Targeted development of a microsatellite marker associated with a true loose smut resistance gene in barley (Hordeum vulgare L.)

Microsatellite markers have many of the properties of an ideal marker, but development of microsatellite markers is tedious, time-consuming and expensive. In the past few years, great efforts have been made to develop, map and utilize microsatellite markers in various crops. It is still a major challenge to find a microsatellite marker associated with an economically important trait. In the present study we report on the targeted development of a microsatellite marker to a barley disease resistance gene. The method includes the following steps: (1) pooling DNA samples from a segregating population based on the principle of bulked-segregant analysis; (2) digesting the pooled DNAs and ligating adaptors; (3) selectively amplifying and identifying polymorphic microsatellites; and (4) developing primers for the microsatellite associated with the targeted trait. Using this method, a microsatellite marker associated with the true loose smut resistance gene (Un8) in the Harrington × TR306 doubled-haploid population was identified. This marker showed polymorphism in four breeding populations segregating for true loose smut resistance. In three of these populations, genetic distance between the microsatellite and the true loose smut resistance gene varied from 8.6 to 10.3 cM. Polymorphism of the microsatellite was tested among three disease resistant lines and 21 susceptible cultivars. Fourteen to eighteen of the 21 susceptible cultivars exhibited a polymorphism for the microsatellite with respect to at least one of the disease-resistant lines. This method for the targeted development of microsatellite markers should have widespread applicability and should efficiently provide highly polymorphic markers for use in breeding programs

Mapping of Barley (Hordeum vulgare L.) Beta -amylase Alleles in which an Amino Acid Substitution Determines Beta -amylase Isoenzyme Type and the Level of Free Beta -amylase

The three beta -amylase genes (Bmy1, 2 and 3) in cultivated barley were mapped to chromosomes 4HL, 2HL And 4HL respectively using RFLP analysis. No recombinants between Bmy1 andBmy3 were detected among 264 DH lines. Polymorphism of the Sd1 and Sd2 isoenzymes of beta -amylase co-segregated with the Bmy loci on chromosome 4HL in a doubled-haploid population of the cross Chebec (Sd2)×Harrington (Sd1). This locus also explained 90·5% of the variation in the level of free enzyme between the two parents. Two cDNAs ofbeta -amylase were isolated by RT-PCR from the developing grains of Harrington (Sd1) and Galleon (Sd2). Alignment of the deduced amino acid sequences identified three amino-acid substitutions between the Sd2 and Sd1 forms of beta -amylase (Arg115 – Cys, Asp165 – Glu, and Val430 – Ala). Three allele-specific PCR primer pairs based on the three amino acid substitutions were used to amplify the beta -amylase genes in genomic DNA of sixteen barley cultivars/lines. Only the Arg115(Sd2)/Cys(Sd1) substitution was consistent with the isoenzyme form. This amino acid replacement reduced the pI of the Sd1 beta -amylase consistent with the fact that the Sd2 form is more basic than the Sd1 form when separated by IEF. The mutation from Arg115 to Cys in the Sd1 form also provides one more -SH group to form S-S-bridges. As bound beta -amylase is linked to the insoluble proteins of the endosperm and its inhibitor via disulphide bridges this could explain the higher level of binding exhibited by Sd1 vs Sd2. Thus a single amino acid substitution determines both the isoenzyme type and beta -amylase binding