ZHANG Ning， ZHANG Qiang， ZHANG Yuna， LI Xin， HUANG Xueqing

(State Key Laboratory of Genetic Engineering， School of Life Sciences， Fudan University， Shanghai 200438， China)

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Dissection of the Genetic Architecture of Plant Height and Ear Height in Maize(ZeamaysL.)

ZHANG Ning， ZHANG Qiang， ZHANG Yuna， LI Xin， HUANG Xueqing

(State Key Laboratory of Genetic Engineering， School of Life Sciences， Fudan University， Shanghai 200438， China)

Maize(ZeamaysL.) is among the crops with the greatest worldwide economic importance. Plant height(PH) and ear height(EH) are two very important traits which are considered necessary in maize breeding and are related to morphology， lodging， and yield. To explore the genetic mechanism of PH and EH， an F3：4recombinant inbred lines(RILs) population with 165 lines was generated from a cross between inbred line(Zheng58) and inbred line(B73). 189 polymorphic simple sequence repeat(SSR) markers were used to map quantitative trait loci(QTLs) for PH and EH. A total of 11 QTLs(5 QTLs for PH and 6 QTLs for EH) detected were located on 8 chromosomes except chromosome 2 and 6. Single QTL explained from 4.3% to 14.2% of the phenotypic variance. Interestingly， the novel plant height QTL(qPH04-01) was specific to the population， which was detected near umc0371 and could explain 8.8% of phenotypic variation. It is worthy of further research and utilization.

maize； plant height； ear height； quantitative trait loci； genetic architecture

1 Introduction

Maize(ZeamaysL.) is one of the most important cereal crops worldwide， and increasing the grain yield and biomass has been the most important goals of maize production[1]. Among the various traits that are normally considered in maize breeding programs， plant height and ear height are two important traits affecting plant architecture and yield potential. On the one hand， they are not only closely correlated with grain yield， leaf number， flowering time[2]and other important agronomic traits， including biomass production and forage yield in maize[3]， but also directly affects and determines resistance to stalk lodging. Low PH and EH can lower the center of gravity of the plant， which is considered to be important in the determination of stalk lodging[4]. On the other hand， excessively high plant will decrease planting density， lodging resistance and harvest index； too short plant will influence the permeability of population， susceptibility to diseases and insect pests and reduce biomass. Excessively high ear height will easily cause lodging； too short ear height will affect the efficient transport of the photosynthetic product to the ear. Therefore， an appropriate plant height and ear height is a prerequisite for attaining the desired yield in maize-breeding programs.

Genetic studies have indicated that plant height and ear height in maize are complex traits controlled by both qualitative genes and quantitative genes. Plant height loci have been cloned and resolved by molecular tagging of large effect alleles often induced by mutagenesis[5-6]. Over 40 maize genes at which mutations have large effects on plant height have been identified. These are involved in hormone synthesis， transport， and signaling pathway[7]. Well-characterized maize height genes include：brachytic2， influencing polar auxin transport[8-9]；dwarf-1(dt)， controlling the three gibberellin-biosynthetic steps[10]；dwarf3， mediating gibberellin synthesis[11]；dwarf8 anddwarf9， regulating DELLA proteins of gibberellin signal transduction pathways[12-13]； andnanaplant1， impacting brassinosteroid synthesis[14]. In addition to the semi-dwarfing and dwarfing genes， the PH and EH variation in maize breeding populations is mostly controlled by a set of quantitative trait loci(QTLs) with minor effects. Over the last two decades， genetic dissection of maize PH and EH by classical QTL mapping using biparental populations has resulted in identification of numerous PH and EH QTLs[15-37]. Nevertheless， different studies provided different results， including QTL number， distribution， and genetic effect. For instance， Lima et al used maize inbred lines L-20-01F and L-02-03D as parental lines， and 9 QTLs for ear height were located on chromosomes 2(two)， 3(two)， 4(one)， 7(one)， 9(two)， and 10(one)[27]， while in the report by Li et al， Mo17 and Huangzao4 were employed as parents， and only 1 QTL was identified on chromosomes 1[34]. Inconsistent detection of QTLs in different research reflects the necessity and importance of QTL mapping with various parents and populations， and in various environments， to reveal the complicated heredity of plant height and ear height. Therefore， taking the complex and polygenic inheritance nature of plant architecture of maize into account， further investigations of the QTLs underlying the phenotypic variance of these traits are needed.

In the present study， an F3：4recombinant inbred lines(RILs) population derived from a cross between inbred line Zheng58 and B73， was used to identify QTLs for two traits affecting plant architecture：plant height and ear height. The objectives were to i) better understand the genetic basis of plant architecture and ii) identify molecular markers for MAS in maize breeding projects.

2 Materials and Method

2.1 Plant materials

The recombination inbred line(RIL) populations were obtained by crossing B73 with Zheng58. The parents were chosen on the basis of their different plant architecture and maize germplasm groups. B73 with the higher plant stature is the common parent in NAM population and has been sequenced. Zheng58 is an elite foundation inbred line with dwarf architecture， which is used broadly in China. From the F2progeny， a single seed descent was applied to generate 165 RILs at the F4generation.

2.2 Field experiments and statistical analyses

The field experiments were performed at the experimental station located in Songjiang District， Shanghai during 2014 and 2015. A randomized complete block design with two replications was applied. Each plot had one row that was 3 m long and 0.67 m wide， with a total of 10 plants at a density of 50000 plants/ha. The field management followed common agricultural practice in maize production in China. Five representative plants from the middle of each plot were chosen to measure the plant height(PH) and ear height(EH) at grain maturity stage. PH was measured as the distance(cm) from the soil surface to the tip of the tassel； EH was measured as the distance(cm) from the soil surface to the node of attachment of the primary ear. The trait value for each RIL was averaged for the five measured plants in each replication.

Based on the means of the phenotypic data of the population， the SPSS20 software was used to perform statistics analysis. For each trait， broad-sense heritability(h2) was estimated as the proportion of variance explained by between RIL(genotypic) variance and RIL by block(error) variance. The correlation coefficients among the traits were obtained with the “cor” function in the SPSS software package.

2.3 Genetic map construction and QTL mapping

Young leaf samples were collected at the seedling stage from the four RIL populations， and genomic DNA was extracted using the CTAB method[38]. The F3population individuals were analyzed using Simple Sequence Repeats(SSRs) markers. In order to select the most informative SSR primer pairs， the parental lines， B73 and Zheng58， and an F1 individual were screened with 393 SSR markers chosen from Maize Genetics and Genomic Database(MaizeGDB) based on their repeat unit and physical position. This resulted in the selection of 189 pairs of SSR markers that clearly show codominant segregation. They were used to genotype the F3 individuals. Primer sequences are available from the MaizeGDB website(http：∥www. maizegdb. org). PCR reactions were run in 10μL total volume and the final concentration of each compositions as follows：1μL 10×PCR buffer， 0.2μmol/L of the forward and reverse primers， and 1.5mmol/L MgCl2， 0.2μmol/L dNTP， and 0.1 units of Taq polymerase， 50—100 ng template DNA， then metered volume to 10μL with ddH2O.

The touchdown PCR(TD PCR) cycling programs were as follows：94℃ for 3min， 94℃ for 30s， 36 cycles with 94℃ for 30s， Tm for 30s， and 72℃ for 30s， and in the first 16 cycles the annealing temperature was reduced by 1℃ per cycle from 65℃ to 50℃， the last 20 cycles run at the constant Tm 50℃， then followed by 72℃ for 10 min.

According to the physical position of the SSR markers obtained from the genome sequencing results of B73， a physical map was constructed through assigning the informative markers to the corresponding chromosome. The software package MapQTL 5.0 was used to identify and locate QTL on the linkage map by using interval mapping and multiple-QTL model(MQM) mapping methods as described in its reference manual(http：∥www. kyazma. nl). In a first step， putative QTL were identified using interval mapping. Thereafter， the closest marker at each putative QTL was selected as a cofactor and the selected markers were used as genetic background controls in the approximate multiple QTL model of MapQTL 5.0.LOD threshold values applied to declare the presence of QTL were estimated by performing permutation tests implemented in MapQTL 5.0 using at least 1000 permutations of the original data set， resulting in a 95% LOD threshold of 2.9. The estimated additive genetic effect and the percentage of variance explained by each QTL and the total variance explained by all the QTL affecting a trait were obtained using MQM mapping.

3 Results

3.1 Analysis plant architecture traits in F3：4population and parental lines

There are significant differences between the two inbred maize varieties B73 and Zheng58 in plant architecture traits used in this study. Zheng58 had dwarf architecture with an average PH of 172.6 and EH of 58.4， whereas B73 displayed a higher plant stature with an average PH of 228.4 and EH of 92.1(Tab.1). Table 1 presents a number of descriptive statistics of the two plant architecture traits for the two parents and the F3：4population. Large differences were found for these traits between the two parents，and the wider range of variation for the traits in the F3：4population， normal distributions with transgressive segregation suggested polygenic inheritance of the traits(
Fig.1). The estimated broad-sense heritability(h2) values for traits were generally high and ranged from 82.5 to 86.4(Tab.1). Additionally， the significant positive correlation was observed between PH and EH.

Tab.1 Descriptive statistics of the plant architecture traits in parental lines (B73 and Zheng58) and the population of F3：4 at grain maturity stage

1) PH， plant height； EH， ear height； 2) All the differences between the two parents are statistically significant at the 0.01 probability level.

3.2 Linkage maps of F3：4populations

A survey of 393 SSR primer pairs identified 189 loci polymorphic between the parents. According to the physical position of the SSR markers obtained from the genome sequencing results of B73， a physical map with 189 SSR markers was constructed through assigning the informative markers to the corresponding chromosome. The number of markers placed in different chromosomes ranged from 13—29 with averages of 18.9. The longest marker distance was 38.98 Mb， and the shortest 1.41 Mb. The average genetic distance of 10.89 Mb between two neighbouring markers and the distribution of markers in all chromosomes was relatively even without crowding(
Fig.2).

3.3 Identification of QTLs for plant architecture traits

Quantitative trait loci analysis of plant architecture traits was conducted using MAPQTL 5.0 software and 11 detected QTLs were distributed on 8 chromosomes except for chromosome 2 and 6(Tab.2，
Fig.2). Among them， two QTLs were detected on each of chromosomes 1(qPH01-01 and qEH01-01)， 3(qPH03-01 and qEH03-01)， 5(qPH05-01 and qEH05-01)； and one QTL was detected on each of chromosomes 4(qPH04-01)， 7(qEH07-01)， 8(qEH08-01)， 9(qEH09-01) and 10(qPH10-01). Some QTLs detected at different traits were found located in the same interval. For example， two QTLs were simultaneously detected in PH and EH and located on chromosome 3(bnlg1035-umc1644 interval) and chromosome 5(umc1155-umc1072 interval)， respectively. It is notable that most QTLs for PH and EH had positive additive effects except for qPH10-01 and qEH07-01 with negative additive effects， indicating that the B73 parent contributed most alleles for increasing plant height and ear height.

Five QTLs on chromosome 1， 3， 4， 5 and 10， respectively， were identified for PH(
Fig.2， Tab.2)， which explained 55.4% of the total phenotypic variance， and single QTL accounted for from 6.4% to 17.2% of the phenotypic variance.

Six QTLs for EH， accounting for 58.8% of the total phenotypic variance， were identified on chromosome 1， 3， 5， 7， 8 and 9， respectively(
Fig.2， Table 2). Single QTL explained from 6.6% to 14.1% of the phenotypic variance.

Tab.2 QTL analysis of maize plant height and ear height in the F3：4 population of B73×Zheng58

Additive effect：effect of the substitution of the Zheng58 allele by the B73 allele. A positive value indicates that the B73 allele increases the value of the trait； A negative value indicates that the Zheng58 allele increases the value of the trait.

4 Discussion

PH and EH are two important agronomic traits in the maize breeding project. They are related to morphology， lodging， and grain yield； therefore， understanding their genetic basis has important theoretical and practical meaning[39]. Quantitative trait locus(QTL) mapping is a well-reasoned solution to realize the genetic basis of traits in crop breeding. In the past few decades， to increase planting density and prevent plants from lodging， studies on the genetic mechanism of plant and ear height were given great attention. Until now， a number of QTL conferring plant height and ear height are reported to be located on all ten chromosomes in maize. In this research， we chose a dwarf-type inbred line Zheng58 and a normal inbred line B73 as the parents of mapping population and detected 11 QTLs on chromosomes 1， 3， 4， 5， 7， 8， 9 and 10 for PH and EH in an F3：4population(
Fig.3). Compared to previous studies， ten of the 11 QTLs for PH and EH were found to have similar chromosomal locations with different mapping experiments or different genetic background， which demonstrated that the chromosome regions for these consistent QTLs might be hot spots for the important QTLs for PH and EH. Also the congruence in QTLs detected in this study with previous reports indicates the robustness of our results. However， in our study， no QTL was detected on chromosome 2 and 6； the cause of this was probably the too small genetic effects or no allelic difference between the two parents. Interestingly， one QTL for PH， qPH04-01， was detected in chromosome 4， which has not been reported in maize by previous researchers. The novel QTL may be due to the specific genetic background from dwarf-type parent Zheng58. Furthermore， newly detected major QTLs may serve a complementary role in revealing the genetic nature of plant-height traits.

The analyses also revealed high phenotypic correlations between plant height and ear height. The genetic basis of such high correlations can largely be explained by the co-localization of the QTLs for the two traits， either due to pleiotropic effects or tight linkage. Examples of such genomic regions included：the interval marked by bnlg1035-umc1644on chromosome 3 and umc1155-umc1072 where QTLs for PH and EH were simultaneously detected. The similar co-localization QTL was widely reported[23-25]. Zhou et al detected 4 pQTLs regions that control both plant height and ear height[37]. The co-localization of the QTLs for PH and EH is of great relevance in understanding the plant architecture. Whether genes in these regions have pleiotropic effects or the plant architecture expression is due to the effect of linked genes needs to be investigated. To achieve this objective， developing heterogeneous inbred families(HIFs) for this QTL region for fine mapping and cloning of these QTLs is in progress. Undoubtedly， this will lead to better understanding of the mechanism of plant architecture in maize.

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玉米株高和穗位高遺傳基礎(chǔ)的QTL剖析

張 寧，張 強，張玉娜，李 鑫，黃雪清

(復(fù)旦大學(xué) 生命科學(xué)學(xué)院 遺傳工程國家重點實驗室，上海 200438)

玉米是世界范圍內(nèi)具有經(jīng)濟重要性的作物之一.株高和穗位高是玉米育種過程中需考慮的2個重要農(nóng)藝性狀，對玉米產(chǎn)量、抗倒伏性及株型等都有較大影響.為進一步明確玉米株高和穗位高的遺傳機制，本研究以B73×Zheng58的含有165個株系的F3：4重組自交系群體為作圖群體，利用覆蓋玉米10條染色體189個SSR標記對株高和穗位高進行QTL定位分析.總共定位到5個株高QTL和6個穗位高QTL；這11個QTL分布在除2號和6號之外的其他8條染色體上.單個QTL表型變異貢獻率的變幅為4.3%～14.2%.其中10個QTL與以前報道過的QTL的位置相近或重疊，而株高QTL(qPH04-01)是新發(fā)現(xiàn)的群體專一性的QTL，最靠近標記umc0371，表型變異貢獻率為8.8%，是值得進一步研究和利用的位點.

玉米； 株高； 穗位高； 數(shù)量性狀位點； 遺傳基礎(chǔ)

0427-7104(2016)05-0605-09

Shanghai Pujiang Program(14PJ1400700)； National Natural Science Foundation of China(31471151)

Q 37 Document code：A

Received date：2016-04-08

Biography：ZHANG Ning(1989—)， female， graduate candidate； Corresponding author：HUANG Xueqing， male， professor， E-mail：xueqinghuang@fudan.edu.cn.