In Arabidopsis hybrids and Hybrid Mimics up-regulation of cell wall biogenesis is associated with increased plant size

Hybrid breeding is of economic importance in agriculture for increasing yield, yet the basis of the heterosis is not well understood. In Arabidopsis, crosses between different accessions produce hybrids with varied levels of heterosis relative to parental phenotypes in biomass. In all hybrids the advantages of the F1 hybrid is lost in the F2 for both phenotypic uniformity and yield gain. Success in generating F5/F6 Hybrid Mimic from the cross between C24 and Landsberg erecta (Ler) demonstrated that the large plant phenotype of the F1 hybrids can be stabilized. Hybrid Mimics selection was applied to Wassilewskija (Ws)/Ler and Col/Ler hybrids. The two hybrids showing different levels of heterosis. At 30 DAS, the Col/Ler hybrid generated Hybrid Mimics with rosette diameter and fresh weight equivalent to the F1 hybrid; Ws/Ler Hybrid Mimics outperformed the F1 hybrids in both the rosette size and biomass. Transcriptome analysis revealed up-regulation of cell wall biosynthesis and expansion genes could be a common pathway in increased size in Arabidopsis hybrids and Hybrid Mimics. Intercross of two independent Hybrid Mimic lines can further increase the biomass gain. Our results encourage the use of Hybrid Mimics for breeding and for investigating the molecular basis of heterosis. Overall design: Arabidopsis hybrid seeds [Wassilewskija (Ws)/Landsberg erecta (Ler), Col/Ler and C24/Ler] were produced by hand-pollination between parental accessions. Ws/Ler and Col/Ler Hybrid Mimic lines and F7 small plant lines were produced from the recurrent selection protocol (Wang et al., 2015, Wang et al., 2017). Sterilized seeds were sown onto plates with Murashige and Skoog (MS) medium (Murashige and Skoog Basal Salts with minimal organics, Sigma-Aldrich, M6899) supplemented with 3% (wt/vol) sucrose and 0.8% wt/vol agar, pH5.7. Seeds were kept at 4 °C in the dark, then transferred into a growth room with conditions of 16h light (22°C)/8h dark (18°C) and light density at 120 -150 µmol photons m-2·s-1. Plate-grown seedlings were transferred into soil at 15 days after sowing. For the sample set of Ws/Ler system, the rosette leaves of 25 day-old parents Ws and Ler, two reciprocal hybrids Ws x Ler and Ler x Ws, seven Hybrid Mimic lines (WL_HM1-7) and two small lines (wl_sml1-2) were collected at time Zeitgeber Time (ZT) = 6-8 (ZT = 0 refers to dawn). For the same set of Col/Ler system, the rosette leaves of 25 day-old parents Col, two reciprocal hybrids Col x Ler and Ler x Col, six Hybrid Mimic lines (CL_HM1-6) and two small lines (cl_sml1-2) were collected. The transcriptomes of Hybrid Mimics CL_HM5 and CL_HM6 were excluded from the analysis due to unsatisfactory phenotypes of plant size at sampling day -25 DAS. For the parents and hybrids, three biological replicates were collected per plant line; rosette leaves from three plants of each genotype were pooled as one biological replicate. For each Hybrid Mimic and small line, rosette leaves of three siblings were collected separately as three biological replicates.