An AFLP marker that differentiates biotypes of the Asian rice gall midge (Orseolia oryzae, Wood-Mason) is sex-linked and also linked to avirulence

In an attempt to identify a specific marker for biotype 2 of the Asian rice gall midge (Orseolia oryzae, Wood-Mason), we used AFLP (amplified fragment length polymorphism) fingerprinting. We identified an AFLP marker that is specifically amplified in biotypes 1, 2 and 5 of the rice gall midge, but not in biotype 4. Biotypes 1, 2 and 5 are avirulent to hosts bearing the Gm2 resistance gene (found in rice variety Phalguna), whereas biotype 4 is virulent to Gm2. Based on the sequence of this AFLP marker, SCAR (sequence characterized amplified region) primers were designed and used in combination with previously developed SCAR primers to distinguish effectively all five biotypes in a multiplex PCR-based assay. The inheritance pattern of this marker in the progenies of inter-biotype crosses between biotypes 1, 2 and 4 shows that the marker can be amplified by PCR from all F₁ females, irrespective of the biotype status of their parents. However, the marker is present only in those male progenies whose mother was of a Gm2 avirulent biotype. The specific amplification of this marker in the avirulent biotypes and its pattern of inheritance show that avirulence with respect to carriers of the Gm2 gene in rice gall midge is sex-linked. Received: 16 August 1999 / Accepted: 27 December 1999     

Identification and potential use of a molecular marker for rust resistance in common bean

Summary The Up ₂ gene of common bean (Phaseolus Vulgaris L.) is an important source of dominant genetic resistance to the bean rust pathogen [Uromyces appendiculatus (Pers. ex Pers.) Unger var appendiculatus [syn U. Phaseoli (Reben) Wint.]. Up ₂ in combination with other rust resistance genes may be used to obtain potentially stable genetic resistance. It is difficult, however, to combine rust resistance genes effective against a single race due to epistatic interactions that frequently occur between them. A strategy that employed bulked DNA samples formed separately from the DNA of three BC₆F₂ individuals with Up ₂ and three without Up ₂ as contrasting near-isogenic lines (NILs) was used to identify random amplified polymorphic DNA fragments (RAPDs) tightly linked to the Up ₂ locus. Only 1 of 931 fragments amplified by 167 10-mer primers of arbitrary sequence in the polymerase chain reaction (PCR) was polymorphic. The RAPD marker (OA14₁₁₀₀) amplified by the 5-TCTGTGCTGG-3 primer was repeatable and its presence and absence easy to score. No recombination was observed between OA14₁₁₀₀ and the dominant Up ₂ allele within a segregating BC₆F₂ population of 84 individuals. This result suggests that OA14₁₁₀₀ and Up ₂ are tightly linked. Andean and Mesoamerican bean germ plasm, with and without the Up ₂ allele, were assayed for the presence of OA14₁₁₀₀. Apparently, the marker is of Andean origin because all Andean lines, with or without the Up ₂ allele, contained the marker, and the marker was absent in all Mesoamerican germ plasm except the lines to which Up-2 had been purposely transferred. These results suggest that OA14₁₁₀₀ will be most useful for pyramiding Up ₂ with other rust resistance genes into germ plasm of Mesoamerican origin where the marker does not traditionally exist. The use of bulked DNA samples may have concentrated resources toward the identification of RAPDs that were tightly linked to the target locus. Marker-based selection may provide an alternative to the time-consuming testcrosses required to pyramid bean rust resistance genes that exhibit epistasis.Research supported by the Michigan Agricultural Research Station and the USDA-ARS. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable     

Patterns of inheritance with RAPD molecular markers reveal novel types of polymorphism in the honey bee

Summary The polymerase chain reaction (PCR) was used to generate random amplified polymorphic DNA (RAPD) from honey bee DNA samples in order to follow the patterns of inheritance of RAPD markers in a haplodiploid insect. The genomic DNA samples from two parental bees, a haploid drone and a diploid queen, were screened for polymorphism with 68 different tennucleotide primers of random sequence. Parents were scored for the presence or absence of individual bands. An average of 6.3 bands and 1.3 polymorphisms for presence/absence were observed per primer between the parents. Thirteen of these primers were used to determine the inheritance of RAPD marker alleles in the resulting progeny and in haploid drones from a daughter queen. Four types of polymorphisms were observed. Polymorphisms for band presence/absence as well as for band brightness were inherited as dominant markers, meeting Mendelian expectations in haploid and diploid progeny. Polymorphisms for fragment-length were also observed. These segregated in a near 11 ratio in drone progeny. The last type of polymorphism was manifested as a diploid-specific band. Mixing of amplification products after PCR showed that the diploid-specific band was the result of heteroduplex formation from the DNA of alternate alleles in heterozygotes. In two of the four cases of heteroduplex formation, the alternative alleles were manifested as small fragment-length polymorphisms, resulting in co-dominant markers. This is the first demonstration that a proportion of RAPD markers are not inherited in a dominant fashion.     

Identification of a codominant amplified polymorphic DNA marker linked to the verticillium wilt resistance gene in tomato

Resistance to verticillium wilt, a vascular disease causing yield losses in many crops, is conferred in tomato by a single dominant allele, Ve. A population segregating for the Ve allele was generated using near-isogenic tomato lines. Analysis of the parental tomato DNA using the polymerase chain reaction and 400 random primers, each 10 deoxyribonucleotides in length, produced 1,880 amplified DNA fragments. Of the four polymorphisms observed between the resistant and susceptible parental genotypes, only one was linked to the Ve gene. No recombination was observed between this DNA marker and the Ve locus, indicating that the linkage is less than 3.5±2.7 cM. The marker detected both the susceptible and resistant alleles, producing amplified DNA fragments of approximately 1,300 and 1,350 bp, respectively. The sequence of the primer, determined from cloned amplified products, was 5 CTCACATGCA 3 instead of the expected 5 CTCACATGCC 3. The marker will be of value to tomato breeding programs because of the tight linkage, Codominant nature, and analytical procedure utilized.     

A molecular marker associated with the H21 Hessian fly resistance gene in wheat

Near-isogenic lines in conjunction with bulked segregant analysis were used to identify a DNA marker in wheat (Triticum aestivum L.) associated with the H21 gene conferring resistance to biotype L of Hessian fly [Mayetiola destructor (Say)] larvae. Near-isogenic lines were developed by backcross introgression BC3F3:4 (Coker 797 * 4 / Hamlet) and differed by the presence or absence of H21 (on 2RL) derived from Chaupon rye (Secale cereale L.). Bulked DNA samples were prepared from near-isogenic lines and BC3F2 population individuals segregating for reaction to Hessian fly biotype L and screened for random amplified polymorphic DNA markers using 46 10mer primers. Random-amplified polymorphic DNA markers from resistant and susceptible individuals and parental lines were scored and these data were used to identify a 3 kb DNA fragment that was related to the occurrence of H21. This fragment was amplified from DNA isolated from Hamlet, a near-isogenic line carrying 2RL, and bulked DNA from resistant BC3F2 individuals, but not from the recurrent parent Coker 797 or DNA bulks from susceptible BC3F2 plants. Analysis of 111 BC3F2 segregating individuals and BC3F2:3 segregants confirmed the co-segregation of the 3 kb DNA marker with the H21 resistance gene to Hessian fly. Use of this marker could facilitate more rapid screening of plant populations for Hessian fly resistance and monitoring the introgression of H21.     

Molecular variation and population structure in Elymus trachycaulus and comparison with its morphologically similar E. alaskanus

Random amplified polymorphic DNA (RAPD) markers were used to determine the levels and pattern of molecular variation in four populations of Elymus trachycaulus, and to estimate genetic similarity among different populations of E. trachycaulus from British Columbia and the Northwest Territories and one population of Elymus alaskanus from the Northwest Territories. Based on 124 RAPD bands (loci), mean percent polymorphic loci for E. trachycaulus (P_P) was 67.4% (a range 41.2% to 86.3%), and mean gene diversity (H_e) for E. trachycaulus species was 0.23 (range 0.18 to 0.27). The total genetic diversity was 0.32. Differentiation among populations was 31% (F_ST = 0.31) with most of the genetic variation found within populations (69%). This pattern of genetic variation was different from that reported for inbred species in general.The authors are very grateful to Michael Bond for excellent Laboratory assistance, to Dr. Mary Barkworth for her encouragement. This study was supported by a Natural Science and Engineering Research Council (NSERC) discovery grant and by a Saint Marys University Internal grant to G.S.