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The mechanism of sex determination in dioecious species of the genus Atriplex (Chenopodiaceae) has not been determined. This paper reports the discovery of a male-specific DNA fragment in the diploid dioecious species A. garrettii. DNA samples extracted individually from ten male and ten female plants were bulked by sex. Random amplified polymorphic DNA (RAPD) fragments were generated in the two bulks in order to identify markers that were polymorphic between male and female plants. A total of 158 decamer primers were tested. A 2075 base-pair (bp) male-specific DNA fragment generated with the OPAF-14 primer was identified. The fragment was cloned and partially sequenced and 24-mer primers that exclusively amplified this fragment were constructed. When 124 male plants, 126 female plants, and one hermaphroditic plant were tested individually, the male-specific 2075-bp DNA fragment was present in the hermaphrodite and all but one of the male plants, and was absent in all female plants. A smaller DNA fragment ([approximately] 1800 bp) that was homologous to the 2075-bp fragment was amplified from the single male plant that lacked the 2075-bp fragment. Cytogenetic analysis revealed no apparent heteromorphic sex chromosomes. These observations suggest that sex determination in A. garrettii is genetic, with no evidence of heteromorphic sex chromosomes. Key words: Atriplex; bulk segregant selection; Chenopodiaceae; dioecy; RAPD (random amplified polymorphic DNA) analysis; repetitive DNA; sex determination.

Karyotypes of six species (in ten populations) of the genus Serjania and one species of the genus Urvillea were studied using root tip mitotic metaphases. All species of Serjania were diploids with 2n = 24 chromosomes and the species of Urvillea was octoploid with 2n = 86 chromosomes. Analysis of the karyotypes of Serjania showed that the species are differentiated by chromosomic structural changes. Chromosome lengths showed great differences among the species that may be explained by chromosomal deletion or addition. The basic chromosomic number is x = 12 for Serjania and x = 11 for Urvillea. In Urvillea the evolutionary trend may be toward polyploidy followed by reductional aneuploidy. The Giemsa (C-band) staining technique was applied in Serjania; however, no clear blocks of heterochromatin were observed. This result agrees with the presence of semireticulate nuclei. The Nucleolar Organizing Region (NOR-banding) technique was applied in S. communis and S. laruotteana, which showed one and two pairs stained, respectively. Variation in the number of nucleoli was observed among the different populations of Serjania. This variation could be explained by the presence of small nucleolar organizer regions that occur scattered along the genome and are not detected by the silver staining methodology.

Dioecy is a common phenomenon among Atriplex species. However, a mechanism of sex determination has not been determined in this group. We discovered a male-specific RAPD DNA fragment of 2075 bp which was identified with the primer OPAF-14 (5'GGTGCGCACT3') in Atriplex garrettii. The 2075 bp fragment was cloned and sequenced. The DNA sequence of the 2075 bp fragment contains several direct repeats, but no inverted repeats. Two 24-mer oligonucleotype primers which exclusively amplified this fragment were designed and used as SCAR primers. Cytogenetic analysis revealed no apparent heteromorphic chromosome suggesting that sex determination in A. garrettii is genetic, with no evidence of heteromorphic sex chromosomes.

Analysis of chromosome morphology was performed in 23 annual and 31 perennial taxa of Atriplex. The taxa analized were predominantly diploid with 2n = 18 chromosomes. However, tetraploid and hexaploid were also common in both annual and perennial species. While annual Atriplex species were highly variable in their karyotype constitution, perennial species had, comparatively, very uniform chromosome patterns. Differences in chromosome morphology and in haploid chromosome length suggested the presence of interspecific variation for DNA content among annual Atriplex species. Karyotype analysis suggested that perennial Atriplex species have evolved predominantly by centromeric shifts and polyploidy while annual Atriplex species differed by addition and deletion of chromatin associated with structural chromosome changes and polyploidy.