四倍体野生种花生A.monticola全基因组SSR的开发与特征分析

doi:10.3864/j.issn.0578-1752.2019.15.002

Abstract

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

WANG YuLong, HUANG BingYan, WANG SiYu, DU Pei, QI FeiYan, FANG YuanJin, SUN ZiQi, ZHENG Zheng, DONG WenZhao, ZHANG XinYou. Development and Characterization of Whole Genome SSR in Tetraploid Wild Peanut (Arachis monticola)[J].Scientia Agricultura Sinica, 2019, 52(15): 2567-2580.

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材料名称 Materials	区组 Section	种别 Species	倍性 Ploidy	基因组 Genomes
PI 219823	花生区组Section arachis	A.duranesis	二倍体Diploid (2n=2x=20)	A
PI 468322	花生区组Section arachis	A.ipaensis	二倍体Diploid (2n=2x=20)	B
PI 219824	花生区组Section arachis	A.moticola	四倍体Tetraploid (2n=2x=40)	AB
Tifrunner	花生区组Section arachis	A.hypogaea	四倍体Tetraploid (2n=2x=40)	AB

材料 Materials	统计量 Statistic	单核苷酸Mono-nucleotide	2核苷酸Di-nucleotide	3核苷酸Tri-nucleotide	4核苷酸Tetra-nucleotide	5核苷酸Penta-nucleotide	6核苷酸Hexa-nucleotide	总计 Total
A.duranesis (AA)	数目Amount	29689	47805	42529	4988	7658	2860	135529
	类型比例 Type ratio (%)	21.91	35.27	31.38	3.68	5.65	2.11	100.00
	平均距离 Average distance (kb)	36.5	22.7	25.5	217.4	141.6	379.1	8.0
A.ipaensis (BB)	数目Amount	57589	75334	45717	6736	11092	3489	199957
	类型比例 Type ratio (%)	28.80	37.68	22.86	3.37	5.55	1.74	100
	平均距离 Average distance (kb)	23.5	18.0	29.6	201.0	122.1	388.0	6.7
A.moticola (AABB)	数目Amount	316197	208181	113794	12170	19552	6984	676878
	类型比例 Type ratio (%)	46.71	30.76	16.81	1.80	2.89	1.03	100.00
	平均距离 Average distance (kb)	8.3	12.6	23.0	215.2	133.9	375.0	3.8
A.moticola (A-subgenome)	数目Amount	114570	81364	46531	4664	7445	2816	257390
	类型比例 Type ratio (%)	44.51	31.61	18.08	1.81	2.89	1.09	100.00
	平均距离 Average distance (kb)	9.0	12.7	22.2	221.2	138.5	366.3	4.0
A.moticola (B-subgenome)	数目Amount	174453	118620	62638	7032	11358	3912	378013
	类型比例 Type ratio (%)	46.15	31.38	16.57	1.86	3.00	1.03	100.00
	平均距离 Average distance (kb)	8.5	12.5	23.7	210.7	130.5	378.8	3.9

重复基元类型 Motif types	A亚基因组特异重复基元 Specific motifs in A-subgenome	B亚基因组特异重复基元 Specific motifs in B-subgenome
四核苷酸 Tetra-nucleotide	AACG/CGTT AGCC/CTGG AGCG/CGCT	AAGC/CTTG AGGC/CCTG
五核苷酸 Penta-nucleotide	ACCTG/AGGTC AGCCT/AGGCT ACGGG/CCCGT AGCCC/CTGGG ACTGG/AGTCC ACGTC/ACGTG	AACGC/CGTTG AACTT/AAGTT AAGGC/CCTTG ACAGT/ACTGT ACATG/ATGTC ACCGT/ACGGT ACTAG/AGTCT AGCGC/CGCTG AGCGG/CCGCT ATGCC/ATGGC
六核苷酸 Hexa-nucleotide	AAACCC/GGGTTT AAAGCT/AGCTTT AACAGG/CCTGTT AACCGT/ACGGTT AACGGT/ACCGTT AACGTC/ACGTTG AACGTT/AACGTT AACTCC/AGTTGG AAGTGT/ACACTT AATACC/ATTGGT AATTCG/AATTCG ACACCG/CGGTGT ACATCG/ATGTCG ACCCAG/CTGGGT ACCCAT/ATGGGT ACCTCG/AGGTCG ACGCCG/CGGCGT ACGGAG/CCGTCT ACTCAT/AGTATG ACTCGG/AGTCCG ACTGCC/AGTGGC AGCATG/ATGCTC AGCCCG/CGGGCT AGCCCT/AGGGCT AGCTAT/AGCTAT	AAACGC/CGTTTG AAAGGT/ACCTTT AACGAC/CGTTGT AACGAT/ATCGTT AACTCT/AGAGTT AACTGG/AGTTCC AAGACC/CTTGGT AAGCAT/ATGCTT AAGGCG/CCTTCG AAGGGC/CCCTTG AAGTAC/ACTTGT AATCCG/ATTCGG AATCGC/ATTGCG AATGAC/ATTGTC AATGCG/ATTCGC AATGGT/ACCATT ACAGAT/ATCTGT ACAGGC/CCTGTG ACAGGG/CCCTGT ACATAG/ATGTCT ACATGC/ATGTGC ACCATG/ATGGTC ACCCTG/AGGGTC ACCGAG/CGGTCT ACCTAG/AGGTCT ACGCGG/CCGCGT ACGCTC/AGCGTG ACGTCC/ACGTGG ACTAGG/AGTCCT ACTCTG/AGAGTC ACTGGC/AGTGCC ACTGGG/AGTCCC AGCCAT/ATGGCT AGGCCG/CCTCGG AGGGAT/ATCCCT ATCGGC/ATGCCG

Development and Characterization of Whole Genome SSR in Tetraploid Wild Peanut (Arachis monticola)

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