龙眼染色质重塑因子Snf2基因家族的进化动力学研究及在体胚发生早期的表达

doi:10.16420/j.issn.0513-353x.2020-0726

摘要/Abstract

摘要：

以龙眼（Dimocarpus longan Lour.）‘红核子’品种早期体细胞胚胎发生的胚性愈伤组织（embryogenic callus,EC）、不完全胚性紧实结构（incomplete embryogenic compact structures,ICpEC）和球形胚（globular embryos,GE）为材料,通过生物信息学方法鉴定和分析其染色质重塑因子DlSnf2家族成员及分子进化特性,并通过转录组和实时荧光定量PCR（real-time quantitative PCR,qRT-PCR）分析其表达模式。龙眼DlSnf2家族包含35个成员,家族扩张受到全基因组或片段复制驱动。选择含有SNF2_N和HELICc结构域的31个成员,根据进化特征分为6大类19个亚族。蛋白互作、microRNA和启动子预测显示,部分成员能够发生互作,大部分基因受到microRNA的潜在调控,且存在与激素、胁迫、光、生长和发育相关顺式作用元件。转录组和qRT-PCR分析显示：大多数基因在ICpEC阶段中高表达,DlCHR18、DlCHR24、DlCHR25、DlCHR29和DlCHR31a从EC到GE的过程中表达逐渐下调,而DlCHR4、DlCHR15和DlCHR42表达逐渐上调。分析显示DlSnf2家族基因响应2,4-D、ABA、乙烯利、茉莉酸甲酯、4 ℃和40 ℃。DlCHR10和DlCHR11的表达被乙烯利上调,而DlCHR28b和DlCHR36被下调,DlCHR11被ABA和高温（40 ℃）下调,DlCHR10被茉莉酸甲酯下调,DlCHR36被低温（4 ℃）下调,DlCHR24和DlCHR35被高温（40 ℃）下调。Snf2基因家族在植物进化过程中可能具有功能的相对保守性及一定物种特异性。在植物生长调节剂和温度处理下,龙眼DlSnf2家族成员可能参与调控体胚发生和抵御高、低温胁迫,且推测DNA甲基化、microRNA和组蛋白修饰等其他表观调控机制参与了这一过程。

关键词: 龙眼, 染色质重塑因子Snf2, 进化, 体胚发生, 表观调控

Abstract:

EC（embryogenic callus）,ICpEC（incomplete embryogenic compact structures）and GE（globular embryos）of the early somatic embryogenesis（SE）of Dimocarpus longan Lour.‘Honghezi’were used as experimental materials. Using bioinformatic methods identified and analyzed the members and its molecular evolutionary characteristic of longan chromatin remodeling factor DlSnf2 family,and the expression patterns of DlSnf2 gene family were analyzed through transcriptome data and real-time quantitative PCR（qRT-PCR）. The longan DlSnf2 family contains 35 members,and expansion of the family is mainly driven by whole-genomic or segmental duplication events. 31 members of containing SNF2_N and HELICc domains were selected and divided into 19 subfamilies under six categories depending on the evolutionary characteristics. Protein interaction,microRNA and promoter prediction showed that partial members can interact each other,almost all genes may be potentially regulated by microRNA and exist a large number of cis-acting elements related to hormone,stress,light,growth and development;Transcriptome and qRT-PCR analysis showed that most genes were highly expressed in ICpEC,and the expressions of DlCHR18,DlCHR24,DlCHR25,DlCHR29 and DlCHR31a were gradually decreased from EC to GE,while DlCHR4,DlCHR15 and DlCHR42 gradually increased;The analysis exhibited that DlSnf2 family genes responded to 2,4-D,ABA,ethrel,Methyl jasmonate,4 ℃ and 40 ℃. Expressions of DlCHR10 and DlCHR11 are up regulated by ethrel,while DlCHR28b and DlCHR36 are down regulated. DlCHR11 is down regulated by ABA and high temperature,DlCHR10 is down regulated by Methyl Jasmonate,DlCHR36 is down regulated by low temperature,and DlCHR24 and DlCHR35 are down regulated by high temperature. The Snf2 gene family may have relatively conservative functions and certain species specificity during plant evolution. Under plant growth regulator and temperature treatments,longan DlSnf2 family members may be involved in regulating somatic embryogenesis and resisting high and low temperature stress,and it is speculated that other epigenetic regulatory mechanisms such as DNA methylation,microRNA and histone modification are involved in the process.

Key words: Dimocarpus longan, chromatin remodeling factor Snf2, evolution, somatic embryogenesis, epigenetic regulation

中图分类号:

S667.2

申序, 陈晓慧, 张婧, 陈荣珠, 徐小萍, 李晓斐, 蒋梦琦, 刘蒲东, 倪珊珊, 林玉玲, 赖钟雄. 龙眼染色质重塑因子Snf2基因家族的进化动力学研究及在体胚发生早期的表达[J]. 园艺学报, 2022, 49(1): 41-61.

SHEN Xu, CHEN Xiaohui, ZHANG Jing, CHEN Rongzhu, XU Xiaoping, LI Xiaofei, JIANG Mengqi, LIU Pudong, NI Shanshan, LIN Yuling, LAI Zhongxiong. Evolutionary Dynamics Investigation and the Expression Analysis of Chromatin Remodeling Factor Snf2 Gene Family During Early Somatic Embryogenesis in Dimocarpus longan[J]. Acta Horticulturae Sinica, 2022, 49(1): 41-61.

图/表 11

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基因 Gene	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
DlCHR2	AACTTCTCAGCATCGGGTGG	CCTCAAGTGGTGGTGGAACA
DlCHR6	CCGGAGGTCTGGGAATCAAC	GGTCTGCCCAAGTCGATGAG
DlCHR8	TTACGGGACTTGATCATGCC	TCTTTGTTCTGCAGTGAGGC
DlCHR10	AAGTCGAGGGAGTTGCTTGG	GTCACGCTCAGAGGACACAA
DlCHR11	TAAGGTACTCCGGCCGTTTC	AGGCGTTTACGTTCTCCACC
DlCHR12	ACACCGTCTCGTCCATCTAT	AACCTGACATGCATTTTGGC
DlCHR14	AGCTTCTTCCATTCCAGCGG	AGCCCAATGCAAACGTAAGG
DlCHR16b	GACCCTGATGATGATGTCCG	ACTGGTGGATGGGCTTAGAT
DlCHR21	ACATTCATCCCACGAGCCAG	GCAGTAGCCTCTTGACCCAG
DlCHR24	GCTGCGAGAACGTATTCAACC	GTGGTGAGCCATCAAAAGCC
DlCHR28b	GCATCTGCAATGATCCCCCT	GTGTGGCCTTGGAAAACACC
DlCHR29	TGATACGCAACAGTACTCGG	TTTCGCAGCTCCTCTACAAC
DlCHR31a	GCTTGATGTTGCATGGAACC	GTCCCGGATGTAATGAGACG
DlCHR35	GCTTTCGGGAACCCTCTACC	CGGTTCACAATAGGCCGAGA
DlCHR36	GCAGCAGGCACATCAACAAT	TGGGTGCGTTGTATGCTCTT
DlCHR42	AGTGCTCCGTGAAAGTCCTG	ATGAACTTCGGTCGAGCCAG
DlEF-1a	GATGATTCCCACCAAGCCCAT	GGGTCCTTCT TCTCAACACT CT
DleIF-4a	TTGTGCTGGATGAAGCTGATG	GGAAGGAGCTGGAAGATATCATAGA
DlFe-SOD	GGTCAGATGGTGAAGCCGTAGAG	GTCTATGCCACCGATACAACAAACCC

基因 Gene	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
DlCHR2	AACTTCTCAGCATCGGGTGG	CCTCAAGTGGTGGTGGAACA
DlCHR6	CCGGAGGTCTGGGAATCAAC	GGTCTGCCCAAGTCGATGAG
DlCHR8	TTACGGGACTTGATCATGCC	TCTTTGTTCTGCAGTGAGGC
DlCHR10	AAGTCGAGGGAGTTGCTTGG	GTCACGCTCAGAGGACACAA
DlCHR11	TAAGGTACTCCGGCCGTTTC	AGGCGTTTACGTTCTCCACC
DlCHR12	ACACCGTCTCGTCCATCTAT	AACCTGACATGCATTTTGGC
DlCHR14	AGCTTCTTCCATTCCAGCGG	AGCCCAATGCAAACGTAAGG
DlCHR16b	GACCCTGATGATGATGTCCG	ACTGGTGGATGGGCTTAGAT
DlCHR21	ACATTCATCCCACGAGCCAG	GCAGTAGCCTCTTGACCCAG
DlCHR24	GCTGCGAGAACGTATTCAACC	GTGGTGAGCCATCAAAAGCC
DlCHR28b	GCATCTGCAATGATCCCCCT	GTGTGGCCTTGGAAAACACC
DlCHR29	TGATACGCAACAGTACTCGG	TTTCGCAGCTCCTCTACAAC
DlCHR31a	GCTTGATGTTGCATGGAACC	GTCCCGGATGTAATGAGACG
DlCHR35	GCTTTCGGGAACCCTCTACC	CGGTTCACAATAGGCCGAGA
DlCHR36	GCAGCAGGCACATCAACAAT	TGGGTGCGTTGTATGCTCTT
DlCHR42	AGTGCTCCGTGAAAGTCCTG	ATGAACTTCGGTCGAGCCAG
DlEF-1a	GATGATTCCCACCAAGCCCAT	GGGTCCTTCT TCTCAACACT CT
DleIF-4a	TTGTGCTGGATGAAGCTGATG	GGAAGGAGCTGGAAGATATCATAGA
DlFe-SOD	GGTCAGATGGTGAAGCCGTAGAG	GTCTATGCCACCGATACAACAAACCC

物种成员 Species member	龙眼基因 Longan gene	Ka	Ks	Ka/Ks		物种成员 Species member	龙眼基因 Longan gene	Ka	Ks	Ka/Ks
物种成员 Species member	龙眼基因 Longan gene	Ka	Ks	Ka/Ks	AtCHR33	物种成员 Species member	龙眼基因 Longan gene	Ka	Ks	Ka/Ks	DlCHR28b	0.40	1.36	0.30	PNT24595	DlCHR19	0.19	0.81	0.23
AtCHR30	DlCHR28b	0.50	1.45	0.35		PNS97818	DlCHR21	0.11	0.87	0.13
AtCHR15	DlCHR15	0.73	1.85	0.39		PNT29744	DlCHR21	0.11	0.85	0.13
AtCHR9	DlCHR9	0.23	2.85	0.08		PNT11818	DlCHR28b	0.32	0.85	0.37
AtCHR37	DlCHR37	0.27	1.50	0.18		PNT39429	DlCHR28b	0.25	0.87	0.28
AtCHR18	DlCHR18	0.17	1.09	0.16		PNT41192	DlCHR25	0.13	0.80	0.16
AtCHR34	DlCHR35	0.45	—	—		PNT41496	DlCHR35	0.20	1.51	0.13
AtCHR35	DlCHR35	0.31	2.02	0.15		PNT20954	DlCHR35	0.23	1.32	0.18
AtCHR6	DlCHR6	0.21	1.82	0.12		PNT56587	DlCHR5	0.12	1.03	0.11
AtCHR36	DlCHR36	0.27	1.38	0.19		PNT42020	DlCHR29	0.19	0.85	0.23
AtCHR10	DlCHR10	0.20	1.34	0.15		PNT42442	DlCHR15	1.16	2.27	0.51
AtCHR2	DlCHR2	0.20	1.31	0.15		PNT19611	DlCHR5	0.12	0.96	0.12
AtCHR25	DlCHR25	0.19	1.18	0.16		PNT05067	DlCHR37	0.22	0.92	0.24
AtCHR12	DlCHR12	0.18	1.70	0.10		PNS94713	DlCHR9	0.14	1.04	0.14
AtCHR11	DlCHR11	0.06	1.67	0.03		PNT09833	DlCHR14	0.24	1.15	0.21
AtCHR7	DlCHR6	0.32	2.14	0.15		ar_scaffold00002.143	DlCHR6	1.12	2.87	0.39
AtCHR29	DlCHR29	0.21	1.29	0.16		ar_scaffold00009.110	DlCHR11	1.11	—	—
AtCHR17	DlCHR11	0.06	1.57	0.04		ar_scaffold00025.389	DlCHR12	0.20	2.16	0.09
AtCHR23	DlCHR12	0.20	1.60	0.12		ar_scaffold00045.118	DlCHR18	0.24	1.80	0.13
AtCHR22	DlCHR36	0.95	2.20	0.43		ar_scaffold00057.182	DlCHR11	0.08	1.91	0.04
AtCHR24	DlCHR24	0.32	1.78	0.18		ar_scaffold00077.105	DlCHR11	1.04	3.04	0.34
AtCHR1	DlCHR1a	0.21	2.17	0.10		ar_scaffold00009.153	DlCHR2	0.28	1.91	0.15
Os02t0762800-00	DlCHR25	0.24	2.35	0.10		ar_scaffold00045.118	DlCHR2	0.95	2.30	0.41
Os02t0527100-01	DlCHR29	0.25	2.42	0.10		ar_scaffold00045.118	DlCHR36	1.00	2.18	0.46
Os02t0650800-00	DlCHR31a	0.56	—	—		ar_scaffold00067.58	DlCHR8	0.26	—	—
Os03t0101700-01	DlCHR10	0.52	2.15	0.24		ar_scaffold00067.58	DlCHR42	1.23	3.10	0.40
Os03t0722400-01	DlCHR1a	0.25	—	—		ar_scaffold00077.105	DlCHR36	0.35	1.67	0.21
Os05t0150300-01	DlCHR11	0.10	3.63	0.03		ar_scaffold00077.105	DlCHR8	1.02	3.20	0.32
Os06t0183800-01	DlCHR6	0.67	2.42	0.28		ar_scaffold00077.105	DlCHR2	1.15	3.54	0.32
Os07t0598300-01	DlCHR14	0.40	2.10	0.19		ar_scaffold00111.13	DlCHR10	0.68	3.82	0.18
Os07t0636200-01	DlCHR18	0.23	1.93	0.12		ar_scaffold00009.153	DlCHR1a	0.72	—	—
PNT15539	DlCHR12	0.13	0.83	0.15		ar_scaffold00077.105	DlCHR1a	0.78	4.74	0.16
PNT13987	DlCHR18	0.12	0.75	0.16		ar_scaffold00029.1	DlCHR28a	0.35	1.40	0.25
PNT14263	DlCHR11	0.04	0.97	0.04		ar_scaffold00053.139	DlCHR21	0.22	2.15	0.10
PNS92200	DlCHR6	0.12	0.91	0.13		ar_scaffold00003.327	DlCHR28b	0.25	1.56	0.16
PNT33877	DlCHR6	0.12	0.95	0.13		ar_scaffold00009.266	DlCHR4	1.17	3.88	0.30
PNT25862	DlCHR11	0.04	0.85	0.05		ar_scaffold00067.58	DlCHR4	0.87	3.48	0.25
PNT24755	DlCHR12	0.13	0.90	0.14		ar_scaffold00077.105	DlCHR28b	1.09	2.01	0.54
PNT25862	DlCHR12	0.64	—	—		ar_scaffold00146.48	DlCHR4	1.10	3.01	0.37
PNT06750	DlCHR20a	0.16	0.79	0.20		ar_scaffold00039.164	DlCHR35	0.89	—	—
PNT07119	DlCHR16a	0.12	0.82	0.14		ar_scaffold00126.55	DlCHR25	0.22	2.06	0.11
PNT07119	DlCHR16b	0.12	0.82	0.14		ar_scaffold00040.267	DlCHR29	0.26	2.39	0.11
PNS90011	DlCHR20a	0.16	0.85	0.19		ar_scaffold00148.60	DlCHR15	0.60	2.25	0.27
PNT17288	DlCHR42	0.27	1.28	0.21		ar_scaffold00009.110	DlCHR39	1.02	3.00	0.34
PNT03745	DlCHR2	0.11	0.75	0.15		ar_scaffold00045.118	DlCHR37	1.09	2.28	0.48
PNT03205	DlCHR10	0.18	0.89	0.20		ar_scaffold00077.105	DlCHR9	0.98	—	—
PNS90743	DlCHR36	0.52	—	—		ar_scaffold00002.143	DlCHR13	1.21	—	—
PNT49991	DlCHR2	0.11	0.81	0.14		ar_scaffold00009.153	DlCHR13	0.93	2.75	0.34
PNT38168	DlCHR8	0.15	0.78	0.19		ar_scaffold00032.254	DlCHR13	0.82	—	—
PNT23278	DlCHR42	0.24	1.04	0.23		ar_scaffold00032.254	DlCHR13	0.82	—	—
PNT35260	DlCHR24	0.21	0.97	0.21		ar_scaffold00009.110	DlCHR31a	1.06	—	—
PNT28173	DlCHR24	0.21	0.89	0.24		ar_scaffold00045.118	DlCHR14	0.83	—	—
PNT15682	DlCHR19	0.13	0.94	0.14		ar_scaffold00110.12	DlCHR14	0.36	2.13	0.17

物种成员 Species member	龙眼基因 Longan gene	Ka	Ks	Ka/Ks		物种成员 Species member	龙眼基因 Longan gene	Ka	Ks	Ka/Ks
物种成员 Species member	龙眼基因 Longan gene	Ka	Ks	Ka/Ks	AtCHR33	物种成员 Species member	龙眼基因 Longan gene	Ka	Ks	Ka/Ks	DlCHR28b	0.40	1.36	0.30	PNT24595	DlCHR19	0.19	0.81	0.23
AtCHR30	DlCHR28b	0.50	1.45	0.35		PNS97818	DlCHR21	0.11	0.87	0.13
AtCHR15	DlCHR15	0.73	1.85	0.39		PNT29744	DlCHR21	0.11	0.85	0.13
AtCHR9	DlCHR9	0.23	2.85	0.08		PNT11818	DlCHR28b	0.32	0.85	0.37
AtCHR37	DlCHR37	0.27	1.50	0.18		PNT39429	DlCHR28b	0.25	0.87	0.28
AtCHR18	DlCHR18	0.17	1.09	0.16		PNT41192	DlCHR25	0.13	0.80	0.16
AtCHR34	DlCHR35	0.45	—	—		PNT41496	DlCHR35	0.20	1.51	0.13
AtCHR35	DlCHR35	0.31	2.02	0.15		PNT20954	DlCHR35	0.23	1.32	0.18
AtCHR6	DlCHR6	0.21	1.82	0.12		PNT56587	DlCHR5	0.12	1.03	0.11
AtCHR36	DlCHR36	0.27	1.38	0.19		PNT42020	DlCHR29	0.19	0.85	0.23
AtCHR10	DlCHR10	0.20	1.34	0.15		PNT42442	DlCHR15	1.16	2.27	0.51
AtCHR2	DlCHR2	0.20	1.31	0.15		PNT19611	DlCHR5	0.12	0.96	0.12
AtCHR25	DlCHR25	0.19	1.18	0.16		PNT05067	DlCHR37	0.22	0.92	0.24
AtCHR12	DlCHR12	0.18	1.70	0.10		PNS94713	DlCHR9	0.14	1.04	0.14
AtCHR11	DlCHR11	0.06	1.67	0.03		PNT09833	DlCHR14	0.24	1.15	0.21
AtCHR7	DlCHR6	0.32	2.14	0.15		ar_scaffold00002.143	DlCHR6	1.12	2.87	0.39
AtCHR29	DlCHR29	0.21	1.29	0.16		ar_scaffold00009.110	DlCHR11	1.11	—	—
AtCHR17	DlCHR11	0.06	1.57	0.04		ar_scaffold00025.389	DlCHR12	0.20	2.16	0.09
AtCHR23	DlCHR12	0.20	1.60	0.12		ar_scaffold00045.118	DlCHR18	0.24	1.80	0.13
AtCHR22	DlCHR36	0.95	2.20	0.43		ar_scaffold00057.182	DlCHR11	0.08	1.91	0.04
AtCHR24	DlCHR24	0.32	1.78	0.18		ar_scaffold00077.105	DlCHR11	1.04	3.04	0.34
AtCHR1	DlCHR1a	0.21	2.17	0.10		ar_scaffold00009.153	DlCHR2	0.28	1.91	0.15
Os02t0762800-00	DlCHR25	0.24	2.35	0.10		ar_scaffold00045.118	DlCHR2	0.95	2.30	0.41
Os02t0527100-01	DlCHR29	0.25	2.42	0.10		ar_scaffold00045.118	DlCHR36	1.00	2.18	0.46
Os02t0650800-00	DlCHR31a	0.56	—	—		ar_scaffold00067.58	DlCHR8	0.26	—	—
Os03t0101700-01	DlCHR10	0.52	2.15	0.24		ar_scaffold00067.58	DlCHR42	1.23	3.10	0.40
Os03t0722400-01	DlCHR1a	0.25	—	—		ar_scaffold00077.105	DlCHR36	0.35	1.67	0.21
Os05t0150300-01	DlCHR11	0.10	3.63	0.03		ar_scaffold00077.105	DlCHR8	1.02	3.20	0.32
Os06t0183800-01	DlCHR6	0.67	2.42	0.28		ar_scaffold00077.105	DlCHR2	1.15	3.54	0.32
Os07t0598300-01	DlCHR14	0.40	2.10	0.19		ar_scaffold00111.13	DlCHR10	0.68	3.82	0.18
Os07t0636200-01	DlCHR18	0.23	1.93	0.12		ar_scaffold00009.153	DlCHR1a	0.72	—	—
PNT15539	DlCHR12	0.13	0.83	0.15		ar_scaffold00077.105	DlCHR1a	0.78	4.74	0.16
PNT13987	DlCHR18	0.12	0.75	0.16		ar_scaffold00029.1	DlCHR28a	0.35	1.40	0.25
PNT14263	DlCHR11	0.04	0.97	0.04		ar_scaffold00053.139	DlCHR21	0.22	2.15	0.10
PNS92200	DlCHR6	0.12	0.91	0.13		ar_scaffold00003.327	DlCHR28b	0.25	1.56	0.16
PNT33877	DlCHR6	0.12	0.95	0.13		ar_scaffold00009.266	DlCHR4	1.17	3.88	0.30
PNT25862	DlCHR11	0.04	0.85	0.05		ar_scaffold00067.58	DlCHR4	0.87	3.48	0.25
PNT24755	DlCHR12	0.13	0.90	0.14		ar_scaffold00077.105	DlCHR28b	1.09	2.01	0.54
PNT25862	DlCHR12	0.64	—	—		ar_scaffold00146.48	DlCHR4	1.10	3.01	0.37
PNT06750	DlCHR20a	0.16	0.79	0.20		ar_scaffold00039.164	DlCHR35	0.89	—	—
PNT07119	DlCHR16a	0.12	0.82	0.14		ar_scaffold00126.55	DlCHR25	0.22	2.06	0.11
PNT07119	DlCHR16b	0.12	0.82	0.14		ar_scaffold00040.267	DlCHR29	0.26	2.39	0.11
PNS90011	DlCHR20a	0.16	0.85	0.19		ar_scaffold00148.60	DlCHR15	0.60	2.25	0.27
PNT17288	DlCHR42	0.27	1.28	0.21		ar_scaffold00009.110	DlCHR39	1.02	3.00	0.34
PNT03745	DlCHR2	0.11	0.75	0.15		ar_scaffold00045.118	DlCHR37	1.09	2.28	0.48
PNT03205	DlCHR10	0.18	0.89	0.20		ar_scaffold00077.105	DlCHR9	0.98	—	—
PNS90743	DlCHR36	0.52	—	—		ar_scaffold00002.143	DlCHR13	1.21	—	—
PNT49991	DlCHR2	0.11	0.81	0.14		ar_scaffold00009.153	DlCHR13	0.93	2.75	0.34
PNT38168	DlCHR8	0.15	0.78	0.19		ar_scaffold00032.254	DlCHR13	0.82	—	—
PNT23278	DlCHR42	0.24	1.04	0.23		ar_scaffold00032.254	DlCHR13	0.82	—	—
PNT35260	DlCHR24	0.21	0.97	0.21		ar_scaffold00009.110	DlCHR31a	1.06	—	—
PNT28173	DlCHR24	0.21	0.89	0.24		ar_scaffold00045.118	DlCHR14	0.83	—	—
PNT15682	DlCHR19	0.13	0.94	0.14		ar_scaffold00110.12	DlCHR14	0.36	2.13	0.17

基因	微小RNA	期望值	抑制方式	基因	微小RNA	期望值	抑制方式	基因	微小RNA	期望值	抑制方式
Gene	MicroRNA	E-value	Inhibition	Gene	MicroRNA	E-value	Inhibition	Gene	MicroRNA	E-value	Inhibition
DlCHR1a	miR5177	3	Cleavage	DlCHR12	miR1312	3	Cleavage		miR1514b	4	Cleavage
	miR166j	4	Cleavage		miR1428e	4	Translation		miR3452	4	Cleavage
	miR5079	4	Cleavage		miR2868	4	Cleavage		miR3637	4	Cleavage
DlCHR2	miR2657a	3	Cleavage		miR396a	4	Cleavage		miR415	4	Cleavage
	miR420	4	Translation	DlCHR13	miR414	3.5	Cleavage		miR5078	4	Translation
	miR4415	4	Cleavage		miR416	3.5	Cleavage	DlCHR24	miR778	3.5	Cleavage
	miR845d	4	Translation		miR847	3.5	Cleavage		miR778	3.5	Cleavage
DlCHR3	miR5200	3	Cleavage		miR850	3.5	Cleavage		miR414	4	Cleavage
	miR1881	3.5	Cleavage		miR860	3.5	Cleavage	DlCHR25	miR2936	3.5	Cleavage
	miR1027a	4	Cleavage		miR869	3.5	Cleavage		miR3631a	4	Cleavage
	miR1044	4	Cleavage		miR1039	4	Translation	DlCHR28a	miR1528	4	Cleavage
	miR1521	4	Cleavage		miR1044	4	Cleavage		miR4249	4	Cleavage
	miR166c	4	Cleavage	DlCHR14	miR4400	2.5	Cleavage		miR4366	4	Cleavage
	miR2089*-3	4	Cleavage		miR1535	4	Cleavage		miR857	4	Cleavage
	miR3707	4	Cleavage	DlCHR15	miR1160	3	Cleavage		miR860	4	Cleavage
DlCHR4	miR1884b	3.5	Cleavage		miR1528	4	Translation	DlCHR28b	miR172a	3.5	Cleavage
	miR2595	4	Cleavage		miR1884b	4	Cleavage		miR1863	3.5	Translation
	miR2615a	4	Translation		miR2611	4	Cleavage		miR419	3.5	Cleavage
	miR2616	4	Translation		miR4223	4	Cleavage		miR833	3.5	Cleavage
	miR416	4	Cleavage	DlCHR16a	miR1863	3	Cleavage	DlCHR29	miR3702	4	Cleavage
	miR860	4	Cleavage		miR1863b	3.5	Cleavage	DlCHR31a	miR2673a	2.5	Cleavage
DlCHR5	miR2673a	2.5	Cleavage		miR1510a	4	Cleavage		miR2118e	3	Cleavage
	miR2673a	4	Translation		miR1871	4	Cleavage		miR4249	3.5	Cleavage
DlCHR6	miR2677	3.5	Cleavage		miR2930	4	Cleavage		miR1052	4	Cleavage
	miR773b	3.5	Cleavage		miR3637	4	Translation		miR2868	4	Cleavage
	miR773b	3.5	Cleavage		miR5016	4	Cleavage	DlCHR35	miR870	3	Translation
	miR1884b	4	Cleavage		miR820a	4	Cleavage		miR156e	4	Cleavage
	miR2105	4	Cleavage	DlCHR16b	miR1863	3	Cleavage		miR4391	4	Cleavage
	miR2642	4	Cleavage		miR1525	3.5	Cleavage		miR5072	4	Cleavage
	miR2928	4	Cleavage		miR1863b	3.5	Cleavage	DlCHR36	miR1028c	3	Cleavage
	miR870	4	Cleavage		miR1510a	4	Cleavage		miR158b	3.5	Cleavage
DlCHR8	miR1863	4	Cleavage		miR1871	4	Cleavage		miR417	3.5	Cleavage
	miR2928	4	Cleavage		miR2930	4	Cleavage		miR1081	4	Translation
	miR4393b	4	Cleavage		miR3637	4	Translation		miR1217	4	Cleavage
	miR5021	4	Cleavage		miR5016	4	Cleavage		miR1919a	4	Cleavage
	miR5200	4	Cleavage		miR820a	4	Cleavage		miR5052	4	Cleavage
DlCHR9	miR1028c	2.5	Cleavage	DlCHR18	miR390a.3	3.5	Cleavage		miR5176	4	Cleavage
	miR3637	3.5	Cleavage		miR536	3.5	Cleavage	DlCHR37	miR1027a	4	Cleavage
	miR393	3.5	Translation		miR536	3.5	Cleavage		miR1878	4	Cleavage
	miR1320	4	Cleavage	DlCHR19	miR5025	3.5	Cleavage	DlCHR39	miR1169	3	Cleavage
	miR2604	4	Cleavage		miR2097	4	Translation		miR1888	4	Cleavage
	miR860	4	Cleavage	DlCHR20a	miR479	3	Cleavage		miR2673a	4	Cleavage
DlCHR11	miR5058	3.5	Cleavage		miR1513	4	Cleavage		miR3706	4	Cleavage
	miR1120	4	Translation		miR2604	4	Cleavage	DlCHR42	miR1875	4	Cleavage
	miR2610a	4	Cleavage		miR413	4	Cleavage		miR2673a	4	Cleavage
	miR2673a	4	Cleavage		miR811d	4	Cleavage		miR3437	4	Cleavage
	miR3435	4	Cleavage	DlCHR21	miR5079	3.5	Cleavage		miR413	4	Translation
	miR4246	4	Cleavage		miR1044	4	Translation