a-Globin is encoded by the two adjacent genes, al and a2. Although it is clearly established that both a-globin genes are expressed, their relative contributions to a-globin messenger RNA (mRNA) and protein synthesis are not fully defined. Furthermore, changes that may occur in a-globin gene activity secondarily to the loss of function of one or more of these genes (a-thalassemia [Thall) have not been directly investigated. This study further defines the expression of the two human a-globin genes by determining the relative levels of al and a2 mRNA in the reticulocytes of normal individuals and in individuals heterozygous for the common 3.7-kilobase deletion within the a-globin gene cluster that removes the a2-globin gene (the rightward type aThal-2 deletion). To quantitate accurately the ratio of the two a-globin mRNAs, we have modified a previously reported S1 nuclease assay to include the use of 32P end-labeled probes isolated from al-and a2-globin complementary DNA recombinant plasmids. In individuals with a normal a-globin genotype (as determined by Southern blot analysis Iaa/aal), a2-globin mRNA is present at an average 2.8-fold excess to al. In individuals heterozygous for the rightward type a-Thal-2 deletion (-a/aa) the ct2/al mRNA ratio is 1:1. These results suggest that the loss of the a2-globin gene in the a-Thal-2 deletion is associated with a 1.8-fold compensatory increase al-globin gene expression.

Verticillium wilt (VW) of alfalfa is a soilborne disease causing severe yield loss in alfalfa. To identify molecular markers associated with VW resistance, we used an integrated framework of genome-wide association study (GWAS) with high-throughput genotyping by sequencing (GBS) to identify loci associated with VW resistance in an F1 full-sib alfalfa population. Phenotyping was performed using manual inoculation of the pathogen to cloned plants of each individual and disease severity was scored using a standard scale. Genotyping was done by GBS, followed by genotype calling using three bioinformatics pipelines including the TASSEL-GBS pipeline (TASSEL), the Universal Network Enabled Analysis Kit (UNEAK), and the haplotype-based FreeBayes pipeline (FreeBayes). The resulting numbers of SNPs, marker density, minor allele frequency (MAF) and heterozygosity were compared among the pipelines. The TASSEL pipeline generated more markers with the highest density and MAF, whereas the highest heterozygosity was obtained by the UNEAK pipeline. The FreeBayes pipeline generated tetraploid genotypes, with the least number of markers. SNP markers generated from each pipeline were used independently for marker-trait association. Markers significantly associated with VW resistance identified by each pipeline were compared. Similar marker loci were found on chromosomes 5, 6, and 7, whereas different loci on chromosome 1, 2, 3, and 4 were identified by different pipelines. Most significant markers were located on chromosome 6 and they were identified by all three pipelines. Of those identified, several loci were linked to known genes whose functions are involved in the plants’ resistance to pathogens. Further investigation on these loci and their linked genes would provide insight into understanding molecular mechanisms of VW resistance in alfalfa. Functional markers closely linked to the resistance loci would be useful for MAS to improve alfalfa cultivars with enhanced resistance to the disease.