Analysis of Codon Usage Bias in the Chloroplast Genome of Ananas comosus

doi:10.3969/j.issn.1000-2561.2022.03.001

Abstract

Abstract:

The codon usage bias of chloroplast genome affects the genes expression efficiency, which provides important information for chloroplast genetic engineering and species genetic improvement. To determine the codon usage characteristic of the chloroplast genome and its main influencing factors in pineapple (Ananas comosus), the codon usage pattern and its preference of pineapple chloroplast genome were analyzed using bioinformatics softwares. The average GC content of codons of pineapple chloroplast genes was 38.31%, the average GC content of codons from the first position to the third position was 46.78%, 39.61% and 28.53%, respectively, and the average GC contents at the first and second positions of codons were significantly higher than that at the third position. The value range of effective number of codons (ENC) was 38.48—61.00, and its average value was 47.21, indicating weak codon bias. GC1 was significantly correlated with GC2, GC_all was significantly positively correlated with GC1, GC2 and GC3, and GC3 was not significantly correlated with GC1 and GC2. ENC was not significantly correlated with GC1, but significantly and extremely significantly correlated with GC2 and GC3, respectively. Codon number (N) was only significantly correlated with GC3, which indicating that the base composition of the third position in the three positions of the codon mainly affects codon number. RSCU analysis showed that among the 29 codons with RSCU>1, 12 codons ended with A, 16 codons ended with U, and 1 codon ended with G. Neutrality plot analysis showed that there was not significant correlation between GC12 and GC3, and the correlation coefficient and regression coefficient was 0.065 and 0.085, respectively. ENC-plot analysis showed most genes were distributed near the standard curve, and most ENC ratios were distributed in the interval of -0.05—0.05. PR2-plot analysis showed that all genes were unevenly distributed in the four regions of the PR2 plan, and the use frequency of pyrimidine T/C at the third position of codon was higher than that of purine A/G. Bias analysis of Neutrality plot, ENC-plot and PR2-plot suggested that natural selection and mutation as the main factors affect the codon usage bias of pineapple chloroplast genome. Analysis of optimal codons and RSCU showed that most of the 29 codons with RSCU>1 and the 18 optimal codons ended with A or U. The results provide scientific basis for codon optimization of exogenous genes and improvement of their expression efficiency.

Key words: pineapple (Ananas comosus), chloroplast genome, codon usage bias, optimal codons

CLC Number:

S668.3

YANG Xiangyan, CAI Yuanbao, TAN Qinliang, QIN Xu, HUANG Xianya, WU Mi. Analysis of Codon Usage Bias in the Chloroplast Genome of Ananas comosus[J]. Chinese Journal of Tropical Crops, 2022, 43(3): 439-446.

Figures/Tables 7

References 28

[1]	ALVES-PEREIRA A, CLEMENT C R, PICANÇO- RODRIGUES D, VEASEY E A, DEQUIGIOVANNI G, RAMOS S L F, PINHEIRO J B, ZUCCHI M I. Patterns of nuclear and chloroplast genetic diversity and structure of manioc along major Brazilian Amazonian rivers[J]. Annals of Botany, 2018, 121(4):625-639. DOI URL
[2]	MEUCCI S, SCHULTE L, ZIMMERMANN H H, STOOF- LEICHSENRING K R, EPP L, BRONKEN EIDESEN P, HERZSCHUH U. Holocene chloroplast genetic variation of shrubs (Alnus alnobetula, Betula nana, Salix sp.) at the siberian tundra-taiga ecotone inferred from modern chloroplast genome assembly and sedimentary ancient DNA analyses[J]. Ecology and Evolution, 2021, 11(5):2173-2193. DOI URL
[3]	WANG K, CUI Y, WANG Y, GAO Z, LIU T, MENG C, QIN S. Chloroplast genetic engineering of a unicellular green alga Haematococcus pluvialis with expression of an antimicrobial peptide[J]. Marine Biotechnology, 2020, 22(4):572-580. DOI URL
[4]	WANNATHONG T, WATERHOUSE J C, YOUNG R E, ECONOMOU C K, PURTON S. New tools for chloroplast genetic engineering allow the synjournal of human growth hormone in the green alga Chlamydomonas reinhardtii[J]. Applied Microbiology and Biotechnology, 2016, 100(12):5467-5477. DOI URL
[5]	LIU H, LU Y, LAN B, XU J. Codon usage by chloroplast gene is bias in Hemiptelea davidii[J]. Journal of Genetics, 2020, 99:8. DOI URL
[6]	KONG W Q, YANG J H. The complete chloroplast genome sequence of Morus cathayana and Morus multicaulis, and comparative analysis within Genus morus L[J]. PeerJ, 2017, 5(31):e3037. DOI URL
[7]	WANG Z, XU B, LI B, ZHOU Q, WANG G, JIANG X, WANG C, XU Z. Comparative analysis of codon usage patterns in chloroplast genomes of six Euphorbiaceae species[J]. PeerJ, 2020, 8:e8251. DOI URL
[8]	QUAX T E, CLAASSENS N J, SÖLL D, OOST J. Codon bias as a means to fine-tune gene expression[J]. Molecular Cell, 2015, 59(2):149-161. DOI URL
[9]	BHATTACHARYYA D, UDDIN A, DAS S, CHAKRABORTY S. Mutation pressure and natural selection on codon usage in chloroplast genes of two species in Pisum L. (Fabaceae: Faboideae)[J]. Mitochondrial DNA, 2019, 30(4):664-673.
[10]	WU S, XU L, HUANG R, WANG Q. Improved biohydrogen production with an expression of codon-optimized hemH and lba genes in the chloroplast of Chlamydomonas reinhardtii[J]. Bioresource Technology, 2011, 102(3):2610-2616. DOI URL
[11]	KWON K C, CHAN H T, LEÓN I R, WILLIAMS- CARRIER R, BARKAN A, DANIELL H. Codon optimization to enhance expression yields insights into chloroplast translation[J]. Plant Physiology, 2016, 172(1):62-77. DOI URL
[12]	CHAKRABORTY S, YENGKHOM S, UDDIN A. Analysis of codon usage bias of chloroplast genes in Oryza species: codon usage of chloroplast genes in Oryza species[J]. Planta, 2020, 252(4):1-20. DOI URL
[13]	TIAN G, LI G, LIU Y, LIU Q, WANG Y, XIA G, WANG M. Polyploidization is accompanied by synonymous codon usage bias in the chloroplast genomes of both cotton and wheat[J]. PLoS One, 2020, 15(11):e0242624.
[14]	REDWAN R M, SAIDIN A, KUMAR S V. Complete chloroplast genome sequence of MD-2 pineapple and its comparative analysis among nine other plants from the subclass Commelinidae[J]. BMC Plant Biology, 2015, 15(196):1-20.
[15]	REDWAN R M, SAIDIN A, KUMAR S V. Erratum to: Complete chloroplast genome sequence of MD-2 pineapple and its comparative analysis among nine other plants from the subclass Commelinidae[J]. BMC Plant Biology, 2015, 15:294. DOI URL
[16]	陈哲, 胡福初, 王祥和, 范鸿雁, 张治礼. 菠萝密码子使用偏好性分析[J]. 果树学报, 2017, 34(8):946-955.
	CHEN Z, HU F C, WANG X H, FAN H Y, ZHANG Z L. Analysis of codon usage bias of Ananas comosus with genome sequencing data[J]. Journal of Fruit Science, 2017, 34(8):946-955. (in Chinese)
[17]	杨祥燕, 蔡元保, 张治礼, 孙光明, 孙伟生, 吴青松, 王运儒. 菠萝MADS-box基因AcMADS2的克隆与表达分析[J]. 植物生理学报, 2016, 52(9):1406-1412.
	YANG X Y, CAI Y B, ZHANG Z L, SUN G M, SUN W S, WU Q S, WANG Y R. Cloning and expression analysis of MADS-box gene AcMADS2 from pineapple (Ananas comosus)[J]. Plant Physiology Journal, 2016, 52(9):1406-1412. (in Chinese)
[18]	蔡元保, 杨祥燕, 孙光明, 黄强, 刘业强, 李绍鹏, 张治礼. 菠萝花发育相关基因AcMADS1的克隆与组织表达特性分析[J]. 植物学报, 2014, 49(6):692-703. DOI
	CAI Y B, YANG X Y, SUN G M, HUANG Q, LIU Y Q, LI S P, ZHANG Z L. Cloning of flowering-related gene AcMADS1 and characterization of expression in tissues of pineapple (Ananas comosus)[J]. Chinese Bulletin of Botany, 2014, 49(6):692-703. (in Chinese)
[19]	PAUL P, MALAKAR A K, CHAKRABORTY S. Codon usage and amino acid usage influence genes expression level[J]. Genetica, 2018, 146(1):53-63. DOI URL
[20]	李显煌, 杨生超, 辛雅萱, 屈亚亚, 杨琳懿, 冯发玉, 范伟, 辛培尧. 灯盏花叶绿体基因组密码子偏好性分析[J]. 云南农业大学学报(自然科学), 2021, 36(3):384-392.
	LI X H, YANG S C, XIN Y X, QU Y Y, YANG L Y, FENG F Y, FAN W, XIN P Y. Analysis of the codon usage bias of chloroplast genome in Erigeron breviscapus (Vant.) Hand- Mazz[J]. Journal of Yunnan Agricultural University (Natural Science), 2021, 36(3):384-392. (in Chinese)
[21]	LI G, PAN Z, GAO S, HE Y, XIA Q, JIN Y, YAO H. Analysis of synonymous codon usage of chloroplast genome in Porphyra umbilicalis[J]. Genes and Genomics, 2019, 41(10):1173-1181. DOI URL
[22]	尚明照, 刘方, 华金平, 王坤波. 陆地棉叶绿体基因组密码子使用偏性的分析[J]. 中国农业科学, 2011, 44(2):245-253.
	SHANG M Z, LIU F, HUA J P, WANG K B. Analysis on codon usage of chloroplast genome of Gossypium hirsutum[J]. Scientia Agricultura Sinica, 2011, 44(2):245-253. (in Chinese)
[23]	王鹏良, 吴双成, 杨利平, 王华宇, 陈乃明, 张照远. 巨桉叶绿体基因组密码子偏好性分析[J]. 广西植物, 2019, 39(12):1583-1592.
	WANG P L, WU S C, YANG L P, WANG H Y, CHEN N M, ZHANG Z Y. Analysis of codon bias of chloroplast genome in Eucalyptus grandis[J]. Guihaia, 2019, 39(12):1583-1592. (in Chinese)
[24]	唐玉娟, 赵英, 黄国弟, 付海天, 宋恩亮, 李日旺, 金刚. 芒果叶绿体基因组密码子使用偏好性分析[J]. 热带作物学报, 2021, 42(8):2143-2150.
	TANG Y J, ZHAO Y, HUANG G D, FU H T, SONG E L, LI R W, JIN G. Analysis on codon usage bias of chloroplast genes from mango[J]. Chinese Journal of Tropical Crops, 2021, 42(8):2143-2150. (in Chinese)
[25]	WANG L, ROOSSINCK M J. Comparative analysis of expressed sequences reveals a conserved pattern of optimal codon usage in plants[J]. Plant Molecular Biology, 2006, 61:699-710. DOI URL
[26]	刘慧, 王梦醒, 岳文杰, 邢光伟, 葛玲巧, 聂小军, 宋卫宁. 糜子叶绿体基因组密码子使用偏性的分析[J]. 植物科学学报, 2017, 35(3):362-371.
	LIU H, WANG M X, YUE W J, XING G W, GE L Q, NIE X J, SONG W N. Analysis of codon usage in the chloroplast genome of Broomcorn millet (Panicum miliaceum L.)[J]. Plant Science Journal, 2017, 35(3):362-371. (in Chinese)
[27]	李冬梅, 吕复兵, 朱根发, 操君喜, 孙映波, 刘海林, 王真. 文心兰叶绿体基因组密码子使用的相关分析[J]. 广东农业科学, 2012, 39(10):61-65.
	LI D M, LV F B, ZHU G F, CAO J X, SUN Y B, LIU H L, WANG Z. Analysis on codon usage of chloroplast genome of Oncidium Gower Ramsey[J]. Guangdong Agricultural Science, 2012, 39(10):61-65. (in Chinese)
[28]	王媛媛, 杨美青. 蒙古韭叶绿体基因组密码子使用偏好性分析[J]. 分子植物育种, 2021, 19(4):1084-1092.
	WANG Y Y, YANG M Q. Analysis of the codon usage bias in the chloroplast genome of Allium mongolicum Regel[J]. Molecular Plant Breeding, 2021, 19(4):1084-1092. (in Chinese)

基因Gene	GC1/%	GC2/%	GC3/%	GC_all/%	ENC	基因Gene	GC1/%	GC2/%	GC3/%	GC_all/%	ENC
psbA	48.87	44.07	32.77	41.90	42.70	rpl20	37.29	41.53	27.97	35.59	49.88
matK	41.21	31.45	26.95	33.20	48.93	clpP	59.31	36.27	32.35	42.65	61.00
atpA	55.91	40.16	27.17	41.08	46.35	psbB	53.83	46.17	28.29	42.76	46.85
atpF	49.73	34.05	30.27	38.02	42.60	petB	48.61	41.20	31.02	40.28	43.24
atpI	48.79	36.29	29.03	38.04	48.16	petD	48.94	39.36	29.26	39.18	40.57
rps2	41.77	44.30	32.91	39.66	50.03	rpoA	46.18	33.53	27.65	35.78	48.19
rpoC2	45.82	37.09	28.14	37.01	49.74	rps11	53.96	55.40	20.14	43.17	38.64
rpoC1	50.36	37.99	28.38	38.91	50.08	rps8	40.74	40.74	22.96	34.81	44.02
rpoB	50.05	38.12	28.52	38.89	48.10	rpl14	55.28	36.59	21.95	37.94	42.39
psbD	52.82	43.50	33.33	43.22	45.51	rpl16	53.28	54.01	24.82	44.04	39.42
psbC	54.01	46.20	33.97	44.73	46.02	rps3	45.21	33.33	24.20	34.25	47.52
rps14	42.57	46.53	29.70	39.60	38.48	rpl22	41.86	37.98	24.81	34.88	51.05
psaB	48.03	42.86	32.38	41.09	48.64	rpl2	50.73	50.73	31.75	44.40	50.07
psaA	52.46	43.28	34.62	43.45	51.97	ycf2	41.71	34.53	37.29	37.84	52.87
ycf3	47.37	39.18	29.82	38.79	56.52	ndhB	41.88	39.33	31.51	37.57	45.63
rps4	49.01	38.61	25.74	37.79	49.46	rps7	52.56	45.51	21.79	39.96	44.01
ndhJ	48.12	37.50	30.00	38.54	48.64	ndhF	37.72	37.58	21.17	32.16	42.88
ndhK	40.96	44.03	31.74	38.91	53.96	ccsA	32.19	38.75	26.88	32.60	49.33
ndhC	50.41	34.71	31.40	38.84	53.28	ndhD	39.44	37.45	30.48	35.79	47.28
atpE	52.59	41.48	31.11	41.73	45.90	ndhE	38.24	32.35	27.45	32.68	45.65
atpB	56.11	41.48	28.86	42.15	47.20	ndhG	41.24	35.03	28.81	35.03	48.47
rbcL	57.50	43.12	28.75	43.12	47.72	ndhI	40.88	35.91	21.55	32.78	45.63
accD	39.47	38.24	26.38	34.70	44.95	ndhA	42.98	38.84	23.97	35.26	45.45
ycf4	45.41	42.16	31.89	39.82	49.56	ndhH	50.76	35.53	27.66	37.99	46.99
cemA	41.30	27.83	32.17	33.77	47.58	ycf1	36.53	29.35	28.12	31.33	49.16
petA	54.52	35.51	28.35	39.46	48.28	平均值	46.78	39.61	28.53	38.31	47.21
rps18	36.27	43.14	25.49	34.97	38.53	平均值	46.78	39.61	28.53	38.31	47.21

基因Gene	GC1/%	GC2/%	GC3/%	GC_all/%	ENC	基因Gene	GC1/%	GC2/%	GC3/%	GC_all/%	ENC
psbA	48.87	44.07	32.77	41.90	42.70	rpl20	37.29	41.53	27.97	35.59	49.88
matK	41.21	31.45	26.95	33.20	48.93	clpP	59.31	36.27	32.35	42.65	61.00
atpA	55.91	40.16	27.17	41.08	46.35	psbB	53.83	46.17	28.29	42.76	46.85
atpF	49.73	34.05	30.27	38.02	42.60	petB	48.61	41.20	31.02	40.28	43.24
atpI	48.79	36.29	29.03	38.04	48.16	petD	48.94	39.36	29.26	39.18	40.57
rps2	41.77	44.30	32.91	39.66	50.03	rpoA	46.18	33.53	27.65	35.78	48.19
rpoC2	45.82	37.09	28.14	37.01	49.74	rps11	53.96	55.40	20.14	43.17	38.64
rpoC1	50.36	37.99	28.38	38.91	50.08	rps8	40.74	40.74	22.96	34.81	44.02
rpoB	50.05	38.12	28.52	38.89	48.10	rpl14	55.28	36.59	21.95	37.94	42.39
psbD	52.82	43.50	33.33	43.22	45.51	rpl16	53.28	54.01	24.82	44.04	39.42
psbC	54.01	46.20	33.97	44.73	46.02	rps3	45.21	33.33	24.20	34.25	47.52
rps14	42.57	46.53	29.70	39.60	38.48	rpl22	41.86	37.98	24.81	34.88	51.05
psaB	48.03	42.86	32.38	41.09	48.64	rpl2	50.73	50.73	31.75	44.40	50.07
psaA	52.46	43.28	34.62	43.45	51.97	ycf2	41.71	34.53	37.29	37.84	52.87
ycf3	47.37	39.18	29.82	38.79	56.52	ndhB	41.88	39.33	31.51	37.57	45.63
rps4	49.01	38.61	25.74	37.79	49.46	rps7	52.56	45.51	21.79	39.96	44.01
ndhJ	48.12	37.50	30.00	38.54	48.64	ndhF	37.72	37.58	21.17	32.16	42.88
ndhK	40.96	44.03	31.74	38.91	53.96	ccsA	32.19	38.75	26.88	32.60	49.33
ndhC	50.41	34.71	31.40	38.84	53.28	ndhD	39.44	37.45	30.48	35.79	47.28
atpE	52.59	41.48	31.11	41.73	45.90	ndhE	38.24	32.35	27.45	32.68	45.65
atpB	56.11	41.48	28.86	42.15	47.20	ndhG	41.24	35.03	28.81	35.03	48.47
rbcL	57.50	43.12	28.75	43.12	47.72	ndhI	40.88	35.91	21.55	32.78	45.63
accD	39.47	38.24	26.38	34.70	44.95	ndhA	42.98	38.84	23.97	35.26	45.45
ycf4	45.41	42.16	31.89	39.82	49.56	ndhH	50.76	35.53	27.66	37.99	46.99
cemA	41.30	27.83	32.17	33.77	47.58	ycf1	36.53	29.35	28.12	31.33	49.16
petA	54.52	35.51	28.35	39.46	48.28	平均值	46.78	39.61	28.53	38.31	47.21
rps18	36.27	43.14	25.49	34.97	38.53	平均值	46.78	39.61	28.53	38.31	47.21

参数Parameter	GC1	GC2	GC3	GC_all	ENC
GC2	0.347^*
GC3	0.128	-0.034
GC_all	0.823^**	0.709^**	0.407^**
ENC	0.041	-0.341^*	0.446^**	0.005
N	-0.133	-0.254	0.285^*	-0.112	0.242

参数Parameter	GC1	GC2	GC3	GC_all	ENC
GC2	0.347^*
GC3	0.128	-0.034
GC_all	0.823^**	0.709^**	0.407^**
ENC	0.041	-0.341^*	0.446^**	0.005
N	-0.133	-0.254	0.285^*	-0.112	0.242

氨基酸 Amino acid	密码子 Codon	基因组 Genome		高表达基因 High expression gene		低表达基因 Low expression gene		ΔRSCU
氨基酸 Amino acid	密码子 Codon	数目Number	RSCU	数目Number	RSCU	数目Number	RSCU	ΔRSCU
Phe	UUU^**	733	1.25	18	1.38	102	1.04	0.34
	UUC	444	0.75	8	0.62	95	0.96	-0.34
Leu	UUA^***	692	1.90	20	2.22	59	1.14	1.08
	UUG^**	466	1.28	17	1.89	77	1.49	0.40
	CUU	463	1.27	12	1.33	77	1.49	-0.16
	CUC	141	0.39	2	0.22	27	0.52	-0.30
	CUA	299	0.82	3	0.33	44	0.85	-0.52
	CUG	121	0.33	0	0	26	0.50	-0.50
Ile	AUU^*	891	1.44	25	1.42	108	1.34	0.08
	AUC	393	0.63	7	0.40	59	0.73	-0.33
	AUA^*	574	0.93	21	1.19	74	0.92	0.27
Val	GUU	430	1.47	15	1.58	52	1.54	0.04
	GUC	124	0.42	1	0.11	22	0.65	-0.54
	GUA^***	452	1.55	20	2.11	37	1.10	1.01
	GUG	162	0.55	2	0.21	24	0.71	-0.50
Ser	UCU	445	1.65	7	0.95	78	1.54	-0.59
	UCC^*	269	1.00	9	1.23	57	1.13	0.10
	UCA	341	1.27	11	1.50	74	1.46	0.04
	UCG	150	0.56	4	0.55	32	0.63	-0.08
	AGU^***	334	1.24	12	1.64	48	0.95	0.69
	AGC	78	0.29	1	0.14	15	0.30	-0.16
Pro	CCU^**	340	1.56	19	1.69	40	1.23	0.46
	CCC^*	181	0.83	10	0.89	25	0.77	0.12
	CCA	253	1.16	12	1.07	42	1.29	-0.22
	CCG	99	0.45	4	0.36	23	0.71	-0.35
Thr	ACU	429	1.56	11	1.29	47	1.36	-0.07
	ACC^*	206	0.75	6	0.71	21	0.61	0.10
	ACA^***	342	1.25	16	1.88	46	1.33	0.55
	ACG	120	0.44	1	0.12	24	0.70	-0.58
氨基酸 Amino acid	密码子 Codon	基因组 Genome		高表达基因 High expression gene		低表达基因 Low expression gene		ΔRSCU
氨基酸 Amino acid	密码子 Codon	数目Number	RSCU	数目Number	RSCU	数目Number	RSCU	ΔRSCU
Ala	GCU	534	1.84	18	1.50	44	1.69	-0.19
	GCC	168	0.58	3	0.25	16	0.62	-0.37
	GCA^**	332	1.15	17	1.42	27	1.04	0.38
	GCG^*	125	0.43	10	0.83	17	0.65	0.18
Tyr	UAU	660	1.59	11	1.47	94	1.61	-0.14
	UAC^*	170	0.41	4	0.53	23	0.39	0.14
His	CAU^**	409	1.54	12	1.85	53	1.45	0.40
	CAC	121	0.46	1	0.15	20	0.55	-0.40
Gln	CAA^*	582	1.51	16	1.60	78	1.33	0.27
	CAG	189	0.49	4	0.40	39	0.67	-0.27
Asn	AAU	793	1.57	21	1.62	127	1.56	0.06
	AAC	216	0.43	5	0.38	36	0.44	-0.06
Lys	AAA^**	796	1.49	32	1.52	106	1.18	0.34
	AAG	270	0.51	10	0.48	74	0.82	-0.34
Asp	GAU	714	1.61	13	1.63	145	1.67	-0.04
	GAC	174	0.39	3	0.38	29	0.33	0.05
Glu	GAA^*	848	1.47	18	1.50	120	1.28	0.22
	GAG	309	0.53	6	0.50	68	0.72	-0.22
Cys	UGU^***	184	1.48	5	2.00	30	1.40	0.60
	UGC	65	0.52	0	0	13	0.60	-0.60
Trp	UGG	386	1.00	10	1.00	54	1.00	0
Arg	CGU^***	294	1.38	19	1.52	22	0.70	0.82
	CGC	76	0.36	3	0.24	13	0.41	-0.17
	CGA	287	1.35	19	1.52	46	1.46	0.06
	CGG	87	0.41	3	0.24	21	0.67	-0.43
	AGA^**	409	1.92	28	2.24	60	1.90	0.34
	AGG	122	0.57	3	0.24	27	0.86	-0.62
Gly	GGU^***	506	1.37	21	1.53	30	0.86	0.67
	GGC	135	0.37	7	0.51	16	0.46	0.05
	GGA	603	1.64	21	1.53	60	1.73	-0.20
	GGG	230	0.62	6	0.44	33	0.95	-0.51