and F(ST) values were very high in microsatellites, compared with the other markers. Microsatellite and RAPD markers reported a higher degree of genetic differentiation than allozymes among the populations depicted by pairwise F(ST)/G(ST), AMOVA, Nei's genetic distance, and UPGMA dendrogram. The three classes of markers demonstrated striking genetic differentiation between pairs of H. brachysoma populations. The data emphasize the need for fishery management, conservation, and rehabilitation of this species.     

Efficiency of RFLP, RAPD, and AFLP markers for the construction of an intraspecific map of the tomato genome.

We have constructed a tomato genetic linkage map based on an intraspecific cross between two inbred lines of Lycopersicon esculentum and L. esculentum var. cerasiforme. The segregating population was composed of 153 recombinant inbred lines. This map is comprised of one morphological, 132 RFLP (restriction fragment length polymorphism, including 16 known-function genes), 33 RAPD (random amplified polymorphic DNA), and 211 AFLP (amplified fragment length polymorphism) loci. We compared the 3 types of markers for their polymorphism, segregation, and distribution over the genome. RFLP, RAPD, and AFLP methods revealed 8.7%, 15.8%, and 14.5% informative bands, respectively. This corresponded to polymorphism in 30% of RFLP probes, 32% of RAPD primers, and 100% of AFLP primer combinations. Less deviation from the 1:1 expected ratio was obtained with RFLP than with AFLP loci (8% and 18%, respectively). RAPD and AFLP markers were not randomly distributed over the genome. Most of them (60% and 80%, respectively) were grouped in clusters located around putative centromeric regions. This intraspecific map spans 965 cM with an average distance of 8.3 cM between markers (of the framework map). It was compared to other published interspecific maps of tomato. Despite the intraspecific origin of this map, it did not show any increase in length when compared to the high-density interspecific map of tomato.     

Comparative Evaluation of RAPD and AFLP Based Genetic Diversity in Brinjal (Solanum melongena)

The present investigation was carried out with an objective of evaluating genetic diversity in brinjal (Solanum melongena) using DNA markers. A total of 38 brinjal accessions including one wild-species, Solanum sisymbrifolium were characterized using random amplified polymorphic DNA (RAP D) and amplified fragment length polymorphism (AFLP) techniques. Out of 45 primers employed to generate RAPD profiles, reproducible patterns were obtained with 32 primers and 30 (93.7%) of these detected polymorphism. A total of 149 bands were obtained, out of which 108 (72.4%) were polymorphic. AFLP analysis was carried out using four primer combinations. Each of these primers was highly polymorphic. Out of 253 fragments amplified from these four primer combinations, 237 (93.6%) were polymorphic. The extent of pair-wise similarity ranged from 0.264 to 0.946 with a mean of 0.787 in RAPD, in contrast to a range of 0.103 to 0.847 with a mean of 0.434 in AFLP. The wild species clustered separately from the brinjal genotypes. In the dendrogram constructed separately using RAPD and AFLP markers, the brinjal genotypes were grouped into clusters and sub-clusters, and the varieties released by IARI remained together on both the dendrograms. All the 30 RAPD primers in combination and each of the four primer pairs in AFLP could distinguish the brinjal accessions from each other. AFLP was thus found to be more efficient than RAPD in estimation of genetic diversity and differentiation of varieties in brinjal.     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

RAPD和ISSR标记对水稻化感种质资源遗传多态性的分析

运用RAPD和ISSR技术分析水稻化感种质资源的遗传多态性。从供试材料中筛选到具有多态性的RAPD引物12条,ISSR引物7条。RAPD引物共扩增到85条清晰的多态性条带,多态性条带比率为69．4％。ISSR引物共扩增到34条清晰的多态性条带,多态性条带比率为53．0％。对两种标记结果进行UPGMA聚类分析,结果极其类似,呈极显著的正相关(r=0．74)。聚类结果表明,地理位置相近的品种聚为一类。部分具有较强化感作用潜力的水稻品种亲缘关系很近,表明控制其化感作用性状的基因可能是等位的相同基因。而部分化感作用潜力差异显著的水稻品种聚为一类,这是由于人类在长期高产品种的定向选择过程中,水稻化感作用性状不被注意而丢失,遗传基础日益狭窄的原因。     

Construction of Genetic Linkage Maps and Comparative Genome Analysis of Catfish Using Gene-Associated Markers

A genetic linkage map of the channel catfish genome (N = 29) was constructed using EST-based microsatellite and single nucleotide polymorphism (SNP) markers in an interspecific reference family. A total of 413 microsatellites and 125 SNP markers were polymorphic in the reference family. Linkage analysis using JoinMap 4.0 allowed mapping of 331 markers (259 microsatellites and 72 SNPs) to 29 linkage groups. Each linkage group contained 3–18 markers. The largest linkage group contained 18 markers and spanned 131.2 cM, while the smallest linkage group contained 14 markers and spanned only 7.9 cM. The linkage map covered a genetic distance of 1811 cM with an average marker interval of 6.0 cM. Sex-specific maps were also constructed; the recombination rate for females was 1.6 times higher than that for males. Putative conserved syntenies between catfish and zebrafish, medaka, and Tetraodon were established, but the overall levels of genome rearrangements were high among the teleost genomes. This study represents a first-generation linkage map constructed by using EST-derived microsatellites and SNPs, laying a framework for large-scale comparative genome analysis in catfish. The conserved syntenies identified here between the catfish and the three model fish species should facilitate structural genome analysis and evolutionary studies, but more importantly should facilitate functional inference of catfish genes. Given that determination of gene functions is difficult in nonmodel species such as catfish, functional genome analysis will have to rely heavily on the establishment of orthologies from model species.     

Applicability of inter-simple sequence repeat polymorphisms in wheat for use as DNA markers in comparison to RFLP and RAPD markers

 Inter-simple sequence repeat polymorphic DNA (ISSR) was evaluated for its applicability as a genetic marker system in wheat. PCR was carried out with primers that annealed to simple sequence repeats. The resultant products were subjected to agarose-gel electrophoresis, and the banding patterns were compared among six wheat accessions containing diploid, tetraploid, and hexaploid members. Out of 100 examined, 33 primers produced distinguishable as well as polymorphic bands in each of the six accessions. Although most of the primers that gave distinct bands (30 primers out of 33) contained dinucleotide repeats, each of the primers with tri-, tetra-, and penta-nucleotide motifs also yielded discrete bands. Primers based on (AC)_n repeats gave the most polymorphic bands. In total, 224 polymorphic bands were found in the comparison between Einkorn wheats whereas, on the average, 120 polymorphic bands were detected between common wheats. ISSR primers produced several times more information than RAPD markers. The extent of band polymorphism was similar to that of RFLP markers, and greater than that of RAPDs. The genetic relationships of wheat accessions estimated by the polymorphism of ISSR markers were identical with those inferred by RFLP and RAPD markers, indicating the reliability of ISSR markers for estimation of genotypes. These polymorphic bands are potential candidates as novel markers for use in linkage-map construction in wheat. The characteristic features of ISSR markers, i.e. polymorphism, generation of information and ease of handling, suggest their applicability to the analysis of genotypes as well as to the construction of PCR-based genome maps of wheats. Received: 15 September 1996 / Accepted: 25 October 1996     

Development of Polymorphic EST Markers Suitable for Genetic Linkage Mapping of Catfish

Expressed sequence tag (EST) markers are important for gene mapping and for marker-assisted selection (MAS). To develop EST markers for use in catfish gene mapping, 100 randomly picked complementary DNAs from the channel catfish (Ictalurus punctatus) pituitary library were sequenced. The EST sequences were used to design primers to amplify channel catfish and blue catfish (I. furcatus) genomic DNAs. Polymerase chain reaction products of the ESTs were analyzed to determine length polymorphism between the channel catfish and blue catfish. Eleven polymorphic EST markers were identified. Five of the 11 EST markers were from known genes and the other six were from unidentified ESTs. Seven ESTs were found to be associated with microsatellite sequences. Analysis of channel catfish gene sequences indicated highly biased codon usage, with 16 codons being preferably used. These codons were more preferably used in highly expressed ribosomal protein genes and in highly expressed pituitary hormone genes. G/C-rich codons are less used in channel catfish than those in other vertebrates suggesting AT-richness of the channel catfish genome. Received June 29, 1998; accepted March 29, 1999.     

云南普通野生稻遗传多样性和亲缘关系

野生稻（Oryza rufipogon）是稻属的重要组成部分,具有许多优良性状,是水稻遗传改良的天然基因库。本研究通过对形态学性状的观测,及ISSR和RAPDUPGMA聚类分析,将云南普通野生稻划分为4个类型,即元江类型、景洪紫杆直立型、景洪绿杆直立型和景洪匍匐型。在供试材料中筛选到具有多态性的ISSR和RAPD引物各11个,ISSR引物扩增出多态带113条,多态性条带比率（PPB）为82．26％,RAPD引物共扩增出多态性条带76条,PPB值为76．77％,两种分子标记的分析结果呈极显著正相关（r=0.951）。此外UPGMA聚类结果表明,云南普通野生稻不同类型与其它地区普通野生稻之间的遗传亲缘关系差异明显。     

AFLP-based genetic linkage map for the red flour beetle (Tribolium castaneum)

The red flour beetle (Tribolium castaneum) is a major pest of stored grain and grain products and a popular model species for a variety of ecological, evolutionary, and developmental biology studies. Development of a linkage map based on reproducible and highly polymorphic molecular markers would greatly facilitate research in these disciplines. We have developed a genetic linkage map using 269 amplified fragment length polymorphism (AFLP) markers. Ten previously known random amplified polymorphic DNA (RAPD) markers were used as anchor markers for linkage group assignment. The linkage map was constructed through genotyping two independent F(2) segregating populations with 48 AFLP primer combinations. Each primer combination generated an average of 4.6 AFLP markers eligible for linkage mapping. The length of the integrated map is 573 cM, giving an average marker resolution of 2.0 cM and an average physical distance per genetic distance of 350 kb/cM. A cluster of loci on linkage group 3 exhibited significant segregation distortion. We have also identified six X-linked and two Y-linked markers. Five mapped AFLP fragments were sequenced and converted to sequence-tagged site (STS) markers.     

利用RAPD分子标记对三组三系杂交水稻及亲本的遗传分析和鉴定

利用RAPD技术,从248个随机寡聚核苷酸（10bp）中筛选出13个引物能在供试的三组三系杂交水稻及亲本间扩增出43条稳定性较好的多态性片段,其中6个引物能在供试材料间扩增出20个强的多态性标记。利用这些标记能有效地区分各组合中不育系、保持系、恢复系和F1,并能看出各组合中不育系与保持系、不育系与恢复系、F1与亲本间的遗传关系。 Abstract:A total of 248 arbitrary 10-mer oligonucleotide primers were screened using RAPD (random amplified polymorphic DNA) techniques with the genome DNA of three groups of three-line hybrid rice and their parents.Thirteen primers produced 43 polymorphism fragments.Six primers of them produced 20 obviously repeatable polymorphic markers among rice lines tested.Using this RAPD markers,the hybrid rice combinations (sterile-line,maintainer-line,restorer-line and F1)can be effectively identified,and the genetic relationship among them can be shown.     

云南普通野生稻遗传多样性和亲缘关系

野生稻(Oryza rufipogon)是稻属的重要组成部分, 具有许多优良性状, 是水稻遗传改良的天然基因库。本研究通过对形态学性状的观测, 及ISSR和RAPD UPGMA聚类分析, 将云南普通野生稻划分为4个类型, 即元江类型、景洪紫杆直立型、景洪绿杆直立型和景洪匍匐型。在供试材料中筛选到具有多态性的ISSR和RAPD引物各11个, ISSR引物扩增出多态带113条, 多态性条带比率(PPB)为82.26%, RAPD引物共扩增出多态性条带76条, PPB值为76.77%, 两种分子标记的分析结果呈极显著正相关(r = 0.951)。此外UPGMA聚类结果表明, 云南普通野生稻不同类型与其它地区普通野生稻之间的遗传亲缘关系差异明显。     

利用RAPD和ISSR标记分析烤烟品种间遗传关系

利用RAPD和ISSR标记对22份烤烟(Nicotiana tabacumL.)品种进行了遗传关系研究。在RAPD分析中筛选到13个引物,共扩增出167条带,其中多态性带50条,多态性比率为29.9%;在ISSR分析中筛选出7个引物,共扩增出96条带,其中多态性带44条,多态性比率为45.8%。两种标记相结合估算出的品种间遗传相似系数在0.881~0.979之间,平均为0.933。单独基于RAPD标记和ISSR标记的聚类结果有一定差异;两种标记结合起来的聚类分析结果与系谱信息吻合程度更高。定向选择可能对烤烟品种间遗传关系有较大影响;国外引进品种与国内育成品种并未完全分开,表明分子水平的遗传关系和地理来源间缺乏必然联系。