The Pea (Pisum sativum L.) Rogue Paramutation is Accompanied by Alterations in the Methylation Pattern of Specific Genomic Sequences

Abstract

Abstract:The spontaneous emergence among common pea (Pisum sativum L.) cultivars of off-type rogue plants exhibiting leaves with narrower and pointed leaflets and stipules and the non-Mendelian inheritance of this new phenotype were first described in the early 20th century. However, so far, no studies at the molecular level of this first identified case of paramutation have been carried out. In this study, we show for the first time that the pea rogue paramutation is accompanied by alterations in the methylation status of specific genomic sequences. Although, no significant differences were observed in the genome-wide DNA methylation in leaves of non-rogue cv. Onward in comparison to its rogue paramutant line JI2723, 22 DNA sequences were identified by methylation-sensitive amplified fragment length polymorphisms (MS-AFLP) analysis as differentially methylated in the two epigenomes. Mitotically inherited through all leaf tissues, the differential methylation patterns were also found to be meiotically inherited and conserved in pollen grains for 12 out of the 22 sequences. Fourteen of the sequences were successfully amplified in cDNA but none of them exhibited significant differential expression in the two contrasting epigenotypes. The further exploitation of the present research results on the way towards the elucidation of the molecular mechanisms behind this interesting epigenetic phenomenon is discussed.

A Non-Rogue Mutant Line Induced by ENU Mutagenesis in Paramutated Rogue Peas (Pisum sativum L.) Is Still Sensitive to the Rogue Paramutation

“…Progreta vs. the SRP1 mutant line of the methylation status of 22 sequences previously identified as differentially methylated in cv. Onward vs. its rogue counterpart line JI2723, was performed as previously described [10].…”

“…One hundred-twenty-five nanograms (125 ng) of each restricted DNA bulk were additionally digested under the same conditions with 1.25 U of EcoRI. An equal volume of a solution containing 5 pMol of EcoRI-adapter, 50 pMol of HpaII/MspI-adapter, 0.5 U of T4 DNA ligase, 2× ligase buffer (Fermentas) was added to each inactivated restriction reaction for ligation of MS-AFLP adapters [10] and the reaction left to proceed overnight at room temperature.…”

“…Amplification primers and PCR conditions for the assessment of the methylation status of the target sequences have been previously described in detail [10].…”

“…More recently, we obtained the first data regarding the differential expression of some genes in rogues vs. non-rogue plants [9] and found that the rogue phenotype is accompanied by specific alterations of the methylation status of specific genome sequence [10]. Strongly inherited through mitosis, the identified alterations in DNA methylation remained, in multiple cases, conserved through meiosis to the pollen grains, suggesting a transgenerational inheritance of these modifications [10].…”

Polyploidy-associated paramutation in Arabidopsis is determined by small RNAs, temperature, and allele structure

“…This led to its description as para-(resembling)-mutation or somatic gene conversion by the pioneers working on maize and tomato in the 1950's [1][2][3]. Even earlier genetic experiments with peas [4] are now interpreted as evidence of paramutation [5]. Numerous cases of paramutation have been reported in several species including metazoans and plants [reviewed in 6,7,8], indicating that paramutation is not just a bizarre exception but may be a general phenomenon among eukaryotes [9].…”