An integrated genetic linkage map for eucalypts using RFLP,RAPD and isozyme markers

An integrated genetic linkage map for E. nitens was constructed in an outbred three-generation pedigree. Analysis of 210 RFLP, 125 RAPD and 4 isozyme loci resulted in 330 markers linked in 12 linkage groups covering 1462 cM (n=11 in eucalypts). The 12th linkage group is comprised of only 5 markers and will probably coalesce with another linkage group when further linked loci are located. Co-dominant RFLP loci segregating in both parents were used to integrate linkages identified in the male and female parents. Differences in recombination frequencies in the two parents were observed for a number of pairs of loci, and duplication of sequences was identified both within and between linkage groups. The markers were distributed randomly across the genome except for the RFLPs in linkage group 10 and for some loci showing segregation distortion, which were clustered into three regions of the map. The use of a large number of co-dominant RFLP loci in this map enables it to be used in other pedigrees of E. nitens and forms a basis for the detection and location of QTL in E. nitens and other eucalypt species.     

Intra- and inter-specific variations in Lens revealed by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate intra- and interspecific variations in the genus Lens (lentil). Twenty cultivars of L. culinaris ssp. culinaris, including 11 microsperma (small-seeded) and nine macrosperma (large-seeded) types, and 16 wild relatives (four accessions each of L. culinaris ssp. orientalis, L. odemensis, L. nigricans and L. ervoides), were evaluated for genetic variability using a set of 40 random 10-mer primers. Fifty reproducibly scorable DNA bands were observed from ten of the primers, 90% of which were polymorphic. Genetic distances between each of the accessions were calculated from simple matching coefficients. A dendrogram showing genetic relationships between them was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). This study revealed that (1) expect for L. ervoides, the level of intraspecific variation in cultivated lentil is lower than that in wild species, (2) L. culinaris ssp. orientalis is the most likely candidate for a progenitor of the cultivated species, and (3) microsperma and macrosperma cultivars were indistinguishable by the RAPD markers identified here.     

Conversion of a RAPD-generated PCR product,containing a novel dispersed repetitive element,into a fast and robust assay for the presence of rye chromatin in wheat

Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye.     

Nature of 2n gamete formation and mode of inheritance in interspecific hybrids of diploid Vaccinium darrowi and tetraploid V. corymbosum

RAPD markers were used to determine the level of heterozygosity transmitted via 2n gametes from V. darrowi selection Florida 4B (Fla 4B) to inter-specific hybrids with tetraploid V. corymbosum cv Bluecrop. The tetraploid hybrid US 75 was found to contain about 70% of Fla 4B's heterozygosity, a value attributed to a first-division restitution (FDR) mode of 2n gamete production. Crossovers during 2n gamete formation were evidenced by the absence of 16 dominant alleles of Fla 4B in US 75, and direct tests of segregation in a diploid population involving Fla 4B. RAPD markers that were present in both Fla 4B and US 75 were used to determine the mode of inheritance in a segregating population of US 75xV. corymbosum cv Bluetta. Thirty-one duplex loci were identified which segregated in a 51 ratio, indicating that US 75 undergoes tetrasomic inheritance.     

An extended map of the sugar beet genome containing RFLP and RAPD loci

An updated map of sugar beet (Beta vulgaris L. ssp. vulgaris var altissima Doell) is presented. In this genetic map we have combined 248 RFLP and 50 RAPD loci. Including the loci for rhizomania resistance Rr1, hypocotyl colour R and the locus controlling the monogerm character M, 301 loci have now been mapped to the nine linkage groups covering 815 cM. In addition, the karyotype of some of the Beta vulgaris chromosomes has been correlated with existing RFLP and RAPD linkage maps.     

Analysis of genetic diversity in common loon Gavia immer using RAPD and mitochondrial RFLP techniques

We used random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLP) of mitochondrial cytochrome b (cyt b ) gene to evaluate the genetic diversity in common loon Gavia immer populations from two regions in the United States: New England (NE) and Michigan (MI). RAPD analysis with 18 primers showed 74% polymorphism in NE and 50% in MI loons (similarity coefficient F = 0.92). Although no population-specific markers were found, the frequencies of some RAPD bands varied between the two populations suggesting geographical differences. RFLP analyses with Bam HI enzyme and a 307-bp mitochondrial cyt b gene showed four haplotypes in the NE loon samples and two in the MI samples. The mtDNA haplotype diversity was 0.74 for NE and 0.51 for MI loons, supporting the RAPD data that NE loons have greater genetic diversity than MI loons.     

Population genetic response to microsite ecological stress in wild barley, Hordeum spontaneum

Genetic diversity was studied in six subpopulations (a total of 60 individuals) of wild barley, Hordeum spontaneum , the progenitor of cultivated barley, sampled from six stations located along a transect of 300 m across the two opposing slopes of 'Evolution Canyon', a Mediterranean microsite at Lower Nahal Oren, Mt Carmel. The two opposing slopes are separated by between 100 and 400 m and designated SFS (South-Facing Slope) and NFS (North-Facing Slope) with each having three equidistant test stations. The SFS, which receives up to 300% more solar radiation, is drier, ecologically more heterogeneous, fluctuating, and more stressful than the NFS. Analysis of 12 RAPD primers, representing a total of 51 putative loci, revealed a significant inter- and intraslope variation in RAPD band polymorphism. A significantly higher proportion of polymorphic RAPD loci was found amongst the subpopulations on the SFS (mean P = 0.909) than on the NFS (mean P = 0.682), on the basis of the presence and absence of 22 strong bands. Polymorphism generally increased upwards from the bottom to the top of the SFS (0.636, 0.773, 0.955) and NFS (0.409, 0.500, 0.545), respectively. Gametic phase disequilibria estimates, D, revealed SFS and NFS unique predominant combinations which sharply differentiated the two slopes and indicated that there is differential interslope selection favouring slope-specific multilocus combinations of alleles, or blocks of genes over tens to hundreds of meters. This suggests that selection overrides migration. RAPD polymorphism appears to parallel allozyme diversity which is climatically adaptive and driven by natural selection in the same subpopulations at the microsite.     

Sequence Tag Site and Host Range Assays Demonstrate that Radapholus similis and R. citraphilus are not Reproductively Isolated

Males of citrus-parasitic Radopholus citrophilus (FL1) were mated with non-citrus-parasitic R. similis (FL5) females. Progeny inherited a 2.4-kb sequence tag site (DK#1) and the ability to reproduce in citrus from the paternal parent (FLl); both traits were absent in the maternal line (FL5). The hybrid progeny produced offspring in roots of citrus seedlings over an 8-month period and therefore were considered reproductively viable. Genomic DNA hybridization studies indicated that one or more copies of DK#1 were present in R. citrophilus FL1. It is not likely that DK#1 represents a citrus parasitism gene because it was amplified from some burrowing nematode isolates that did not parasitize citrus and because DK#1 contains no open reading frames. Inability to reliably test individual nematodes for their ability to parasitize citrus was a constraint to obtaining F2 data required for definitive genetic characterization of citrus parasitism in burrowing nematodes, and alternate approaches will be required. Although the physical relationship of DK#1 and the citrus parasitism locus remains undefined, results of controlled mating studies using these parameters as genetic markers enabled us to identify hybrid F₁ progeny. Therefore, R. similis and R. citrophilus are not sibling species since gene flow between the two does not appear to be restricted via geographic isolation (sympatric in Florida) or by genetics.     

玉米CMS材料线粒体DNA遗传多型性的研究

选用１１×４＝４４个探针／酶组合,５０个（１０ｍｅｒ）随机序列引物对２５种不同胞质来源的ＣＭＳ玉米,５种正常胞质玉米线粒体ＤＮＡ进行ＲＦＬＰ和ＲＡＰＤ研究。研究结果表明：（１）４５％的探针／酶组合可检测到玉米线粒体ＤＮＡ的多型性,共表现１５种ＲＦＬＰ类型,其中Ｓ组ＣＭＳ材料内有７种,正常胞质材料内有２种;８０％的随机引物可检测到ＲＡＰＤ。（２）基于ＲＦＬＰ资料的聚类分析结果,可将３０种胞质明确地划分为Ｔ、Ｃ、Ｓ、Ｎ４组,其结果与恢复专效性测定结果一致。其中ｐＨＪ２－７－１／ＢａｍＨＩ的ＲＦＬＰ类型可成为利用ＲＦＬＰ技术进行胞质分组的鉴定体系。（３）“双”型胞质线粒体ＤＮＡ常表现Ｓ＋Ｃ胞质的ＲＦＬＰ图谱。     

Identification of somaclonal variants of sugarcane (Saccharum spp.) resistant to sugarcane mosaic virus via RAPD markers

Somaclonal variants resistant to sugarcane mosaic virus (SCMV) were obtained from susceptible sugarcane cv PR62258 through somatic embryogenesis by increasing the number of subcultures of the embryogenic callus tissue in MS medium with 3 mg/L 2,4-dichlorophenoxyacetic acid. Transfers were made at 30-day intervals for 1, 2 or 3 subcultures. Two somaclones, namely AT626 and BT627, were selected by their resistance to SCMV. These subclones have maintained the resistance trait over seven years of testing in the field. In this report we identified the somaclonal SCMV resistant variants from the maternal line and the nonresistant somaclones, using the RAPD technique.