Biological and genomic analysis of a symbiotic nitrogen fixation defective mutant in Medicago truncatula

Abstract

Medicago truncatula has been selected as one of the model legume species for gene functional studies. To elucidate the functions of the very large number of genes present in plant genomes, genetic mutant resources are very useful and necessary tools. Fast Neutron (FN) mutagenesis is effective in inducing deletion mutations in genomes of diverse species. Through this method, we have generated a large mutant resource in M. truncatula. This mutant resources have been used to screen for different mutant using a forward genetics methods. We have isolated and identified a large amount of symbiotic nitrogen fixation (SNF) deficiency mutants. Here, we describe the detail procedures that are being used to characterize symbiotic mutants in M. truncatula. In recent years, whole genome sequencing has been used to speed up and scale up the deletion identification in the mutant. Using this method, we have successfully isolated a SNF defective mutant FN007 and identified that it has a large segment deletion on chromosome 3. The causal deletion in the mutant was confirmed by tail PCR amplication and sequencing. Our results illustrate the utility of whole genome sequencing analysis in the characterization of FN induced deletion mutants for gene discovery and functional studies in the M. truncatula. It is expected to improve our understanding of molecular mechanisms underlying symbiotic nitrogen fixation in legume plants to a great extent.

Keywords: Medicago truncatula (barrel medic); legume; nodule development; symbiotic nitrogen fixation (SNF); whole genome sequencing (WGS).

Figure 1

M. truncatula FN007 mutant phenotype. (A) Growth of FN007 (right) and wild type (WT, left) seedlings 16 days post inoculation (dpi) with S. medicae ABS7 strain, showing dwarf phenotype of FN007 mutant. (B) leaf phenotype of FN007 mutant (right) and WT (left), yellowish leaves are the sign of nitrogen deficiency phenotype. Nodules developed on (C) (WT) and (D) (FN007 mutant) were pink and white color, respectively. Close-up view of (E), WT and (F), FN007 mutant nodules. Scale bar: (A), 1 cm; (B), 50 mm; (C, D), 10 mm; (E, F), 1 mm.

Figure 2

Time-course observations of nodule phenotypes. M.truncatula WT and FN007 mutant seedlings were inoculated with S. medicae ABS7 strain and inoculated root nodules were observed in a time-course assay. Representative images of (A), WT and (F), FN007 mutant roots 6 dpi; (B), WT and (G), FN007 mutant 9 dpi; (C), WT and (H), FN007 mutant 12 dpi; (D), WT and (I), FN007 mutant 15 dpi; (E), WT and (J), FN007 mutant 19 dpi. These observations suggest that FN007 mutant has an abnormal nodule development. Scale bar: 500 μm.

Figure 3

LacZ stained sections observation of nodule development. M.truncatula WT and FN007 mutant seedlings were inoculated with S. medicae ABS7 strain harboring hemA::LacZ reporter gene. Longitudinal sections of nodules were observed under a MVX10+DP74 microscope and representative images were shown. (A), WT and (B), FN007 mutant nodules 12 dpi; (C), WT and (D), FN007 mutant nodules 15 dpi. Scale bar: 200 μm.

Figure 4

Laser confocal microscopic analysis of nodule development. M.truncatula WT and FN007 mutant seedlings were inoculated with S. meliloti Sm2011 strain harboring hemA::mGFP reporter gene. Longitudinal sections of nodules were observed under a MVX10+DP74 microscope and representative images were shown. (A), WT and (B), FN007 mutant nodules 11 dpi; (C), WT and (D), FN007 mutant nodules 14 dpi. Scale bar: 200 μm.

Figure 5

Semithin sections of WT and FN007 mutant nodules. M. truncatula, (A), WT and D, FN007 mutant nodules 13 days post inoculation (dpi) were sectioned longitudinally and stained with toluidine blue, showing development of meristem (I), infection (II) and transition zones (IZ), and infected cells in both WT and FN007 mutant. Compared with WT nodules, there was no fixation zone (III) in FN007 mutant, but a clearly senescence zone (IV) was emerged. Close-up view of (B), WT and (E), FN007 infected cells. Magnification view of (C), WT and (F), FN007 infected cells, showing degradation of symbiosomes in FN007 infected cells. Scale Bar: (A, D): 100 μm; (B, E): 20 μm; (C, F): 10 μm.

Figure 6

Accumulation of starch granules in FN007 mutant nodules. Potassium iodide staining of starch granules in infected cells in WT nodules (A–C) and FN007 mutant nodules (D–F). In WT nodules, starch granules was deposited in large amounts only in the II-III interval zone and in small amounts in the IV zone, while in FN007 mutant nodules, the starch granules deposition was greatly expanded. Scale bar: (A): 100 μm; (D):200 μm; (B, E): 20 μm; (C, F): 10 μm.

Figure 7

Live/dead staining of nodule cells. Live/dead staining of bacteroids in FN007 mutant compared with WT nodules (13 dpi). (A–C), wild type (WT) and (D–F), FN007 mutant. Live bacteroids have a green signal (GFP) and dying bacteroids have a red signal from propidium iodide (PI). PI can only enter bacteroids with compromised membranes. FN007 mutant nodules fail to sustain differentiated bacteroids. Scale bar: (A, D): 200 μm; (B, E): 50 μm; (C, F): 10 μm.

Figure 8

Base error rate distribution graph. Horizontal coordinates represent the base positions on reads, and vertical coordinates represent the average error rate of single bases; the left side shows the base quality distribution of R1 Reads in pair-end sequencing, and the right side shows the base quality distribution of R2 Reads.

Figure 9

Base percentage distribution. Horizontal coordinates represent base position on the reads, and vertical coordinates represent the proportion of single bases, A,T,C,G,N, and GC, respectively.

Figure 10

SNP & InDel Variant Detection in FN007 mutant (A) SNP substitution type statistics in FN007 mutant. Horizontal coordinates represent SNP substitution type, vertical coordinates are the mean, maximum and minimum values for each sample, and colors indicate whether SNP substitution type is a transition or a transversion. (B) InDel substitution type length statistics in FN007 mutant. Horizontal coordinates show the mean, maximum and minimum values of the InDel numbers, vertical coordinates show substitution base lengths, and colors indicate whether the substitution type is insertion or deletion. (C) Circos visualization of identified deletions in FN007 mutant. SNP and InDel distributions on individual chromosomes, where the first circle represents the FN007 chromosome, the purple dots in the second circle represent SNP density, and blue dots in the third circle represent InDel density.

Figure 11

Identification of copy number variation (CNV) in FN007 mutant. (A) Visualization of the causal deletion using IGV. The reads alignment for WT and FN007 mutant. The sequencing showed a large deletion at the corresponding position in FN007 mutant; (B) Coordinate positions between four primers are designed for the identification of deletion border in FN007 mutant; (C) PCR detection for deletion junction through the flanking sequencing primers in FN007 mutant; (D) Tail-PCR was carried out, and the corresponding band were extracted for sequencing; (E) Deletion border confirmed by PCR in FN007 mutant. A pair of primer (FN007F/FN007R) was designed at deletion junction and amplified in FN007 mutant and WT. The results showed that FN007F/R could amplify the target band in FN007 mutant but not in WT.

Figure 12

Genetic linkage analysis of FN007 mutant A genetic linkage analysis of FN007 F2 segregating population. (A) PCR analysis was carried out using primers spanning the deletion border in FN007 F2 population; (B) Shown are PCR amplification of deletion border in F2 mutant pools (mutant like plants, 10 plants per pool) and in wild type-like pools (WT like plants, 10 plants per pool). M, DL2000, molecular weight markers. There were no specific bands in F2 mutant pool by MDB-F and DB-R primer sets, while in WT like pools, all of pools could amplify the correct band. These results indicate that the deletion region has linkage with FN007 mutant phenotype; (C) A putative 348 kb deletion segment in FN007 mutant.