Genome survey of Chinese fir (Cunninghamia lanceolata): Identification of genomic SSRs and demonstration of their utility in genetic diversity analysis

Abstract

Chinese fir (Cunninghamia lanceolata) is an important coniferous species that accounts for 20-30% of the total commercial timber production in China. Though traditional breeding of Chinese fir has achieved remarkable success, molecular-assisted breeding has made little progress due to limited availability of genomic information. In this study, a survey of Chinese fir genome was performed using the Illumina HiSeq Xten sequencing platform. K-mer analysis indicated that Chinese fir has a large genome of approximately 11.6 Gb with 74.89% repetitive elements and is highly heterozygous. Meanwhile, its genome size was estimated to be 13.2 Gb using flow cytometry. A total of 778.02 Gb clean reads were assembled into 10,982,272 scaffolds with an N50 of 1.57 kb. In total, 362,193 SSR loci were detected with a frequency of 13.18 kb. Dinucleotide repeats were the most abundant (up to 73.6% of the total SSRs), followed by trinucleotide and tetranucleotide repeats. Forty-six polymorphic pairs were developed, and 298 alleles were successfully amplified from 199 Chinese fir clones. The average PIC value was 0.53, indicating that the identified genomic SSR (gSSR) markers have a high degree of polymorphism. In addition, these breeding resources were divided into three groups, and a limited gene flow existed among these inferred groups.

Figure 1

Estimation of genome size in Chinese fir by flow cytometry. G_0/1 nuclei peaks (indicated by the arrows) were obtained after flow cytometric analysis of PI-stained nuclei suspensions prepared from leaf samples. Each sample was determined with 10 replicates, and the CV of each peak was set below 5%. (a) G_0/1 nuclei peak of Chinese fir (2 C = 26.99 pg, fluorescence intensity 3.67 × 10⁶). (b) G_0/1 nuclei peak of barley (2 C = 10.43 pg, fluorescence intensity 1.38 × 10⁶). (c) G_0/1 nuclei peak of P. trichocarpa (2 C = 0.98 pg, fluorescence intensity 1.38 × 10⁵).

Figure 2

K-mer (K = 17) analysis for estimating the genome size of Chinese fir. The X-axis is depth (×) and the Y-axis is the proportion that represents the frequency at that depth divided by the total frequency of all depths. The peak indicated by the black arrow is the main peak corresponding to the expected K-mer depth. The genome size is estimated according to the formula: genome size = total K-mer number/peak depth (total K-mer number = 347,935,256,501). Atha. ×39 represents the depth of Arabidopsis thaliana is 39. H0.02 and H0.021 means that heterozygous rate is 2% and 2.1%, respectively. The peaks of Atha. ×39 were used as references.

Figure 3

Guanine plus cytosine (GC) content and average sequencing depth of the genome data. The X-axis is the GC content percent across every 10-kb non-overlapping sliding window. The Y-axis represents the average sequencing depth. The distribution of GC content is at the top, while the distribution of sequence depth is on the right side.

Figure 4

Distributions of di- to hexanucleotide motif types with different repeat numbers (from 4 to 14) in the assembled genomic sequences of Chinese fir.

Figure 5

Frequency distribution of different dinucleotide and trinucleotide repeat motifs in the assembled genomic sequences of Chinese fir. (a) Frequency distribution of different dinucleotide repeat motifs. (b) Frequency distribution of different trinucleotide repeat motifs.

Figure 6

Allelic variation of gSSR marker CLgSSR1 among 12 Chinese fir clones. To reveal the allelic variation, PCR products of CLgSSR1 primer pairs were analyzed and visualized by capillary electrophoresis. The symbols, such as M33, Cl77, Cl80, etc., on the left of the picture represent different Chinese fir clones. The peaks 1, 2 and 3 represent the 160 bp, 163 bp and 166 bp, respectively.

Figure 7

Neighbor-joining dendrogram of 199 Chinese fir clones based on Nei’s 1983 genetic distances. To compare with population structure revealed by STRUCTURE, those clones belonging to the different groups inferred by STRUCTURE analysis were indicated by different colored dots, respectively.

Figure 8

Population structure analysis of 199 Chinese fir clones using STRUCTURE software. (a), ΔK was calculated to determine the most appropriate K value for population structure estimation. The modal value of this distribution is the true K (*) or the uppermost level of structure, here three (K = 3) clusters. (b) Population structure of 199 Chinese fir clones. These clones were assigned into three groups (Groups I, II and III) as indicated by the different coloured blocks. Each clone was represented by a single colored line. The greater proportion of a color, the greater the possibility that the represented individual belongs to the group indicated by that color.