Genome-Wide Analysis of Simple Sequence Repeats in Cabbage (Brassica oleracea L.)

Abstract

Cabbage (Brassica oleracea L. var. capitata) accounts for a critical vegetable crop belonging to Brassicaceae family, and it has been extensively planted worldwide. Simple sequence repeats (SSRs), the markers with high polymorphism and co-dominance degrees, offer a crucial genetic research resource. The current work identified totally 64,546 perfect and 93,724 imperfect SSR motifs in the genome of the cabbage 'TO1000.' Then, we divided SSRs based on the respective overall length and repeat number into different linkage groups. Later, we characterized cabbage genomes from the perspectives of motif length, motif-type classified and SSR level, and compared them across cruciferous genomes. Furthermore, a large set of 64,546 primer pairs were successfully identified, which generated altogether 1,113 SSR primers, including 916 (82.3%) exhibiting repeated and stable amplification. In addition, there were 32 informative SSR markers screened, which might decide 32 cabbage genotypes for their genetic diversity, with level of polymorphism information of 0.14-0.88. Cultivars were efficiently identified by the new strategy designating manual diagram for identifying cultivars. Lastly, 32 cabbage accessions were clearly separately by five Bol-SSR markers. Besides, we verified whether such SSRs were available and transferable in 10 Brassicaceae relatives. Based on the above findings, those genomic SSR markers identified in the present work may facilitate cabbage research, which lay a certain foundation for further gene tagging and genetic linkage analyses, like marker-assisted selection, genetic mapping, as well as comparative genomic analysis.

Keywords: SSR; cabbage; genetic diversity; genome; manual cultivar identification diagram; molecular makers.

Figure 1

Distribution of SSR repeated motif length across 12 cruciferous genomes.

Figure 2

Characterization of perfect SSRs in the cabbage genome. (A) The changeable rule from mononucleotides to hexanucleotide motifs. (B) The frequency of repeat classes (class I>30nt, class II 20–30nt, class III <20nt). (C) The distribution of motif type within each class.

Figure 3

Distribution of the major repeat motifs in cabbage genome.

Figure 4

Intra-chromosomal distribution of SSRs. (A) Relationship between the SSR number and chromosome length in the cabbage genome. (B) The frequency of mono- to hexanucleotide motifs in cabbage chromosomes. (C) Number and density of the SSR motifs across nine chromosomes of the cabbage reference genome.

Figure 5

Distribution of microsatellite sizes in the cabbage genome. (A) Non-triplet SSR vs. triplet SSR in both perfect and imperfect motifs. (B) Distribution of repeat types within perfect and imperfect SSR motifs. (C) A comparison between di- and trinucleotide repeats in both the gene space and full genomic region.

Figure 6

Functional analysis (gene ontology) of cabbage genes containing SSRs. (A) GO classification. (B) Revigo summary of “biological process” and “molecular function” enriched terms.

Figure 7

Genetic diversity analysis of 32 cabbage accessions with SSR markers. Amplification of 32 genotypes with the SSR primers Bol-SSR32 (A) Bol-SSR24 (B) and Bol-SSR12 (C) by polyacrylamide gel electrophoresis, and the UPGMA dendrogram of 32 cabbage genotypes based on 32 new SSR markers (D) M: 50bp DNA ladder. The genotype name of numbers (1–32) is listed in Supplementary Table S1.

Figure 8

MCID analysis of the 32 cabbage genotypes with the DNA fingerprints of five SSR primers. The number above each horizontal line in the diagram denotes the size of the polymorphic bands used to separate the genotypes following the line. (+) or (−) denotes the presence or absence of the polymorphic band.

Figure 9

Identification of related species in the Brassicaceae family (A) and amplified results by SSR primers (B). (a) Brassica campestris subsp. chinensis (AA); (b) Brassica oleracea var. capitata (CC); (c) B. oleracea var. italica (CC); (d) Brassica rapa ssp. pekinensis (AA); (e) B. oleracea var. botrytis (CC); (f) B. oleracea var. caulorapa; (g) B. alboglabra; (h) B. parachinensis; (i) B. napus (AACC); (j) B. campestris L. ssp. chinensis.