关键词: 花生, Arachis monticola, 全基因组序列, SSR位点, 基序

Abstract:

【Objective】We aimed to identify simple sequence repeat (SSR) loci throughout the genome of tetraploid wild peanut Arachis monticola (AABB, 2n = 4x = 40), to identify their distribution characteristics, and to develop and validate SSR primers. These markers have potential uses in genetic evolution analyses and in the development of molecular markers for important traits in peanut.【Method】Using the bioinformatics software MISA, we searched for SSR loci in the whole genome sequence of A. monticola, which was downloaded from the GigaScience database of the BGI. One hundred SSR loci were randomly selected and primers were designed and synthesized. The primers were used to amplify products from four different Arachis genomes, and the products were analyzed by polyacrylamide gel electrophoresis (PAGE).【Result】 A total of 676 878 SSRs were found in the genome of tetraploid wild peanut A. monticola in 5 127 scaffolds (average, one SSR per 3.8 kb). The SSRs ranged from single nucleotides to hexanucleotides. Single nucleotide SSRs were significantly more abundant than hexanucleotide SSRs. Single, double, and triple nucleotide SSRs were predominant, accounting for 94.28% of all the SSRs. Single nucleotide SSRs accounted for the largest proportion of total SSRs (46.71%) and showed the highest density. Hexanucleotide SSRs accounted for the smallest proportion and showed the sparsest density. Most SSRs were located in intergenic regions, and most of the SSRs in gene sequences were located in introns. A total of 395 different repeat motifs were identified in the whole genome, of which 342 were in the A-subgenome and 356 were in the B-subgenome. The most abundant repeat motif was A/T. The most abundant repeat motifs for SSRs with 1-6 nucleotides were A/T, AT/AT, AAT/ATT, AAAT/ATTT, AAAAT/ATTTT, and AAAAAT/ATTTTT, respectively. There were less than 50 of each type of SSR repeat motif, but the number of each type of SSR motif varied greatly. The number of each type of SSRs repeat motif decreased with increasing number of nucleotides in the motif. Chromosome B03 had the most SSRs, and chromosome A08 showed the highest density of SSRs. We designed 192 303 pairs of SSR primers, and the detection rate of single-locus SSR markers was 50.35%. The distribution of SSR markers in the genome was dense at both ends and sparse in the middle. Among the 100 synthesized primer pairs, 90 pairs amplified stable and clear bands from A. monticola genomic DNA. The bands amplified from four different peanut genomic DNAs showed different characteristics. 【Conclusion】The A. monticola genome was rich in SSRs ranging from single nucleotides to hexanucleotides. Single nucleotide repeats were the most abundant and densely distributed, and hexanucleotides showed the lowest frequency and the sparsest distribution. There was no strict correlation between the frequency of different repeats and the repeat type. The A-subgenome and B-subgenome had their own specific SSRs. The AT-enriched repeat motifs were the most abundant, while GC-enriched repeat motifs showed much lower frequencies. The number of SSRs with the same type of repeat motif decreased with increasing numbers of nucleotides in the motif. Compared with the genomes of the two diploid wild species, the tetraploid genome of A. monticola had more SSRs, a higher density of SSRs, and a different SSR distribution pattern. Preliminary validation analyses showed that the SSR primers designed in this study shared certain universal properties among four Arachis genomes.

Key words: peanut, Arachis monticola, whole genome sequence, SSR locus, motif