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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (04): 542-553.doi: 10.3724/SP.J.1006.2018.00542

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

Genome-wide Association Analysis of Some Phytotoxicity Related Traits at Seedling Stage in Rapeseed under Glufosinate Stress

Dong-Liang CHEN1,2,**, Cui CUI1,**, Yi-Ying REN1, Qian WANG1, Jia-Na LI1, Zhang-Lin TANG1, Qing-Yuan ZHOU1,*()   

  1. 1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
    2 Oil Research Institute in Guizhou Academy of Agricultural Sciences, Guiyan 550006, Guizhou, China
  • Received:2016-12-15 Accepted:2017-11-21 Online:2018-01-19 Published:2017-12-20
  • Contact: Dong-Liang CHEN,Cui CUI,Qing-Yuan ZHOU E-mail:E-mail: zhouqy2005@163.com
  • Supported by:
    This study was supported by the National Key Technology Support Program of China (2013BAD01B03-12), the China Agriculture Research System (CARS-12), the Science and Technology Committee of Chongqing (cstc2016shmszx0756), and the Doctoral Start-up Fund of Southwestern University (swu113064).

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Abstract:

With planting rape in large area and weeds growing seriously in the field, glufosinate has been used in controlling field weeds tentatively. However glufosinate also produced phytotoxicity to the main cultivated rapeseed more or less, affecting the normal physiological and morphological activities, even yield. In this study, glufosinate of 200 mg L-1 was used at stress concentration 506 representative Brassica napus varieties (lines) were genotyped using the Brassica 60k Illumina Infinium SNP array by genome-wide association studies of herbicide resistance coefficient of dry weight per unit leaf area (CLW), herbicide resistance coefficient of chlorophyll content (CCC) and comprehensive phytotoxicity coefficient (CPC). A total of 16 SNP sites related to the killing mechanism of glufosinate were detected. Six SNP sites associated with CLW were detected, with phenotypic contribution rate ranging from 6.53% to 10.04%, and 22 SNP sites associated with CCC, with the phenotypic from 4.97% to 6.20%, 98 SNP sites associated with CPC over A subgenome and C subgenome, with the rate range from 3.25% to 18.66%. After analysising the genes in the linkage disequilibrium (LD) interval of significant SNP, we found 18 glufosinate tolerance related candidate genes. Eleven candidate genes were found in the LD interval of SNPs related to CCC, among them nine were involved in acyltransferase activity, two were involved in acetyl-CoA transferase activity, seven candidate genes were found in the LD interval of SNPs related to CPC, one of them was associated with glutamine transferase activity, participating in glutamine metabolism, and other six genes were involved in acetyl CoA transferase activity. The physiological and biochemical process regulated by these genes related to the changes of leaf dry weight, chlorophyll content and the killing mechanism of glufosinate. Mining association sites and candidate genes provides a reference for the research of gene regulation mechanism in the physiological and morphological process under glufosinate stress.

Key words: Brassica napus L., seedling stage, glufosinate, genome-wide association studies

Fig. 1

Physiological and morphological differences under glufosinate in different concentration on seedling"

Fig. 2

Phenotype frequency distribution of CLW, CCC, and CPC CLW: coefficient of dry weight per unit leaf area; CCC: coefficient of chlorophyll content; CPC: comprehensive phytotoxicity coefficient."

Table 1

Phenotypic statistics of related traits"

性状
Trait		 均值±标准差
Mean±SD		 变幅范围
Range (%)		 变异系数
CV (%)
单位叶面积干重系数CLW 37.30±6.210** 21.1-68.8 16.649
叶绿素含量系数CCC 80.24±8.323** 54.2-100.0 10.373
综合药害指数CPC 58.93±14.144** 0.490-95.000 24.001

Table 2

Correlation analysis of CLW, CCC, and CPC"

测定指标Index 单位叶面积干重系数CLW 综合药害指数CPC 叶绿素含量系数CCC
单位叶面积干重系数CLW 1.000
综合药害指数CPC -0.134** 1.000
叶绿素含量系数CCC 0.185** -0.314** 1.000

Fig. 3

Estimation of the ∆K value in the natural population"

Fig. 4

Distribution of pairwise kinship of different B. napus lines in natural population"

Fig. 5

LD decay of different chromosomes for A and C subgenomes in B. napus"

Fig. 6

Manhattan plots of tolerance traits of Brassica napus on seedling growth by Q+K model a1, a2: Manhattan plot and QQ plot of CLW; b1, b2: Manhattan plot and QQ plot of CCC; c1,c2: Manhattan plot and QQ plot of CPC."

Table 3

Associations of SNP sites"

性状
Trait		 位点名称
SNP		 染色体
Chr.		 P值
P-value		 贡献率
R2 (%)		 位点名称
SNP		 染色体
Chr.		 P 值
P-value		 贡献率
R2 (%)
单位叶面积干重系数
CLW		 Bn-A01-p1704123 A01 2.36×10-6 5.362 Bn-A07-p593034 A07 2.45×10-5 6.526
Bn-A01-p4549945 A01 3.86×10-6 7.723 Bn-C01-p38105098 C01 1.35×10-6 8.348
Bn-A01-p5849310 A01 1.94×10-5 6.766 Bn-C06-p17529141 C06 2.90×10-5 6.526
叶绿素含量系数
CCC		 Bn-A01-p21360797 A01 4.25×10-6 5.418 Bn-A06-p20421942 A06 1.83×10-5 5.179
Bn-A02-p4940608 A02 1.35×10-5 5.313 Bn-A07-p3477622 A07 3.81×10-6 5.883
Bn-A03-p21755979 A03 9.06×10-6 5.493 Bn-A08-p9184409 A08 1.90×10-6 6.195
Bn-A03-p4171667 A03 2.37×10-5 5.063 Bn-A08-p984273 A08 5.34×10-6 5.730
Bn-A04-p16495266 A04 1.06×10-5 5.423 Bn-A09-p16498800 A09 2.14×10-6 6.142
Bn-A04-p7873782 A04 1.64×10-5 5.228 Bn-A09-p15859343 A09 5.27×10-6 5.737
Bn-A05-p10742077 A05 1.52×10-5 5.261 Bn-A09-p4171667 A09 1.77×10-5 5.193
Bn-A06-p20575568 A06 4.27×10-6 5.831 Bn-C01-p25343230 C01 9.59×10-6 5.468
Bn-A06-p20526477 A06 4.78×10-6 5.780 Bn-C07-p41693853 C07 1.41×10-5 5.294
Bn-A06-p20546803 A06 4.83×10-6 5.775 Bn-C08-p32299051 C08 2.55×10-5 5.030
Bn-A06-p20602779 A06 1.79×10-5 5.187 Bn-C09-p40117533 C09 2.89×10-5 4.974
综合药
害指数
CPC		 Bn-A01-p986711 A01 2.30×10-5 9.031 Bn-A09-p6931495 A09 1.41×10-7 7.465
Bn-A01-p994322 A01 3.35×10-5 4.360 Bn-A10-p12019156 A10 2.21×10-6 7.783
Bn-A02-p6931495 A02 2.75×10-5 12.645 Bn-A10-p19586200 A10 6.82×10-6 4.996
Bn-A02-p6901239 A02 1.11×10-6 5.712 Bn-A10-p10124750 A10 2.88×10-6 5.236
Bn-A02-p6976201 A02 2.36×10-7 18.659 Bn-A10-p14852876 A10 1.28×10-7 5.632
Bn-A02-p6371001 A02 8.58×10-6 4.896 Bn-A10-p15935269 A10 3.45×10-5 7.852
Bn-A02-p8005692 A02 1.39×10-9 10.455 Bn-A10-p15524190 A10 1.86×10-5 4.891
Bn-A02-p8945120 A02 4.27×10-9 7.960 Bn-C01-p9201350 C01 1.84×10-7 8.123
Bn-A02-p145473 A02 1.75×10-5 4.513 Bn-C01-p9611053 C01 9.24×10-6 4.866
Bn-A02-p121255 A02 1.01×10-5 4.838 Bn-C01-p9965323 C01 2.01×10-6 5.624
Bn-A02-p9652140 A02 4.23×10-6 4.020 Bn-C01-p9604036 C01 5.12×10-6 5.108
Bn-A02-p9570320 A02 3.69×10-5 4.181 Bn-C01-p32514001 C01 2.32×10-10 8.583
Bn-A02-p965241 A02 2.06×10-5 5.342 Bn-C01-p33876059 C01 9.97×10-7 5.798
Bn-A02-p974395 A02 1.02×10-6 5.745 Bn-C02-p41927269 C02 8.48×10-5 4.122
Bn-A02-p3483764 A02 1.87×10-5 4.337 Bn-C02-p41639953 C02 9.25×10-9 8.390
Bn-A02-p3546981 A02 3.69×10-5 4.321 Bn-C03-p55465412 C03 5.56×10-5 4.391
Bn-A02-p6627159 A02 5.02×10-7 12.607 Bn-C03-p57991205 C03 4.04×10-6 5.938
Bn-A03-p9845120 A03 1.96×10-8 16.363 Bn-C03-p57106187 C03 2.87×10-9 8.559
Bn-A03-p9468697 A03 1.39×10-5 4.706 Bn-C03-p57326280 C03 1.52×10-8 7.918
Bn-A03-p9999846 A03 4.75×10-7 4.842 Bn-C04-p6292605 C04 2.30×10-6 5.720
Bn-A03-p9451200 A03 2.96×10-5 3.958 Bn-C04-p6200125 C04 5.97×10-6 5.387
Bn-A03-p21867237 A03 8.88×10-6 4.472 Bn-C04-p6312067 C04 5.11×10-6 5.460
Bn-A04-p16524169 A04 2.94×10-5 4.735 Bn-C04-p621851 C04 5.98×10-6 5.426
Bn-A04-p16714914 A04 3.05×10-8 7.548 Bn-C04-p6313229 C04 5.51×10-5 4.388
Bn-A04-p16732201 A04 4.77×10-6 5.505 Bn-C04-p6985469 C04 4.38×10-6 5.512
Bn-A04-p1983502 A04 3.09×10-8 7.544 Bn-C04-p20152051 C04 3.48×10-5 4.623
Bn-A04-p1945120 A04 1.24×10-6 6.115 Bn-C04-p9140899 C04 2.70×10-5 4.689
Bn-A04-p9002613 A04 1.03×10-8 8.011 Bn-C05-p7747904 C05 8.26×10-7 6.148
性状
Trait		 位点名称
SNP		 染色体
Chr.		 P值
P-value		 贡献率
R2 (%)		 位点名称
SNP		 染色体
Chr.		 P 值
P-value		 贡献率
R2 (%)
综合药
害指数
CPC		 Bn-A05-p19765662 A05 2.77×10-5 4.675 Bn-C05-p7765985 C05 1.89×10-10 9.835
Bn-A05-p19693221 A05 7.18×10-5 4.364 Bn-C05-p3235412 C05 8.74×10-5 4.207
Bn-A06-p15660438 A06 3.14×10-5 4.626 Bn-C05-p3698257 C05 3.25×10-5 4.656
Bn-A06-p14512365 A06 5.24×10-6 5.467 Bn-C05-p36474843 C05 5.81×10-5 4.140
Bn-A06-p15261863 A06 2.47×10-5 4.741 Bn-C05-p21774776 C05 2.47×10-6 18.623
Bn-A06-p15365248 A06 4.82×10-7 6.452 Bn-C05-p2564789 C05 6.21×10-5 4.119
Bn-A07-p23001203 A07 1.00×10-8 8.026 Bn-C06-p24235721 C06 7.16×10-5 4.061
Bn-A07-p23418663 A07 2.23×10-5 4.812 Bn-C06-p32728327 C06 4.25×10-5 4.069
Bn-A07-p4480260 A07 2.25×10-6 4.260 Bn-C07-p22140411 C07 5.55×10-7 10.771
Bn-A07-p20935217 A07 2.44×10-6 5.263 Bn-C07-p12256348 C07 2.45×10-5 4.487
Bn-A07-p23340612 A07 7.48×10-5 4.260 Bn-C07-p22118892 C07 2.87×10-7 6.745
Bn-A07-p28542122 A07 2.77×10-5 4.865 Bn-C07-p20147854 C07 1.33×10-5 4.756
Bn-A08-p10182282 A08 1.81×10-5 4.869 Bn-C08-p13638153 C08 2.39×10-9 5.369
Bn-A08-p13250140 A08 7.39×10-5 4.362 Bn-C08-p12356824 C08 8.26×10-6 4.912
Bn-A08-p7652410 A08 2.12×10-7 6.728 Bn-C08-p19139658 C08 3.25×10-8 6.952
Bn-A08-p7505807 A08 7.75×10-8 7.289 Bn-C08-p19568928 C08 1.37×10-6 5.627
Bn-A09-p2201470 A09 1.12×10-7 7.154 Bn-C09-p40117533 C09 4.16×10-7 6.356
Bn-A09-p2176198 A09 9.27×10-6 5.182 Bn-C09-p46985366 C09 6.42×10-5 5.632
Bn-A09-p16521452 A09 7.81×10-6 5.211 Bn-C09-p12891995 C09 2.99×10-5 3.265
Bn-A09-p15882405 A09 2.91×10-5 4.721 Bn-C09-p12356923 C09 3.54×10-5 4.631
Bn-A09-p6598420 A09 1.90×10-5 5.304 Bn-C09-p13003243 C09 1.67×10-5 4.365

Table 4

A summary of candidate genes associated with herbicide tolerance related traits"

性状
Trait		 位点
Site		 染色体
Chr.		 物理位置
Physical interval
(bp)		 拟南芥基因
Arabidopisis genes		 候选基因
Candidate genes
in the LD interval
单位叶面积干重系数 Bn-A01-p1704123 A01 1633033-1633658 AT4G33467 BnaA01g03470D
CWL Bn-A01-p4549945 A01 4548199-4552433 AT4G18490 BnaA01g09330D
叶绿素含量系数 Bn-A01-p21360797 A01 21153560-21155842 AT3G11290 BnaA01g31070D
CCC Bn-A02-p4940608 A02 4607681-4610486 AT5G55050 BnaA02g09320D
Bn-A02-p4940608 A02 459901-4602406 AT5G55070 BnaA02g09300D
Bn-A02-p4940608 A02 5204578-5206797 AT5G53470 ACBP1
Bn-A03-p21755979 A03 21582364-21584595 AT4G17250 BnaA03g42990D
Bn-A03-p21755979 A03 21958105-21965340 AT2G07680 ATABCC13
Bn-A03-p21755979 A03 21953245-21955187 AT4G18070 BnaA03g43580D
Bn-A03-p4171667 A03 4122660-4124952 AT5G59960 BnaA03g09150D
Bn-A04-p16495266 A04 16368845-16371398 AT2G37500 BnaA04g21470D
Bn-A04-p16495266 A04 16372246-16376604 AT2G37520 BnaA04g21480D
Bn-A04-p16495266 A04 16498173-16499643 AT2G20625 BnaA04g21710D
Bn-A04-p16495266 A04 16508877-16511523 AT2G37960 BnaA04g21730D
Bn-A04-p7873782 A04 7808158-7809504 AT5G67150 BnaA04g08790D
Bn-A05-p10742077 A05 10774154-10779061 LACS2 BnaA05g16170D
Bn-A07-p3477622 A07 3235720-3238222 AT2G16270 BnaA07g03580D
Bn-A09-p16498800 A09 16425283-16427170 AT1G33270 BnaA09g23800D
Bn-C07-p41693853 C07 41701416-41703232 AT2G25150 BnaC07g42100D
Bn-C07-p41693853 C07 41797435-41799523 AT4G30060 BnaC07g42290D
综合药害指数 Bn-A02-p6901239 A02 6771193-6774102 AT1G66860 BnaA02g12600D
CPC Bn-A04-p1983502 A04 2101543-2102589 AT2G23060 BnaA04g03220D
Bn-A04-p1983502 A04 2110543-2111592 AT3G57100 BnaA04g03240D
Bn-A07-p4480260 A07 4562018-4563915 AT2G06025 BnaA07g04340D
Bn-A08-p10182282 A08 10187701-10189221 AT4G34520 BnaA08g11130D
Bn-A08-p13250140 A08 13047661-13049407 AT4G37580 BnaA08g15700D
Bn-C07-p22140411 C07 22179972-22180303 AT5G67160 EPS1
Bn-C07-p22140411 C07 22181878-22183299 AT5G67150 BnaC07g16220D
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