Image_1_Genome-Wide Identification of TCP Family Transcription Factors in Medicago truncatula Reveals Significant Roles of miR319-Targeted TCPs in Nodule Development.pdf

<p>TCP proteins, the plant-specific transcription factors, are involved in the regulation of multiple aspects of plant development among different species, such as leaf development, branching, and flower symmetry. However, thus far, the roles of TCPs in legume, especially in nodulation are still not clear. In this study, a genome-wide analysis of TCP genes was carried out to discover their evolution and function in Medicago truncatula. In total, 21 MtTCPs were identified and classified into class I and class II, and the class II MtTCPs were further divided into two subclasses, CIN and CYC/TB1. The expression profiles of MtTCPs are dramatically different. The universal expression of class I MtTCPs was detected in all organs. However, the MtTCPs in CIN subclass were highly expressed in leaf and most of the members in CYC/TB1 subclass were highly expressed in flower. Such organ-specific expression patterns of MtTCPs suggest their different roles in plant development. In addition, most MtTCPs were down-regulated during the nodule development, except for the putative MtmiR319 targets, MtTCP3, MtTCP4, and MtTCP10A. Overexpression of MtmiR319A significantly reduced the expression level of MtTCP3/4/10A/10B and resulted in the decreased nodule number, indicating the important roles of MtmiR319-targeted MtTCPs in nodulation. Taken together, this study systematically analyzes the MtTCP gene family at a genome-wide level and their possible functions in nodulation, which lay the basis for further explorations of MtmiR319/MtTCPs module in association with nodule development in M. truncatula.</p

Construction of Whole Genome Radiation Hybrid Panels and Map of Chromosome 5A of Wheat Using Asymmetric Somatic Hybridization

<div><p>To explore the feasibility of constructing a whole genome radiation hybrid (WGRH) map in plant species with large genomes, asymmetric somatic hybridization between wheat (<em>Triticum aestivum</em> L.) and <em>Bupleurum scorzonerifolium</em> Willd. was performed. The protoplasts of wheat were irradiated with ultraviolet light (UV) and gamma-ray and rescued by protoplast fusion using <em>B. scorzonerifolium</em> as the recipient. Assessment of SSR markers showed that the radiation hybrids have the average marker retention frequency of 15.5%. Two RH panels (RHPWI and RHPWII) that contained 92 and 184 radiation hybrids, respectively, were developed and used for mapping of 68 SSR markers in chromosome 5A of wheat. A total of 1557 and 2034 breaks were detected in each panel. The RH map of chromosome 5A based on RHPWII was constructed. The distance of the comprehensive map was 2103 cR and the approximate resolution was estimated to be ∼501.6 kb/break. The RH panels evaluated in this study enabled us to order the ESTs in a single deletion bin or in the multiple bins cross the chromosome. These results demonstrated that RH mapping via protoplast fusion is feasible at the whole genome level for mapping purposes in wheat and the potential value of this mapping approach for the plant species with large genomes.</p> </div

Alignment of RH map to physical map.

<p>Comparison between wheat chromosome 5A RH linkage group (right) with deletion map (left). The solid lines between markers show the consistent order between RH map and deletion map. The broken lines show the inconsistent marker order between two maps.</p

Identification of hybrid cell clones.

<p>A: RAPD amplification pattern generated using primer <i>OPV-07</i>. W: Wheat; B: <i>B. scorzonerifolium</i>; 1–21: Regenerated cell clones; arrowhead: <i>B. scorzonerifolium</i> characteristic band; arrow: Wheat characteristic band; cell clone No.7 was identified as a hybrid due to the presence of both parental specific bands. B-E: GISH analysis. B: Wheat; C: <i>B. scorzonerifolium</i>; D: Hybrid cell clone No.10 in WB5K showing two chromosomal fragments of wheat inserted into the genome of <i>B. scorzonerifolium</i>; E: Hybrid cell clone No.7 in WB60 showing three recombined chromosomes; arrow: Chromosomal fragments of wheat; <i>Bar</i>: 5 µm. F–I: Differentiation of hybrid cell clones. F: The shoots regenerated from hybrid clone No.64 in WB120; G: The plantlet of intermediate type regenerated from hybrid cell clone No.88 in WB60; H: The plantlet resembled <i>B. scorzonerifolium</i> regenerated from hybrid cell clone No.41 in WB60; I: Diverse morphology of regenerated shoots in combination WB120; <i>Bar</i>: 1cm.</p

Genotyping of nuclear SSR markers and the RH map of wheat chromosome 5A.

<p>A: Genotyping of SSR loci <i>Xgwm186</i>, <i>Xbarc1</i>, <i>Xcfa2250</i> and <i>Xcfa2155</i> in RH panel. W: Wheat; B: <i>B. scorzonerifolium</i>; 47–138: Radiation hybrids; <i>M</i>: 100 bp DNA ladder; arrow: SSR-PCR products of wheat; Solid squares represent retained markers, open squares represent eliminated markers and asterisks represent undetermined data. B: Wheat chromosome 5A RH map (right) as compared with the genetic (left) and the deletion map (middle). The black lines on the deletion map refer to bins assigned to the chromosome 5A. The RH map has an estimated total length of 2103 cR covered by 68 SSR markers.</p

Consistency between RH map and virtual gene order of chromosome 5AL.

<p>The black lines above refer to deletion bins assigned to the chromosome 5AL. Each EST is represented by a bar and positioned according to the results of RH mapping. In total 46 ESTs are ordered on chromosome 5AL. The ESTs marked by arrowheads showed the inconsistent order with the virtual gene order.</p

Hybrid number,frequency and differentiation capacity of different hybridization combinations.

a<p>no differentiation ability, ^bdifferentiate into leaves and shoots, ^cdifferentiate into complete plantlet.</p

The somatic hybridization combinations between wheat and <i>B. scorzonerifolium</i>.

<p>The somatic hybridization combinations between wheat and <i>B. scorzonerifolium</i>.</p

Individual marker retention frequency and donor DNA retention frequency of wheat in radiation hybrids.

<p>A: Individual marker retention frequencies in 155 radiation hybrids derived from seven asymmetric hybridization combinations. Forty-two microsatellite loci across the wheat genome are listed to the left of the figure. B–D: Donor DNA retention frequency of wheat in hybrids. B: Average donor DNA retention frequencies of A, B, D genome of wheat in each hybridization combination; C: Average donor DNA retention frequencies of seven chromosome groups of wheat in each of hybridization combinations; D: Average donor DNA retention frequencies of seven asymmetric hybridization combinations and control combination.</p