Production and Molecular Identification of Interspecific Hybrids between Phaius mishmensis (Lindl. and Paxton) Rchb. f. and Phaius tankervilliae (Banks) Blume

This study aimed at assessing the hybridization feasibility and evaluating genetic fidelity of the hybrid seedlings originated from Phaius mishmensis (Lindl. and Paxton) Rchb. f. and P. tankervilliae (Banks) Blume. Intra- and interspecific hybridization between Phaius mishmensis (Lindl. and Paxton) Rchb. f. and P. tankervilliae (Banks) Blume were examined to establish the primary hybrid, observe their cross ability and identify the F1 hybrids using sequence-characterized amplified region (SCAR) markers. Self-incompatibility and cross ability of P. mishmensis and P. tankervilliae were tested before starting the breeding program. Results showed that they were self-compatible orchids. The interspecific hybridization between P. mishmensis and P. tankervilliae was achieved with the highest pod setting (80%), seed germination percentage (94.8%) and the rate of protocorm development into mature seedlings (stage 6) (10.6%), but the smallest size of embryo with width 46.5 μm, length 67.3 μm was also observed when P. mishmensis was taken as the female parent. A comparative study on leaf morphology and anatomy of plantlets regenerated from intra- and interspecific hybrids of P. mishmensis and P. tankervilliae showed a transitional character to the parental species. Herein, the presence of interspecific hybrids between P. mishmensis and P. tankervilliae, as well as their reciprocal cross, was verified using Pmis524 SCAR markers developed by the decamer primer.

A New and Improved Automated Technology for Early Sex Determination of Ginkgo biloba

Random amplified polymorphic DNA (RAPD) technique with male associated decamer primer S1478 was used to amplify DNA from 72 leaf samples collected from Ginkgo biloba trees with known sexual determinism in the canton of Geneva, Switzerland. This marker was found to be male-specific and was lacking in all female plants. Automated random polymorphic DNA analysis (ARPA), a new automated technology developed in the frame of this work, proved highly effective in distinguishing males and females with 100% efficiency and successful in male and female discrimination from a collection of young seedlings derived from a sexual cross. Our findings provide unambiguous evidence that ARPA combined with the male-associated decamer primer S1478 could be considered an efficient, rapid, and easy method to make an early sex determination in the dioecious tree Ginkgo biloba.

Genetic mapping in Eucalyptus urophylla and Eucalyptus grandis using RAPD markers

Two single-tree linkage maps were constructed for Eucalyptus urophylla and Eucalyptus grandis, based on the segregation of 480 random amplified polymorphic DNA (RAPD) markers in a F1 interspecific progeny. A mixture of three types of single-locus segregations were observed: 244 1:1 female, 211 1:1 male, and 25 markers common to both, segregating 3:1. Markers segregating in the 1:1 ratio (testcross loci) were used to establish separate maternal and paternal maps, while markers segregating in the 3:1 ratio were used to identify homology between linkage groups of the two species maps. An average of 2.8 polymorphic loci were mapped for each arbitrary decamer primer used in the polymerase chain reaction. The mean interval size beween framework markers on the maps was 14 cM. The maps comprised 269 markers covering 1331 cM and 236 markers covering 1415 cM, in 11 linkage groups, for E. urophylla (2n = 2x = 22) and E. grandis (2n = 2x = 22), respectively. A comparative mapping analysis with two other E. urophylla and E. grandis linkage maps showed that RAPDs could be reliable markers for establishing a consensus species map. RAPD markers were automatically and quantitatively scored with an imaging analyzer. They were classified into four categories based on their optical density. A fragment intensity threshold is proposed to optimize the selection of reliable RAPD markers for future mapping experiments. Key words : genetic linkage map, Eucalyptus urophylla, Eucalyptus grandis, random amplified polymorphic DNA, RAPD, automated data collection.

RAPD Markers Flanking the Are Gene for Anthracnose Resistance in Common Bean

Incorporation of the dominant gene Are, of Middle American origin, into commercial cultivars of Phaseolus vulgaris L., has been the main disease control strategy of plant breeders to limit the potential damage of Colletotrichum lindemuthianum (Sacc. & Magnus.) Lams.-Scrib. A random amplified polymorphic DNA (RAPD) marker designated OQ41440, generated by a 5′-AGTGCGCTGA-3′ decamer primer, was found tightly linked in coupling with the Are gene. OQ41440 mapped at 2.0 ± 1.4 centimorgans (cM) from the Are allele in the Andean genetic background and at 5.5 ± 2.3 CM in the Middle American background. A second coupling phase RAPD marker B3551000, generated by the 5′-GTATGGGGCT 3′ primer mapped at 5.4 ± 2.3 cM from the Are allele in the Andean genetic background and at 7.7 ± 2.7 CM in the Middle American background. Based on a recombination distance of 7.0 ± 1.9 cM between the two markers, OQ41440 and B3551000 RAPDs appear to flank the Are gene. The bracketing molecular markers allowed tagging of the Are allele with a selection fidelity of 99%. Use of the OQ41440 and B3551000 RAPD markers for marker-based selection will afford the opportunity to retain the Are anthracnose resistance gene in bean germplasm, as other epistatic resistance genes are characterized, and incorporated into contemporary bean cultivars.