基于叶绿体DNA变异的山荆子种质遗传多样性和系统演化

doi:10.3864/j.issn.0578-1752.2020.03.012

Abstract

Abstract:

【Objective】Malus baccata (L.) Borkh is the most widely distributed native species in China. The non-coding region of chloroplast genome by maternal inheritance is suitable for the systematic study of lower taxonomic levels (such as families and genera). The non-coding regions of cpDNA of 215 germplasms were sequenced, and their genetic variation was analyzed. In this study, the genetic diversity of Malus baccata and the phylogenetic relationship among different populations were explored from the perspective of maternal inheritance, which provided a theoretical basis for origin and genetic evolution, collection and protection of Malus baccata germplasm resources in China.【Method】Four non-coding regions (trnH-psbA, trnS-trnG spacer + intron, trnT-5'trnL and 5'trnL-trnF) of 215 germplasms were amplified by four primers. After manually proofreading sequences obtained through forward and backward sequencing, MEGA 7.0 was used for sequence splicing and alignment, and based on genetic distance, the Neighbour-Joining phylogenetic tree was constructed among different populations of Malus baccata. DnaSP ver5.10.01 was used to calculate the genetic diversity parameters of chloroplast DNA, gene flow and gene differentiation among different populations. Arlequin v3.5 was used to analyze standard molecular variation (AMOVA), and NetWork 4.6.1.2 was used to construct Median-Joining network for cpDNA haplotypes among intraspecific populations of Malus baccata.【Result】The length of four non-coding regions of chloroplast DNA was 3 777 bp after sequencing, splicing, alignment and merging, and 171 variable sites were detected, which included 1 singleton variable sites, 20 parsimony informative sites and 150 insertion-deletion gaps. Among 215 accessions of Malus baccata, the number of variable sites of region trnH-psbA, trnS-trnG spacer + intron, trnT-5'trnL and 5'trnL-trnF were 26, 32, 103 and 10, respectively. The number of haplotypes for four regions were 8, 8, 6 and 4, respectively, and after four regions merged, the haplotypes of chloroplast DNA fragments were 24. The region with highest nucleotide diversity was trnT-5'trnL (Pi=0.01174), and the region with highest haplotype diversity was trnS-trnG spacer + intron (Hd=0.599), and the haplotype diversity of 5'trnL-trnF was the lowest (Hd=0.228). The cpDNA diversity of Malus baccata was high (Hd=0.727, Pi=0.00577). Tajima’s test showed all Tajima’s D values were not statistical at different levels, which indicated that variation of those chloroplast regions followed natural theory of molecular evolution. AMOVA showed that genetic variation mainly existed within populations.【Conclusion】The four non-coding regions of chloroplast DNA were suitable for the analysis of genetic diversity and phylogenetics of Malus baccata. At the cpDNA level, it was not natural selection, but mutation pressure and genetic drift that led to population evolution of Malus baccata. The genetic differentiation among populations was not completely correlated with their geographical distance. Malus baccata might originate from several sites, and the three possible origins, including Heilongjiang and Jilin, Inner Mongolia, Gansu and Shanxi, were inferred.

Key words: Malus baccata, chloroplast DNA, non-coding region, genetic diversity, phylogenetics

GAO Yuan,WANG DaJiang,WANG Kun,CONG PeiHua,ZHANG CaiXia,LI LianWen,PIAO JiCheng. Genetic Diversity and Phylogenetics of Malus baccata (L.) Borkh Revealed by Chloroplast DNA Variation[J].Scientia Agricultura Sinica, 2020, 53(3): 600-611.

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References 51

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编号 Code	cpDNA间区 cpDNA intergenic regions	F序列（5′-3′） Forward sequence (5′-3′)	R序列（5′-3′） Reverse sequence (5′-3′)	扩增片段长度 Amplified fragment length
CP20	trnH-psbA	CGCGCATGGTGGATTCACAATCC	GTTATGCATGAACGTAATGCTC	~400
CP21	trnS-trnG spacer + intron	AGATAGGGATTCGAACCCTCGGT	GTAGCGGGAATCGAACCCGCATC	~1500
CP22	trnT-5' trnL	CATTACAAATGCGATGCTCT	TCTACCGATTTCGCCATATC	~1200
CP23	5' trnL-trnF	ATTTGAACTGGTGACACGAG	CGAAATCGGTAGACGCTACG	~1000

编号 Code	扩增区域 Region	PCR反应条件 PCR condition	退火温度 Annealing temperature
CP20	trnH-psbA	80℃ 5 min;94℃ 30 s,56℃ 30 s,72℃ 1 min,35个循环（35 cycles）;72℃ 10 min	56℃
CP21	trnS-trnG spacer + intron	80℃ 5 min;96℃ 10 s,50℃ 5 s,60℃ 4 min,30个循环（30 cycles）;60℃ 10 min	50℃
CP22	trnT-5' trnL	96℃ 5 min;96℃ 1 min,57℃ 2 min,72℃ 2.5 min,34个循环（34 cycles）;72℃ 10 min	57℃
CP23	5' trnL-trnF	80℃ 5 min;96℃ 10 s,50℃ 5 s,60℃ 4 min,30个循环（30 cycles）;60℃ 10 min	50℃

叶绿体DNA区域 cpDNA regions	片段长度 Length of fragment (bp)	变异位点 Variable sites (Vs)	单一突变位点 Singleton variable sites (Ss)	简约信息位点 Parsimony informative sites (Ps)	插入-缺失位点 Insertion-deletion gaps (Is)	核苷酸多样性 Nucleotide diversity (Pi)	平均核苷酸差异 Average number of nucleotide difference (K)
trnH-psbA	285	26	0	2	24	0.00913	3.13236
trnS-trnG spacer+intron	1383	32	1	5	26	0.00311	4.52649
trnT-5' trnL	1140	103	0	11	92	0.01174	15.68937
5' trnL-trnF	965	10	0	2	8	0.00045	0.44216
组合 Combined	3773	171	1	20	150	0.00577	23.79039

cpDNA区域 cpDNA Regions	单倍型数目 Number of haplotypes (h)	单倍型（基因）多样性 Haplotype (Gene) diversity (Hd)	单倍型多样性方差 Variance of haplotype Diversity (Vh)	单倍型多样性标准差 Standard deviation of haplotype diversity (Sh)
trnH-psbA	8	0.4762	0.00159	0.040
trnS-trnG spacer + intron	8	0.599	0.00111	0.033
trnT-5' trnL	6	0.372	0.00168	0.041
5' trnL-trnF	4	0.228	0.00133	0.037
组合 Combined	24	0.727	0.00084	0.029

cpDNA区域 cpDNA regions	Tajima's D值 Tajima's D	显著性 Significance
trnH-psbA	-0.78313	P>0.10
trnS-trnG spacer+ intron	-0.45018	P>0.10
trnT-5' trnL	-0.29083	P>0.10
5' trnL-trnF	-1.70516	0.05<P<0.10
组合 Combined	-0.54177	P>0.10

Genetic Diversity and Phylogenetics of Malus baccata (L.) Borkh Revealed by Chloroplast DNA Variation

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试材编号Code of accessions	来源地Origion	个体数量Number of accessions	群体代码Code of populations
MB1-MB83	黑龙江 Heilongjiang	83	MBHLJ
MB84-MB101	吉林 Jilin	18	MBJL
MB102-MB143	内蒙古 Inner Mongolia	42	MBNM
MB144-MB197	河北 Hebei	54	MBHB
MB198-MB210	山西 Shanxi	13	MBSX
MB211-MB214	甘肃 Gansu	4	MBGS
MB215	辽宁 Liaoning	1	MBLN

变异来源 Source of variation	自由度 df	平方和 Sum of square	变异组分 Variance component	变异百分比 Percentage of variation	概率值 P
居群间Among population	6	201.966	0.84873	6.97	<0.001
居群内Within population	206	2334.987	11.33487	93.03	<0.001
总数 Total	212	2536.948	12.18360	100	<0.001

6个山荆子群体 6 populations of Malus baccata	MBGS	MBHB	MBHLJ	MBJL	MBNM	MBSX
MBGS	0.000
MBHB	0.184	0.000
MBHLJ	0.206	0.042	0.000
MBJL	0.216	0.035	0.002	0.000
MBNM	0.209	0.166	0.061	0.079	0.000
MBSX	-0.024	0.120	0.097	0.056	0.104	0.000

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