Sex-specific SCAR markers in the dioecious plant Rumex nivalis (Polygonaceae) and implications for the evolution of sex chromosomes

Abstract

We developed SCAR primers based on isolated and sequenced male-specific fragments as identified in an AFLP analysis of the dioecious plant Rumex nivalis. PCR amplification using these primers on females and males resulted in fragments exclusively present in males. Coamplification of the nuclear rDNA internal transcribed spacer 2 together with the male-specific fragment was applied as an internal control for successful PCR reactions to avoid false-negative sex scoring. With a length of about 164 bp, the AFLP fragment was of a similar size as the tandemly arranged, repetitive sequences of 180 bp located on the Y chromosomes of Rumex acetosa. The genetic distances between the Y-chromosomal sequences of R. nivalis and R. acetosa, both members of the section Acetosa, were substantial. We found intra-individual divergence among cloned sequences of the male-specific fragment in R. nivalis. The patterns of interspecific and intra-individual sequence variation found are in accordance with proposed modes of the evolution of sex chromosomes. Y chromosomes possibly arose only once in the genus Rumex and consist mainly of heterochromatic DNA. Due to the almost complete absence of selection on them, Y chromosomes are likely to accumulate large numbers of mutations.

Mechanisms Governing Sex-Ratio Variation in Dioecious Rumex Nivals

“…To determine whether seeds and vegetative individuals sampled from the field and in the glasshouse experiment (described below) were female or male, we used male-specific sequence-characterized amplified-regions (SCAR) primers following the methods detailed in Stehlik and Blattner (2004). To avoid false negative scoring of sex, we co-amplified the nuclear rDNA internal transcribed spacer 2 (ITS2) together with the male-specific fragment as an internal control for successful PCR reactions.…”

“…To avoid false negative scoring of sex, we co-amplified the nuclear rDNA internal transcribed spacer 2 (ITS2) together with the male-specific fragment as an internal control for successful PCR reactions. We subjected genomic DNA amounts of 3 ng to PCR in 12.5 l reaction volumes with 1.25 l 10ϫ PCR reaction buffer, 3 l of 10 mM MgCl 2 , 1 l of 10 mM dNTPs, 0.1 l of 25 pM of RnivYf and RnivYr (SCAR primers; Stehlik and Blattner 2004), 0.1 l of 5 pM ITS-B and ITS-D (Blattner 1999), 0.05 l 100ϫ BSA and 0.1 l 5 U/l Taq, adjusted with ddH 2 O. Cycling conditions were: 4 min at 94ЊC followed by 35 cycles of 45 sec at 92ЊC, 45 sec at 48ЊC, and 30 sec at 72ЊC and a final extension time of 10 min at 72ЊC. We visualized PCR products on 1.6% agarose gels stained with ethidium bromide.…”

“…At each site, we sampled plants from early-and late-melting snowbed microsites to investigate how both local conditions, as well as broader elevational gradients might influence sex-ratio variation. Having established that female-biased sex ratios are a general feature of flowering portions of R. nivalis populations, we then investigated the sex of vegetative individuals using male-specific sequence-characterized amplified-regions (SCAR) primers (Stehlik and Blattner 2004). This marker allowed us to determine whether sex ratios change during the species' life history.…”

Seed sexing revealed female bias in two Rumex species

“…Results obtained this way can be questioned, however, because of the lack of information about the sex of ungerminated seeds. Information on Y chromosome-specific DNA sequences in Rumex (Shibata et al 1999;Stehlik and Blattner 2004) has been used to develop more convenient PCR-based methods of sexing sorrel seeds. Korpelainen (2002) showed that the primary sex ratio in Finnish populations of R. acetosa did not significantly differ from 1:1, and Stehlik and Barrett (2005) and Stehlik et al (2008) revealed a small but evident female bias in seeds of alpine R. nivalis from Switzerland.…”

“…Korpelainen (2002) showed that the primary sex ratio in Finnish populations of R. acetosa did not significantly differ from 1:1, and Stehlik and Barrett (2005) and Stehlik et al (2008) revealed a small but evident female bias in seeds of alpine R. nivalis from Switzerland. The observed dissimilarity may conceivably be due to genetic differences between these plants: analysis of Y chromosome-specific sequences in R. nivalis showed a relatively large genetic difference between it and R. acetosa (Stehlik and Blattner 2004).…”