The application of restriction landmark genome scanning method for surveillance of non-mendelian inheritance in f(1) hybrids

Abstract

We analyzed inheritance of DNA methylation in reciprocal F(1) hybrids (subsp. japonica cv. Nipponbare x subsp. indica cv. Kasalath) of rice (Oryza sativa L.) using restriction landmark genome scanning (RLGS), and detected differing RLGS spots between the parents and reciprocal F(1) hybrids. MspI/HpaII restriction sites in the DNA from these different spots were suspected to be heterozygously methylated in the Nipponbare parent. These spots segregated in F(1) plants, but did not segregate in selfed progeny of Nipponbare, showing non-Mendelian inheritance of the methylation status. As a result of RT-PCR and sequencing, a specific allele of the gene nearest to the methylated sites was expressed in reciprocal F(1) plants, showing evidence of biased allelic expression. These results show the applicability of RLGS for scanning of non-Mendelian inheritance of DNA methylation and biased allelic expression.

Figure 1

RLGS [MspI] (NotI-MspI-BamHI) patterns of rice genomic DNA. Comparison of Nipponbare and Kasalath patterns revealed Nipponbare and Kasalath specific spots. (a) Nipponbare pattern. Spots 200 and 231 were detected at diminished spot intensities and are indicated by closed arrowheads. (b) Kasalath pattern. Neither spots 200 nor 231 were not detected.

Figure 2

RLGS [MspI] (NotI-MspI-BamHI) combination patterns of the parents, their selfed progeny, and their reciprocal F₁ hybrids. Spot 200 (arrowhead) was detected in the [MspI] patterns (Figure 1) and [HpaII] (NotI-HpaII-BamHI) patterns (data not shown) of Nipponbare and its selfed progeny. The presence or absence of the spot segregated in both F₁ populations (NKF₁ and KNF₁). The spot intensity of this spot was half that of the others.

Figure 3

Location of spot 200. Schematic of the region of chromosome 11 containing the restriction enzyme sites located in the region 5′ to the transcription start site of the non-protein coding transcript (Os11g0417300). The DNA fragments were digested at the NotI (N) and MspI/HpaII (M) sites and fractionated by one dimensional electrophoresis. Next, the DNA fragments that were digested at the BamHI (B) sites were fractionated by two dimensional electrophoresis, which allowed detection of the B-N fragment as an RLGS spot. Spot 200 corresponds to the fragment between the N and B sites. The N and M sites were identified in the parental Nipponbare and Kasalath, and in both reciprocal hybrids. The B site was only absent in Kasalath, resulting in the absence of spot 200 in the RLGS pattern.

Figure 4

Expression analysis of the nearest gene to the methylated site. (a) RT-PCR showed that a non-protein coding transcript (Os11g0417300) was expressed in leaf blade and sheath of Nipponbare, Kasalath, NKF₁, and KNF₁ plants. (b) Sequence analysis of the RT-PCR products of the expressed Os11g0417300 allele. The single nucleotide polymorphism between Nipponbare (C) and Kasalath (T) is indicated in the RT-PCR products by arrowheads. Specific expression of the Nipponbare allele was confirmed by detection of base C in both NK5 and KN5 plants. (c) Sequence analysis of RT-PCR products of the expressed Os01g0327900 allele. The single nucleotide polymorphism in RT-PCR products between Nipponbare (C) and Kasalath (A) is indicated by arrowheads. Specific expression of the Kasalath allele was confirmed by detection of base A in both NK7 and KN10 plants.