Morphological and Molecular Genetic Analysis of a Dwarf and Small Grain Rice Variety Xiaoxiang’ai

doi:10.16819/j.1001-7216.2017.6156 238

Abstract

Abstract:

【Objective】To uncover the genetic mechanism of a dwarf and small grain variety Xiaoxiang’ai (XXA), and lay an important theoretical foundation in rice dwarfing breeding.【Method】Rice variety XXA characterized as a dwarf and small grain phenotype was derived from natural mutation under breeding process. The agronomic traits of XXA were investigated. Its sensibility to phytohormone was analyzed under GA₃ and BR treatments at various concentrations. Genetic analysis of XXA with different varieties and fine mapping of xxa gene from the F₂ population derived from the cross between near-isogenic lines NIL(NIP) and XXA were done. Furthermore, complementary assay was performed to confirm the candidate gene.【Result】The XXA shows short spikes and compact panicles, with significant decrease in 1000-grain weight under GA₃ and BR treatments, we found that XXA is partially sensitive to GA₃, whereas insensitive to BR. The segregation behavior in each of the derived F₂ populations between XXA and either varieties was consistent with the Mendelian monogenic ratio of 3:1. The xxa gene was finally narrowed down to a 70 kb physical region between the InDel markers F81 and F82 on chromosome 5. Within this region, eight open reading frames (ORFs) were predicted, of which, ORF5 (LOC_Os05g26890) has been annotated as a Dwarf1 gene (D1). Furthermore, sequence analysis of all the region revealed that there are only two mutation sites including one 1-bp nonsense mutation in the 5th exon and a 3-bp deletion that resulted in a lysine deletion in the 12th exon of D1. The complementary assay showed that the abnormal phenotype of XXA can be recovered.【Conclusion】The dwarf and small grain phenotype is probably associated with GA₃ metabolism. The abnormal phenotype is mainly controlled by a single recessive gene. The D1 indisputably corresponds to mutation phenotype of XXA.

Key words: rice, dwarf and small grain, gene mapping, mechanized propagation

摘要：

【目的】研究揭示潇湘矮矮秆小粒的遗传机制,为潇湘矮的育种利用提供理论基础。【方法】利用水稻育种过程中自然突变而得到的稳定遗传的矮秆小粒水稻材料潇湘矮,对其进行农艺性状考查、赤霉素(GA₃)和油菜素内酯(BR)敏感性分析、遗传学分析,并利用潇湘矮与其近等基因系NIL(NIP)衍生的F₂群体对控制矮秆小粒的基因xxa进行图位克隆,最终利用转基因互补试验验证候选基因。【结果】潇湘矮除表现为矮秆小粒外,其穗长变短,穗型紧凑,千粒重极显著下降。不同浓度梯度的赤霉素(GA₃)和油菜素内酯(BR)处理,发现潇湘矮对GA₃部分敏感,而对BR不敏感。遗传分析发现其符合孟德尔3:1分离规律。图位克隆将xxa基因定位于第5染色体InDel标记F81和F82之间约70 kb区间的物理距离内。该区间包含8个开放阅读框(ORF)。其中,第5个ORF(LOC_Os05g26890)被注释为水稻株高基因D1。序列分析发现,潇湘矮的D1基因在第5和12外显子分别有1个碱基的无义替换和3个碱基的缺失,其中第12外显子3个碱基的缺失导致1个赖氨酸的缺失。转基因互补显示D1可以恢复潇湘矮的表型。【结论】潇湘矮控制株高的途径可能与GA₃代谢有关;潇湘矮矮秆小粒表型符合单隐性核基因控制的遗传规律;潇湘矮矮秆小粒性状是由D1基因突变所致。

关键词: 水稻, 矮秆小粒, 图位克隆, 机械化制种

CLC Number:

S511.024

Yusong LÜ, Yunfeng XIE, Zhonghua SHENG, Yawen WU, Shaoqing TANG, Peisong HU, Xiangjin WEI. Morphological and Molecular Genetic Analysis of a Dwarf and Small Grain Rice Variety Xiaoxiang’ai[J]. Chinese Journal OF Rice Science, 2017, 31(3): 238-246.

吕育松, 谢耘丰, 圣忠华, 邬亚文, 唐绍清, 胡培松, 魏祥进. 矮秆小粒水稻潇湘矮的形态学与分子遗传学分析[J]. 中国水稻科学, 2017, 31(3): 238-246.

Figures/Tables 7

Table 1 Primers for mapping in the study.

引物 Marker name	前引物序列 Forward(5′-3′)	后引物序列 Reverse(5′-3′)
RM18408	GGCACTCTGGTTCCTCAATGG	TCTGTATCATCAACGGCAACTCC
RM5140	GGCACTCGTATTTCTCAACTTCTCC	GGGTGTATCAGGAGTACAGGTTGC
RM18457	ATCCTCCACCGCTCAAGAACACG	CGAGGCCATTCATCGAACAAAGC
RM18402	TTATGAGGCAGCCCGTAATGTTGC	GCAGCGGTGTCAACAGCTTCC
RM18450	AAGGCTCCATGGTTGGTTGC	CGATGGACAGACAGTGTGTAGTGG
F78	GGGACGAATTCTTTTCGATTAC	CGTGGACCAATTTTGGTAACTG
F79	AATTATTCCACTATGCACATGT	ATTTTCTTCCATCGCCTCTTGC
F81	GTAAACTATCGACTTGCTATGT	ACTAGTGCAGACTGTTTTCCTG
F82	AGATGATTGGATGAGAATTTAA	ACCCAGAAACCATCTAGTAATT
F84	GGGTGGCTCCTTACGACATTAC	TTTCATATTTTAGCGGTGCTCT
F85	CCTCGACCCACTGCATCATCAG	TGGTCGGTCTCCTCCCTCTTCA
F86	CAGTATTCGAGTAAGTTCACAA	CCTCGCCCCTATTCATCCTCTT

Fig. 1. Phenotype analysis of XXA and NIL(NIP). A, 5-weeks-old seedlings of NIL(NIP)(left) and XXA(right); B, Phenotypes of NIL(NIP) and XXA plants at the mature stage in paddy field; C, Comparison of main panicles between NIL(NIP) and XXA at the mature stage; D, Grain size of NIL(NIP) and XXA at the mature stage; E-J, Plant height(E), number of panicles per plant(F), seed setting rate(G), 1,000-grain weight(H), grain length(I) and grain width(J) between NIL(NIP) and XXA. Bar=10 cm in Figures A, B and C; Bar=5 mm in Figure D. Data in E-J are shown as Mean±SD from 10 individual replicates. The asterisks indicate statistical significance, as determined by the Student’s t-test(*P<0.05, **P<0.01).

Fig. 2. The internode and panicle length comparison in XXA and NIL(NIP). A, Differences in panicles and internode length for NIL(NIP)(left) and XXA(right) plants. P, Panicle. Those from I to V indicate the corresponding internodes from the top to bottom, bar = 15 cm; B, Internode and young panicle length analysis of NIL(NIP) and XXA at the heading stage. Data in B are shown as Mean±SD from 10 individual replicates. The asterisks indicate statistical significance, as determined by the t-test(**P<0.01).

Fig. 3. Elongation of the second leaf sheath in response to gibberellin(A) and brassinolide(B) in NIL(NIP) and XXA plants. Data in A, B are shown as Mean±SD from 10 individual replicates.

Fig. 4. Fine mapping and positional cloning of the candidate gene. A, Fine mapping of the xxa gene; B, The structure of candidate gene LOC_Os05g26890 and the mutation sites in XXA. Black arrows show 1-bp nonsense mutation in the 5th exon and a 3-bp deletion that resulted in a lysine deletion in the 12th exon of D1.

Table 2 Segregation of combination Nipponbare/XXA , Reyan 1/XXA, 0248/XXA and Y01/XXA.

F₁组合 Combination of F₁	总植株数 Total No. of plants	正常植株数 No. of half-dwarfing plants	矮秆小粒植株数 No. of dwarfing plants	理论比 Expressed ratio	χ²
日本晴/潇湘矮 Nipponbare/XXA	3223	2341	782	3:1	0.078
热研1号/潇湘矮 Reyan 1/XXA	2964	2249	725	3:1	0.420
0248/潇湘矮 0248/XXA	3744	2812	932	3:1	0.937
Y01/潇湘矮 Y01/XXA	3416	2560	856	3:1	0.937

Fig. 5. Phenotype analysis of complementary plants(D1/XXA). A-D, Phenotypes of the complementary plants of D1/XXA-1(right) and XXA(left) at harvesting time, including plant type(A), panicle(B), grain size(C) and internode(D) of D1/XXA-1 and XXA; E, Relative expression levels of D1 gene in XXA and D1/XXA lines. F-I, Internode length(F), plant height(G), grain length(H) and grain width(I) of D1/XXA-1 and XXA(Bar=10 cm in A, B and D; Bar=5 mm in C). Data in E are shown as Mean±SD from 3 individual replicates. Data in F-I are shown as Mean±SD from 10 individual replicates. The asterisks indicate statistical significance, as determined by the Student’s t-test(*P<0.05; **P<0.01).

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