中国农业科学 ›› 2021, Vol. 54 ›› Issue (18): 3932-3944.doi: 10.3864/j.issn.0578-1752.2021.18.012
宋春晖1(),陈晓菲1(
),王枚阁1,郑先波1,宋尚伟1,焦健1,王苗苗1,马锋旺2,白团辉1(
)
收稿日期:
2021-01-28
接受日期:
2021-04-13
出版日期:
2021-09-16
发布日期:
2021-09-26
联系方式:
宋春晖,E-mail: songchunhui060305@126.com。|陈晓菲,E-mail: chenxiaofei0312@163.com。
基金资助:
SONG ChunHui1(),CHEN XiaoFei1(
),WANG MeiGe1,ZHENG XianBo1,SONG ShangWei1,JIAO Jian1,WANG MiaoMiao1,MA FengWang2,BAI TuanHui1(
)
Received:
2021-01-28
Accepted:
2021-04-13
Published:
2021-09-16
Online:
2021-09-26
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。
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.
表1
样品测序质量及与参考基因组的匹配情况"
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 |
表2
SLAF标签和多态性SLAF标签在染色体分布"
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 |
表4
苹果砧木耐涝性状的关联分析"
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 |
Chr17 | 1839746 | 1853187 | 0.01 | 3 | |
SNP-index法 SNP-index method | Chr10 | 12955610 | 13906230 | 0.95 | 45 |
Chr10 | 1942245 | 7959407 | 6.02 | 445 | |
交集 Intersection | Chr10 | 1942245 | 3252362 | 1.31 | 120 |
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 |
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