关键词:水稻; 粒形; 基因 GW8; 功能标记; 单体型 Development of Functional Markers and Identification of Haplotypes for Rice Grain Shape Gene GW8 YI Chuan-Deng*, WANG De-Rong, JIANG Wei, Li Wei, CHENG Xiao-Jun, WANG Ying, ZHOU Yong, LIANG Guo-Hua, GU Ming-Hong Jiangsu Key Laboratory of Crop Genetics and Physiology, Co-Innovation Center for Modern Production Technology of Grain Crops, Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou 225009, China Fund:This study was supported by the National Natural Science Foundation of China (31571624, 31071382), National Basic Research Program of China (973 program) (2010CB125904, 2013CBA01405), the Key Natural Science Project in University of Jiangsu Province (15KJA210004), and the Priority Academic Program Development of Jiangsu Higher Education Institutions AbstractRice grain shape including grain length, grain width, grain thickness, and grain length-to-width ratio is a major character affecting rice yield and quality. On the basis of the alignment of gene GW8 genomic DNA sequence, three functional markers were developed on the 10-bp indel in promoter region and two missense polymorphism sites (A/C and T/G) in the exon3, respectively. Subsequently the markers were used to identify the genotypes of gene GW8 in the 294 accessions of rice mini-core collection and 65 japonica varieties certificated in Jiangsu province from 2007 to 2013. We found that the allelic variations of the three target loci had significant or very significant effects on most traits of grain shape. On the bais of the three variation sites of gene GW8, eight haplotypes (combinations) were found to have very significant effect on the four grain shape traits in the rice mini-core collection. The main haplotypes were Hap3, Hap6, and Hap1, contained in 126, 85, and 58 representative varieties, respectively; Hap7 has the most strongest effect on grain length, ratio of length to width and 1000-grain weight. While the only two haplotypes, Hap6 and Hap2, contained in 63 and 2 japonica varieties certificated in Jiangsu provice, respectively. The results paved a way to apply the useful allelic variations or haplotypes of gene GW8 in rice yield and quality breeding programs.
表1 基于基因GW8启动子区和第3外显子DNA序列变异设计的PCR引物 Table 1 PCR primers based on the GW8 DNA sequence variations in promotor and exon3
标记名称 Marker
变异位点所在区域a Region of sequence variations a
基因型 Genotype
引物序列 Primer sequence (5° -3° )
标记类型 Marker type
GW8-1
Promotor(-23 bp)
+/-b
F: AAAGAGACAGCCACGGAATC R: ATCTTGAGATCCCACTCCATG
Sequencing
GW8-2
Exon3 (821 bp)
C/A
F: AGGCGAGATCAGCTTCGTCA R: GCTACCGTCTTCAGAAGTGGC
dCAPs-Hind II
GW8-3
Exon3 (1092 bp)
G/T
F: ATGTTCTCCGATGGTGGGTT R: TGAAGGCCAGAGATGAGAGG
CAPs-Nco I
a: genomic DNA sequence (LOC_Os08g41940) of gene GW8 as a reference; b: +/- means 10-bp insertion/deletion in the promoter region of gene GW8. a: 以基因GW8的基因组序列LOC_Os08g41940作为参照; b: +/- 表示基因GW8启动子区10 bp (GAGCTGAGCT)序列的有/无。
表1 基于基因GW8启动子区和第3外显子DNA序列变异设计的PCR引物 Table 1 PCR primers based on the GW8 DNA sequence variations in promotor and exon3
图1 基因GW8不同变异位点特异引物的PCR鉴定 A: 是引物GW8-1的扩增产物电泳图; B和C: 分别是引物GW8-2和GW8-3的扩增产物酶切电泳图。M: DL2000 DNA marker (TaKaRa), 泳道1~4: 分别为水稻品种Basmati、日本晴、9311和华粳籼74。Fig. 1 PCR identification of specific primers for corresponding mutation sites in geneGW8 A: PCR amplification products of primer GW8-1; B and C: enzyme-digested PCR amplification products of primer GW8-2(B) and GW8-3(C). M: DL2000 DNA marker (TaKaRa); lanes 1-4: Basmati, Nipponbare, 9311, and Huajingxian 74, respectively.
表4 基因GW8不同单体型对水稻粒形性状的差异显著性分析 Table 4 Analysis of the difference of grain-related traits based on different haplotypes in gene GW8
单体型Haplotype
GW8-1
GW8-2
GW8-3
样品数 Number
粒长 Grain length (mm)
粒宽 Grain width (mm)
粒厚 Grain thickness (mm)
长宽比 Ratio of length to width
千粒重 1000-grain weight (g)
Hap1
+
C
T
58
8.40± 0.11 a
2.99± 0.05 ab
2.08± 0.02 b
2.87± 0.07 a
25.22± 0.54
Hap2
+
C
G
4
8.46± 0.22 a
2.99± 0.14 ab
2.12± 0.04 ab
2.85± 0.18 ab
25.86± 1.20
Hap3
+
A
T
126
8.44± 0.08 a
2.91± 0.03 b
2.09± 0.01 b
2.95± 0.05 a
24.43± 0.38
Hap4
+
A
G
2
7.34± 0.08 b
2.94± 0.25 ab
2.10± 0.10 ab
2.51± 0.19 ab
22.58± 1.72
Hap5
-
C
T
9
7.61± 0.27 b
3.20± 0.05 a
2.20± 0.03 a
2.39± 0.10 b
25.09± 1.02
Hap6
-
C
G
85
7.59± 0.08 b
3.18± 0.03 a
2.17± 0.02 a
2.42± 0.05 b
24.74± 0.34
Hap7
-
A
T
9
8.69± 0.39 a
2.94± 0.07 ab
2.15± 0.04 ab
2.98± 0.16 a
25.96± 0.96
Hap8
-
A
G
1
All data about grain-related traits are given as mean± SE. a, b: means Multiple comparisons based on least significant difference (LSD) test. 表中籽粒相关性状的数据为平均数± 标准误。a、b表示基于最小显著差数法的多重比较。
表4 基因GW8不同单体型对水稻粒形性状的差异显著性分析 Table 4 Analysis of the difference of grain-related traits based on different haplotypes in gene GW8
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