Sequence Variation Analysis of the Parthenogeny-inducing Gene BBM1 in Rice

doi:10.16819/j.1001-7216.2024.231210

Abstract

Abstract:

【Objective】Ectopic expression of BBM1 in rice egg cells can induce parthenogenesis. Exploring superior allelic variants of BBM1 and functionally similar genes is of great significance for heterosis fixation in rice.【Method】By utilizing third-generation sequencing data from 383 core rice accessions, we conducted an analysis of structural variations, sequence polymorphisms, and haplotypes of BBM1. Additionally, we screened for genes with similar sequences and expression patterns to identify functional analogues of BBM1.【Results】Our findings revealed the presence of two copies of the BBM1 gene in three rice accessions, along with MITEs insertion variants observed in two O. rufipogon accessions. The BBM1 sequence exhibited a clear indica-japonica differentiation, with 94.80% of japonica accessions and 3.80% of indica accessions experiencing premature termination due to single base mutations, resulting in the loss of 26 amino acid residues. Furthermore, the coding region of BBM1 is highly conserved, and most rice accessions share the same haplotype. We also identified LOC_Os06g44750 as a candidate gene with a similar sequence and expression pattern to BBM1, which may potentially contribute to inducing parthenogenesis.【Conclusion】This study provides insights into the variation present within the BBM1 gene in rice while also identifying important haplotypes and potential candidate genes with functional similarities. This foundational knowledge serves to enhance the frequency of parthenogenesis in rice.

Key words: rice, BBM1, parthenogenesis, sequence variation

摘要：

【目的】在水稻卵细胞中异位表达BBM1能够诱导孤雌生殖，挖掘BBM1优异等位变异和功能类似基因对水稻杂种优势固定具有重要意义。【方法】利用383份水稻核心种质的三代测序数据，对BBM1基因进行结构变异分析、序列多态性分析、单倍型分析，并根据序列相似度以及表达模式相似度筛选BBM1功能类似基因。【结果】BBM1基因在3份种质中存在两个拷贝，在2份野生稻中存在微型颠倒重复转座元件（MITEs）插入变异；具有非常明显的籼粳分化，94.80%的粳稻和3.80%的籼稻中BBM1因单碱基变异造成翻译提前终止，缺少26个氨基酸残基；BBM1编码区序列十分保守，绝大多数材料中具有相同的单倍型；筛选到BBM1序列和表达模式相似基因LOC_Os06g44750，其可能具有BBM1诱导孤雌生殖的功能。【结论】明确了水稻中BBM1基因变异情况，挖掘出重要单倍型和功能类似候选基因，为进一步提高水稻孤雌生殖频率奠定基础。

关键词: 水稻, BBM1, 孤雌生殖, 序列变异

WANG Qing, WANG Yanru, ZHANG Xiuli, LÜ Qiming. Sequence Variation Analysis of the Parthenogeny-inducing Gene BBM1 in Rice[J]. Chinese Journal OF Rice Science, 2024, 38(6): 627-637.

汪晴, 王艳茹, 张秀丽, 吕启明. 水稻孤雌生殖诱导基因BBM1序列变异分析[J]. 中国水稻科学, 2024, 38(6): 627-637.

Figures/Tables 10

Fig. 1. Structural variation of BBM1 A, B, C show the BBM1 structural variants of TG12, wild12 and NH190, blue represents the BBM1 flanking large fragment repeat sequence and red represents the BBM1 gene sequence. D: Map of BBM1 gene structure of CW13 and CW16, burgundy represents exons, yellow represents introns, and ▲ represents MITE insertion sites.

Fig. 2. Nucleotide diversity of BBM1 sequences in different rice accessions A, B and C show the nucleotide polymorphisms of BBM1 sequence in wild rice, indica and japonica rice, respectively. The BBM1 exon is shown in burgundy and the BBM1 intron in orange.

Table 1. Analysis of BBM1 sequence polymorphism in different rice accessions

水稻类型 Rice type	对比序列数Number of sequences used	多态性位点Number of polymorphic (segregating) sites	突变总数 Total number of mutations	单倍体数Number of haplotypes	单倍型多样性Haplotype (gene) diversity	单倍型多态性方差 Variance of haplotype diversity	单倍型多态性标准差 Standard deviation of haplotype diversity	核苷酸多态性Nucleotide diversity
核心种质 Core collections	383	268	285	69	0.868	0.0001	0.010	0.00506
野生稻 Wild rice	30	196	200	29	0.998	0.00009	0.009	0.00623
籼稻 indica	204	91	94	22	0.786	0.00045	0.021	0.00333
粳稻 japonica	103	77	79	11	0.318	0.00359	0.060	0.00131

Fig. 3. Evolutionary relationship analysis of BBM1 sequences in 383 rice accessions

Fig. 4. Haplotype analysis

Fig. 5. Schematic representation of the BBM1 domain BBM1 contains two AP2 domains, the AP2 domain in red, the amino acid sequence in blue, and the amino acid position in numbers.

Table 2. Nonsynonymous mutation sites in different haplotypes

单倍型 Haplotype	数量 Number	16	67	69	107	124	142	146	147	147.1	258	351	445	493	521	537	561
Hap-1	109	S	T	A	M	V	G	A	A	-	R	T	T	P	D	L	stop
Hap-36	92	S	T	A	M	A	D	A	A	-	R	I	T	P	D	V	+26AA
Hap-37	51	S	M	A	M	A	G	A	A	-	R	T	T	P	D	V	+26AA
Hap-22	29	S	T	A	M	A	G	A	A	-	R	T	T	Q	D	V	+26AA
Hap-17	17	S	T	A	M	A	G	A	A	-	R	T	T	P	D	V	+26AA
Hap-40	12	S	T	A	M	A	D	A	A	-	R	I	I	P	D	V	+26AA
Hap-4	10	S	T	A	M	A	G	-	-	-	R	T	T	P	N	V	+26AA
Hap-6	5	S	T	A	M	A	G	-	-	-	R	T	T	P	D	V	+26AA
Hap-43	4	Y	T	D	M	V	G	A	A	A	R	T	T	P	D	L	stop
Hap-23	3	S	T	A	M	A	G	A	A	-	R	T	T	P	D	V	+26AA
Hap-8	2	S	T	A	M	A	G	-	-	-	\	\	\	\	\	\	\
Hap-44	2	S	T	A	T	A	G	A	A	-	R	T	T	P	D	V	+26AA

Fig. 6. Validation of different types of BBM1 sequences A, Distribution map of different BBM1 types in 2941 rice accessions, cyan indicates the occurrence of premature termination of BBM1, and green indicates normal BBM1; B, Results of agarose electrophoresis of full-length amplified BBM1 coding region sequences in NIPP and R498; C, Sequencing peak map of BBM1 coding region in NIPP; D, Sequencing peak map of BBM1 coding region in R498.

Fig. 7. Phylogenetic tree of the AP2/ERF family in rice

Fig. 8. Tissue expression profiles of candidate genes

References 38

[1]	Ye X X, Ma Y B, Sun B. Influence of soil type and genotype on Cd bioavailability and uptake by rice and implications for food safety[J]. Journal of Environmental Sciences, 2012, 24(9): 1647-1654.
[2]	任光俊, 颜龙安, 谢华安. 三系杂交水稻育种研究的回顾与展望[J]. 科学通报, 2016, 61(35): 3748-3760.
	Ren G J, Yan L A, Xie H A. Retrospective and perspective on indica three-line hybrid rice breeding research in China[J]. Chinese Science Bulletin, 2016, 61(35): 3748-3760. (in Chinese with English abstract)
[3]	袁隆平. 杂交水稻的育种战略设想[J]. 杂交水稻, 1987(1): 1-3.
	Yuan L P. Strategy of hybrid rice breeding[J]. Hybrid Rice, 1987(1): 1-3. (in Chinese with English abstract)
[4]	Bicknell R A, Koltunow A M. Understanding apomixis: Recent advances and remaining conundrums[J]. The Plant Cell, 2004, 16: 228-245.
[5]	Koltunow A M. Apomixis: Embryo sacs and embryos formed without meiosis or fertilization in ovules[J]. The Plant Cell, 1993, 5(10): 1425-1437.
[6]	蔡得田, 陈冬玲. 论无融合生殖固定水稻杂种优势的策略[J]. 杂交水稻, 1989(6): 1-3.
	Cai D T, Chen D L. Trying discussion in the tactics of fixing hybrid vigor of rice by apomixes[J]. Hybrid Rice, 1989(6): 1-3. (in Chinese with English abstract)
[7]	蔡得田, 马平福, 关和新, 姚家琳, 王灶安, 祝虹. 高频率无融合生殖水稻的研究[J]. 华中农业大学学报, 1991, 10(3): 223-227.
	Cai D T, Ma P F, Gan H X, Yao J L, Wang Z A, Zhu H. The study of high frequency of apomictic rice material (HDAR). Journal of Huazhong Agricultural University, 1991, 10(3): 223-227. (in Chinese with English abstract)
[8]	Jefferson R A. Apomixis: A social revolution for agriculture[J]. Biotechnology and Development Monitor, 1994, 19: 14-16.
[9]	Spillane C, Curtis M D, Grossniklaus U. Apomixis technology development-virgin births in farmers' fields?[J]. Nature Biotechnology, 2004, 22(6): 687-691.
[10]	Ondřej P, Michal S, Martin D. Reciprocal hybridization between diploid Ficaria calthifolia and tetraploid Ficaria verna subsp verna: Evidence from experimental crossing, genome size and molecular markers[J]. Botanical Journal of the Linnean Society, 2019, 189(3): 293-310.
[11]	Vijverberg K, Ozias-Akins P, Schranz M E. Identifying and engineering genes for parthenogenesis in plants[J]. Frontiers in Plant Science, 2019, 10: 128.
[12]	Brukhin V. Molecular and genetic regulation of apomixis[J]. Russian Journal of Genetics, 2017, 53(9): 943-964.
[13]	Mieulet D, Jolivet S, Rivard M, Cromer L, Vernet A, Mayonove P, Pereira L, Droc G, Courtois B, Guiderdoni E, Mercier R. Turning rice meiosis into mitosis[J]. Cell Research, 2016, 26(11): 1242-1254.
[14]	Rashid M, He G Y, Yang G X, Hussain J, Xu Y. AP2/ERF transcription factor in rice: Genome-wide canvas and syntenic relationships between monocots and eudicots[J]. Evolutionary Bioinformatics Online, 2012, 8: 321-355.
[15]	Xie W, Ding C, Hu H, Dong G, Zhang G, Qian Q, Ren D. Molecular events of rice AP2/ERF transcription factors[J]. International Journal of Molecular Sciences, 2022, 23(19): 12013.
[16]	Chen B, Maas L, Figueiredo D, Zhong Y, Reis R, Li M, Horstman A, Riksen T, Weemen M, Liu H, Siemons C, Chen S, Angenent G C, Boutilier K. BABY BOOM regulates early embryo and endosperm development[J]. Proceedings of the National Academy of Sciences of the United States of America, 2022, 119(25): e2201761119.
[17]	Anderson S N, Johnson C S, Chesnut J, Jones D S, Khanday I, Woodhouse M, Li C, Conrad L J, Russell S D, Sundaresan V. The zygotic transition is initiated in unicellular plant zygotes with asymmetric activation of parental genomes[J]. Developmental Cell, 2017, 43: 349-358.
[18]	Anderson S N, Johnson C S, Jones D S, Conrad L J, Gou X, Russell S D, Sundaresan V. Transcriptomes of isolated Oryza sativa gametes characterized by deep sequencing: Evidence for distinct sex-dependent chromatin and epigenetic states before fertilization[J]. Plant Journal, 2013, 76(5): 729-741.
[19]	Conner J A, Mookkan M, Huo H, Chae K, Ozias-Akins P. A parthenogenesis gene of apomict origin elicits embryo formation from unfertilized eggs in a sexual plant[J]. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112: 11205-11210.
[20]	Conner J A, Podio M, Ozias-Akins P. Haploid embryo production in rice and maize induced by PsASGR-BBML transgenes[J]. Plant Reproduction, 2017, 30: 41-52.
[21]	Khanday I, Skinner D, Yang B, Mercier R, Sundaresan V. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds[J]. Nature, 2019, 565(7737): 91-95.
[22]	Vernet A, Meynard D, Lian Q, Mieulet D, Gibert O, Bissah M, Rivallan R, Autran D, Leblanc O, Meunier A C, Frouin J, Taillebois J, Shankle K, Khanday I, Mercier R, Sundaresan V, Guiderdoni E. High-frequency synthetic apomixis in hybrid rice[J]. Nature Communications, 2022, 13(1): 7963.
[23]	Wei X, Liu C, Chen X, Lu H, Wang J, Yang S, Wang K. Synthetic apomixis with normal hybrid rice seed production[J]. Molecular Plant, 2023, 16(3): 489-492.
[24]	Wang C, Liu Q, Shen Y, Hua Y, Wang J, Lin J, Wu M, Sun T, Cheng Z, Mercier R, Wang K. Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes[J]. Nature Biotechnology, 2019, 37(3): 283-286.
[25]	Wang C C, Yu H, Huang J, Wang W S, Faruquee M, Zhang F, Zhao X Q, Fu B Y, Chen K, Zhang H L, Tai S S, Wei C, McNally K L, Alexandrov N, Gao X Y, Li J, Li Z K, Xu J L, Zheng T Q. Towards a deeper haplotype mining of complex traits in rice with RFGB v2.0[J]. Plant Biotechnology Journal, 2020, 18(1): 14-16.
[26]	Wang W, Mauleon R, Hu Z, Chebotarov D, Tai S, Wu Z, Li M, Zheng T, Fuentes R R, Zhang F, Mansueto L, Copetti D, Sanciangco M, Palis K C, Xu J, Sun C, Fu B, Zhang H, Gao Y, Zhao X, Shen F, Cui X, Yu H, Li Z, Chen M, Detras J, Zhou Y, Zhang X, Zhao Y, Kudrna D, Wang C, Li R, Jia B, Lu J, He X, Dong Z, Xu J, Li Y, Wang M, Shi J, Li J, Zhang D, Lee S, Hu W, Poliakov A, Dubchak I, Ulat V J, Borja F N, Mendoza J R, Ali J, Li J, Gao Q, Niu Y, Yue Z, Naredo M E B, Talag J, Wang X, Li J, Fang X, Yin Y, Glaszmann J C, Zhang J, Li J, Hamilton R S, Wing R A, Ruan J, Zhang G, Wei C, Alexandrov N, McNally K L, Li Z, Leung H. Genomic variation in 3,010 diverse accessions of Asian cultivated rice[J]. Nature, 2018, 557(7703): 43-49.
[27]	Lü Q, Li W, Sun Z, Ouyang N, Jing X, He Q, Wu J, Zheng J, Zheng J, Tang S, Zhu R, Tian Y, Duan M, Tan Y, Yu D, Sheng X, Sun X, Jia G, Gao H, Zeng Q, Li Y, Tang L, Xu Q, Zhao B, Huang Z, Lu H, Li N, Zhao J, Zhu L, Li D, Yuan L, Yuan D. Resequencing of 1,143 indica rice accessions reveals important genetic variations and different heterosis patterns[J]. Nature Communication, 2020, 11(1): 4778.
[28]	Yu H, Kou L, Li J. 10k-level integrated rice database shows power for exploiting rare variants[J]. Journal of Integrative Plant Biology, 2023, 65(12): 2539-2540.
[29]	Shang L, Li X, He H, Yuan Q, Song Y, Wei Z, Lin H, Hu M, Zhao F, Zhang C, Li Y, Gao H, Wang T, Liu X, Zhang H, Zhang Y, Cao S, Yu X, Zhang B, Zhang Y, Tan Y, Qin M, Ai C, Yang Y, Zhang B, Hu Z, Wang H, Lv Y, Wang Y, Ma J, Wang Q, Lu H, Wu Z, Liu S, Sun Z, Zhang H, Guo L, Li Z, Zhou Y, Li J, Zhu Z, Xiong G, Ruan J, Qian Q. A super pan-genomic landscape of rice[J]. Cell Research, 2022, 32(10): 878-896.
[30]	Qin P, Lu H, Du H, Wang H, Chen W, Chen Z, He Q, Ou S, Zhang H, Li X, Li X, Li Y, Liao Y, Gao Q, Tu B, Yuan H, Ma B, Wang Y, Qian Y, Fan S, Li W, Wang J, He M, Yin J, Li T, Jiang N, Chen X, Liang C, Li S. Pan-genome analysis of 33 genetically diverse rice accessions reveals hidden genomic variations[J]. Cell, 2021, 184(13): 3542-3558.
[31]	Zhang F, Xue H, Dong X, Li M, Zheng X, Li Z, Xu J, Wang W, Wei C. Long-read sequencing of 111 rice genomes reveals significantly larger pan-genomes[J]. Genome Research, 2022, 32(5): 853-863.
[32]	项聪英, 蔡年俊, 李静, 羊健, 陈剑平, 张恒木. 一个水稻小热休克蛋白基因的克隆和鉴定[J]. 中国水稻科学, 2016, 30(6): 587-592.
	Xiang C Y, Cai N J, Li J, Yang J, Chen J P, Zhang H M. Cloning and characterization of a small heat shock protein (SHSP) gene in rice plant[J]. Chinese Journal of Rice Science, 2016, 30(6): 587-592. (in Chinese with English abstract)
[33]	Tamura K, Stecher G, Kumar S. MEGA11: Molecular evolutionary genetics analysis version 11[J]. Molecular Biology and Evolution, 2021, 38(7): 3022-3027.
[34]	Rozas J, Ferrer-Mata A, Sánchez-DelBarrio J C, Guirao-Rico S, Librado P, Ramos-Onsins S E, Sánchez-Gracia A. DnaSP 6: DNA sequence polymorphism analysis of large data sets[J]. Molecular Biology Evolution, 2017, 34(12): 3299-3302.
[35]	Kawahara Y, de la Bastide M, Hamilton J P, Kanamori H, McCombie W R, Ouyang S, Schwartz D C, Tanaka T, Wu J, Zhou S, Childs K L, Davidson R M, Lin H, Quesada-Ocampo L, Vaillancourt B, Sakai H, Lee S S, Kim J, Numa H, Itoh T, Buell C R, Matsumoto T. Improvement of the Oryza sativa Nipponbare reference genome using next generation sequence and optical map data[J]. Rice, 2013, 6(1): 4.
[36]	Chen C, Chen H, Zhang Y, Thomas H R, Frank M H, He Y, Xia R. TBtools: An integrative toolkit developed for interactive analyses of big biological data[J]. Molecular Plant, 2020, 13(8): 1194-1202.
[37]	赵凤利. 水稻全基因组拷贝数变异研究揭示复制基因的非对称进化[D]. 北京: 中国农业科学院, 2021.
	Zhao F L. A genome-wide survey of copy number variations reveals an asymmetric evolution of duplicated genes in rice[D]. Beijing: Agricultural Genomics Institute Graduate School, 2021. (in Chinese with English abstract)
[38]	Guillet-Claude C, Birolleau-Touchard C, Manicacci D, Rogowsky P M, Rigau J, Murigneux A, Martinant J P, Barrière Y. Nucleotide diversity of the ZmPox3 maize peroxidase gene: Relationships between a MITE insertion in exon 2 and variation in forage maize digestibility[J]. BMC Genetics, 2004, 16(5): 19.