improvement of yield and its related traits for backbone hybrid rice parent minghui 86 using...

Journal of Integrative Agriculture2013, 12(4): 561-570 April 2013

© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.doi:10.1016/S2095-3119(13)60273-7

RESEARCH ARTICLE

Improvement of Yield and Its Related Traits for Backbone Hybrid Rice ParentMinghui 86 Using Advanced Backcross Breeding Strategies

ZHANG Hong-jun1*, WANG Hui1, 2*, YE Guo-you3, QIAN Yi-liang1, 4, SHI Ying-yao4, XIA Jia-fa2, LI Ze-fu2,ZHU Ling-hua1, GAO Yong-ming1 and LI Zhi-kang1, 3

1 National Key Facility for Crop Gene Resources and Genetic Improvement, Ministry of Agriculture & National Development and ReformCommission/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China

2 Rice Research Institute, Anhui Academy of Agricultural Sciences, Anhui 230031, P.R.China3 International Rice Research Institute, DAPO Box 7777, Philippines4 College of Agriculture, Anhui Agricultural University, Anhui 230036, P.R.China

Abstract

How to overcome yield stagnation is a big challenge to rice breeders. An effective method for quickly developing newcultivars is to further improve an outstanding cultivar. In this study, three advanced backcross populations under yieldselection that consist of 123 BC2F2:4 introgression lines (ILs) were developed by crossing Minghui 86 (recurrent parent,RP) with three high-yielding varieties (donors), namely, ZDZ057, Fuhui 838, and Teqing, respectively. The progenytesting allowed the identification of 12 promising ILs that had significantly higher mean grain yields than Minghui 86 intwo environments. A total of 55 QTLs that affect grain yield and its related traits were identified, which included 50 QTLsthat were detected using the likelihood ratio test based on stepwise regression (RSTEP-LRT) method, and eight grain yieldper plant (GY) QTLs were detected using chi-squared (c2) test. Among these QTLs, five QTLs were simultaneouslydetected in different populations and 22 QTLs were detected in both environments. The beneficial donor alleles forincreased GY and its related traits were identified in 63.6% (35 out of 55) of the QTLs. These promising ILs and QTLsidentified will provide the elite breeding materials and genetic information for further improvement of the grain yield forMinghui 86 through pyramiding breeding.

Key words: rice (Qryza sativa L.), quantitative trait locus (QTL), selective introgression population, yield

Received 1 July, 2012 Accepted 17 September, 2012ZHANG Hong-jun, E-mail: [email protected]; WANG Hui, E-mail: [email protected]; Correspondence GAO Yong-ming, Tel: +86-10-82106697,Fax: +86-10-82108559, E-mail: [email protected]* These authors contributed equally to this study.

INTRODUCTION

The increase of population size and the decrease ofarable lands and investments in research caused a wide-spread concern on food security all the time (Rosegrantand Cline 2003). Therefore, high production is alwaysthe primary objective of rice breeding programsworldwide.

Backbone parents refer to breeding materials that

were widely utilized, and made great contribution tothe development of new varieties in different ecologicalregions. In the last several decades, numerous back-bone parents in different crops including rice, wheat,and corn, have been developed. As many as 156 Chi-nese rice cultivars were derived from Aizizhan owingto its dwarf genes (Wei et al. 2010). In the case ofwheat, Abbondanza, Funo, and Nanda 2419 contrib-uted to 217, 165, and 110 varieties, respectively (Ge et al.2009). These backbone parents played a crucial role in

562 ZHANG Hong-jun et al.

© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.

cultivar development by providing elite “genetic back-bone” and beneficial genes. Therefore, to further im-proving these backbone parents had been the big chal-lenges to breeders.

Minghui 86, an elite restorer line developed by theSanming Institutes of Agricultural Sciences in Fujian,China, is one of the most widely used backbone par-ents in the 1990s in China. It is characterized by highgrain yield, high restoring ability and good special com-bining ability with many male sterile lines. Its crossIIYouMing 86 was one of the first 25 super-rice variet-ies dominated by the Ministry of Agriculture of Chinain 2005. The donors ZDZ057, Fuhui 838, and Teqing,three high-yielding restorer lines, were commonly char-acterized by larger panicle with numerous grains (Yanget al. 1990; Deng et al. 1996; Wang et al. 1997) andhave been extensively used as the parents in many hy-brid rice breeding programs in China, especially fordonors Fuhui 838 and Teqing (http://www.ricedata.cn/variety/; www.ricedata.cn/variety/index.htm).

In comparison with conventional strategies for QTLidentification, the advanced backcross QTL (AB-QTL)analysis integrates variety improvement and QTL de-tection into a single process, reduces linkage drag andundesirable alleles and makes QTL effect more predict-able in breeding program (Tanksley and Nelson 1996).In this study, the modified AB-QTL method (Li et al.2005; Zhang et al. 2011) was used to introgress desir-able alleles from three modern elite restorer lines,namely, ZDZ057, Fuhui 838, and Teqing into Minghui 86for further improving its grain yield and also identifybeneficial QTLs associated with yield traits from donors.

RESULTS

Performances of introgression lines andevaluation of selection efficiency

Trait performances for three populations under twoenvironments were present in Appendix A. Positivetransgressive segregations were found for all nine traitsin all three populations under two testing environments.For 2007 Anhui (07AH) trial, the average performanceof all selected ILs from each population was higherthan RP in plant height (PH), panicle number (PN), filled

grains per panicle (FGP), filled grains rate (FGR), andgrain yield per plant (GY). In contrast, only spikeletsper panicle (SPP) had higher mean values than RP in2008 Hainnan (08HN) environment. 07AH trial hadhigher mean values for FGP and SPP but lower for FGR.Comparing with the other two populations, Minghui 86/ZDZ057 population (population I) had stable performancefor all nine traits except heading date (HD), FGP, and1 000-grain weight (TGW) across two environments.A total of six, two, and four of ILs from populations I,Minghui 86/Fuhui 838 (population II), and Minghui86/Teqing (population III), respectively, had stably and sig-nificantly higher mean GY than recurrent parent Minghui86 by progeny testing in the two environments (Table 1).

Across-environment ANOVA for three populations wasgiven in Appendix B. The variation between genotype(ILs) was highly significant for all traits in all threepopulations, suggesting that considerable genetic varia-tion was maintained even if high selection intensity hadimpacted on BC2F2 populations. Significant G×E interac-tion was observed also for all traits except PN and TGW.

Significant positive correlations were observed be-tween yield and its related traits except TGW for threepopulations under two environments (Appendix C).Panicle number had significant negative correlationswith FGP and SPP. In comparison with the populationII, the other two populations had stable performancesfor each trait across different environments.

QTL identification

QTL identification using RSREP-LRT method Atotal of 50 QTLs associated with grain yield and itsrelated traits were detected in three populations acrosstwo environments using RSTEP-LRT method, whichmainly distributed on 12 chromosomes except 4 and 9.Phenotypic variation explained ranging from 3.10-30.5%by each QTL. For these QTLs, three of them werecommon detected in different populations (Fig. 1). Thefirst one was the locus qPN-2-1 with the allele fromZDZ057 decreasing PN by 0.77, but the allele fromTeqing increasing PN by 0.73. The 2nd one was QTLqFGP-2-1 with the allele from ZDZ057 increasing FGPby 12.8, while the allele from Teqing decreased FGP by10.8. The last one, QTL qTGP-2-1 with ZDZ057 allelehad positive additive effect for increased TGP by 15.4,

Improvement of Yield and Its Related Traits for Backbone Hybrid Rice Parent Minghui 86 Using Advanced Backcross 563


conversely, the Teqing allele had negative additive ef-fect for decreased TGP by 14.4 (Table 2). Among 50QTLs, 22 of them were identified in both environments,which included two QTLs, i.e., qPH-1-1 and qPH-3-1associated with PH that were detected from popula-tions I and II, respectively, and donor alleles averagelydecreased PH by 2.11 and 2.52 cm at these two loci,

respectively; one QTL qPL-3-1 affected panicle length(PL) in population II; five QTLs controlled PN in threepopulations, and the donor alleles at two QTLs(qPN-2-2 and qPN-11-1) had positive additive effectsfor increased PN; three QTLs (qFGP-2-1, qFGP-3-1,and qFGP-3-2) associated with FGP in three populations,and donor alleles at these three loci averagely increased

Table 1 Phenotypic values of 12 ILs with significantly higher mean yields identified based on progeny testing of 123 ILs in two environmentsRP and IL Env.1) HD2) PH PL PN FGP SPP FGR TGW GYMinghui 86/ZDZ057 population (I) Minghui 86 07AH - 119.28 28.08 5.46 145.51 210.28 70.27 28.14 22.36

08HN 110.17 111.05 23.25 6.60 114.79 135.30 84.87 30.15 22.55Mean - 115.17 25.67 6.03 130.15 172.79 77.57 29.15 22.46

WD152 07AH - 128.73** 27.40 6.66 154.32 230.68** 67.13 29.33 30.21*

08HN 108.67 117.87* 23.07 7.33 102.22 118.25 86.45 32.70** 24.44Mean - 123.30* 25.24 7.00 128.27 174.47 73.52 31.02 27.33*

WD153 07AH - 124.50** 26.93 7.13 136.23 178.79 76.72 32.47** 31.71**

08HN 111.67 114.80 22.83 7.33 87.69 103.34 85.07 35.63** 23.05Mean - 119.65 24.88 7.25* 111.96 141.07 79.37 34.05** 27.38*

WD156 07AH - 121.07 26.97 5.18 172.05* 247.61** 69.72 31.07** 27.70*

08HN 111.00 110.00 23.00 5.67 141.78* 160.16 88.09 33.07** 26.25Mean - 115.54 24.99 5.43 156.92* 203.89** 76.96 32.07* 26.98*

WD160 07AH - 126.10** 28.83 6.52 151.76 209.49 72.05 25.37 24.8908HN 109.33 124.47** 24.87 6.93 147.21** 170.00** 86.61 27.80 28.13*

Mean - 125.29** 26.85 6.70 149.49 189.75 78.78 26.59 26.51*

WD163 07AH - 126.40** 26.63 5.80 132.83 217.12 61.49 34.80** 26.7208HN 109.00 119.13* 21.83 6.67 106.07 130.01 82.00 35.37** 27.53*

Mean - 122.77* 24.23 6.23 119.45 173.57 68.82 37.09** 27.13*

WD175 07AH - 122.77 26.80 5.85 206.16** 232.41** 88.84** 26.27 31.67**

08HN 110.00 113.27 23.67 5.33 153.28** 165.13** 92.87** 28.60 22.88Mean - 118.02 25.24 5.59 179.72** 198.77* 90.42** 27.44 27.28*

Minghui 86/Fuhui 838 population (II) Minghui 86 07AH - 120.26 29.24 4.82 136.82 204.31 67.17 28.02 18.46

08HN 109.33 113.15 23.85 6.45 124.13 135.70 91.43 30.48 24.16Mean - 116.71 26.55 5.64 130.48 170.01 79.30 29.25 21.31

WD210 07AH - 124.17 28.23 5.88* 159.47 198.71 80.74** 28.43 27.01**

08HN 111.67 113.20 23.77 6.73 126.10 141.00 89.43 33.00** 27.02Mean - 118.69 26.00 6.35 142.79 169.86 84.06* 30.72 27.02*

WD226 07AH - 121.70 28.93 6.87** 146.28 190.25 76.84** 27.37 27.52**

08HN 108.00 111.60 24.87 7.47 128.24 144.43 89.03 29.87 28.11Mean - 116.65 26.90 7.17** 137.26 167.34 82.02 28.62 27.82*

Minghui 86/Teqing population (III) Minghui 86 07AH - 121.00 30.15 5.57 129.95 185.96 70.06 28.30 20.48

08HN 109.67 114.20 23.55 6.20 121.47 132.51 91.68 30.28 22.80Mean - 117.60 26.85 5.89 125.71 159.24 80.87 29.29 21.64

WD-240 07AH - 124.12 26.60 8.63** 111.52 157.48 70.82 30.47 29.33**

08HN 107.00 113.87 23.60 8.73* 94.07 109.62 85.88 33.87** 27.10*

Mean - 119.00 25.10 8.68** 102.80 133.55 76.97 32.17** 28.22*

WD-245 07AH - 114.73 26.40 5.92 155.00* 229.88** 67.35 28.23 25.93*

08HN 110.67 109.20 22.50 7.73 118.29 141.74 83.40 29.80 27.01*

Mean - 111.97 24.45 6.83 136.65 185.81* 73.54 29.02 26.47*

WD-253 07AH - 123.50 27.63 6.47 156.54* 216.60* 72.67 29.00 29.35**

08HN 105.67 116.27 23.87 6.40 118.51 144.19 81.79 32.47* 23.93Mean - 119.89 25.75 6.44 137.53 180.40* 76.24 30.74 26.64*

WD-258 07AH - 125.90 29.47 6.37 181.73** 259.14** 69.85 27.23 31.55**

08HN 110.33 117.60 22.70 6.13 140.46 * 170.46* 82.36 28.27 24.06Mean - 121.75 26.09 6.25 161.10** 214.80** 75.00 27.75 27.81*

1) 07AH, 2007 Anhui; 08HN, 2008 Hainan. The same as below.2) HD, heading date; PH, plant height; PL, panicle length; PN, panicle number; FGP, filled grains per panicle; SPP, spikelets per panicle; FGR, filled grains rate; TGW, 1 000-grain weight, and GY, grain yield per plant. The same as below.-, data missing. *, P<0.05, **, P<0.01.



Fig. 1 QTLs that affect grain yield and its related traits identified in three populations across two environments. Among 12 chromosomes,10 were drawn since QTLs were identified in these chromosomes only. The underlined QTLs represent ones that were commonly identifiedin both environments. Black arrows indicate the common QTLs identified in the same population using the RSTEP-LRT and chi-squared (c2)tests methods. Roman numerals I, II, and III denote Minghui 86/ZDZ057 population, Minghui 86/Fuhui 838 population, and Minghui 86/Teqing population, respectively.

FGP by 12.8, 24.8, and 10.8, respectively; one QTL qSPP-6-2 affected SPP in population I; four QTLs associated

with FGR in populations I and III, donor alleles at three, i.e.,qFGR-1-1, qFGR-3-1, and qFGR-12-1 increased FGR



Table 2 QTLs that affect yield and its related traits were identified in three BC2F2:4 populations in two environments using RSTEP-LRTmethodTrait QTL Chr. Marker Pop.1) Env. Increased effects2) LOD PVE (%) Add.3)

HD qHD-2-1 2 RM110 III 08HN Teqing 4.58 18.7 0.82qHD-6-1 6 RM340 III 08HN Minghui 86 4.38 16.0 -0.80qHD-10-1 10 RM222 II 08HN Minghui 86 5.78 25.0 -1.65qHD-10-2 10 RM258 III 08HN Teqing 4.14 19.0 1.39qHD-11-1 11 RM3133 III 08HN Teqing 4.67 17.8 1.05

PH qPH-1-1 1 RM200 I 07AH Minghui 86 2.74 8.80 -2.141 RM200 I 08HN Minghui 86 4.89 12.0 -2.07

qPH-3-1 3 RM231 III 07AH Minghui 86 2.65 21.2 -2.513 RM231 III 08HN Minghui 86 3.44 26.7 -2.53

qPH-8-1 8 RM331 II 07AH Fuhui 838 3.43 20.8 3.89qPH-10-1 10 RM222 II 08HN Minghui 86 4.25 17.4 -2.54

PL qPL-2-1 2 RM279 II 08HN Fuhui 838 4.55 21.0 0.78qPL-3-1 3 RM55 II 07AH Minghui 86 2.78 14.2 -0.69

3 RM55 II 08HN Minghui 86 6.14 23.3 -1.17PN qPN-2-1 2 RM110 I 07AH Minghui 86 8.36 20.9 -0.77

2 RM110 III 07AH Teqing 3.25 18.1 0.752 RM110 III 08HN Teqing 3.35 11.9 0.72

qPN-2-2 2 RM3850 II 07AH Fuhui 838 5.73 16.8 0.512 RM3850 II 08HN Fuhui 838 2.74 3.10 0.30

qPN-3-1 3 RM416 III 08HN Minghui 86 8.39 22.7 -1.19qPN-5-1 5 RM3476 I 07AH Minghui 86 11.5 29.5 -0.76

5 RM3476 I 08HN ZDZ057 3.42 12.0 0.44qPN-7-1 7 RM481 II 07AH Minghui 86 3.77 9.70 -0.27

7 RM481 II 08HN Minghui 86 17.4 30.5 -0.93qPN-11-1 11 RM254 II 07AH Fuhui 838 2.90 8.60 0.57

11 RM254 II 08HN Fuhui 838 6.24 11.4 0.92FGP qFGP-2-1 2 RM110 I 07AH ZDZ057 2.73 9.40 10.1

2 RM110 I 08HN ZDZ057 5.67 27.4 15.52 RM110 III 07AH Minghui 86 2.61 23.6 -10.22 RM110 III 08HN Minghui 86 4.82 20.2 -11.3

qFGP-3-1 3 RM3779 II 07AH Fuhui 838 2.14 8.20 19.73 RM3779 II 08HN Fuhui 838 9.83 26.8 29.9

qFGP-3-2 3 RM16 I 07AH ZDZ057 7.39 24.3 15.33 RM16 I 08HN ZDZ057 2.54 4.40 6.43

qFGP-5-1 5 RM440 II 08HN Fuhui 838 9.54 25.5 15.1qFGP-11-1 11 RM3133 III 07AH Teqing 5.68 27.5 18.5qFGP-12-1 12 RM270 II 08HN Minghui 86 9.71 26.3 -17.5

SPP qSPP-2-1 2 RM110 I 08HN ZDZ057 6.76 26.0 15.72 RM110 III 08HN Minghui 86 4.19 21.8 -14.4

qSPP-3-1 3 RM3779 II 07AH Fuhui 838 4.09 8.80 27.2qSPP-3-2 3 RM55 II 07AH Minghui 86 5.44 14.8 -12.8qSPP-6-1 6 RM275 II 07AH Fuhui 838 3.78 10.5 11.4qSPP-6-2 6 RM3307 I 07AH ZDZ057 3.80 12.7 10.3

6 RM3307 I 08HN ZDZ057 2.79 5.00 4.87qSPP-12-1 12 RM511 II 08HN Fuhui 838 3.22 15.6 11.9

FGR qFGR-1-1 1 RM86 I 07AH ZDZ057 3.40 7.40 3.231 RM86 I 08HN ZDZ057 7.64 10.0 2.66

qFGR-2-1 2 RM535 I 07AH Minghui 86 6.95 14.9 -4.322 RM535 I 08HN Minghui 86 13.8 19.7 -3.47

qFGR-3-1 3 RM16 I 07AH ZDZ057 9.21 27.9 7.383 RM16 I 08HN ZDZ057 9.07 11.6 3.67

qFGR-3-2 3 RM55 II 07AH Fuhui 838 4.06 25.5 5.44qFGR-3-3 3 RM85 III 07AH Minghui 86 7.29 21.7 -6.03qFGR-11-1 11 RM3133 III 07AH Teqing 10.73 26.3 6.08qFGR-12-1 12 RM6973 III 07AH Teqing 6.55 23.7 7.82

12 RM6973 III 08HN Teqing 3.48 21.9 7.76qFGR-12-2 12 RM270 II 08HN Minghui 86 12.6 27.1 -9.72

TGW qTGW-3-1 3 RM16 I 07AH Minghui 86 10.5 23.1 -1.863 RM16 I 08HN Minghui 86 3.72 8.10 -1.31

qTGW-7-1 7 RM18 II 07AH Fuhui 838 8.15 25.2 1.237 RM18 II 08HN Fuhui 838 2.27 10.4 0.74

qTGW-8-1 8 RM310 I 07AH ZDZ057 5.79 8.40 1.178 RM310 I 08HN ZDZ057 4.01 9.60 1.35

(Continued on next page)



by 2.94, 5.02, and 8.29%, respectively; and all locicontrolled TGW in three populations, and donor allelesat three loci (qTGW-7-1, qTGW-8-1, and qTGW-12-1)had positive additive effects for increased TGW.QTL identification using chi-squared (ccccc2) test methodbased on genetic hitchhiking A total of eight QTLsthat affect GY were detected in three populations usingchi-squared (c2) test method. It included six QTLs thatwere identified in the population I, one QTL in the popu-lation II, and three QTLs in the population III (Table 3).Among these QTLs, qGY-2-1 and qGY-6-1 were com-mon identified in populations I and III, and three of themwere consistent with those detected using RSTEP-LRTmethod in the same population.

DISCUSSION

The improvement of Minghui 86 has been a big chal-

lenge to rice breeder for it had high restoring ability andgood special combining ability already (http://www.ricedata.cn/). In this study, a total of 39 (31.7%) ILswith high GY than RP were repeatedly observed basedon progeny testing for 123 ILs in two environments,and 12 of which (9.8%) had significant higher meanGY than RP. Phenotypic analysis showed that theseILs with higher GY mainly attributed to the significantimprovement of PN and FGP or SPP (Table 1). Thisfinding indicated that the first round yield selection onBC2F2 population showed high efficiency on improvingMinghui 86. However, the improvement efficiency var-ied with donors. Of these, ZDZ057 was the best donorused in this study, next is Teqing. Fuhui 838 was thelast choice as a donor to improve the yield of Minghui 86.

Previous studies confirmed that the landraces andmodern cultivars were powerful stocks for increasingrice yield potential under modern cultivation as theyhave been over decades subjected to domestication and

Table 2 (Continued from preceding page)Trait QTL Chr. Marker Pop.1) Env. Increased effects2) LOD PVE (%) Add.3)

qTGW-10-1 10 RM216 III 07AH Minghui 86 3.62 24.5 -0.6810 RM216 III 08HN Minghui 86 5.91 28.1 -1.05

qTGW-12-1 12 RM270 I 07AH ZDZ057 13.9 27.3 1.6312 RM270 I 08HN ZDZ057 4.33 9.00 1.02

GY qGY-2-1 2 RM110 I 07AH Minghui 86 4.14 9.10 -1.58qGY-2-2 2 RM3850 II 08HN Minghui 86 3.24 9.80 -1.87qGY-3-1 3 RM16 II 07AH Fuhui 838 4.35 14.7 1.41qGY-5-1 5 RM405 I 07AH ZDZ057 9.34 22.6 1.80qGY-6-1 6 RM3 I 08HN Minghui 86 4.58 19.1 -2.57qGY-8-1 8 RM331 II 07AH Fuhui 838 3.70 12.1 1.70qGY-11-1 11 RM3133 III 07AH Teqing 3.14 21.9 4.09qGY-12-1 12 RM511 II 07AH Fuhui 838 4.41 15.5 2.11

1) I, Minghui 86/ZDZ057 population; II, Minghui 86/Fuhui 838 population; and III, Minghui 86/Teqing population. The same as below.2) Increase effect is the source of allele causing an increase in trait measurement (whether favorable or unfavorable).3) Add, additive effect was estimated according to the following formulas, Add=(MD-MR)/2, where MD and MR are the mean phenotypic values of the donor homozygote and recurrent parent homozygote, respectively.

Table 3 QTLs that affect grain yield per plant were detected by chi-square (c2) tests using 39 high-yielding ILs selected from threepopulations based on progeny testing in two environments

QTL Chr. Marker Pop. Frequency1) c2 value P-value

B H AqGY-1-1 1 RM86 I 0.185 0.259 0.556 49.24 0.0000qGY-1-2 1 RM488 III 0.143 0.286 0.571 30.50 0.0000qGY-2-1 2 RM110 I 0.111 0.148 0.741 12.25 0.0002

2 RM110 II 0.417 0.083 0.500 26.41 0.0000qGY-3-1 3 RM16 I 0.040 0.200 0.760 24.17 0.0000qGY-5-1 5 RM405 I 0.185 0.185 0.630 23.55 0.0000qGY-6-1 6 RM527 I 0.185 0.222 0.593 35.17 0.0000

6 RM527 III 0.154 0.231 0.615 17.70 0.0001qGY-7-1 7 RM481 I 0.154 0.192 0.654 23.33 0.0000qGY-11-1 11 RM3133 III 0.643 0.000 0.357 40.98 0.0000

1) B, H, and A, frequencies of the donor homozygote, heterozygote, and introgression, respectively.



long-term artificial selection process (Li et al. 2005; Aliet al. 2006; He et al. 2010; Chen et al. 2011). In thisstudy, as many as 63.6% (35 out of 55) favorable alle-les associated with yield and its components were fromdonors, and these favorable alleles mainly focused onPN, FGP, SPP, FGR, and GY (Table 2). Among thefavorable QTL alleles, 23 (65.7%) were carried in 12 ILswith significantly higher mean GY (Table 4). Forinstance, loci qPN-2-1 with favorable alleles fromFuhui 838 was detected in two environments and lociqFGP-2-1 with beneficial alleles from ZDZ057 wasdetected in both environments. qSPP-6-2 controlledspikelets per panicle with beneficial allele from donorZDZ057 were also found in two environments. Inaddition, qGY-1-1, qGY-1-2, qGY-2-1, qGY-3-1, qGY-5-1, qGY-6-1, qGY7-1, qGY-11-1, qGY-12-1 that af-fect GY with favorable alleles were from donorsZDZ057 or Fuhui 838 or Teqing (Tables 2 and 3). Itcan be infered that these loci from the donors played akey role in improving the grain yield of Minghui 86.This finding indicated that advanced backcross intro-gression method under selection is an effective and fea-sible ways for simultaneously improving target traits

and detecting favorable alleles affecting these targettraits.

In this study, 20.0% (11 out of 55) of the QTLsidentified were also reported in the previous studies(www.gramene.org). For HD, QTL qHD-2-1 nearRM110 was also identified by Septiningsih et al. (2003).QTL qPH-1-1 near RM200 affecting PH was consis-tent with the previous mapping results (Brondani et al.2002). Likewise, QTL qPN-11-1 linked with RM254shared common region as ppl11.2 found by Septiningsihet al. (2003). Two QTLs (qFGP-3-2 and qFGP-12-1)controlling FGP were also reported by Marri et al. (2005)and Temnykh et al. (2001). For TGW, three QTLsassociated with RM16, RM18, and RM216 were si-multaneously reported by Fan et al. (2006), Zhuang et al.(2001) and Thomson et al. (2003), respectively.Actually, QTL qTGW-3-1 strongly associated with GS3gene that controlled a grain weight and size cloned byFan et al. (2006). In addition, four QTLs (qGY-3-1,qGY-5-1, qGY-6-1, and qGY-8-1) for GY shared com-mon genomic regions with previous studies (Brondaniet al. 2002; Thomson et al. 2003; Jiang et al. 2004).

The allelic diversities and their functional diversities

Table 4 Summary of favorable donor alleles that affect yield and its related traits carried in the 12 significantly high-yielding ILsIL code Env. HD PH PL PN FGP SPP FGR TGW GY1)

Minghui86/ZDZ057 population (I) WD152 07AH qFGR-1-1 qGY-1-1, qGY-5-1

08HN qFGR-1-1 qGY-1-1, qGY-5-1 WD153 07AH qFGR-1-1 qTGW-12-1 qGY-1-1, qGY-7-1

08HN qFGR-1-1 qTGW-12-1 qGY-1-1, qGY-7-1 WD156 07AH qSPP-6-2

08HN qSPP-6-2 WD160 07AH qFGP-2-1 qGY-2-1, qGY-7-1

08HN qFGP-2-1 qSPP-2-1 qGY-2-1, qGY-7-1 WD163 07AH qTGW-8-1 qGY-5-1, qGY-6-1

08HN qTGW-8-1 qGY-5-1, qGY-6-1 WD175 07AH qFGP-2-1 qSPP-6-2 qFGR-1-1 qTGW-12-1 qGY-1-1, qGY-2-1, qGY-6-1

08HN qFGP-2-1 qSPP-2-1, qSPP-6-2 qFGR-1-1 qTGW-12-1 qGY-1-1, qGY-2-1, qGY-6-1Minghui 86/Fuhui 838 population (II) WD210 07AH qFGR-3-2 qTGW-7-1 qGY-2-1

08HN qPL-2-1 qTGW-7-1 qGY-2-1 WD226 07AH qGY-2-1, qGY-3-1

08HN qPL-2-1 qSPP-12-1 qGY-2-1, qGY-12-1Minghui 86/Teqing population (III) WD240 07AH qPN-2-1 qGY-1-2

08HN qHD-2-1 qPN-2-1 qGY-1-2 WD245 07AH qGY-1-2

08HN qGY-1-2 WD253 07AH qGY-1-2

08HN qGY-1-2 WD258 07AH qFGP-11-1 qFGP-11-1 qGY-11-1

08HN qHD-11-1 qGY-11-1

1) QTLs in bold represent ones that were simultaneously detected by the RSTEP-LRT and chi-squared (c2) test methods.



were widely revealed in the previous studies (Xue et al.2008; Zhang et al. 2009; Wang et al. 2011). In thisstudy, five loci showed multi-alleles as they existed thedifferent alleles among donors. Furthermore, threeQTLs that were detected using RSTEP-LRT methodhad different allelic effects. Although the large varia-tion in QTL effect among different alleles in the samegenetic background may suggest the presence of truefunctional allelic diversity at these QTLs, they remainindistinguishable from cases resulting from linkage,epistasis and/or QTL×E interaction.

CONCLUSON

By the progeny testing on 123 yield introgression linesin two environments, which allowed the identificationof 12 promising ILs that had significantly higher grainyields than Minghui 86. A total of 55 QTLs that affectyield and its related traits were identified using thelikelihood ratio test based on stepwise regression(RSTEP-LRT) method and using chi-squared (c2) test.Among these QTLs, the beneficial donor alleles forincreased GY and its related traits were identified in63.6% (35 out of 55) of the QTLs. These promisingILs and favorable QTLs detected from the donors willprovide the elite breeding materials and genetic infor-mation for further improvement of the grain yield forMinghui 86.

METERAILS AND METHODS

Construction of experimental populations

Three advanced backcross populations were constructedusing Minghui 86 as the recurrent parent (RP) and threehigh-yielding varieties, namely, ZDZ057, Fuhui 838, andTeqing as the donors, respectively. Single crosses weremade between RP and the donors in the spring of 2004 inHainan Province, China, to produce three F1s, which werebackcrossed with Minghui 86 to produce BC1F1s in thesummer of 2005 in Anhui. Twenty-five randomly chosenplants from each BC1F1 population were backcrossed withRP again to produce 25 BC2F1 lines in the following seasonin Hainan. Seeds collected from plants of the 25 BC2F1lines were bulked to form a single bulk BC2F2 population.500 seeds from each BC2F2 population were sowed for se-lecting plants with higher yields than Minghui 86 by mor-

phological evaluation and measuring yield and its relatedtraits in Anhui in 2006. Finally, 51, 46, and 26 introgressionlines (ILs) were selected from the Minghui 86/ZDZ057 popu-lation (population I), Minghui 86/Fuhui 838 population(population II), and Minghui 86/Teqing population(population III), respectively. For seed increase, these ILswere grown to generate IL families (to BC2F2:3) in the springof 2007 in Hainan.

Field trial and phenotypic evaluation

Three BC2F2:4 populations were tested in field trials using arandomized complete block design with three replications.Recurrent parent, Minghui 86 was arranged three times (inboth ends and middle) in each replication, which is conve-nient to accurately select high-yield ILs by comparing themean of Minghui 86 with each IL from three replicationsfor each population. The trial was conducted at the AnhuiExperiment Station of the Anhui Agricultural University inHefei in summer 2007 (07AH). Anhui is in the middle ofChina with a sub-tropic and warm-temperate climate type.In the spring of 2008 trial was conducted at the Experimen-tal Station of the Chinese Academy of Agricultural Sci-ences in Hainan (08HN). It is an island in the south ofChina having a tropical climatic type, dry from Decemberto May and wet from June to November. The seeds ofeach IL family were sowed in the seedbeds and transplantedinto the plots (10 plants per plot) with a space of 26.4 cm×16.5 cm after 30 d after sowing.

Five plants randomly chosen from each plot were evalu-ated for nine traits: (1) heading date (HD, in d) was re-corded as the days from sowing until 50% of the panicleshad headed; (2) plant height (PH, in cm) was measured asthe average height of the five plants in each plot from thesoil surface to the tip of the tallest panicle; (3) paniclelength (PL, in cm) was measured as the average lengthfrom the panicle neck to the panicle tip for all panicles fromfive plants; (4) panicle number (PN) was the average num-ber of five plants except panicles having less than twoseeds; (5) filled grains per panicle (FGP) was calculated asgrain yield per plant divided by the 1 000-grain weight andpanicle number per plant then multiplied by 1 000; (6) spike-lets per panicle (SPP) were calculated by FGP plus unfilledgrains per panicle; (7) filled grains rate (FGR, in %) wascalculated as the number of filled grains per panicle di-vided by spikelets per panicle; (8) 1 000-grain weight (TGW,in g) was the average weight of 200 filled grains taken frombulk harvested seeds of five plants (three times), then con-verted to 1 000-grain weight; and (9) grain yield per plant(GY, in g) was the average weight of five plants.

Genotyping analysis

For each population, leaf samples were collected from 10



individuals and bulked for DNA extraction using the CTABmethod. Polymerase chain reaction (PCR) were conductedas follows: the total reaction system was 20 μL, and thethermal cycle profile was used, the initial denaturing stepwas 95°C for 5 min, subsequent denaturing was at 95°C for1 min, annealing was 55-67°C for 1 min, and extension was72°C for 2 min, and from steps 2 to 4 were repeated for atotal of 35 cycles with a final extension at 72°C for 10 min.PCR products were run on 8% polyacrylamide gels.

About 700 simple sequence repeat markers (SSR) fromCornell University (Temnykh et al. 2000; McCouch et al.2002) were used to screen polymorphisms betweenMinghui 86 and the donors. A total of 126, 110, and 114polymorphic markers were identified for populations I, II,and III, respectively. For these polymorphic markers, 75,67, and 60 SSR markers that were evenly distributedthroughout the whole genome and covered all polymor-phic genomic regions of the three populations were cho-sen for genotyping.

Data analysis

Analysis of variance (ANOVA) was conducted across-en-vironment using SAS PROC GLM (SAS Institute). Traitcorrelations were analyzed for each environment separatelyusing IL family means. In order to fully detect the favor-able alleles from donors, two mapping methods based onthe different theoretic basis were used in this study. First,QTLs that affect the measured traits were identified by thelikelihood ratio test based on stepwise regression (RSTEP-LRT) method (Wang et al. 2006). The markers with LODscore equal to or larger than 3.0 were considered as exist-ence of a QTL in order to make QTL identified more reliable.QTLs were also considered significant in two environmentswhen they were detected in at least one environment withan LOD score of 3.0, and less than 3.0 but larger than 2.5 inthe supporting environment. Second, the genetic hitch-hiking happened while the target trait selection imposedon a population. Chi-squared (c2) test based on the ge-netic hitchhiking effects was also used to identify QTLsassociated with GY using those ILs that had high GY thanRP tested in two environments repeatedly (Li et al. 2005;Zhang et al. 2011). An experiment-wise significance levelof P<0.0005 was chosen to declare the presence of a puta-tive QTL in a given genomic region.

AcknowledgementsThe study was funded by the National High-Tech R&DProgram of China (2010AA101806) and the Bill & MelindaGates Foundation, USA (OPP51587).

Appendix associated with this paper can be available onhttp://www.ChinaAgriSci.com/V2/En/appendix.htm

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