Interpretation of randomly amplified polymorphic DNA marker data for fingerprinting sweet potato (Ipomoea batatas L.) genotypes

In this paper we present a method for the generation of randomly amplified polymorphic DNA (RAPD) markers for sweet potato. These were applied to produce genetic fingerprints of six clonal cultivars and to estimate genetic distances between these cultivars. The level of polymorphism within the species was extremely high. From the 36-decamer random primers used, 170 fragments were amplified, of which 132 (77.6%) were polymorphic. Ten primers resulted in no detected amplification. Of the remaining 26 primers for which amplification was achieved, only one did not reveal polymorphism. Six primers used alone enabled the discrimination of all six genotypes. Pattern analysis, which employed both a classification and ordination method, enabled the grouping of cultivars and the identification of primers which gave greatest discrimination among the cultivars.     

Genetic transformation and regeneration of transgenic plants in grapevine (Vitis rupestris S.)

Isolated somatic embryos from petiole-derived callus cultures ofVitis rupestris Scheele have been employed in experiments on genetic transformation. Co-cultivation of somatic embryos during embryogenesis induction withAgrobacterium tumefaciens strain LBA4404, which contains the plasmid pBI121 carrying the neomycin phosphotranspherase and the-glucuronidase genes, produced transformed cellular lines capable of recurrent somatic embryogenesis. Precocious selection for high levels of kanamycin (100 mgl^-1) was an important part of our transformation protocol. Transformed lines still have strong-glucuronidase expression as well as stable insertion of the marker genes after 3 years of in-vitro culture, during which they have maintained their capacity to organize secondary embryos and to regenerate transgenic plants with an agreeable efficiency (13%).     

Genetic variation of trypsin and chymotrypsin inhibitors in pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives

Variation in the trypsin inhibitors (TIs) and the chymotrypsin inhibitors (CIs) among 69 pigeonpea [Cajanus cajan (L.) Millsp.] strains from a wide geographical distribution and among 17 accessions representing seven wild Cajanus species was studied by electrophoretic banding pattern comparisons and by spectrophotometric activity assays. The TI and CI electrophoretic migration patterns among the pigeonpea strains were highly uniform but varied in the inhibitor band intensities. The migration patterns of the inhibitors in the wild Cajanus species were highly species specific. The mean TI activity of pigeonpea strains (2279 units) was significantly higher than that of the wild Cajanus species (1407 units). However, the mean CI activity in the pigeonpea strains (62 units) was much lower than that in the wild species (162 units). Kenya 2 and ICP 9151 were the lowest and the highest, respectively, in both the TI and CI activities among all the pigeonpea strains used in this study. A highly-significant positive correlation was observed between the TI and CI activities. The Bowman-Birk type inhibitors with both TI and CI activities were identified in all the pigeonpea strains and also in the accessions of all the wild species except C. volubilis (Blanco) Blanco. The C. volubilis accession ICPW 169 was found to be null for both CI bands and CI activity. Environment, strain, and environment x strain interaction showed highly-significant effects on both the TI and CI activities. Growing the pigeonpea strains at a different environment from their area of adaptation increased TI and CI activities and also altered the maturity period.     

A simple derivation of the partitioning of genetic differentiation within subdivided populations

The genetic differentiation within a subdivided population can be partitioned into two proportions, one due to differentiation within sub-populations and the other due to differentiation among subpopulations. A simple mathematical derivation of this procedure, known as Nei's G _ST -statistics, is presented. The significance of considering the differing relative sizes of subpopulations is stressed. Possible fields of applications for breeders and conservationists who are concerned with the establishment of genetically diverse populations are mentioned.     

Inheritance of restriction fragment length polymorphisms and random amplified polymorphic DNAs in coastal Douglas-fir

A total of 225 new genetic loci [151 restriction fragment length polymorphisms (RFLP) and 74 random amplified polymorphic DNAs (RAPD)] in coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] have been identified using a three-generation outbred pedigree. The Mendelian inheritance of 16 RFLP loci and 29 RAPD loci was demonstrated based on single-locus segregation in a sample of F₂ progeny. One RFLP locus, PtIFG2025, showed segregation distortion. Probe pPtIFG2025 is a loblolly pine cDNA probe encoding for rbcS. The 16 RFLP loci and 23 allozyme loci were also assayed in a sample of 16 Douglas-fir seed-orchard clones. Allelism was determined at 11 of the 16 RFLP loci. RFLPs were able to detect slightly more variation (4.0 alleles per locus) than allozymes (3.1 alleles per locus). The inheritance of an additional 80 RAPD loci was determined based on haploid segregation analysis of megagametophytes from parent tree 013-1. Once 200–300 markers are identified and placed on a genetic map, quantitative trait loci affecting bud phenology will be mapped.     

Mapping of a Magnaporthe grisea locus affecting rice (Oryza sativa) cultivar specificity

Magnaporthe grisea causes rice blast, the most important fungal disease of rice. The segregation of genes controlling virulence of M. grisea on rice was studied to establish the genetic basis of cultivar specificity in this host-parasite interaction. Full-sib progeny and parent isolates Guy11 and 2539 of M. grisea were inoculated onto rice (Oryza sativa) cultivar CO39 and five near-isogenic lines (NILs) of CO39. Each NIL contained a different single gene affecting resistance to specific isolates of M. grisea. No differential interactions between NILs and progeny or parents were observed; parents and progeny pathogenic on CO39 were pathogenic on all five NILs. Segregation ratios of 101 full-sib progeny, 117 progeny from full-sib parents, and 109 backcross progeny, indicated a common single gene affecting pathogenicity on CO39 and the five NILs. A subset of the above 327 isolates (43 fullsib progeny, 37 progeny from full-sib parents, and 32 backcross progeny) were inoculated onto rice cultivar 51583; all were pathogenic, indicating that cultivar specificity to CO39 was segregating in this population of isolates. The locus controlling cultivar specificity, named avrCO39, was mapped to chromosome 1 using a subset of the progeny previously used to construct an RFLP map of M. grisea. The closest reported RFLP markers were 11.8 (estimated 260 kb) and 17.2 cM (estimated 380 kb) away and provide starting points on either side of the locus for a chromosome walk to clone the locus.     

Introgressions fromLycopersicon pennellii can improve the soluble-solids yield of tomato hybrids

RFLP-defined chromosome segments covering the entire tomato genome were introgressed from the wild green-fruited speciesLycopersicon pennellii into the cultivated tomato (L. esculentum cv M82; Eshed et al. 1992). SixL. pennellii chromosome segments were selected for a detailed evaluation based on previous observations of their effects on the two yield components, fresh tomato yield and total soluble-solids content (Brix). Differences in the quantitative traits measured between M82 and the introgression lines, or their hybrids with different inbred parents, can be attributed to the alien chromosome segments. Replicated field trials, grown at wide and dense spacing, identified three quantitative trait loci (QTLs) for solublesolids content on chromosomes 1, 5 and 7. In plants heterozygous for the chromosome-5 locus there was a 50% increase in soluble-solids yield in wide but not in dense spacing. Plants heterozygous for the chromosome-1 QTL/s were tested over a 2-year period, in three genetic backgrounds, and showed a significant 16% elevation in soluble-solids yield only in dense spacing. These results demonstrate that wild tomato germplasm can be used to improve the yield of the cultivated crop.     

Ecogeographical distribution and differential adaptedness of multilocus allelic associations in Spanish Avena sativa L.

We determined the nine-locus isozyme genotype of 267 landrace accessions of Avena sativa from 31 provinces of Spain. Our results establish that level of genetic variability is usually high both within and among accessions of this heavily self-fertilizing hexaploid grass and that multilocus genetic structure differs in various ecogeographical regions of Spain. We concluded that selection favoring different multilocus genotypes in different environments was the main integrating force that shaped the internal genetic structure of local populations as well as the overall adaptive landscape of A. sativa in Spain. Implications in genetic resource conservation and utilization are discussed.     

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.     

Linkages among RFLP,RAPD, isozyme,disease-resistance,and morphological markers in narrow and wide crosses of cucumber

An algorithm of automatic classification is proposed and applied to a large collection of perennial ryegrass wild populations from France. This method is based on an ascendant hierarchical clustering using the Euclidian distance from the principal components extracted from the variance-covariance matrix between 28 agronomic traits. A contiguity constraint is imposed: only those pairs of populations which are defined as contiguous are grouped together into a cluster. The definition of contiguity is based on a geostatistical parameter: the range of the variogramme, i.e. the largest distance above which the variance between pairs of population no longer increases. This method yields clusters that are generally more compact than those obtained without constraint. In most cases the contours of these clusters fit well with known ecogeographic regions, namely, for macroclimatic homogeneous conditions. This suggests that selective factors exert a major influence in the genetic differentiation of ryegrass populations for quantitatively inherited adaptive traits. It is proposed that such a method could provide useful genetic and ecogeographic bases for sampling a core collection in widespread wild species such as forage grasses.Institut National de la Recherche Agrononique