Acccepted:September 27, 2017
Corresponding author: Yutaka Okumoto (okumoto3@kais.kyoto-u.ac.jp)
QTL analysis of VGI (Vigorously growing plant in IM294) mutants related traits in rice
Chong XU
1), Takuji Tsukiyama
2), Masayoshi Teraishi
1),Takatoshi Tanisaka
3), Yutaka Okumoto
1)1）Graduate school of Agriculture, Kyoto University (Kitashirakawa- Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan) 2）Faculty of Agriculture, Kindai University (3327-204 Nakamachi, Nara City, Nara, 631-8505, Japan)
3）Department of Agricultural Regional Vitalization, Kibi International University
(370-1, shichisareo, Minamiawaji, Hyogo, 656-0484, Japan)
Summary: In a rice variety `Gimbozu` genome, transposition of miniature Ping ( mPing) often causes the modification of gene expression and the disruption of gene function. IM294 is a slender-glume mutant line induced from Gimbozu by gamma-ray irradiation, and its mutant phenotype is caused by an insertion of mPing at the fourth exon of Rurm1 (rice ubiquitin related modifi er-1) gene. Inserted mPing is excised from the Rurm1 gene and slender-glume mutant plant reverts to the original normal-glume plant. At the time of this reverse mutation, some of the revertants exhibited a vigorous growth. We named this revertant as a vigorously growing plant in IM294 (VGI). Still we do not know any molecular clue regarding the mechanism of vigorous growth of VGI. In this study, we investigated nine agronomic traits including the grain shape and the grain number per panicle of a progeny plant of a VGI. QTL analysis was conducted to determine the chromosome region, which might be corresponding to the genetic factors modifi ed by mPing insertion.
Key words: Oryza sativa L., Transposon, mPing, Linkage mapping, QTL analysis
Introduction
miniature Ping (mPing) belongs to type II transposable element (TE) and is actively transposing in a rice variety `Gimbozu` genome. mPing is frequently excised from an original position and is inserted into the other chromosome region (Naito et al., 2006). As a result, the transposition of mPing often causes the modifi cation of gene expression and the disruption of gene function (Naito et al., 2009). IM294 is a mutant line induced from Gimbozu by gamma-ray irradiation, and its mutant phenotype of the slender glume is caused by an insertion of mPing in exon 4 of Rurm1 (rice ubiquitin related modifier-1) gene. When inserted mPing is excised from the Rurm1 gene, a slender-glume mutant plant reverts to the original normal-glume plant (Nakazaki et al., 2003). While most of the revertants exhibit the similar agronomic trait as Gimbozu, a few revertants exhibit a vigorous growth (Horibata & Yamagata 2000, Yamagata & Shakudo 1968). We named these revertants as a vigorously growing plant in IM294 (VGI). VGI often exhibits tall plant height, large tiller number, long panicle length, and large glume size. The fake-revertants caused by out-crossing can be easily detected by using the IM294 specifi c Ping element as a molecular marker. Segregated normal-glume plant heterozygous for this Ping element can be regarded as a fake-revertant originated from the out-crossing. As mPing is highly
activated in VGI according to the results of transposon display (Tsukiyama, 2013 unpublished), the vigorous growth of VGI might be closely related to the genomic modifi cation induced by mPing transposition. Thus, identification of genetic factors modified by the mPing in the VGI will lead to clarify the molecular mechanism of the vigorous growth of VGI and will provide the useful information for the breeding of rice varieties with the high biomass productivity.
In this study, we investigate the genetic factors contributing to the traits related with the vigorous growth of VGI. We made a cross between one of the progeny plants derived from a single VGI and Nipponbare. Using the F2 population derived from this cross, we investigated grain size and the grain number per panicle. Then, QTL analysis was conducted to determine the chromosome region, which may be corresponding to the genetic factors modified by mPing insertion. Genetic map was constructed with SSR markers and mPing-SCAR markers (Monden et al., 2009).
Materials and methods
Plant materials and DNA extraction
In addition to rice variety Gimbozu and Nipponbare, we used a slender-glume mutant line IM294 and a selfed-progeny line derived from a VGI plant. For the genetic analysis of VGI related traits, we used F2 population derived from the cross between Nipponbare and VG-15. In 2008, a single revertant
(normal grain) was segregated in IM294 (slender glume mutant line). As this revertant exhibited vigorous growth, we tentatively regarded this plant as VGI. Then, selfed-seed derived from this VGI plant were harvested to raise the progeny line. In 2013, we grew Nipponbare, Gimbozu, IM294 in addition to the progeny line of VGI in the field. The progeny line was segregating for plant height, tiller number, panicle length, and glume size. Among those segregating plants, we selected a single plant (VG-15). VG-15 had taller plant height, larger panicle length and larger grain size when it was compared with Nipponbare. We crossed VG-15 with Nipponbare to conduct QTL analysis of VGI related traits. In 2014, a total of 94 F2 plants were transplanted to the Wagner s pots with one plant per one pot. Each pot was fertilized with 2.38g (NH4)2SO4, 0.83g Ca(H2PO4)2·H2O+CaSO4 and 2.86g KCl. At the maturing time, we harvested selfed-seed of all F2 plant. In addition, three plants of Nippobare and the progeny of VG-15 were also planted in the Wagner's pots in the same condition as F2 plants. To determine the genotype of SSR markers and mPing-SCAR markers of all F2 plants, we extracted DNA from the bulked eight-seedlings grown from the harvested seeds of every F2 plant with CTAB method.
PCR analysis
We used nine SSR markers and forty eight mPing-SCAR markers (Monden et al., 2009) to construct a genetic linkage map using 94 F2 plants. For SSR markers, PCR is performed in 5 μl containing 2.5μl 2×Go taq (Promega), 0.75μl STDW, 0.25μl DMSO, 1μl genomic DNA and 0.5μl of each primer. PCR is conducted as follows: 94℃ for 10 min; 35 cycles of 94℃ for 30s, 50℃ for 1 min and 72℃ for 30s; 72℃ for 7 min. For mPing-SCAR markers, PCR is performed in 5 μl containing 2.5μl 2×Go taq (Promega), 0.75μl STDW, 0.25μl DMSO, 1μl genomic DNA and 0.5μl of each primer. PCR is conducted as follows: 94℃ for 3 min; 35 cycles of 94℃ for 30s, 57℃ for 45s and 72℃ for 1 min; 72℃ for 5 min.
Construction of genetic map and QTL analysis of nine agronomic traits
Genotype data of 94 F2 plants were used to construct genetic map using Mapmarker ver.3.0. We measured nine agronomic traits of all the F2 plants; namely number of fi lled spikelet per panicle (NFS), panicle length (PAL), number of primary branches per panicle (NPB), number of secondary branches per panicle (NSB), 100-seed-weight (HSW), air-dried weight above ground part (DGW), surface area size of glume (SAG), glume length (GLH), and glume width (GWH). QTL analysis of above nine traits was conducted using QTL Cartographer 2.0. The threshold LOD score were determined after computing 500 times permutation tests.
Results
Nipponbare, Gimbozu and the progeny line of VGI were planted in the fi eld in 2013. All agronomic traits were observed until harvest. In the progeny line of VGI, the most of plants exhibited taller plant height comparing to Nipponbare. Furthermore, VG-15, which is one of the segregating plants in the progeny line, exhibited a longer panicle, larger number of grain per panicle and bigger glume size comparing to Nipponbare.
In the F2 population of the cross between VG-15 and Nipponbare, frequency distribution of NFS, PAL, NPB, NSB HSW, DGW, SAG, GLH, and GWH exhibited continuous distribution (Fig. 1). Mean values of parental line of Niponnbare and the progeny line of VG-15 were also shown in Fig. 1. In PAL, HSW and DGW, there was no difference between parents. The mean values of NFS and NBP in Nipponbare were larger than those in the progeny line of VG-15. In NSB, SAG, GLH and WH, VG-15 exhibited larger values than Nipponbare. In NFS, PAL, NPB, NSB, HSW, GLH, SAG and GLH, apparent transgressive segregation was observed. In DGW, the most of the plants showed larger value comparing to their parents. In SAG and GWH, the most F2 plants were distributed between parental lines.
QTL analysis was conducted using the F2 population. Genetic map was constructed by Mapmarker ver.3.0 with 9 SSR markers and 48 mPing-SCAR markers. The total map-length was 1780.5cM including twelve linkage group with the average inter-marker distance of 31.2cM (Fig.2). As the results of the interval mapping, nine QTLs were detected in seven traits. There were 3 QTLs (qHSW1, qHSW2, qHSW3) for 100-seed-weight on Chr. 1, Chr. 3, Chr. 5, respectively. A single QTL for air-dried weight of above ground part is located on Chr. 7 (qDGW). A QTL for number of fi lled spikelet per panicle (qNFS) and for number of secondary branches per panicle are located on Chr.2 (qNSB). A QTL for surface area size of glume (qSAG) and for glume length is located are located on Chr. 8 (qGLH). A QTL for glume width is located on Chr.5 (qGWH) (Table. 1).
qSAG and qGLH are located at the same position on Chr.8. In this case, a genetic factor contributes the surface area size of glume through the the glume length. qHSW3 and qGWH were found on Chr.2. Because both QTLs are closely linked to marker XC5_18, this QTL control the 100-seed-wieght through the grain width. Both qNFS and qNSB were found on the Chr.2. However, they are located at totally different position. Furthermore, in this cross combination, there are no correlation between number of fi lled spiklets per panicle and number of secondary branches per panicle.
0 5 10 15 20 25 30 35 No. of line 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 40 0 2 4 6 8 10 12 14 16 18 20 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 35 40 16 17 18 19 20 >20 ) 0 5 10 15 20 25 3.15 3.2 3.25 3.3 3.35 3.4 >3.4 VGI NB VGI NB VGI NB VGI NB VGINB VGINB NB NB NB VGI VGI VGI 0 5 10 15 20 25 30 6.7 6.9 7.1 7.3 7.5 7.7 7.9 8.1
a. No. of filled spiklets per panicle b. Panicle length (cm) c. No. of primary branches per panicle
d. No. of secondary branches per panicle e. 100-seed-weight (g) f. Air-dried above ground part weight (g)
g. Surface area size of glume (mm2) h. Glume length (mm) i. Glume width (mm)
Fig1. Frequency distributions of nine agronomic traits segregating in the F2 population of the cross between Nipponbare and VG-15
Discussion
Rice is one of the most important and stable cereal crops and is widely cultivated in the world. In recent decades, more attention has been paid to increasing the rice yield. This is considered to be one of the most effective ways to improve the crop productivity. Rice yield is determined by many components, such as the number of productive tillers, panicle length, the number of filled spikelets per panicle, the number of primary branches per panicle and secondary branches per panicle, 1000-seed-weight, grain size and so on.
Under the field condition, VG-15 showed larger value than Nipponbare in glume size. However, the biomass and yield did not show any significant difference between Nipponbare and VG-15. In F2 population between Nipponbare and VG-15, observed nine traits showed a continuous distribution. In addition, plant height, panicle length, the number of filled spikelets per panicle, plant dry weight and 100-seed-weight showed transgressive segregation.
Today, several QTLs related to grain size have been identifi ed such as GS3 for glume length on Chr.3 (Fan et al., 2006), GS5 for glume width on Chr.5 (Li et al., 2011), and SLG7 for glume shape on Chr.7 (Zhou et al., 2015). In this study, we detected a new QTL (qGLH) for glume length on Chr.8. However, qDWH for glume width on Chr.5 seems to be the same as GS5. So qGLH supposed to be a novel QTL. We also detected a QTL (qGDW) related to biomass on Chr. 7. According to Gramene QTL Database (http://archive.gramene.org/qtl/), there have been already reported about a QTL for air-dried weight above ground part qDGW in the same position on Chr.7. In Chr. 2, it is reported that a QTL for the number of secondary branches per panicle was detected in position 111.2-114.2cM. We also detected a QTL (qNSB) on Chr. 2, however it is located nearby MK2_2, which is located at 38.52cM on Chr. 2. Thus, qNSB supposed to be a novel QTL.
In this study, we could successfully conduct a QTL analysis using mPing-SCAR markers. We found two new QTLs for glume length qGLH and for number of secondary branch per
panicle qNSB. These traits are closely related to a typical VGI. Further researches should be done to clarify the relationship between the mPing transposition and modification of corresponding genes.
References
Fan C.C., Y.Z. Xing, H. L. Mao, T. T. Lu, B. Han, C. G. Xu, X. H. Li and Q. F. Zhang (2006) GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein. Theor Appl Genet 112: 1164.
Horibata, A. and H. Yamagata (2000) Reversion Mutability of a Slender-glume Gene slg and its Infl uence on the Activity of a Mutagenic Factor in Rice Breeding Research 2 :125-132 Li Y. B., C. C. Fan, Y. Z. Xing, Y. H., Jiang, L. J. Luo, L. Sun, D.
Shao, C. J. Xu, X. H. Li, J. H. Xiao, Y. Q. He and Q. F. Zhang (2011) Natural variation in GS5 plays an important role in regulating grain size and yield in rice. Nature Genetics 43, 1266–1269
Monden Y.. K. Naito, Y. Okumoto, H. Saito, N. Oki, T. Tsukiyama, O. Ideta, T. Nakazaki, S.R. Wessler and T. Tanisaka (2009) High Potential of a Transposon mPing as a Marker System in japonica × japonica Cross in Rice. DNA Res 16 (2): 131-140.
Nakazaki T., Y. Okumoto, A. Horibata, S. Yamahira, M. Teraishi, H. Nshida, H. Inoue and T. Tanisaka (2003) Mobilization of a transposon in the rice genome. Nature vol.421 170-172 Naito, K., E. Cho, G. Yang, M. A. Campbell, K. Yano, Y.
Okumoto, T. Tanisaka, S. R. Wessler (2006) Dramatic amplification of a rice transposable element during recent domestication. PNAS 103:17620-17625.
Naito, K., F. Zhang, T. Tsukiyama, H. Saito, C. N. Hancock, A.O. Richardson, Y. Okumoto, T. Tanisaka, S. R. Wessler (2009) Unexpected consequences of a sudden and massive transposon amplifi cation on rice gene expression. Nature 461: 1130-1135.
Yamagata, H. and K. Syakudo (1968) A mutable gene system
induced by radiation in rice. Proc. XII Internat. Cong. Genet. 1: 106.
Zhou Y., J. Miao, H. Y. Gu, X. R. Peng, M. Leburu, F. H. Yuan, H. W. Gu, Y. Gao, Y. J. Tao, J. Y. Zhu, Z. Y. Gong, C. D. Yi,
イネ強勢突然変異系統 VGI における強勢形質の QTL 解析
許 冲
1）・築山拓司
2）・寺石政義
1）・谷坂隆俊
3）・奥本 裕
1） 1） 京都大学大学院農学研究科（〒 606-8502 京都市左京区北白川追分町） 2） 近畿大学農学部（〒 631-8505 奈良県奈良市中町 3327-204） 2） 吉備国際大学地域創成農学部（〒 656-0484 兵庫県南あわじ市志知佐礼尾 370-1）要旨：本研究で対象とした miniature-Ping （mPing）はイネの非自律性転移因子 MITE の一種であり，品種銀坊主において 1,000 コ ピー数以上存在し，自然条件下でも活発に転移している．銀坊主種子へのγ線照射より誘発された細粒突然変異系統 IM294 では， ユビキチン様タンパク質をコードする Rurm1 遺伝子が mPing 挿入により機能を喪失しており，細粒形質，低発芽率，低草丈，低 稔性などさまざまな生育異常を示す．さらに，IM294 の自殖後代には Rurm1 からの mPing の正確な切り出しによって粒形が正常 粒に復帰する個体が分離する．復帰個体の中には，原品種銀坊主よりも旺盛に生育する強勢個体 ( 以下 VGI：a vigorously growing plant in IM294 とする ) が含まれる．VGI では mPing 転移頻度が顕著に上昇し，新たに多数の mPing 挿入が観察される．このこと から，mPing の新規挿入と VGI との関連が推察されるが，復帰に伴って強勢個体が分離出現する分子機構は未解明である．本研 究では，VGI 個体の後代と日本晴との交雑によって得られた F2 集団を用いて，VGI に関連する特性に関する QTL 解析を行い， 強勢形質を制御する遺伝因子が存在する染色体領域の同定を試みた． キーワード：イネ，トランスポゾン，mPing，連鎖地図，QTL 解析 作物研究 63 号（2018） 連絡責任者：奥本 裕（okumoto3@kais.kyoto-u.ac.jp） M. H. Gu, Z. F. Yang and G. H. Liang (2015) Natural Variations in SLG7 Regulate Grain Shape in Rice. Genetics vol. 201 no. 4 1591-1599;
