基于SLAF-seq技术鉴定苹果砧木耐涝候选基因

doi:10.3864/j.issn.0578-1752.2021.18.012

摘要/Abstract

摘要：

【背景】苹果（Malus×domestica Borkh）是我国主要栽培果树树种之一,但部分苹果产区由于夏、秋季的大量集中降雨和排水不良等造成果园涝害频繁发生,导致苹果树叶片黄化、脱落,果实品质和产量下降。【目的】鉴定苹果耐涝相关基因,为苹果耐涝分子标记辅助育种和优质高产栽培提供依据。【方法】以耐涝苹果砧木G41和不耐涝苹果砧木新疆野苹果（M. sieverii (Ledeb) Roem.）及其构建的包含495个F₁杂交后代为材料,从F₁杂交群体中挑选出耐涝和不耐涝株系各50株,构建两个极端性状DNA混池,采用简化基因组测序（SLAF-seq）技术,开发SLAF标签和SNP标记,结合苹果基因组信息和遗传关联性分析,对苹果耐涝基因进行定位及候选基因预测,并对候选基因在耐涝差异的株系中进行淹水胁迫下的表达分析。【结果】以‘金冠’苹果为参考基因组,共开发119 072个SLAF标签,其中多态性SLAF有11 133个。通过序列分析和检测SNP位点,共获得6 237 071个SNP,其中高质量SNP有170 617个。通过ED和SNP-index方法关联分析,获得一个与耐涝性状紧密关联的候选区域,位于苹果第10号染色体1.94—3.25 Mb,关联区域大小为1.31 Mb,关联区域内包含120个基因。对该区域内基因进行功能注释,发现一个与呼吸代谢相关的基因—乙醇脱氢酶基因ADH1（MD10G1014500）,在淹水处理后1、2、4和6 d,该基因在耐涝植株中的表达量显著高于不耐涝植株。【结论】将苹果耐涝基因定位于第10号染色体1.94—3.25 Mb处,筛选到可能与苹果耐涝相关的候选基因MD10G1014500,可用于苹果耐涝基因的克隆和功能解析。

关键词: 苹果, 耐涝性, SLAF, SNP, 候选基因

Abstract:

【Background】 Apple (Malus×domestica Borkh) is one of the most cultivated fruit crops in China. While apple trees frequently encounter waterlogging stress mainly due to excess rainfall and poor soil drainage, resulting in yellowing leaf, declined fruit quality and yield in summer and autumn. 【Objective】 The aim of this study was to identify waterlogging-tolerant genes of apple, so as to provide a basis for waterlogging tolerance molecular marker assisted breeding, high-quality and high-yield cultivation of apple.【Method】In this study, 50 waterlogging-tolerant plants and 50 waterlogging-sensitive plants were selected to construct two bulked DNA pools from the 495 F₁ population, which were derived from a cross of the waterlogging-tolerant apple rootstock G41 and the waterlogging-tolerant M. sieverii (Ledeb) Roem. Specific-locus amplified fragment (SLAF) labels and single nucleotide polymorphism (SNP) markers were developed by SLAF-seq technique. The mapping and candidate gene prediction of waterlogging tolerance in apple were carried out by combining apple genome and genetic association analysis, and the relative expression of the candidate gene was also analyzed in different waterlogging tolerance plants under waterlogging stress.【Result】A total of 119 072 SLAF labels were obtained, of which, 11 133 were polymorphic between both parents. A total 16 237 071 SNPs were identified by sequence analysis, including 170 617 SNPs with polymorphic. By association analysis with Eudidean distance (ED) and SNP-index, a candidate locus was found to be strongly associated with waterlogging tolerance, which was a region of 1.94-3.25 Mb on apple chromosome 10. The associated region was 1.31 Mb that contained 120 genes, and functional annotation of the genes in this region revealed a gene MD10G1014500, alcohol dehydrogenase gene (ADH1) related to respiratory metabolism. The expression level of ADH1 gene in waterlogging tolerant plants was significantly higher than that in waterlogging sensitive plants at 1, 2, 4 and 6 days after waterlogging treatment. 【Conclusion】 One gene (MD10G1014500) was identified as a potential candidate gene involved waterlogging tolerance of apple, which located in a 1.94-3.25 Mb interval on chromosome 10. These findings laid the foundation for gene cloning and functional analysis for waterlogging tolerance of apple.

Key words: apple, waterlogging tolerance, SLAF, SNP, candidate gene

宋春晖,陈晓菲,王枚阁,郑先波,宋尚伟,焦健,王苗苗,马锋旺,白团辉. 基于SLAF-seq技术鉴定苹果砧木耐涝候选基因[J]. 中国农业科学, 2021, 54(18): 3932-3944.

SONG ChunHui,CHEN XiaoFei,WANG MeiGe,ZHENG XianBo,SONG ShangWei,JIAO Jian,WANG MiaoMiao,MA FengWang,BAI TuanHui. Identification of Candidate Genes for Waterlogging Tolerance in Apple Rootstock by Using SLAF-seq Technique[J]. Scientia Agricultura Sinica, 2021, 54(18): 3932-3944.

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表5

定位区间基因功能预测"

编号No	基因编号Gene ID	注释信息 Function annotation
1	MD10G1014300	生长素输出载体家族蛋白 Auxin efflux carrier family protein
2	MD10G1014400	核小分子RNA71 SnoR71
3	MD10G1014500	乙醇脱氢酶1 Alcohol dehydrogenase 1
4	MD10G1014600	5S核糖体RNA 5S rRNA
5	MD10G1014700	5S核糖体RNA 5S rRNA
6	MD10G1014800	植物硬脂酰酰基载体蛋白去饱和酶家族蛋白 Plant stearoyl-acyl-carrier-protein desaturase family protein
7	MD10G1014900	管状分子分化相关6 Tracheary element differentiation-related 6
8	MD10G1015000	表皮毛双折射7 TRICHOME BIREFRINGENCE-LIKE 7
9	MD10G1015100	表皮毛双折射7 TRICHOME BIREFRINGENCE-LIKE 7
10	MD10G1015200	植物U3 Plant U3
11	MD10G1015300	抗病蛋白（TIR-NBS-LRR类） Disease resistance protein (TIR-NBS-LRR class)
12	MD10G1015400	非编码RNA ncRNA
13	MD10G1015500	丝氨酸羧肽酶42 Serine carboxypeptidase-like 42
14	MD10G1015600	三角状五肽重复序列（PPR）超家族蛋白 Pentatricopeptide repeat (PPR) superfamily protein
15	MD10G1015700	三角状五肽重复序列（PPR）超家族蛋白 Pentatricopeptide repeat (PPR) superfamily protein
16	MD10G1015800	植物糖原蛋白样淀粉起始蛋白2 Plant glycogenin-like starch initiation protein 2
17	MD10G1015900	植物糖原蛋白样淀粉起始蛋白2 Plant glycogenin-like starch initiation protein 2
18	MD10G1016000	液泡蛋白分选45 Vacuolar protein sorting 45
19	MD10G1016100	液泡蛋白分选45 Vacuolar protein sorting 45
20	MD10G1016200	5S核糖体RNA 5S rRNA
21	MD10G1016300	功能未知的蛋白（DUF506） Protein of unknown function (DUF506)
22	MD10G1016400	高尔基核苷酸糖转运蛋白1 Golgi nucleotide sugar transporter 1
23	MD10G1016500	ABC转运蛋白家族蛋白 ABC transporter family protein
24	MD10G1016600	功能未知的蛋白质（DUF300） Protein of unknown function (DUF300)
25	MD10G1016700	snoZ221 snoR21b
26	MD10G1016800	发病相关家族蛋白 Pathogenesis-related family protein
27	MD10G1016900	核糖体RNA 5S rRNA 5S
28	MD10G1017000	snoZ221 snoR21b
29	MD10G1017100	未知 Unknown
30	MD10G1017200	5S核糖体RNA 5S rRNA
31	MD10G1017300	发病相关家族蛋白 Pathogenesis-related family protein
32	MD10G1017400	糖脂转移蛋白2 Glycolipid transfer protein 2
33	MD10G1017500	未表征的蛋白质 Uncharacterized protein
34	MD10G1017600	真核生物翻译起始因子6-2 Eukaryotic translation initiation factor 6-2
35	MD10G1017700	功能未知的蛋白质 Protein of unknown function
36	MD10G1017800	Dof型锌指DNA结合家族蛋白 Dof-type zinc finger DNA-binding family protein
37	MD10G1017900	ABC转运G家族成员22 ABC transporter G family member 22-like
38	MD10G1018000	MAP激酶底物1 MAP kinase substrate 1
39	MD10G1018100	核内体靶向BRO1样结构域含蛋白 Endosomal targeting BRO1-like domain-containing protein
编号No	基因编号Gene ID	注释信息 Function annotation
40	MD10G1018200	RING / FYVE / PHD锌指超家族蛋白 RING/FYVE/PHD zinc finger superfamily protein
41	MD10G1018300	伴刀豆球蛋白A样凝集素蛋白激酶家族蛋白 Concanavalin A-like lectin protein kinase family protein
42	MD10G1018400	抗病蛋白（TIR-NBS-LRR类）家族 Disease resistance protein (TIR-NBS-LRR class) family
43	MD10G1018500	抗病蛋白（TIR-NBS-LRR类） Disease resistance protein (TIR-NBS-LRR class)
44	MD10G1018600	抗病蛋白（TIR-NBS-LRR类） Disease resistance protein (TIR-NBS-LRR class)
45	MD10G1018700	嘌呤生物合成4 Purine biosynthesis 4
46	MD10G1018800	未知 Unknown
47	MD10G1018900	抗病蛋白（TIR-NBS-LRR类） Disease resistance protein (TIR-NBS-LRR class)
48	MD10G1019000	未知 Unknown
49	MD10G1019100	铜转运蛋白2 Copper transporter 2-like
50	MD10G1019200	抗病蛋白（TIR-NBS-LRR类） Disease resistance protein (TIR-NBS-LRR class)
51	MD10G1019300	小核糖核蛋白家族蛋白 Small nuclear ribonucleoprotein family protein
52	MD10G1019400	伴刀豆球蛋白A样凝集素蛋白激酶家族蛋白 Concanavalin A-like lectin protein kinase family protein
53	MD10G1019500	伴刀豆球蛋白A样凝集素蛋白激酶家族蛋白 Concanavalin A-like lectin protein kinase family protein
54	MD10G1019600	伴刀豆球蛋白A样凝集素蛋白激酶家族蛋白 Concanavalin A-like lectin protein kinase family protein
55	MD10G1019700	抗病蛋白（TIR-NBS-LRR类）家族 Disease resistance protein (TIR-NBS-LRR class) family
56	MD10G1019800	伴刀豆球蛋白A样凝集素蛋白激酶家族蛋白 Concanavalin A-like lectin protein kinase family protein
57	MD10G1019900	伴刀豆球蛋白A样凝集素蛋白激酶家族蛋白 Concanavalin A-like lectin protein kinase family protein
58	MD10G1020000	抗病蛋白（TIR-NBS-LRR类） Disease resistance protein (TIR-NBS-LRR class)
59	MD10G1020100	抗病蛋白（TIR-NBS-LRR类） Disease resistance protein (TIR-NBS-LRR class)
60	MD10G1020200	未知 Unknown
61	MD10G1020300	伴刀豆球蛋白A样凝集素蛋白激酶家族蛋白 Concanavalin A-like lectin protein kinase family protein
62	MD10G1020400	含LIM结构域的蛋白质 LIM domain-containing protein
63	MD10G1020500	抗病蛋白（TIR-NBS-LRR类） Disease resistance protein (TIR-NBS-LRR class)
64	MD10G1020600	非编码RNA ncRNA
65	MD10G1020700	线粒体底物载体家族蛋白 Mitochondrial substrate carrier family protein
66	MD10G1020800	含LIM结构域的蛋白质 LIM domain-containing protein
67	MD10G1020900	抗病蛋白（TIR-NBS-LRR类） Disease resistance protein (TIR-NBS-LRR class)
68	MD10G1021000	抗病蛋白（TIR-NBS-LRR类） Disease resistance protein (TIR-NBS-LRR class)
69	MD10G1021100	功能未知的蛋白（DUF506） Protein of unknown function (DUF506)
70	MD10G1021200	β-酮酰基还原酶1 Beta-ketoacyl reductase 1
71	MD10G1021300	具有 FYVE 锌指结构域的染色体浓缩（RCC1）家族调节因子 Regulator of chromosome condensation (RCC1) family with FYVE zinc finger domain
72	MD10G1021400	植物U-box 29 Plant U-box 29
73	MD10G1021500	RNA结合蛋白2 RNA-binding protein 2-like
74	MD10G1021600	核苷酸结合 Nucleotide binding
75	MD10G1021700	未知 Unknown
76	MD10G1021800	非编码RNA ncRNA
77	MD10G1021900	ACT样蛋白酪氨酸激酶家族蛋白 ACT-like protein tyrosine kinase family protein
78	MD10G1022000	碳酸酐酶1 Carbonic anhydrase 1
79	MD10G1022100	F盒家族蛋白 F-box family protein
编号No	基因编号Gene ID	注释信息 Function annotation
80	MD10G1022200	碳酸酐酶2 Carbonic anhydrase 2
81	MD10G1022300	未知 Unknown
82	MD10G1022400	碳酸酐酶2 Carbonic anhydrase 2
83	MD10G1022500	RmlC样铜蛋白超家族蛋白 RmlC-like cupins superfamily protein
84	MD10G1022600	RmlC样铜蛋白超家族蛋白 RmlC-like cupins superfamily protein
85	MD10G1022700	未知 Unknown
86	MD10G1022800	未知 Unknown
87	MD10G1022900	RmlC样铜蛋白超家族蛋白 RmlC-like cupins superfamily protein
88	MD10G1023000	综合调控因子2 General regulatory factor 2
89	MD10G1023100	样铜蛋白超家族蛋白 RmlC-like cupins superfamily protein RmlC
90	MD10G1023200	TCP-1/cpn60伴侣蛋白家族蛋白 TCP-1/cpn60 chaperonin family protein
91	MD10G1023300	含LIM结构域的蛋白质 LIM domain-containing protein
92	MD10G1023400	泛素载体蛋白7 Ubiquitin carrier protein 7
93	MD10G1023500	胚芽蛋白样蛋白2 Germin-like protein 2
94	MD10G1023600	磷酸吡rid醛（PLP）依赖性转移酶 Pyridoxal phosphate (PLP)-dependent transferases superfamily protein
95	MD10G1023700	5S 核糖体RNA 5S rRNA
96	MD10G1023800	VIRE2相互作用蛋白 1VIRE2-interacting protein 1
97	MD10G1023900	Tho复合亚基7 / Mft1p Tho complex subunit 7/Mft1p
98	MD10G1024000	液泡铁转运蛋白（VIT）家族蛋白 Vacuolar iron transporter (VIT) family protein
99	MD10G1024100	真核翻译起始因子2（eIF-2）家族蛋白 Eukaryotic translation initiation factor 2 (eIF-2) family protein
100	MD10G1024200	非编码RNA ncRNA
101	MD10G1024300	泛素相互作用基序蛋白 Ubiquitin interaction motif-containing protein
102	MD10G1024400	果糖-1,6-二磷酸酶 Fructose-1,6-bisphosphatase
103	MD10G1024500	小核糖核蛋白家族蛋白 Small nuclear ribonucleoprotein family protein
104	MD10G1024600	小核糖核蛋白家族蛋白 Small nuclear ribonucleoprotein family protein
105	MD10G1024700	U6
106	MD10G1024800	序列特异性DNA结合转录因子 Sequence-specific DNA binding transcription factors
107	MD10G1024900	非编码RNA ncRNA
108	MD10G1025000	未知 Unknown
109	MD10G1025100	Trihelix转录因子 Trihelix transcription factor
110	MD10G1025200	Trihelix转录因子 Trihelix transcription factor
111	MD10G1025300	结瘤素MtN21/EamA样转运蛋白家族蛋白 Nodulin MtN21/EamA-like transporter family protein
112	MD10G1025400	U6
113	MD10G1025500	DEAD-box ATP依赖性RNA解旋酶21 DEAD-box ATP-dependent RNA helicase 21-like
114	MD10G1025600	桶状样F-box蛋白8（LOC103444645）,mRNA Tubby-like F-box protein 8 (LOC103444645), mRNA
115	MD10G1025700	核小分子RNA118 snoR118
116	MD10G1025800	未知 Unknown
117	MD10G1025900	功能未知的蛋白质（DUF793） Protein of unknown function (DUF793)
118	MD10G1026000	非编码RNA ncRNA
119	MD10G1026100	HSP20样伴侣蛋白超家族蛋白 HSP20-like chaperones superfamily protein
120	MD10G1026200	铜氧还蛋白超家族蛋白 Cupredoxin superfamily protein

表5

图5

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样本 Sample	干净reads Clean reads	干净碱基 Clean base	Q30 (%)	GC (%)	总比对率 Total mapped (%)	正确比对率 Properly mapped (%)
G41	38 365 575	9 666 919 570	85.02	41.1	97.55	87.64
新疆野苹果M. sieversii	39 983 972	10 067 312 448	87.82	41.61	97.48	85.77
耐涝混池 Tolerant pool	10 873 511	2 174 702 200	89.47	39.89	94.94	82.30
不耐涝混池 Sensitive pool	10 279 180	2 055 836 000	88.94	39.86	93.01	80.64

染色体 Chromosome	SLAF 数目 SLAF number	多态性 SLAF Polymorphic SLAF
Chr 01	3 809	354
Chr 02	4 892	530
Chr 03	4 628	436
Chr 04	3 224	251
Chr 05	4 304	495
Chr 06	3 595	308
Chr 07	3 620	383
Chr 08	3 880	394
Chr 09	4 790	455
Chr 10	4 474	423
Chr 11	4 717	435
Chr 12	4 234	418
Chr 13	5 244	486
Chr 14	4 038	405
Chr 15	6 297	567
Chr 16	3 011	281
Chr 17	3 214	337
Chr 00	47 101	4175
总计Total	119 072	11133

样本 Sample	总SNP Total SNP	SNP数目 SNP number	杂合率 Heterozygous ratio (%)
G41	5 093 595	3 313 306	35.79
新疆野苹果M. sieversii	5 093 595	4 208 748	53.41
耐涝混池 Tolerant pool	1 143 476	1 104 683	51.19
不耐涝混池 Sensitive pool	1 143 476	1 090 476	49.99

方法 Method	染色体 Chromosome	起始位置 Start	终止位置 End	大小 Size (Mb)	基因数量 Gene number
欧式距离法 Euclidean distance	Chr10	1942245	3252362	1.31	121
欧式距离法 Euclidean distance	Chr17	1839746	1853187	0.01	3
SNP-index法 SNP-index method	Chr10	12955610	13906230	0.95	45
SNP-index法 SNP-index method	Chr10	1942245	7959407	6.02	445
交集 Intersection	Chr10	1942245	3252362	1.31	120