Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (18): 3435-3444.doi: 10.3864/j.issn.0578-1752.2018.18.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Fine Mapping of Grain Length Associated QTL, qGL12 in Wild Rice (Oryza sativa L.) Using a Chromosome Segment Substitution Line

YingBin DING1,2(), LiZhen ZHANG1,2, Rui XU2, YanYan WANG2, XiaoMing ZHENG2, LiFang ZHANG2, YunLian CHENG2, Fan WU2, QingWen YANG2(), WeiHua QIAO2(), JinHao LAN1()   

  1. 1College of Agriculture of Qingdao Agricultural University, Qingdao 266109, Shandong
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2018-04-11 Accepted:2018-05-26 Online:2018-09-16 Published:2018-09-16

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Abstract:

【Objective】Fine mapping of a grain length QTL from wild rice using a chromosome segment substitution line and secondary population, exploring new genes affecting grain length and providing genetic materials and gene resources for rice breeding.【Method】Our laboratory had preliminary mapped a grain length related QTL, qGL12 using the chromosome segment substitution line (CSSL) population. On this basis, we choose one CSSL, CSSL141 which harbors qGL12 substitution segment and has significant difference of grain length compared with receptor parent 9311. CSSL141 was backcrossed with 9311. The secondary separation population was constructed for fine mapping of qGL12. Detection of length of glumes cells was performed by scanning electron microscope.【Result】CSSL141 has 4 introgressive segments from wild rice, its grain length, grain width and grain weight was significantly higher than 9311 under multi-conditions. Using CSSL141/9311 F2 population, qGL12 was localized to the interval between RM5479 and RM28621 on chromosome 12. qGL12 affected grain length, grain width and grain weight, and the phenotypic variation explained of grain length was 44.61%. Seven polymorphic molecular marker primers in the location interval were designed, F3 plants which had heterozygous genotype in the target interval were investigated. qGL12 was mapped to a 50 kb region between RM5479 and RM28586. Four polymorphic molecular marker primers were designed in this interval, individuals which harbor heterozygous genotype in this interval were selected for next generation F4. Eventually qGL12 was narrowed to a 15.69 kb region between DYB9.1 and RM28586 on chromosome 12. There are 3 genes in the interval. Two candidate genes, Os12g39650 and Os12g39660, which encode a tubulin protein and a calcium-transporting ATPase respectively, with variations in their coding regions. The results of electron microscopes scanning of glumes cell showed that the length and width of 9311’s glumes cell was smaller than CSSL141, indicated that qGL12 regulate grain size of rice by controlling the size of glumes cells.【Conclusion】The wild rice grain length associated QTL qGL12 was fine mapped to a 15.69 kb region on chromosome12. qGL12 control grain length via regulate the size of glumes cells. Two candidate genes, Os12g39650 and Os12g39660, were found and would be used for further research.

Key words: rice, chromosome segment substitution line, grain size, QTL mapping

Table 1

The primers information of fine mapping"

标记名称
Marker name		 标记类型
Marker type		 片段大小
Fragment size (bp)		 正向引物
Forward primer (5′-3′)		 反向引物
Reverse primer (5′-3′)
DYB1.1 InDel 272 GGAAGGAATCCGCTTTACA GGGAATATTGCAGGGTTGA
DYB6.1 InDel 259 GCCAGAATCTCCACAGATCC GTTTGGTGGGAAGGTCATCT
DYB7.1 InDel 283 GAATCTTTGCAGCTTGGGAA AGGTGGTTTGGGCATAGTGA
DYB9.1 InDel 246 GTTTCAGTTTAGTTTTGGATTGCT TACTCTCTTATTATCAGCCTA

Table 2

Detection of qGL12 under different environments"

位点
QTL		 标记
Marker		 染色体
Chr.		 环境
Environment		 LOD
Threshod		 LOD 贡献率
PVE (%)		 加性效应
Add
2014年南京Nanjing, 2014 3.35 6.40 19.87 0.34
2015年北京Beijing, 2015 3.14 3.24 7.14 0.17
qGL12 RM28621 12 2015年南京Nanjing, 2015 3.41 9.70 24.29 0.32
2015年三亚Sanya, 2015 3.17 5.94 14.85 0.27

Fig. 1

The distribution of substitution segments of CSSL141"

Fig. 2

Comparison of kernel traits between 9311and CSSL141 a: The seeds of 9311 and CSSL141; b: 10 Comparison of grain length; c:Comparison of grain traits under multiple environments. **significantly different at P<0.001"

Fig. 3

Frequency distribution diagram of grain length, grain width and 1000 grain weight in CSSL141/9311 F2 population"

Table 3

Mapping of QTLs associated with grain traits using CSSL141/9311 F2 population"

群体
Population		 性状
Trait		 染色体
Chr.		 标记区间Intervals 南京 Nanjing
左标记
Left marker		 右标记
Right marker		 LOD 贡献率
PVE (%)		 加性
Add		 上位性
EPI
粒长Grain length 12 RM5479 RM28621 16.78 44.61 0.20 0.0154
CSSL141 粒宽Grain width 12 RM5479 RM28621 3.60 11.30 0.02 0.0081
千粒重Thousand grain weight 12 RM5479 RM28621 7.35 24.08 0.81 0.3998

Fig. 4

Fine mapping of qGL12"

Fig. 5

The genotype and phenotype of recombinants in the interval of RM5338-RM28642 S: Short Grain; L: Long grain; White region: The genotype of 9311; Black region: The genotype of wild rice; Grey region: Heterozygous genotype. The same as below"

Fig. 6

The genotype and phenotype of recombinants in the interval of RM5479-RM28586"

Fig. 7

Comparison of length of lemma cell a: CSSL141; b: 9311; c: Individual has wild rice genotype in the qGL12 interval; d: Individual has 9311 genotype in the qGL12 interval"

Table 4

Candidate genes of qGL12"

基因名称
Locus name		 基因注释
Gene product name
LOC_Os12g39650 微管蛋白Tubulin, putative, expressed
LOC_Os12g39660 质膜钙转运ATP酶Calcium-transporting ATPase, plasma membrane-type, putative, expressed
LOC_Os12g39670 表达蛋白Expressed protein
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