Identification of Homozygous Male Plants by Quantitative Analysis of a Nucleotide Sequence Linked to the Sex-determination Locus in Asparagus officinalis L.

Abstract

Discrimination of homologous and heterozygous plants at the male-determination locus in asparagus (Asparagus officinalis L.) was examined by quantitative analyses of a diagnostic marker for male asparagus. The quantitative difference of a sequence, AspTaq1, derived from the Asp1-T7 diagnostic marker (Jamsari et al., 2004), was measured using the real-time monitoring PCR and normalized based on the quantitative difference of a sequence, AODEF-Taq1, derived from a single copy gene, AODEF. The normalized difference was higher in established supermales than in regular males grown from commercially available seeds. The averaged values in respective groups are in the approximate ratio of 2 : 1. Homozygous male (MM), heterozygous male (Mm), and female (mm) genotypes were obtained by selfing of an andromonoecious line. The progeny were divided into three groups by the normalized difference; plants showing high values and intermediate values were thought to be homozygous and heterozygous males, respectively. Significant amplification of AspTaq1 was not detected in several plants; therefore, they were judged to be females. Then, to identify supermales, pollen from male plants was crossed separately with female plants, and the sex of the next generation was determined. Pollen from two male plants showing high values of the normalized difference generated only males in the next generation, while pollen from 12 male plants showing intermediate values generated both female and male plants. Pollen from one male plants that showed intermediate values generated only male plants. The normalized difference in homozygous males was comparable to the value obtained using a recombinant sequence that contains an equal number of AspTaq1 and AODEF-Taq1. These results indicate that homozygous and heterozygous males can be identified by quantitative analysis of a nucleotide sequence linked to the sex-determination locus and that the use of an authentic sequence for calibration facilitates the discrimination process of those genotypes in asparagus plants.

Short communication. Employment of molecular markers to develop tetraploid “supermale” asparagus from andromonoecious plants of the landrace ‘Morado de Huétor’

“…1). The primers designed by Horiuchi et al (2011) amplif ied a fragment of 97 bp in all our samples, indicating that there were no false negatives in the amplification produced by Asp1-T7. These results indicate that the sex-linked marker Asp1-T7 can be successfully used to determine sex in the tetraploid landrace 'Morado de Huétor', shortening the time necessary to detect "supermales".…”

“…AB180962; Park et al, 2003) was used as a positive control for DNA quality to discard the presence of false negatives in the amplification of the sex-linked marker, Asp1-T7. This sequence was amplified using the primers designated by Horiuchi et al (2011) (AODEF-Taq1F and AODEF-Taq1R). We followed the protocols of Nakayama et al (2006) and Horiuchi et al (2011) for PCR procedures.…”

“…This sequence was amplified using the primers designated by Horiuchi et al (2011) (AODEF-Taq1F and AODEF-Taq1R). We followed the protocols of Nakayama et al (2006) and Horiuchi et al (2011) for PCR procedures.…”