金花菜叶绿体基因组特征及密码子偏好性分析

尹明华; 胡佳欣; 李瑶瑶; 刘曼情; 欧阳克蕙

doi:10.11829/j.issn.1001-0629.2022-0992

金花菜叶绿体基因组特征及密码子偏好性分析

尹明华^{1, 2, 3, 4},
胡佳欣¹,
李瑶瑶¹,
刘曼情¹,
欧阳克蕙⁵

1.
上饶师范学院生命科学学院, 江西上饶 334001
2.
上饶农业技术创新研究院, 江西上饶 334001
3.
上饶市药食同源植物资源保护与利用重点实验室, 江西上饶 334001
4.
上饶市薯芋类作物种质保存与利用重点实验室, 江西上饶 334001
5.
江西农业大学动物科学技术学院, 江西南昌 330045

基金项目: 国家自然科学基金资助目(31860084、31960079、32060092)；江西省科技厅重点研发计划一般项目(20202BBG73010)；江西省教育厅科学技术研究项目(GJJ201704、GJJ211729)；江西省现代农业产业技术体系建设专项(JXARS-13-赣东站)；上饶市科技局平台载体建设项目(2020J001)

摘要:

本研究对金花菜(Medicago polymorpha, JHC, 江苏)叶绿体基因组进行BGISEQ-500平台测序，对其基因进行Noveplastys组装和GeSeq注释，最后通过MISA、REPuter、Gview、VISTA tools、IRscope和DNADnaSP6.0等软件对其序列特征、基因种类、密码子偏好性以及系统发育进行分析。结果表明，JHC叶绿体基因组大小为124 163 bp，无四分体结构，基因组总GC含量为34.09%，共注释出111个基因(76个CDS 基因、30个tRNA基因、4个rRNA基因、1个假基因)、91个SSR位点和148个长重复序列；JHC叶绿体基因组的第三位密码子偏好以A和U作为末尾碱基，UUA、GCU、UAA、ACU、CCU、GAA、GUA、AUU、UGU、GGU、UUU、CUU为JHC叶绿体基因组的最优密码子；JHC与MZ772862 (云南)亲缘关系较近，与MW971560 (美国)、NC_042848 (美国)关系较远，表明中国金花菜与美国金花菜的起源存在差异。

关键词:

English

Sequence characteristic analyses of the Medicago polymorpha complete chloroplast genome and its codon usage bias

1.
College of Life Sciences, Shangrao Normal University, Shangrao 334001, Jiangxi, China
2.
Shangrao Agricultural Technology Innovation Research Institute, Shangrao 334001, Jiangxi, China
3.
Key Laboratory of protection and utilization of medicinal and edible plant resources in Shangrao City, Shangrao 334001, Jiangxi, China
4.
Key Laboratory of germplasm conservation and utilization of potato and taro crops in Shangrao City, Shangrao 334001, Jiangxi, China;
5.
College of animal science and technology, Jiangxi Agricultural University, Nanchang 330045, Jiangxi, China

Received Date: 2022-12-24
Accepted Date: 2023-05-15
Available Online: 2024-03-20

Abstract:

We performed BGISEQ-500 platform sequencing of the chloroplast genome of Medicago polymorpha (JHC, Jiangsu) and assembled its genes using Noveplastys assembly and GeSeq annotation. we then analyzed its sequence characteristics, gene types, codon preferences, and phylogeny using MISA, REPuter, Gview, VISTA tools, IRscope, and DNADnaSP6.0. The JHC chloroplast genome size was 124163 bp. No tetrad structure was observed, and total genomic GC content was 34.09%. A total of 111 genes, including 76 CDS genes, 30 tRNA genes, 4 rRNA genes, and 1 pseudogene, were annotated from the chloroplast genome of JHC. A total of 91 SSR loci and a total of 148 long repeat sequences were also detected. The third position in JHC chloroplast genome codons preferentially used A and U, and UUA, GCU, UAA, ACU, CCU, GAA, GUA, AUU, UGU, GGU, UUU, CUU were the optimal codons of the chloroplast genome of JHC. JHC was closely related to MZ772862 (Yunnan), and was more distantly related to MW971560 (the United States) and NC_ 042848 the United States), indicating that the origins of American and Chinese Medicago polymorpha strains are different.

Keywords:

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参考文献

[1]	陈莉敏, 李达旭, 白史且, 张玉, 毛德才, 廖兴勇, 杨晓玲. ⁶⁰Co-γ 射线辐射对金花菜种子生物学效应的影响. 核农学报, 2022, 36(9): 1701-1706. CHEN L M, LI D X, BAI S Q, ZHANG Y, MAO D C, LIAO X Y, YANG X L. The biological effect of ⁶⁰Co-γ ray radiation on Medicago polymorpha L. seeds. Journal of Nuclear Agricultural Sciences, 2022, 36(9): 1701-1706.
[2]	刘晓云, 郭振国, 李乔仙, 刘桂霞, 薛世明, 王易鹏. 南苜蓿高效共生根瘤菌土壤的筛选. 生态学报, 2011, 31(14): 4034-4041. LIU X Y, GUO Z G, LI Q X, LIU G X, XUE S M, WANG Y P. Screening of highly effective rhizobial strains on Alfalfa (Medicago polymorpha) in soil. Acta Ecologica Sinica, 2011, 31(14): 4034-4041.
[3]	中国科学院植物研究所. 中国植物志. 北京: 科学出版社, 1998. Institute of Botany, Chinese Academy of Sciences. Flora of China. Beijing: Science Press, 1998.
[4]	董磊, 王栋麟, 王琳, 刘大林. 外源水杨酸缓解金花菜高温胁迫的生理响应. 扬州大学学报(农业与生命科学版), 2022, 43(4): 129-136. DONG L, WANG D L, WANG L, LIU D L. Physiological effects of exogenous salicylic acid on relatively high-temperature stress in Jinhua. Journal of Yangzhou University (Agricultural and Life Science Edition), 2022, 43(4): 129-136.
[5]	DENTON M D, HILL C R, BELLOTTI W D, COVENTRY D R. Nodulation of Medicago truncatula and Medicago polymorpha in two pastures of contrasting soil pH and rhizobial populations. Applied Soil Ecology, 2007, 35(2): 441-448. doi: 10.1016/j.apsoil.2006.08.001
[6]	贾雪杰, 游明鸿, 李达旭, 雷雄, 任小英, 熊晓兰, 杜静, 陈丽敏, 董志晓, 张建波, 马啸. 减量施肥对金花菜-水稻轮作系统中产量和土壤养分的影响. 草地学报, 2023, 31(3): 876-883. JIA X J, YOU M H, LI D X, LEI X, REN X Y, XIONG X L, DU J, CHEN L M, DONG Z X, ZHANG J B, MA X. Effects of nutrient coordination on yield and soil nutrients in a burclover-rice rotation system. Acta Agrestia Sinica, 2023, 31(3): 876-883.
[7]	魏臻武, 任海龙, 武自念, 刘国志, 陈祥, 乔志宏. 金花菜新品种“淮扬金花菜”. 园艺学报, 2015, 42(11): 2335-2336. WEI Z W, REN H L, WU Z N, LIU G Z, CHEN X, QIAO Z H. New Medicago polymorpha cultivar, Huaiyang Jinhuacai. Acta Horticulturae Sinica, 2015, 42(11): 2335-2336.
[8]	洪森荣, 朱盈盈, 李紫莹, 胡明艳, 欧阳克蕙. 盐胁迫下金花菜和紫花苜蓿试管苗的转录组分析及其耐盐基因筛选. 中国农学通报, 2023, 39(3): 111-118. doi: 10.11924/j.issn.1000-6850.casb2022-0235 HONG S R, ZHU Y Y, LI Z Y HU M Y, OUYANG K H. Plantlets of Medicago polymorpha and Medicago sativa under salt stress: transcriptome analysis and salt tolerance gene screening. Chinese Agricultural Science Bulletin, 2023, 39(3): 111-118. doi: 10.11924/j.issn.1000-6850.casb2022-0235
[9]	TANG D F, WEI F, KASHIF M H, KHAN A, LI Z Q, SHI Q Q, JIA R X, XIE H Y, ZHANG L, LI B, CHEN P, ZHOU R Y. Analysis of chloroplast differences in the leaves of rice isonuclear alloplasmic lines. Protoplasma, 2018, 255(3): 863-871. doi: 10.1007/s00709-017-1189-6
[10]	LI G L, PAN Z L, GAO S C, HE Y Y, XIA Q Y, JIN Y, YAO H P. Analysis of synonymous codon usage of chloroplast genome in Porphyra umbilicalis. Genes & Genomics, 2019, 41(10): 1173-1181.
[11]	DURET L. Evolution of synonymous codon usage in metazoans. Current Opinion in Genetics & Development, 2002, 12(6): 640-649.
[12]	HERSHBERG R, PETROV D. Selection of codon bias. Annual Review of Genetics, 2008, 42: 287-299.
[13]	WANG H J, MENG T, WEI W Q. Analysis of synonymous codon usage bias in helicase gene from Autographa californica multiple nucleopolyhedrovirus. Genes & Genomics, 2018, 40(7): 767-780.
[14]	LONG S Y, YAO H P, WU Q, LI G L. Analysis of compositional bias and codon usage pattern of the coding sequence in the Banna virus genome. Virus Research, 2018, 258: 68-72. doi: 10.1016/j.virusres.2018.10.006
[15]	FITTER J T, THOMAS M R, ROSE R J, SCOTT N S. Heteroplasmy of the chloroplast genome ofMedicago sativa L. ‘Regen S’ was confirmed by sequence analysis. Theoretical and Applied Genetics, 1996, 93(5-6): 685-690.
[16]	杨国锋, 苏昆龙, 赵怡然, 宋智斌, 孙娟. 蒺藜苜蓿叶绿体密码子偏好性分析. 草业学报, 2015, 24(12): 171-179. doi: 10.11686/cyxb2015016 YANG G F, SU K L, ZHAO Y R, SONG Z B, SUN J. Analysis of codon usage in the chloroplast genome of Medicago truncatula. Acta Prataculturae Sinica, 2015, 24(12): 171-179. doi: 10.11686/cyxb2015016
[17]	喻凤, 韩明. 紫花苜蓿叶绿体基因组密码子偏好性分析. 广西植物, 2021, 41(12): 2069-2076. YU F, HAN M. Analysis of codon usage bias in alfalfa chloroplast genome of alfalfa (Medicago sativa). Guihaia, 2021, 41(12): 2069-2076.
[18]	孙志轩, 敖平星, 毕玉芬, 赵雁. ‘德钦’紫花苜蓿叶绿体基因组序列及特征分析. 草地学报, 2022, 30(2): 320-328. SUN Z X, AO P X, BI Y F, ZHAO Y. Complete chloroplast genome sequence and characteristics analysis of Medicago sativa ‘Deqin’. Acta Agrestia Sinica, 2022, 30(2): 320-328.
[19]	田春育, 李志勇, 刘倩, 于林清, 武自念. 苜蓿属不同物种叶绿体基因组结构比较及亲缘关系分析. 中国草地学报, 2021, 43(10): 1-8. TIAN C Y, LI Z Y, LIU Q, YU L Q, WU Z N. Comparison of chloroplast genome structure and phylogenetic analysis of different species of Medicago. Chinese Journal of Grassland, 2021, 43(10): 1-8.
[20]	赵嫚, 陈仕勇, 李亚萍, 周青平, 陈有军, 常馨丹. 外源 GABA 对盐胁迫下金花菜种子萌发及幼苗抗氧化能力的影响. 江苏农业学报, 2021, 37(2): 310-316. ZHAO M, CHEN S Y, LI Y P, ZHOU Q P, CHEN Y J, CHANG X D. Influence of exogenous γ-aminobutyric acid ( GABA) on seed germination and antioxidant protection of Medicago polymorpha under salt stress. Jiangsu Journal of Agricultural Sciences, 2021, 37(2): 310-316.
[21]	江舟, 陈丰, 王东军, 魏臻武, 唐晨阳. 金花菜与燕麦间作对牧草产量与品质的影响. 中国草地学报, 2020, 42(5): 127-135. JIANG Z, CHEN F, WANG D J, WEI Z W, TANG C Y. Effects of intercropping burr medics with oats on foyield and quality. Chinese Journal of Grassland, 2020, 42(5): 127-135.
[22]	周克友, 李争艳, 陈祥, 任海龙, 江舟, 魏臻武. 不同光周期条件下金花菜F2代农艺性状的变异特征. 草地学报, 2017, 25(6): 1300-1307. ZHOU K Y, LI Z Y, CHEN X, REN H L, JIANG Z, WEI Z W. Study on variation characteristics of Medicago polymorpha F2 generation under different photoperiods. Acta Agrestia Sinica, 2017, 25(6): 1300-1307.
[23]	JANSEN R K, RUHLMAN T A. Plastid genomes of seed plants. //BOCK R, KOOP V. Genomics of Chloroplasts and Mitochondria. Dordrecht: Springer Netherlands, 2012: 103-126.
[24]	MOWER J P, VICKREY T L. Structural Diversity Amongplastid Genomes of Land Plants. //CHAW S M, JANSEN R K. Advances in Botanical Research. London: Academic Press, 2018: 263-292.
[25]	BRUNEAU A, DOYLE J J PALMER J. Chloroplast DNA inversion is a subtribal characteristic in Phaseoleae (Leguminosae). Systematic Botany, 1990, 15(3): 378-386. doi: 10.2307/2419351
[26]	KOLODNER R, TEWARI K K. Inverted repeats in chloroplast DNA from higher plants. Proceedings of the National Academy of Sciences of the United States of America, 1979, 76(1): 41-45.
[27]	SCHWARZ E N, RUHLMAN T A, WENG M L, KHIYAMI M A, SABIR J S M, HAJARAH N H, ALHARBI N S, RABAH S O, JANSEN R K. Plastome-wide nucleotide substitution rates reveal accelerated rates in Papilionoideae and correlations with genome features across legume subfamilies. Journal of Molecular Evolution, 2017, 84(4): 187-203. doi: 10.1007/s00239-017-9792-x
[28]	王晓娟, 董文攀, 周世良. 基于叶绿体基因组分析我国苜蓿属植物演化路径. 生态学报, 2022, 42(15): 6125-6136. WANG X J, DONG W P, ZHOU S L. The evolutionary path of Medicago in China was based on chloroplast genome analysis. Acta Ecologica Sinica, 2022, 42(15): 6125-6136.
[29]	HONG C P, PARK J, LEE Y, LEE M, PARK S G, UHM Y, LEE J, KIM C. accD nuclear transfer of Platycodon grandiflorum and the plastid of early Campanulaceae. BMC Genomics, 2017, 18(1): 1-13. doi: 10.1186/s12864-016-3406-7
[30]	MILLEN R S, OLMSTEAD R G, ADAMS K L, PALMER J D, LAO N T, HEGGIE L, KAVANAGH T A, HIBBERD J M, GRAY J C, MORDEN C W, CALIE P J, JERMIIN L S, WOLFE K H. Many parallel losses of infA from chloroplast DNA occur during angiosperm evolution, with multiple independent transfers to the nucleus. Plant Cell, 2001, 13(3): 645-658. doi: 10.1105/tpc.13.3.645
[31]	SASKI C, LEE S B, DANIELL H, WOOD T C, TOMKINS J, KIM H G, JANSEN R K. Complete chloroplast genome sequence of Gycine max and comparative analyses with other legume genomes. Plant Molecular Biology, 2005, 59(2): 309-322. doi: 10.1007/s11103-005-8882-0
[32]	LI B, ZHENG Y. Dynamic evolution and phylogenomic analysis of the chloroplast genome in Schisandraceae. Scientific Reports, 2018, 8(1): 9285. doi: 10.1038/s41598-018-27453-7
[33]	SALTONSTALL K, LAMBERTINI C. Value of repetitive sequences in chloroplast DNA for phylogeographic inference: A comment on Vachon and Freeland (2011). Molecular Ecology Resources, 2012, 12(4): 581-589.
[34]	曾令霜, 张晨晨, 张敬, 徐彬. 多年生黑麦草种质SSR分子标记遗传多样性分析. 草业科学, 2022, 39(1): 75-84. doi: 10.11829/j.issn.1001-0629.2021-0364 ZENG L S, ZHANG C C, ZHANG J, XU B. Genetic diversity analysis of perennial ryegrass germplasm using SSR molecular markers. Pratacultural Science, 2022, 39(1): 75-84. doi: 10.11829/j.issn.1001-0629.2021-0364
[35]	张婷婷, 张鹤山, 宋康杰, 赵泽宇, 许本波, 刘洋. 白三叶转录组 SSR 位点特征分析及引物开发. 草业科学, 2023, 40(9): 2266-2275. doi: 10.11829/j.issn.1001-0629.2022-0841 ZHANG T T, ZHANG H S, SONG K J, ZHAO Z Y, XU B B, LIU Y. Analysis of SSR site characteristics of Trifolium repens transcriptome and primer development. Pratacultural Science, 2023, 40(9): 2266-2275. doi: 10.11829/j.issn.1001-0629.2022-0841
[36]	JIANG Y, DENG F, WANG H L. Extensive analysis of global codon usage patterns in baculoviruses. Archives of Virology, 2008, 153(12): 2273-2282. doi: 10.1007/s00705-008-0260-1
[37]	QUAX T F, CLAASSENS N, SÖLL D, OOST J. Codon bias as a means to fine-tune gene expression. Molecular Cell, 2015, 59(2): 149-161. doi: 10.1016/j.molcel.2015.05.035
[38]	WANG L, ROOSSINCK M J. Comparative analysis of expressed sequences reveals a conserved pattern of optimal codon usage in plants. Plant Molecular Biology, 2006, 61(4-5): 699-710. doi: 10.1007/s11103-006-0041-8

图 1 金花菜叶绿体基因组图谱

Figure 1. Medicago polymorpha chloroplast genome map

下载: 全尺寸图片幻灯片

图 2 金花菜及其9个近缘种叶绿体基因组变异圈图

物种从内向外第3圈开始依次为：Medicago radiata NC_042854、花苜MW703984、刺果苜蓿NC_042851、南苜蓿MW971560、金花菜JHC、蒺藜苜蓿JX512022、木本苜蓿NC_042856、Medicago marina NC_042845、野苜蓿MW271003、紫花苜蓿MN218692。右上角GC和GC skew是最内两圈，CDS、rRNA、tRNA圆圈中心位置。

Figure 2. Chloroplast genome variation circle diagram for Medicago polymorpha and nine closely related species

Species start from inner to outer ring: Medicago radiata NC_042854, M. ruthenica MW703984, M. intertexta NC_042851, M. polymorpha MW971560, M. polymorpha (JHC), M. truncatula JX512022, M. arborea NC_042856, M. marina NC_042845, M. falcata MW271003, M. sativa MN218692. GC and GC skew are the two innermost circles in the upper right corner; CDS, rRNA, and tRNA circles are in the center.

下载: 全尺寸图片幻灯片

图 3 金花菜及其9个近缘种叶绿体基因组Pi多样性指数分析

Figure 3. Pi diversity index analysis of the chloroplast genomes of Medicago polymorpha and nine related species

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图 4 金花菜及其9个近缘种叶绿体基因组密码子的偏性分析

物种同图2。密码子第1、2、3位的GC含量分别用GC₁、GC₂、GC₃表示；密码子总GC含量用GC_all表示。表4同。

Figure 4. Partial chloroplast genome codon analysis for Medicago polymorpha and nine closely related species

The species are the same as Figure 2. GC content at first, second, and third codon positions are represented as GC₁, GC₂, and GC₃, respectively; Total codon GC content is represented by GC_all. This is applicable for Table 4 as well.

下载: 全尺寸图片幻灯片

图 5 基于叶绿体基因组的金花菜(JHC)及其45个近缘种和2个外类群物种的系统发育树

Figure 5. Phylogenetic tree of Medicago polymorpha (JHC) and its 45 closely related species and 2 species level taxa based on their chloroplast genomes

下载: 全尺寸图片幻灯片

表 1 金花菜45个近缘种和2个外类群物种的产地或来源

Table 1 Origin or source of 45 closely related species and 2 species level taxa of Medicago polymorpha

序号 Number	近缘种或外类群物种 Related species or outgroup species	产地或来源 Origin or source	序号 Number	近缘种或外类群物种 Related species or outgroup species	产地或来源 Origin or source	序号 Number	近缘种或外类群物种 Related species or outgroup species	产地或来源 Origin or source
1	紫花苜蓿 Medicago sativa KU321683	中国 China	17	蒺藜苜蓿 Medicago truncatula JX512023	美国 USA	33	Medicago intertexta NC_042851	美国 USA
2	紫花苜蓿 Medicago sativa MN218692	云南省德钦县 Deqin County, Yunnan Province	18	蒺藜苜蓿 Medicago truncatula JX512024	美国 USA	34	Medicago orbicularis NC_042850	美国 USA
3	紫花苜蓿 Medicago sativa NC_042841	美国 USA	19	蒺藜苜蓿 Medicago truncatula JX512022	美国 USA	35	天蓝苜蓿 Medicago lupulina NC_042847	美国 USA
4	紫花苜蓿 Medicago sativa MZ983396	中国 China	20	蒺藜苜蓿 Medicago truncatula f. tricycla KF241982	美国 USA	36	Medicago tenoreana NC_057658	美国 USA
5	野苜蓿 Medicago falcata MW271002	中国 China	21	Medicago scutellata MZ895077	中国 China	37	Medicago disciformis NC_057655	美国 USA
6	野苜蓿 Medicago falcata NC_032066	中国 China	22	早花苜蓿 Medicago praecox NC_057657	美国 USA	38	小苜蓿 Medicago minima NC_042849	美国 USA
7	野苜蓿 Medicago falcata MK460490	美国 USA	23	金花菜 Medicago polymorpha MZ772862	中国 China	39	Medicago coronata NC_057660	美国 USA
8	野苜蓿 Medicago falcata MW271003	中国 China	24	金花菜 Medicago polymorpha MW971560	美国 USA	40	Medicago suffruticosa NC_042843	美国 USA
9	Medicago tetraprostra NC_042844	美国 USA	25	金花菜 Medicago polymorpha NC_042848	美国 USA	41	Medicago biflora NC_042415	美国 USA
10	紫花苜蓿 Medicago sativa subsp. Glomerata MK460494	美国 USA	26	褐斑苜蓿 Medicago arabica MT584354	美国 USA	42	毛荚苜蓿 Medicago edgeworthii NC_042415	中国 China
11	Medicago hybrida NC_027153	加拿大 Canada	27	褐斑苜蓿 Medicago arabica NC_057661	美国 USA	43	花苜蓿 Medicago ruthenica NC_053371	中国 China
12	Medicago papillosa NC_027154	加拿大 Canada	28	褐斑苜蓿 Medicago arabica MZ905469	中国 China	44	Medicago radiata NC_042854	美国 USA
13	Medicago cretacea NC_042842	美国 USA	29	Medicago secundiflora NC_057656	美国 USA	45	Medicago monspeliaca NC_042855	美国 USA
14	Medicago marina NC_042845	美国 USA	30	Medicago blancheana NC_042852	美国 USA	46	白花草木樨 Melilotus albus NC_041419	中国 China
15	木本苜蓿 Medicago arborea NC_042856	美国 USA	31	Medicago laciniata NC_042853	美国 USA	47	胡卢巴 Trigonella foenum-graecum NC_042857	美国 USA
16	Medicago pironae NC_042846	美国 USA	32	Medicago sauvagei NC_057659	美国 USA

下载: 导出CSV

表 2 金花菜叶绿体基因的类型分析

Table 2 Type analysis of chloroplast genes in Medicago polymorpha

基因功能 Gene function	基因类型 Gene type	基因名 Gene name	基因数量 Number of genes
光合作用 Photosynthesis	光系统Ⅰ photosystem Ⅰ	psaA, psaB, psaC, psaI, psaJ	5
	光系统Ⅱ photosystem Ⅱ	psaJ, psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbK, psbL, psbM, psbT, psbZ, psbN	15
	NADH 脱氢酶 NADH dehydrogenase	ndhA, ndhB, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK	11
	细胞色素b /f 复合体 cytochrome b/f complex	petA, petB, petD, petG, petL, petN	6
	ATP 合成酶 ATP synthase	atpA, atpB, atpE, atpF, atpH, atpI	6
自我复制 Self-replication	核糖体大亚基蛋白质 Proteins of large ribosomal subunit	rpl14, rpl16, rpl2, rpl20, rpl23, rpl32, rpl33, rpl36	8
	核糖体小亚基蛋白质 Proteins of small ribosomal subunit	rps11, rps12, rps14, rps15, rps18, rps19, rps2, rps3, rps4, rps7, rps8	11
	核糖体大亚基 Large subunit of rubisco	rbcL	1
	RNA 聚合酶 RNA polymerase	rpoA, rpoB, rpoC1, rpoC2	4
	核糖体 RNA Ribosomal RNAs	rrn16, rrn23, rrn4.5, rrn5	4
	转运 RNA Transfer RNAs	trnA-UGC, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC, trnH-GUG, trnI-CAU, trnI-GAU, trnK-UUU, trnL-CAA, trnL-UAA, trnL-UAG, trnM-CAU, trnN-GUU, trnP-UGG, trnQ-UUG, trnR-ACG, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC, trnV-UAC, trnW-CCA, trnY-GUA, trnfM-CAU	30
其他基因 Other genes	成熟酶 Maturase	matK	1
	蛋白酶 Protease	clpP1	1
	囊膜蛋白 Envelope membrane protein	cemA	1
	乙酰辅酶A 羧化酶 Acetyl-CoA carboxylase	accD	1
	c-型细胞色素合成基因 c-type cytochrome synthesis gene	ccsA	1
	翻译起始因子 Translation initiation factor	infA	1
未知功能基因 Unknown function gene	保守假设叶绿体阅读框架 Conserved hypothetical chloroplast Reading Frames	ycf1, ycf2, ycf3, ycf4	4

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表 3 金花菜叶绿体基因组中简单重复序列的类型及分布

Table 3 Type and distribution of simple repeat sequences within the Medicago polymorpha chloroplast genome

重复单元碱基类型 Repetitive unit base type	重复单元重复次数 Repeat number of repeat units															总数 Total
重复单元碱基类型 Repetitive unit base type	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	总数 Total
A/T	−	−	−	−	−	33	15	10	8	5	−	2	−	1	1	75
C/G	−	−	−	−	−	−	−	1	−	−	−	−	−	−	−	1
AT/AT	−	11	1	1	−	−	−	−	−	−	−	−	−	−	−	13
AAT/ATT	1	−	−	−	−	−	−	−	−	−	−	−	−	−	−	1
ATC/ATG	1	−	−	−	−	−	−	−	−	−	−	−	−	−	−	1

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表 4 金花菜叶绿体基因组GC_all、GC₁、GC₂、GC₃和ENC的相关性分析

Table 4 Correlation analysis of the chloroplast genome GC_all, GC₁, GC₂, GC₃, and ENC in Medicago polymorpha

指标 Parameter	GC_all	GC₁	GC₂	GC₃	ENC
GC_all	1
GC₁	0.807 080 845^**	1
GC₂	0.751 657 748^**	0.379 814 149^*	1
GC₃	0.453 207 251^*	0.124 498 687	0.072 925 809	1
ENC	0.002 157 050	−0.074 635 309	−0.074 619 818	0.239 857 869^*	1
和分别表示相关性在0.05和0.01水平上显著相关；ENC 表示有效密码子数。　 and ** indicate significant correlation at the 0.05 and 0.01 levels, respectively; ENC indicates the number of valid codons.

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表 5 金花菜叶绿体基因组密码子的相对同义密码子使用度

Table 5 Relative synonymouscodon usagec (RSCU) of chloroplast genome codons in Medicago polymorpha

密码子 Codon	氨基酸 Amino acid	相对同义密码子使用度 Relative synonymous codon usage (RSCU)		密码子 Codon	氨基酸 Amino acid	相对同义密码子使用度 Relative synonymous codon usage (RSCU)
密码子 Codon	氨基酸 Amino acid	频度 Frequency	数量 Number	密码子 Codon	氨基酸 Amino acid	频度 Frequency	数量 Number
GCA	Ala	1.143 330	349	AAA	Lys	1.556 100	957
GCC	Ala	0.573 301	175	AAG	Lys	0.443 902	273
GCG	Ala	0.412 776	126	AUG	Met	1.992 030	500
GCU	Ala	1.870 600	571	GUG	Met	0.007 968	2
AGA	Arg	1.768 370	369	UUC	Phe	0.588 417	381
AGG	Arg	0.666 134	139	UUU	Phe	1.411 580	914
CGA	Arg	1.389 780	290	CCA	Pro	1.280 700	292
CGC	Arg	0.383 387	80	CCC	Pro	0.671 053	153
CGG	Arg	0.388 179	81	CCG	Pro	0.447 368	102
CGU	Arg	1.404 150	293	CCU	Pro	1.600 880	365
AAC	Asn	0.445 076	235	AGC	Ser	0.314 803	84
AAU	Asn	1.554 920	821	AGU	Ser	1.315 430	351
GAC	Asp	0.388 708	179	UCA	Ser	1.195 500	319
GAU	Asp	1.611 290	742	UCC	Ser	0.824 485	220
UGC	Cys	0.489 627	59	UCG	Ser	0.520 924	139
UGU	Cys	1.510 370	182	UCU	Ser	1.828 860	488
CAA	Gln	1.613 070	617	UAA	Ter	1.815 790	46
CAG	Gln	0.386 928	148	UAG	Ter	0.434 211	11
GAA	Glu	1.560 400	930	UGA	Ter	0.750 000	19
GAG	Glu	0.439 597	262	ACA	Thr	1.231 600	343
GGA	Gly	1.645 470	608	ACC	Thr	0.667 864	186
GGC	Gly	0.341 001	126	ACG	Thr	0.445 242	124
GGG	Gly	0.581 867	215	ACU	Thr	1.655 300	461
GGU	Gly	1.431 660	529	UGG	Trp	1.000 000	383
CAC	His	0.453 441	112	UAC	Tyr	0.335 749	139
CAU	His	1.546 560	382	UAU	Tyr	1.664 250	689
AUA	Ile	0.952 549	629	GUA	Val	1.530 180	469
AUC	Ile	0.519 435	343	GUC	Val	0.440 457	135
AUU	Ile	1.528 020	1 009	GUG	Val	0.486 134	149
CUA	Leu	0.834 693	324	GUU	Val	1.543 230	473
CUC	Leu	0.314 298	122
CUG	Leu	0.350 365	136
CUU	Leu	1.231 430	478
UUA	Leu	2.060 970	800
UUG	Leu	1.208 240	469

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表 6 金花菜叶绿体基因组密码子ENC比值频数分布

Table 6 Codon ENC ratio frequency distribution in the Medicago polymorpha chloroplast genome

区间 Section	组限 Group limit	频数 Number	频率 Frequency
−0.1～0.0	−0.1	4	0.533 3
0.0～0.1	0	6	0.080 0
0.1～0.2	0.1	38	0.506 7
0.2～0.3	0.2	20	0.266 7
0.3～0.4	0.3	3	0.040 0
> 0.4	0.4	4	0.053 3
合计 Total		75	1.000 0

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表 7 金花菜叶绿体基因的最优密码子

Table 7 Optimal codons in the Medicago polymorpha chloroplast genome

密码子 Codon	氨基酸 Amino acid	相对同义密码子使用度 Relative synonymous codon usage (RSCU)		RSCU高表达 RSCU high expression		RSCU低表达 RSCU low expression		RSCU差值 ΔRSCU
密码子 Codon	氨基酸 Amino acid	频度 Frequency	数量 Number	频度 Frequency	数量 Number	频度 Frequency	数量 Number	RSCU差值 ΔRSCU
UUA*	Leu	2.060 970	800	1.370 080	58	1.615 380	7	0.245 300
AUG	Met	1.992 030	500	1.000 000	45	1.000 000	6	0.000 000
GCU*	Ala	1.870 600	571	1.600 000	26	1.875 000	5	0.275 000
UCU	Ser	1.828 860	488	1.631 070	56	1.166 670	7	−0.464 400
UAA*	Ter	1.815 790	46	1.200 000	3	1.800 000	3	0.600 000
AGA	Arg	1.768 370	369	1.808 820	41	1.200 000	3	−0.608 820
UAU	Tyr	1.664 250	689	1.674 420	72	1.500 000	3	−0.174 420
ACU*	Thr	1.655 300	461	1.234 040	29	1.714 290	8	0.480 250
GGA	Gly	1.645 470	608	1.706 670	32	0.857 143	9	−0.849 527
CAA	Gln	1.613 070	617	1.371 430	48	1.000 000	2	−0.371 430
GAU	Asp	1.611 290	742	1.639 340	100	1.000 000	6	−0.639 340
CCU*	Pro	1.600 880	365	1.292 930	32	2.142 860	5	0.849 930
GAA*	Glu	1.560 400	930	1.292 310	84	1.666 670	5	0.374 360
AAA	Lys	1.556 100	957	1.364 160	118	1.166 670	7	−0.197 490
AAU	Asn	1.554 920	821	1.594 200	110	1.000 000	8	−0.594 200
CAU	His	1.546 560	382	1.489 360	35	0.666 667	1	−0.822 693
GUU	Val	1.543 230	473	1.733 330	39	1.666 670	10	−0.066 660
GUA*	Val	1.530 180	469	0.933 333	21	1.666 670	10	0.733 337
AUU*	Ile	1.528 020	1 009	1.411 760	80	1.560 000	13	0.148 240
UGU*	Cys	1.510 370	182	1.153 850	15	1.600 000	4	0.446 150
GGU*	Gly	1.431 660	529	1.013 330	19	1.142 860	12	0.129 530
UUU*	Phe	1.411 580	914	1.182 390	94	1.294 120	11	0.111 730
CGU	Arg	1.404 150	293	0.750 000	17	0.800 000	2	0.050 000
CGA	Arg	1.389 780	290	1.235 290	28	1.200 000	3	−0.035 290
AGU	Ser	1.315 430	351	1.135 920	39			−1.135 920
CCA	Pro	1.280 700	292	1.535 350	38	0.428 571	1	−1.106 779
ACA	Thr	1.231 600	343	1.361 700	32	0.857 143	4	−0.504 557
CUU*	Leu	1.231 430	478	1.417 320	60	1.615 380	7	0.198 060
UUG	Leu	1.208 240	469	1.322 830	56	1.153 850	5	−0.168 980
UCA	Ser	1.195 500	319	1.368 930	47	1.000 000	6	−0.368 930
GCA	Ala	1.143 330	349	1.230 770	20	0.375 000	1	−0.855 770
表示最优密码子。　 represents the optimal codon.

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参考文献(38)

[1]	陈莉敏, 李达旭, 白史且, 张玉, 毛德才, 廖兴勇, 杨晓玲. ⁶⁰Co-γ 射线辐射对金花菜种子生物学效应的影响. 核农学报, 2022, 36(9): 1701-1706. CHEN L M, LI D X, BAI S Q, ZHANG Y, MAO D C, LIAO X Y, YANG X L. The biological effect of ⁶⁰Co-γ ray radiation on Medicago polymorpha L. seeds. Journal of Nuclear Agricultural Sciences, 2022, 36(9): 1701-1706.
[2]	刘晓云, 郭振国, 李乔仙, 刘桂霞, 薛世明, 王易鹏. 南苜蓿高效共生根瘤菌土壤的筛选. 生态学报, 2011, 31(14): 4034-4041. LIU X Y, GUO Z G, LI Q X, LIU G X, XUE S M, WANG Y P. Screening of highly effective rhizobial strains on Alfalfa (Medicago polymorpha) in soil. Acta Ecologica Sinica, 2011, 31(14): 4034-4041.
[3]	中国科学院植物研究所. 中国植物志. 北京: 科学出版社, 1998. Institute of Botany, Chinese Academy of Sciences. Flora of China. Beijing: Science Press, 1998.
[4]	董磊, 王栋麟, 王琳, 刘大林. 外源水杨酸缓解金花菜高温胁迫的生理响应. 扬州大学学报(农业与生命科学版), 2022, 43(4): 129-136. DONG L, WANG D L, WANG L, LIU D L. Physiological effects of exogenous salicylic acid on relatively high-temperature stress in Jinhua. Journal of Yangzhou University (Agricultural and Life Science Edition), 2022, 43(4): 129-136.
[5]	DENTON M D, HILL C R, BELLOTTI W D, COVENTRY D R. Nodulation of Medicago truncatula and Medicago polymorpha in two pastures of contrasting soil pH and rhizobial populations. Applied Soil Ecology, 2007, 35(2): 441-448. doi: 10.1016/j.apsoil.2006.08.001
[6]	贾雪杰, 游明鸿, 李达旭, 雷雄, 任小英, 熊晓兰, 杜静, 陈丽敏, 董志晓, 张建波, 马啸. 减量施肥对金花菜-水稻轮作系统中产量和土壤养分的影响. 草地学报, 2023, 31(3): 876-883. JIA X J, YOU M H, LI D X, LEI X, REN X Y, XIONG X L, DU J, CHEN L M, DONG Z X, ZHANG J B, MA X. Effects of nutrient coordination on yield and soil nutrients in a burclover-rice rotation system. Acta Agrestia Sinica, 2023, 31(3): 876-883.
[7]	魏臻武, 任海龙, 武自念, 刘国志, 陈祥, 乔志宏. 金花菜新品种“淮扬金花菜”. 园艺学报, 2015, 42(11): 2335-2336. WEI Z W, REN H L, WU Z N, LIU G Z, CHEN X, QIAO Z H. New Medicago polymorpha cultivar, Huaiyang Jinhuacai. Acta Horticulturae Sinica, 2015, 42(11): 2335-2336.
[8]	洪森荣, 朱盈盈, 李紫莹, 胡明艳, 欧阳克蕙. 盐胁迫下金花菜和紫花苜蓿试管苗的转录组分析及其耐盐基因筛选. 中国农学通报, 2023, 39(3): 111-118. doi: 10.11924/j.issn.1000-6850.casb2022-0235 HONG S R, ZHU Y Y, LI Z Y HU M Y, OUYANG K H. Plantlets of Medicago polymorpha and Medicago sativa under salt stress: transcriptome analysis and salt tolerance gene screening. Chinese Agricultural Science Bulletin, 2023, 39(3): 111-118. doi: 10.11924/j.issn.1000-6850.casb2022-0235
[9]	TANG D F, WEI F, KASHIF M H, KHAN A, LI Z Q, SHI Q Q, JIA R X, XIE H Y, ZHANG L, LI B, CHEN P, ZHOU R Y. Analysis of chloroplast differences in the leaves of rice isonuclear alloplasmic lines. Protoplasma, 2018, 255(3): 863-871. doi: 10.1007/s00709-017-1189-6
[10]	LI G L, PAN Z L, GAO S C, HE Y Y, XIA Q Y, JIN Y, YAO H P. Analysis of synonymous codon usage of chloroplast genome in Porphyra umbilicalis. Genes & Genomics, 2019, 41(10): 1173-1181.
[11]	DURET L. Evolution of synonymous codon usage in metazoans. Current Opinion in Genetics & Development, 2002, 12(6): 640-649.
[12]	HERSHBERG R, PETROV D. Selection of codon bias. Annual Review of Genetics, 2008, 42: 287-299.
[13]	WANG H J, MENG T, WEI W Q. Analysis of synonymous codon usage bias in helicase gene from Autographa californica multiple nucleopolyhedrovirus. Genes & Genomics, 2018, 40(7): 767-780.
[14]	LONG S Y, YAO H P, WU Q, LI G L. Analysis of compositional bias and codon usage pattern of the coding sequence in the Banna virus genome. Virus Research, 2018, 258: 68-72. doi: 10.1016/j.virusres.2018.10.006
[15]	FITTER J T, THOMAS M R, ROSE R J, SCOTT N S. Heteroplasmy of the chloroplast genome ofMedicago sativa L. ‘Regen S’ was confirmed by sequence analysis. Theoretical and Applied Genetics, 1996, 93(5-6): 685-690.
[16]	杨国锋, 苏昆龙, 赵怡然, 宋智斌, 孙娟. 蒺藜苜蓿叶绿体密码子偏好性分析. 草业学报, 2015, 24(12): 171-179. doi: 10.11686/cyxb2015016 YANG G F, SU K L, ZHAO Y R, SONG Z B, SUN J. Analysis of codon usage in the chloroplast genome of Medicago truncatula. Acta Prataculturae Sinica, 2015, 24(12): 171-179. doi: 10.11686/cyxb2015016
[17]	喻凤, 韩明. 紫花苜蓿叶绿体基因组密码子偏好性分析. 广西植物, 2021, 41(12): 2069-2076. YU F, HAN M. Analysis of codon usage bias in alfalfa chloroplast genome of alfalfa (Medicago sativa). Guihaia, 2021, 41(12): 2069-2076.
[18]	孙志轩, 敖平星, 毕玉芬, 赵雁. ‘德钦’紫花苜蓿叶绿体基因组序列及特征分析. 草地学报, 2022, 30(2): 320-328. SUN Z X, AO P X, BI Y F, ZHAO Y. Complete chloroplast genome sequence and characteristics analysis of Medicago sativa ‘Deqin’. Acta Agrestia Sinica, 2022, 30(2): 320-328.
[19]	田春育, 李志勇, 刘倩, 于林清, 武自念. 苜蓿属不同物种叶绿体基因组结构比较及亲缘关系分析. 中国草地学报, 2021, 43(10): 1-8. TIAN C Y, LI Z Y, LIU Q, YU L Q, WU Z N. Comparison of chloroplast genome structure and phylogenetic analysis of different species of Medicago. Chinese Journal of Grassland, 2021, 43(10): 1-8.
[20]	赵嫚, 陈仕勇, 李亚萍, 周青平, 陈有军, 常馨丹. 外源 GABA 对盐胁迫下金花菜种子萌发及幼苗抗氧化能力的影响. 江苏农业学报, 2021, 37(2): 310-316. ZHAO M, CHEN S Y, LI Y P, ZHOU Q P, CHEN Y J, CHANG X D. Influence of exogenous γ-aminobutyric acid ( GABA) on seed germination and antioxidant protection of Medicago polymorpha under salt stress. Jiangsu Journal of Agricultural Sciences, 2021, 37(2): 310-316.
[21]	江舟, 陈丰, 王东军, 魏臻武, 唐晨阳. 金花菜与燕麦间作对牧草产量与品质的影响. 中国草地学报, 2020, 42(5): 127-135. JIANG Z, CHEN F, WANG D J, WEI Z W, TANG C Y. Effects of intercropping burr medics with oats on foyield and quality. Chinese Journal of Grassland, 2020, 42(5): 127-135.
[22]	周克友, 李争艳, 陈祥, 任海龙, 江舟, 魏臻武. 不同光周期条件下金花菜F2代农艺性状的变异特征. 草地学报, 2017, 25(6): 1300-1307. ZHOU K Y, LI Z Y, CHEN X, REN H L, JIANG Z, WEI Z W. Study on variation characteristics of Medicago polymorpha F2 generation under different photoperiods. Acta Agrestia Sinica, 2017, 25(6): 1300-1307.
[23]	JANSEN R K, RUHLMAN T A. Plastid genomes of seed plants. //BOCK R, KOOP V. Genomics of Chloroplasts and Mitochondria. Dordrecht: Springer Netherlands, 2012: 103-126.
[24]	MOWER J P, VICKREY T L. Structural Diversity Amongplastid Genomes of Land Plants. //CHAW S M, JANSEN R K. Advances in Botanical Research. London: Academic Press, 2018: 263-292.
[25]	BRUNEAU A, DOYLE J J PALMER J. Chloroplast DNA inversion is a subtribal characteristic in Phaseoleae (Leguminosae). Systematic Botany, 1990, 15(3): 378-386. doi: 10.2307/2419351
[26]	KOLODNER R, TEWARI K K. Inverted repeats in chloroplast DNA from higher plants. Proceedings of the National Academy of Sciences of the United States of America, 1979, 76(1): 41-45.
[27]	SCHWARZ E N, RUHLMAN T A, WENG M L, KHIYAMI M A, SABIR J S M, HAJARAH N H, ALHARBI N S, RABAH S O, JANSEN R K. Plastome-wide nucleotide substitution rates reveal accelerated rates in Papilionoideae and correlations with genome features across legume subfamilies. Journal of Molecular Evolution, 2017, 84(4): 187-203. doi: 10.1007/s00239-017-9792-x
[28]	王晓娟, 董文攀, 周世良. 基于叶绿体基因组分析我国苜蓿属植物演化路径. 生态学报, 2022, 42(15): 6125-6136. WANG X J, DONG W P, ZHOU S L. The evolutionary path of Medicago in China was based on chloroplast genome analysis. Acta Ecologica Sinica, 2022, 42(15): 6125-6136.
[29]	HONG C P, PARK J, LEE Y, LEE M, PARK S G, UHM Y, LEE J, KIM C. accD nuclear transfer of Platycodon grandiflorum and the plastid of early Campanulaceae. BMC Genomics, 2017, 18(1): 1-13. doi: 10.1186/s12864-016-3406-7
[30]	MILLEN R S, OLMSTEAD R G, ADAMS K L, PALMER J D, LAO N T, HEGGIE L, KAVANAGH T A, HIBBERD J M, GRAY J C, MORDEN C W, CALIE P J, JERMIIN L S, WOLFE K H. Many parallel losses of infA from chloroplast DNA occur during angiosperm evolution, with multiple independent transfers to the nucleus. Plant Cell, 2001, 13(3): 645-658. doi: 10.1105/tpc.13.3.645
[31]	SASKI C, LEE S B, DANIELL H, WOOD T C, TOMKINS J, KIM H G, JANSEN R K. Complete chloroplast genome sequence of Gycine max and comparative analyses with other legume genomes. Plant Molecular Biology, 2005, 59(2): 309-322. doi: 10.1007/s11103-005-8882-0
[32]	LI B, ZHENG Y. Dynamic evolution and phylogenomic analysis of the chloroplast genome in Schisandraceae. Scientific Reports, 2018, 8(1): 9285. doi: 10.1038/s41598-018-27453-7
[33]	SALTONSTALL K, LAMBERTINI C. Value of repetitive sequences in chloroplast DNA for phylogeographic inference: A comment on Vachon and Freeland (2011). Molecular Ecology Resources, 2012, 12(4): 581-589.
[34]	曾令霜, 张晨晨, 张敬, 徐彬. 多年生黑麦草种质SSR分子标记遗传多样性分析. 草业科学, 2022, 39(1): 75-84. doi: 10.11829/j.issn.1001-0629.2021-0364 ZENG L S, ZHANG C C, ZHANG J, XU B. Genetic diversity analysis of perennial ryegrass germplasm using SSR molecular markers. Pratacultural Science, 2022, 39(1): 75-84. doi: 10.11829/j.issn.1001-0629.2021-0364
[35]	张婷婷, 张鹤山, 宋康杰, 赵泽宇, 许本波, 刘洋. 白三叶转录组 SSR 位点特征分析及引物开发. 草业科学, 2023, 40(9): 2266-2275. doi: 10.11829/j.issn.1001-0629.2022-0841 ZHANG T T, ZHANG H S, SONG K J, ZHAO Z Y, XU B B, LIU Y. Analysis of SSR site characteristics of Trifolium repens transcriptome and primer development. Pratacultural Science, 2023, 40(9): 2266-2275. doi: 10.11829/j.issn.1001-0629.2022-0841
[36]	JIANG Y, DENG F, WANG H L. Extensive analysis of global codon usage patterns in baculoviruses. Archives of Virology, 2008, 153(12): 2273-2282. doi: 10.1007/s00705-008-0260-1
[37]	QUAX T F, CLAASSENS N, SÖLL D, OOST J. Codon bias as a means to fine-tune gene expression. Molecular Cell, 2015, 59(2): 149-161. doi: 10.1016/j.molcel.2015.05.035
[38]	WANG L, ROOSSINCK M J. Comparative analysis of expressed sequences reveals a conserved pattern of optimal codon usage in plants. Plant Molecular Biology, 2006, 61(4-5): 699-710. doi: 10.1007/s11103-006-0041-8