近年來隨著定序技術的進步，分子標誌密度提高且價格相對低廉，可在開發 分子標誌同時完成基因型分析，建立更為完整之連鎖圖譜。從 2005 年完成水稻 全基因體定序 (IRGSP 2005) 以來，相關資料庫的建立與更新加速了水稻相關研 究的進展，不論是特定基因功能研究、基因定位等都有相當大的幫助。本研究利 用兩組野生稻導入系統，以 RAD-seq 進行高通量基因型分型，定位野生稻插入 片段之位置，相較於先前的 SSR，在圖譜中分子標誌數量及密度皆有明顯改善，
與其對基本農藝性狀、產量相關性狀、穗與穀粒性狀及抗病表現相關之基因座，
能得到更精確之染色體區間供後續研究與利用。
本次研究結果仍有許多方面需要改進，如 (1) 材料取得與紀錄更加詳細，
避免發生如親本葉片樣本錯錯之結果。AC 族群基因型太過相似，多個樣品於分
析時被排除而降低效率，可擴大收集 BC2F1後代，以增加帶有野生稻插入片段之
機率；(2) 樣品定序資源分配不均，造成多個樣品因讀序不足而排除；(3) 定序 結果未能最有效之運用，讀序大量集中於栽培稻之基因體，需針對野生稻選用不 同限制酶進行酶切，以減少因野生稻片段讀序深度不足，無法用於分析當中而遭 到剔除之結果；(4) 分析方法之選擇，對於本次使用之四種方法皆有其特有或共 同之 SNP 存在，如何更有效率地運用資源可做進一步調整，減少因使用不同方 法而造成的誤判，或只採用最適合研究目標族群之單一方法進行後續分析。本研 究結果未來可提供以 O. nivara 及 O. officinalis 與栽培稻產生之族群較精細之 SNP，
針對族群相關性狀如抗褐飛蝨、稻熱病、白葉枯病等抗性基因，或是篩選出帶有 優良產量相關性狀基因座之個體，達到分子標誌輔助選種之效果。
未來隨著定序方法的發展與更新，一次能夠獲得更多序列資訊，加上更有效 率之分析方法與整合平台的出現，對於不同野生近緣稻種皆能建立相關之參考序 列，降低育種學家們分析之難度，生物資訊將大幅提升育種效率與相關研究之進 行，加速水稻相關遺傳育種研究與利用。
表一、農藝性狀調查項目與方法
Table 1. Agronomic traits phenotyping and methods
性狀 縮寫 單位 調查法 AA AC
09-1: 1st cropping season in 2009; 09-2: 2nd cropping season in 2009; 15-1: 1st cropping season in 2015; 15-2: 2nd cropping season in 2015.
表一、農藝性狀調查項目與方法 (續)
Table 1. Agronomic traits phenotyping and methods (Continued)
性狀 縮寫 單位 調查法 AA AC
09-1 09-2 15-1 15-2 穗長
(Panicle length) PL cm 測量每一株中各穗由穗頸至穗尖的長度，取樣 3 株取平均值。 V V V
穗重
(Panicle weight) PW g 測量單株所有穗數的總重量，除以穗數。 V V V
穗著粒密度
(Panicle density) PD grain/cm 測量每穗之穎花數除以穗的長度。 V V V
劍葉長
(Flag leaf length) FLL cm 抽穗後每系統測量 5 株取平均。 V V
劍葉寬
(Flag leaf width) FLW cm 抽穗後每系統測量 5 株取平均。 V V
劍葉葉面積
(Flag leaf area) FLA cm2 抽穗後每系統測量 5 株取平均，葉長×葉寬×0.75 求得劍葉葉面積。 V V 榖粒長
(Grain length) GL mm 稻穀烘乾後，每系統取 10 粒稻穀以投影方式測量並求其平均。 V V
榖粒寬
(Grain width) GW mm 稻穀烘乾後，每系統取 10 粒稻穀以投影方式測量並求其平均。 V V
榖粒型
(Grain shape) GS - 以粒長除以粒寬求得粒型。 V V
09-1: 1st cropping season in 2009; 09-2: 2nd cropping season in 2009; 15-1: 1st cropping season in 2015; 15-2: 2nd cropping season in 2015.
表一、農藝性狀調查項目與方法 (續)
Table 1. Agronomic traits phenotyping and methods (Continued)
性狀 縮寫 調查法 AA AC
葉綠素含量
SPAD1 以葉綠素計 (Minolta chlorophyll meter, SPAD 502) 於最高分蘖期測量 5 株各
一成熟葉片之 SPAD 值。 09-1、09-2
SPAD2 以葉綠素計於抽穗日數測量 5 株各一成熟葉片之 SPAD 值。 09-1、09-2
SPAD3 以葉綠素計於黃熟期測量 5 株各一成熟葉片之 SPAD 值。 09-1、09-2
SPAD4 以抽穗日數 SPAD 值減去成熟期 SPAD 值，代表該系統於成熟時葉綠素含量
的變化程度。 09-1、09-2
褐飛蝨抗性檢定
(Brown plant hopper) BPH
待感蟲對照品種枯萎時，再按其被害情況分級紀錄。每系統重複 3 次，求平 均為該系統之抗性等級，調查級數與抗性反應之對應如下：0－5 為抗 (R)；
介於 5－7 之間為中抗 (MR)；大於 7 以上為感 (S)。
08-2、09-1 14-1、14-2 15-1、15-2
09-1: 1st cropping season in 2009; 09-2: 2nd cropping season in 2009; 15-1: 1st cropping season in 2015; 15-2: 2nd cropping season in 2015.
表一、農藝性狀調查項目與方法 (續)
Table 1. Agronomic traits phenotyping and methods (Continued)
性狀 縮寫 調查法 AA AC
稻熱病抗性檢定
(Rice blast) BL
依據國際稻熱病圃 (International Rice Blast Nursery, IRBN) 方法，以肉眼依照調查標 準分 0－9 級記載，每品系重複 2 次，求平均為該品系之抗性等級，葉稻熱病檢定
09-1: 1st cropping season in 2009; 09-2: 2nd cropping season in 2009; 15-1: 1st cropping season in 2015; 15-2: 2nd cropping season in 2015.
表二、2015 年第一期作 AC 族群之農藝性狀彙總
Table 2. Summary of AC agronomic traits collected during 1st cropping season in 2015.
Trait
Low-level fertilizer High-level fertilizer
TNG67 AC population TNG67 AC population
Mean Mean ± SD Min Max Mean Mean ± SD min Max
GWP 32.7 20.6 ± 6.4 07.0 40.0 27.8 23.8 ± 8.1 09.1 44.3
SP 1618.0 852 ± 299 0395.0 1862.0 1697.0 1051± 421 0337.0 2507.0
FER 90.8 89.8 ± 9.3 48.6 97.4 78.4 87.3 ± 9.4 53.4 96.3
TGW 22.1 27.9 ± 3.7 18.6 36.0 20.9 26.8 ± 4.2 15.9 37.9
SPP 0123.0 89.6 ± 19.8 44.4 0130.0 0143.0 97.8 ± 25.5 42.0 0163.0
PN 13.0 9.4 ± 2.3 4.30 15.3 12.3 10.8 ± 2.8 06.0 17.0
PL 18.9 19.5 ± 1.8 15.0 23.1 19.9 20.5 ± 1.8 15.6 24.2
PW 02.6 2.3 ± 0.5 01.0 03.2 02.6 2.4 ± 0.5 01.1 03.4
PD 06.5 4.6 ± 1.0 02.3 07.1 07.2 4.8 ± 1.2 02.2 08.3
GWP, grain weight (g) per plant; SP, No. of spikelet per plant; FER, fertility (%); TGW, 1,000-grain weight (g);
SPP, No. of spikelet per panicle; PN, No. of panicle; PL, panicle length (cm); PW, panicle weight (g); PD, panicle density
表三、AC 族群病蟲害抗性表現
Table 3. Summary of AC population biotic resistance traits
Traits Year Treatment
(Fertilization)
Mean ± SD
BL 2014-1 No 3.0 ± 2.4 a
2015-2 No 5.9 ± 2.6 b
BPH 2014-1 No 5.8 ± 1.4 a
2014-2 No 4.9 ± 1.4 b 2015-1 No 5.7 ± 1.5 a 2015-2 No 6.2 ± 1.5 a BB_XF-115 (cm) 2015-2 Low 9.7 ± 3.5 a
2015-2 High 11.5 ± 4.7 b BB_XF-89b (cm) 2015-2 Low 7.6 ± 2.7 a 2015-2 High 8.3 ± 3.0 a SBPH (cm) 2015-2 Low 94.3 ± 9.1 a
2015-2 High 102 ± 11.0 b SBAve (cm) 2015-2 Low 47.6 ± 12.9 a
2015-2 High 67.5 ± 12.9 b SBPer (%) 2015-2 Low 51.6 ± 16.8 a
2015-2 High 67.0 ± 15.9 b
BL, rice blast (t-test, α= 0.05);
BPH, brown plant hopper (LSD, α= 0.05 );
BB, bacterial blight (2 race, XF-115 and XF-89b, t-test, α= 0.05);
SB, sheath blight (PH, plant height; Ave, spot size; Per, percentage; t-test,α= 0.05)
表四、各流程讀序分析
Table 4. Analysis of reads using four pipelines
Pipeline Raw reads Good reads 1 Alignment 2 Pstacks 872,452,735 781,820,779 (89.7%) 97.9%
Ustacks 872,452,735 781,820,779 (89.7%) blastn 3 GBS 873,744,923 822,878,842 (94.2%) 99.9%
GBSv2 873,744,923 675,074,549 (77.3%) 89.6%
1 Complete, identifiable reads (with barcode) from raw reads, numbers in parentheses indicate the proportion of good reads in raw reads.
2 Use Bowtie2 or BWA to align the reads on reference genome.
3 Use blastn to identify the location of polymorphic markers on the reference genome called by Ustacks.
表五、SNP 解析彙總
Table 5. Summary of SNP called using four pipelines (a) AA population
Pipeline All Polymorphic 1 Good markers 2 Pstacks 105,424 36,271 (34.4%) 3,376 (3.2%) Ustacks 42,892 18,150 (42.3%) 1,698 (4.0%)
(b) AC population
Pipeline All Polymorphic 1 Good markers 2 Pstacks 190,192 5,392 (2.8%) 190 (0.1%) Ustacks 22,351 20,541 (92.0%) 2,834 (12.7%)
GBS 49,332 34,016 (69.0%) 9,995 (20.3%) GBSv2 91,535 70,800 (77.3%) 16,607 (18.1%)
1 Polymorphic markers (MAF >0), numbers in parentheses indicate the proportion of good markers among initial all markers.
2 Call rate≧0.9, MAF≧0.1, heterozygosity≦0.05, numbers in parentheses indicate the proportion of good markers among initial all markers.
表六、AA 族群連鎖圖譜摘要 Table 6. Summary of AA linkage map
Chr No. of LG
No. of Markers
Total length (cM)
Average interval (cM)
Max interval (cM)
1 3 569 337.24 0.56 12.97
2 3 390 280.79 2.90 19.43
3 3 15 48.11 4.01 10.86
4 2 205 137.73 0.35 15.54
5 3 281 256.32 1.23 16.53
6 4 53 32.27 0.98 7.59
7 2 113 142.38 1.45 13.65
8 5 151 103.77 0.79 13.76
9 3 100 97.00 3.44 9.58
10 3 311 121.18 1.08 13.19
11 4 283 171.20 0.63 15.21
12 3 18 15.30 0.76 4.71
Overall 38 2489 1743.3 0.71 19.43
表七、AA 族群 QTL 分析結果 (株高、抽穗日數與產量相關性狀)
Table 7. QTL mapping results for plant height, days to flowering and yield related traits in AA population
3 Percentage of phenotypic variation explained by the locus
4 Additive effect of substituting a “TNG71” allele for a “852T034” allele
5 95% confidence interval, defined as the interval of peak LOD-1
DTH, days to heading; PH, plant height (cm); TN, tiller number; FER, fertility (%);
TGW, 1000-grain weight (g); SPP, No. of spikelet per panicle
表八、AA 族群 QTL 分析結果 (穗部性狀)
Table 8. QTL mapping results for panicle-related traits in AA population
Trait1 LG2 Position (cM) LOD Var (%)3 Add4 CI (cM)5
3 Percentage of phenotypic variation explained by the locus
4 Additive effect of substituting a “TNG71” allele for a “852T034” allele
5 95% confidence interval, defined as the interval of peak LOD-1 PN, No. of panicle; PL, panicle length (cm); PW, panicle weight (g);
PD, panicle density
表九、AA 族群 QTL 分析結果 (榖粒性狀)
Table 9. QTL mapping results for grain-related traits in AA population
Trait1 LG2 Position (cM) LOD Var (%)3 Add4 CI (cM)5
表九、AA 族群 QTL 分析結果 (榖粒性狀) (續)
Table 9. QTL mapping results for grain-related traits in AA population
Trait1 LG2 Position (cM) LOD Var (%)3 Add4 CI (cM)5 GS1 1.3 39.0 18.4 26.1 -0.07 34 - 41
1.3 199.6 4.5 5.1 -0.03 135 - 206 8.5 42.0 3.5 4.0 0.03 4 - 43 11.2 4.0 8.5 10.4 -0.04 0 - 29 11.4 46.0 7.4 9.0 0.04 0 - 49 GS2 1.2 59.6 3.9 3.2 0.02 55 - 62 1.3 1.4 8.5 7.3 -0.04 0 - 7 1.3 54.9 13.7 12.8 -0.05 52 - 59 7.2 61.0 6.3 5.3 0.03 54 - 84 11.2 6.1 10.6 9.5 -0.04 4 - 20 11.4 7.5 9.2 8.0 0.04 4 - 14
1 Trait: 1 means 1st cropping season, 2 means 2nd cropping season in 2009
2 Linkage group
3 Percentage of phenotypic variation explained by the locus
4 Additive effect of substituting a “TNG71” allele for a “852T034” allele
5 95% confidence interval, defined as the interval of peak LOD-1 GL, grain length (cm); GGW, grain width (cm); GS, grain shape
表十、AA 族群 QTL 分析結果 (病蟲害抗性表現)
Table 10. QTL mapping results for BPH and BL in AA population
Trait1 LG2 Position (cM) LOD Var (%)3 Add4 CI (cM)5 Peak marker BPH2 1.3 203.0 7.57 15.9 1.10 185 - 208 S1_41232475
4.1 37.0 6.83 14.2 -1.04 34 - 70 S4_13847936 10.1 46.0 5.10 10.3 -0.91 40 - 56 S10_3087335 BL1 4.1 26.9 6.99 7.3 0.47 23 - 28 S4_6691553
4.1 51.5 8.98 9.6 1.01 48 - 58 S4_14731007 BL2 1.3 85.8 3.80 5.2 0.42 68 - 102 RM265
4.1 41.0 27.11 52.7 1.33 39 - 43 S4_11593078
1 Trait: 1 means 1st cropping season, 2 means 2nd cropping season in 2009
2 Linkage group
3 Percentage of phenotypic variation explained by the locus
4 Additive effect of substituting a “TNG71” allele for a “852T034” allele
5 95% confidence interval, defined as the interval of peak LOD-1 BPH, brown plant hopper resistance
BL, rice blast resistance
表十一、AC 族群 QTL 分析結果 (產量相關性狀)
Table 11. Single marker regression for yield-related traits in AC population Trait1 Chr Pos (Mbp) LOD PV (%)2 Add3 L_SP 3 27.81 4.69 27.3 162.1
11 6.53 8.66 15.2 -101.7 H_SP 2 21.99 4.66 3.7 -287.6 3 34.57 6.84 8.1 -334.7 11 25.26 5.42 7.9 -97.2 H_FER 2 0.22 5.27 19.6 11.3 7 15.42 4.28 6.5 2.5 L_TGW 3 27.68 5.66 15.3 -1.6 4 16.64 4.02 1.6 -0.7 8 26.91 4.23 8.3 -1.1 H_TGW 3 28.52 4.15 25.9 -2.0 L_SPP 3 27.81 9.46 29.1 11.7 4 20.55 4.91 3.7 4.8 9 0.75 3.96 13.6 7.4 H_SPP 3 27.81 8.83 30.7 15.4 4 20.55 3.78 7.1 8.2
1 L: low N; H: high N
2 Percentage of phenotype variation explained by QTL
3 Additive effect of substituting a “TNG67” allele for an “O. officinalis” allele SP, No. of spikelet per plant; FER, fertility (%); TGW, 1,000-grain weight (g);
SPP, No. of spikelet perpanicle
表十二、AC 族群 QTL 分析結果 (穗部性狀)
Table 12. Single marker regression for panicle-related traits in AC population Trait1 Chr Pos (Mbp) LOD PV (%)2 Add3
2 Percentage of phenotype variation explained by QTL
3 Additive effect of substituting a “TNG67” allele for an “O. officinalis” allele PL, panicle length (cm); PW, panicle weight (g); PD, panicle density
表十三、AC 族群 QTL 分析結果 (病蟲害抗性表現)
Table 13. Single marker regression results for biotic resistance in AC population Trait1 Chr Pos (Mbp) LOD PV (%)2 Add3
2 Percentage of phenotype variation explained by QTL
3 Additive effect of substituting a “TNG67” allele for an “O. officinalis” allele BL, rice blast resistance; BPH, brown plant hopper resistance;
Ave, spot size infected by SB (cm); Per, length (%) = Ave ×100 / PH
圖一、栽培稻與野生稻 O. nivara 種間導入系統 AA 族群建立流程
Figure 1. The procedure for the establishment of AA introgression lines between Asian cultivated rice and O. nivara.
圖二、栽培稻與野生稻 O. officinalis 種間導入系統 AC 族群建立流程 Figure 2. The procedure for the establishment of AC introgression lines between Asian cultivated rice and O. officinalis.
圖三、AC 族群 2015 年一期作農藝性狀分布圖
Figure 3. Distribution of AC population agronomic traits collected during the 1st cropping season in 2015.
GWP, grain weight per plant; SP, spikelet per plant; FER, fertility L, low N; H, high N
圖三、AC 族群 2015 年一期作農藝性狀分布圖 (續)
Figure 3. Distribution of AC population agronomic traits collected during the 1st cropping season in 2015 (cont.).
TGW, 1,000-grain weight; SPP, spikelet per panicle; PN, panicles per plant;
L, low N; H, high N
圖三、AC 族群 2015 年一期作農藝性狀分布圖 (續)
Figure 3. Distribution of AC population agronomic traits collected during the 1st cropping season in 2015 (cont.).
PL, panicle length; PW, panicle weight; PD, panicle density L: low N; H: high N.
圖四、AC 族群產量相關性狀與穗、穀粒性狀之表型相關
Figure 4. Phenotypic correlation between yield-related traits and grain-related traits in AC population.
GWP, grain weight per plant; SP, spikelet per plant; FER, fertility;
TGW, 1,000-grain weight; SPP, spikelet per panicle; PN, panicles per plant;
PL, panicle length; PW, panicle weight; PD, panicle density L, low N; H, high N
圖五、AC 族群稻熱病及褐飛蝨抗性表現分布
Figure 5. Distribution of rice blast (BL) and brown plant hopper (BPH) scores in AC population.
圖六、AC 族群白葉枯病抗性表現分布
Figure 6. Distribution of bacterial blight (BB) scores in AC population.
XF115 and F89b are two physiological races of bacterial blight.
圖七、AC 族群紋枯病抗性表現分布
Figure 7. Distribution of Sheath blight (SB) in AC population.
PH, plant height; Ave, spot size infected by SB (cm);
Per, length (%) = Ave ×100 / PH
圖八、讀序數曲線圖
Figure 8. Reads distribution across AA and AC.
(a) No. of reads per sample
(b) average reads number per barcode ,each barcode has been used by 6~8 samples
0.00E+00
P1-01 P1-04 P1-06 P1-08 P1-10 P1-12 P1-15 P1-17 P1-23 P1-25 P1-27 P1-30 P1-34 P1-36 P1-38 P1-40 P1-45 P1-48 P1-50 P1-52 P1-54 P1-56 P1-58 P1-60
Reads
Barcode (b)
(a)
圖九、AA 族群之物理圖譜與遺傳圖譜之比較
Figure 9. physical map compare with genetic map (AA population)
圖十、AC 族群之物理圖譜
Figure 10. physical map in AC population
圖十一、AA 族群使用 Psatcks 及 Ustacks 獲得之 SNP 於物理圖譜之分布 Figure 11. Distribution on physical map for SNP calling using Pstacks and Ustacks Red, Pstacks pipeline; Blue, Ustacks pipeline
圖十二、AA 族群 SNP+SSR 物理圖譜與 SSR 物理圖譜比較
Figure 12. Comparison of AA population RAD-SNP+SSR map with SSR only map
圖十三、AC 族群之物理圖譜
Figure 13. Physical map in AC populations
Different color bar indicate markers from different pipeline.
Blue, Tassel 5.0 GBSv2 pipeline Green, Tassel 3.0 GBS pipeline
Red, Stacks pipeline (Pstacks + Ustacks)
圖十四、AA 及 AC 族群褐飛蝨抗性 QTL 定位之結果
Figure 14. QTL mapping of brown plant hopper resistant in AA and AC population.
BPH6 (Qiu et al. 2010), BPH12 (Yang et al. 2002), BPH15 (Yang et al. 2004), BPH17 (Sun et al. 2005), BPH20 (Rahman et al. 2009), bph21(t) (Yang et al. 2011), BPH27 (Huang et al. 2013)
圖十五、AA 及 AC 族群稻熱病抗性 QTL 定位之結果
Figure 15. QTL mapping of blast resistant in AA and AC population.
Pit (Hayashi et al. 2006), Pish (Fukuta et al. 2004), Pi(t) & Pizh (Causse et al. 1994), Pikur1 (Goto 1988), Pi17(t) (Iwata 1996), Pi21 (Fukuoka and Okuno 2001), Pi24(t)
& Pi29(t) (Sallaud et al. 2003), Pi27(t) (Zhu 2004), Pi33 (Berruyer et al. 2003), Pi36 (Liu et al. 2005), Pi37 (Chen et al. 2004), Pi39(t) (Liu et al. 2007)
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