identification of qtl affecting protein and amino acid ... · determination of protein content...

Rice Science, 2011, 18(3): 187−195 Copyright © 2011, China National Rice Research Institute Published by Elsevier BV. All rights reserved

Identification of QTL Affecting Protein and Amino Acid Contents in Rice

ZHONG Ming1, *, WANG Ling-qiang1, *, YUAN De-jun1, LUO Li-jun2, XU Cai-guo1, HE Yu-qing1 (1National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan) and National Center of

Crop Molecular Breeding, Huazhong Agricultural University, Wuhan 430070, China; 2Shanghai Agrobiological Gene Center,

Shanghai 201106, China; *The authors contributed equally to this paper )

Abstract: The phenotypes of protein and amino acid contents were measured in an F9 recombinant inbred line population

derived from a cross between Zhenshan 97B and Delong 208. A total of 48 and 64 QTLs were identified in 2004 and 2005,

respectively. The contribution of each QTL to the phenotypic variation ranged from 4.0% to 43.7%. Most QTLs co-localized,

forming 29 QTL clusters on the chromosomes with three major ones detected in both years, which were mapped on

chromosomes 1, 7 and 9, respectively. The two QTL clusters for amino acid content, qAa1 and qAa7, influenced almost all

the traits with the allele from Zhenshan 97B, and the third QTL cluster for amino acid content, qAa9, increased the lysine

content with the allele from Delong 208. A wide coincidence was found between the QTL detected under this study and the

loci involved in amino acid metabolism pathways in nitrogen assimilation and transport, or protein biosynthesis. The results

would facilitate the identification of candidate genes and could be used in marker-assisted selection for the favorable allele

in rice quality improvement.

Key words: amino acid content; protein content; quantitative trait locus; grain quality; Oryza sativa

As one of the two major food cereals, rice

provides a large proportion of the total nourishment of

the world population (Juliano, 1985). However,

studies on rice chemistry and technology have lagged

behind as compared to those on wheat. Protein is the

second most abundant constituent of milled rice,

following starch. As a staple food, rice is one of the

most important protein sources for people. Attempts

to improve the nutritional value of rice have been

concentrated on protein content and quality. Breeding

efforts have yielded very limited success so far

because of the complex inheritance and the large

effect of environment on protein content (Coffman

and Juliano, 1985).

The recent advances in molecular marker

technology and the development of high-density

molecular marker linkage maps in rice have provided

a powerful tool for elucidating the genetic basis of

quantitatively inherited traits, including most of

important agronomy traits (Tanksley 1993; Harushima

et al, 1998). Many important traits have been genetically

dissected through identification of quantitative trait

loci (QTL) (Anh et al, 1993b; Tan et al, 1999, 2000

and 2001; Fan et al, 2005; Tian et al, 2005). Many

QTL genes have been cloned to date including those

affecting fruit shape, grain shape, heading date and

grain protein content (Joppa et al, 1997; Yano et al,

1997; Yamamoto et al, 1998 and 2000; Ku et al, 1999;

Liu et al, 2002; Lin et al, 2003; Distelfeld et al, 2004;

Fan et al, 2006). In many cases, QTL mapping

positions have a good agreement with the tagged or

cloned genes, indicating that the mapped QTLs are

accurate (Adam, 2006). Therefore, some scientists

suggested that the results of rough QTL mapping or

fine mapping could be applied in breeding immediately

without costly gene cloning (Salvi and Tuberosa,

2005). Such an approach is particularly useful for

traits that are difficult to measure such as protein

content, amino acid content and fat content.

Quite a few chromosomal regions have been

identified to affect protein content in brown rice using

various rice populations (Tan et al, 2001; Yoshida et al,

2002; Wu et al, 2003; Aluko et al, 2004; Hu et al,

2004; Li et al, 2004; Li et al, 2006; Yu et al, 2006),

and only four have been reported in milled rice (Weng

et al, 2006). The likely reason is that the protein

content is low and not as easy to determine in milled

rice. Protein content is usually calculated from Kjedahl Received: 28 November 2010; Accepted: 21 February 2011 Corresponding author: HE Yu-qing ([email protected])

Rice Science, Vol. 18, No. 3, 2011 188

nitrogen, multiplied by the factor 5.95 (Sotelo et al,

1990), and this method can be used to determine

protein content but not protein quality (i.e. amino acid

content). In fact, there is only one report on QTL

analysis of amino acid content in rice grain to date

(Wang et al, 2008).

In this study, QTLs for 19 traits including protein,

17 amino acids, and total amino acid contents were

investigated simultaneously in a recombinant inbred

line (RIL) population using data from two years. This

study aimed to comprehensively characterize the

genetic basis of protein quality of rice, in order to

facilitate breeding of high-quality rice varieties.

MATERIALS AND METHODS

Rice materials and field arrangements

An RIL population consisting of 188 lines derived

from the cross of two indica varieties, Zhenshan 97B

and Delong 208, were used. Zhenshan 97B is an elite

maternal parent of hybrid Shanyou 63, which is

planted extensively throughout China, and Delong 208

is a local variety from Yunnan Province of Southwest

China with a number of characteristics desirable for

cooked rice quality.

The field experiments were conducted in the rice-

growing seasons of 2004 and 2005 at the experimental

farm of Huazhong Agricultural University, Wuhan,

China. The sowing dates were 25 May in both years.

Ten plants per line were transplanted in a single row

with 16.5 cm between plants and 26.4 cm between

rows. Field management essentially followed normal

agricultural practice with fertilizer applied (per hectare)

as follows: 48.8 kg N, 58.5 kg P and 93.8 kg K as the

basal fertilizer; 86.3 kg N at the tillering stage; 27.6

kg N at the booting stage. The lines were harvested

individually at maturity.

Preparation of milled rice flour

Ten plants in each row were threshed in bulk and

rough rice was air-dried and stored at room temperature

for three months and then stored at 4 ºC. Hulls were

removed from 50 g of rough rice from each line using

a huller (Houston, TX, USA) to yield brown rice. The

embryo and the bran layer were removed from the

brown rice by passing the grains through a miller.

Then the milled rice was further ground into flour

with a Udy Cyclone Sample Mill (Udy Corporation,

Co., USA), passed through a 0.5 mm sieve and stored

at -20 ºC until analysis.

Determination of protein content

Crude protein content was measured using the

Kjeldahl method. The chemical composition was

determined by standard methods (Cunniff, 1995):

Removing moisture by drying in an oven at 105 ºC for

1 h until constant weight; about 0.1 g of sample was

subjected to carbonization with 2 mL of H2SO4 and 1

mL of ddH2O for 12 h, then assimilation at 380 ºC by

an infrared ray digestion equipment (SCP science)

until the digestion solution is limpid. The content of

NH4 in the digestion solution was diluted to 1−10

mg/L. About 5 mL of the dilution was placed into an

auto-sampler cannulation and analyzed using a

segmented continuous flow analyzer (Futura Ⅱ

Alliance). The amount of total N present in each

sample (mg/L) was calculated using a standard sample.

Grain protein content was calculated from the

percentage of total N multiplied by a conversion

factor of 5.95 (Sotelo et al, 1990). The assay for each

line was conducted with three replicates.

Determination of amino acid content

About 100 mg of each sample was hydrolyzed

with 6 mL of 6 mol/L HCl in a sealed air-evacuated

tube at 110 ºC for 22 h. The hydrolysate was diluted to

50 mL, and then 1 mL of the diluted hydrolysate was

transferred to a centrifuge tube and put into a rotary

evaporator to remove HCl and water. The residue was

completely dissolved with vigorous shaking in 1 mL

of 0.02 mol/L HCl, followed by centrifugation at 14 000

r/min for 15 min. About 0.8 mL of supernatant was

put into an auto-sampler bottle and analyzed using an

amino acid auto-analyzer (Model L-8800 Hitachi).

The amount of each amino acid present in the sample

(mg/g) was calculated using a standard sample. The

assay for each line was conducted with three replicates.

Data analysis

The estimation of mean and variance for each

trait was based on the RIL families. The molecular

ZHONG Ming, et al. Identification of QTL Affecting Protein and Amino Acid Contents in Rice 189

linkage map was constructed using Mapmaker/EXP

3.0 (Lincoln et al, 1992). The average of the

measurements for each line in each year was used for

QTL analysis. The means of the traits were used to

identify QTL with QTLMapper 1.6 based on a mixed

linear model approach (Zhu and Weir, 1998). The

likelihood ratio (LR) value corresponding to P=0.005

was used as the threshold for claiming the presence of

putative main QTLs. The significance of QTL effects,

including additive effect, was further tested by

running the sub-menu of the Bayesian test (P<0.005).

The peak points of the LR in the linkage map were

taken as the putative positions of the effects, and

additive effects were taken from the points showing

the largest effect. The relative contribution of a

genetic component was calculated as the proportion of

phenotypic variance explained by that component in

the selected model.

RESULTS

Phenotypic variation in parents and population

Significant phenotypic differences were detected

between the parents using a t-test at the 0.01 probability

level for protein content, 17 amino acid contents and

total amino acid content. Zhenshan 97B had higher

values for protein content, all of the 17 amino acid

contents and total amino acid content than those of

Delong 208 (Table 1). The RIL population showed

transgressive segregation in both directions for all

amino acid contents and total amino acid content. This

might indicate that the protein content and each amino

acid content are quantitative traits, and the positive

alleles could come from either parent.

Correlation analysis

Significantly positive correlations were found

between 2004 and 2005 for the same trait (Table 2).

The correlation of the protein content was the least,

followed by tyrosine, glysine, total amino acid,

threonine, alanine and glutamic acid. The relatively

lower heritability of protein content in milled rice

implied that the heritability of protein content was

more sensitive to environmental change than those of

amino acid contents. Addtionally, significantly

positive correlations were found between all amino

acid contents and protein content.

Linkage map

A linkage map was constructed with 180 SSR

markers, spanning a total of 1 817.2 cM, with an

average interval of 11.8 cM between adjacent markers

(Fig. 1). The marker orders in the map agreed well

with those of Temnykh et al (2000 and 2001).

QTL for protein content

QTLs for protein content (P=0.005) in milled rice

are presented in Table 3. Four QTLs were detected for

protein content in 2004 and 2005, accounting for

Table 1. Means of protein and amino acid contents of milled rice of mapping population in 2004 and 2005. %

2004 2005 Trait

Population range Mean±σ Zhenshan 97B Delong 208 Population range Mean±σ Zhenshan 97B Delong 208Protein 56.90–97.56 78.58±7.62 85.25±2.20 65.62±0.86 43.91–93.29 69.39±10.19 74.29±1.73 65.34±1.90Total amino acid 102.39–161.03 125.95±11.29 135.20±0.87 118.93±0.95 83.92–123.80 104.05±9.35 112.15±1.91 98.09±1.63Lysine 3.54–5.80 4.52±0.40 5.09±0.04 4.28±0.04 3.18–4.48 3.79±0.28 4.38±0.06 3.61±0.06Isoleucine 4.18–6.81 5.23±0.49 6.17±0.13 5.45±0.03 3.31–5.13 4.26±0.40 4.57±0.09 4.02±0.06Leucine 8.71–16.34 11.10±1.14 11.76±0.10 10.51±0.05 6.63–10.90 8.96±0.88 9.85±0.16 8.57±0.17Methionine 1.02–3.55 2.41±0.39 2.26±0.07 2.20±0.03 1.83–2.94 2.37±0.24 2.37±0.04 2.44±0.10Phenylalanine 5.10–9.24 7.14±0.69 8.11±0.06 7.09±0.02 4.53–7.10 5.86±0.57 6.52±0.12 5.50±0.09Threonine 3.68–5.80 4.59±0.38 4.77±0.05 4.30±0.03 3.00–4.52 3.84±0.33 4.17±0.08 3.71±0.05Valine 5.88–9.47 7.39±0.69 8.19±0.16 7.36±0.03 4.69–7.31 6.06±0.55 6.66±0.14 5.82±0.09Aspartic acid 9.22–15.09 11.57±1.07 12.81±0.15 10.73±0.07 7.62–11.29 9.31±0.87 11.02±0.13 8.76±0.13Alanine 5.70–9.23 7.23±0.65 7.61±0.06 6.82±0.05 4.11–7.39 5.74±0.61 6.64±0.11 5.69±0.10Tyrosine 2.92–8.56 5.27±0.80 5.15±0.12 4.38±0.26 3.22–5.35 4.31±0.44 4.17±0.12 4.09±0.33Serine 5.10–9.10 6.44±0.66 6.48±0.05 5.79±0.01 4.05–6.31 5.24±0.47 5.50±0.09 4.83±0.06Glycine 4.54–7.26 5.63±0.51 6.12±0.05 5.33±0.04 3.73–6.03 4.91±0.47 5.25±0.07 4.40±0.07Cystine 2.21–3.86 2.74±0.25 2.87±0.29 2.35±0.08 2.41–3.50 2.94±0.22 1.19±0.77 2.81±0.07Glutamic acid 20.26–32.48 25.57±2.43 27.21±0.24 24.42±0.14 14.98–25.42 20.80±2.14 22.65±0.38 19.47±0.32Histidine 2.55–4.45 3.23±0.36 3.86±0.03 3.47±0.04 2.10–3.19 2.61±0.22 2.80±0.05 2.40±0.05Arginine 8.24–13.80 10.30±1.03 11.00±0.12 9.31±0.20 6.61–10.17 8.40±0.83 9.62±0.11 7.74±0.21Proline 4.37–6.85 5.43±0.51 5.73±0.05 5.13±0.06 3.34–5.48 4.51±0.43 4.80±0.07 4.23±0.07


49.2% and 27.0% of the phenotypic variation in each

year, respectively (Table 3, Fig. 1). Two QTLs, qPr1

and qPr7, were detected in both years. qPr1 is located

at the RM493−RM562 interval on chromosome 1 and

qPr7 at the RM445−RM418 interval. The allele of

qPr1 from Zhenshan 97B decreased the protein

content whereas qPr7 increased the protein content of

milled rice.

QTLs for amino acid content

A total of 48 and 64 QTLs were detected for amino

acid content in 2004 and 2005, respectively (Table 4,

Fig. 1). Most QTLs co-localized, forming 12 QTL

clusters on the chromosomes, and three QTL clusters

were detected in both years. qAa1 located at RM493−

RM562 on chromosome 1, qAa7 located at MRG186−

MRG4499 on chromosome 7, and qAa9 located at

RM328−RM107 on chromosome 9. The Zhenshan

97B alleles of qAa1 and qAa9 decreased the amino

acid content whereas the Zhenshan 97B allele of qAa7

increased the amino acid content of milled rice.

Table 2. Coefficients of pairwise correlations of rice grain protein and amino acid contents in Zhenshan 97B/Delong 208 population between 2004 and 2005 (r0.05=0.2050, r 0.01=0.2673).

Trait Asp Lys Thr Ile Met Val Leu Ala Tyr Phe Ser Gly Cys Glu His Arg Pro Total Protein

Asp 0.436 a Lys 0.934 b 0.453 0.870 c Thr 0.920 0.853 0.490 0.958 0.879 Ile 0.943 0.880 0.926 0.485 0.942 0.844 0.958 Met 0.541 0.514 0.612 0.629 0.446 0.717 0.636 0.759 0.784 Val 0.917 0.844 0.912 0.975 0.667 0.464 0.913 0.816 0.939 0.984 0.757 Leu 0.878 0.826 0.865 0.884 0.551 0.855 0.442 0.949 0.828 0.970 0.978 0.750 0.969 Ala 0.966 0.875 0.928 0.953 0.551 0.922 0.894 0.498 0.901 0.831 0.907 0.899 0.680 0.849 0.906 Tyr 0.441 0.402 0.531 0.562 0.605 0.647 0.422 0.427 0.248 0.771 0.642 0.786 0.783 0.708 0.779 0.783 0.677 Phe 0.912 0.810 0.899 0.937 0.582 0.928 0.747 0.928 0.597 0.472 0.957 0.834 0.977 0.982 0.764 0.971 0.993 0.903 0.797 Ser 0.835 0.735 0.764 0.832 0.578 0.816 0.734 0.835 0.521 0.835 0.471 0.940 0.841 0.976 0.916 0.709 0.895 0.947 0.868 0.800 0.953 Gly 0.978 0.920 0.927 0.953 0.560 0.924 0.887 0.985 0.408 0.915 0.830 0.283 0.823 0.732 0.868 0.867 0.685 0.905 0.886 0.639 0.703 0.888 0.849 Cys 0.682 0.566 0.758 0.724 0.711 0.718 0.663 0.736 0.604 0.680 0.688 0.738 0.366 0.646 0.582 0.738 0.699 0.666 0.733 0.691 0.539 0.842 0.716 0.741 0.718 Glu 0.934 0.781 0.909 0.926 0.580 0.908 0.818 0.955 0.473 0.938 0.894 0.936 0.765 0.532 0.963 0.827 0.980 0.972 0.756 0.961 0.993 0.901 0.785 0.991 0.961 0.887 0.704 His 0.537 0.490 0.638 0.649 0.586 0.666 0.511 0.517 0.725 0.654 0.635 0.523 0.688 0.607 0.320 0.912 0.925 0.935 0.915 0.679 0.908 0.925 0.861 0.737 0.919 0.902 0.831 0.675 0.921 Arg 0.960 0.900 0.900 0.938 0.622 0.936 0.874 0.949 0.505 0.893 0.811 0.968 0.735 0.906 0.513 0.421 0.956 0.874 0.975 0.971 0.770 0.962 0.976 0.899 0.828 0.981 0.950 0.883 0.729 0.976 0.939 Pro 0.935 0.815 0.881 0.898 0.517 0.855 0.854 0.940 0.340 0.851 0.879 0.935 0.771 0.942 0.476 0.909 0.483 0.954 0.856 0.984 0.955 0.768 0.922 0.967 0.934 0.763 0.970 0.956 0.825 0.689 0.973 0.915 0.961 Total 0.962 0.874 0.947 0.976 0.666 0.969 0.888 0.965 0.600 0.950 0.890 0.962 0.773 0.964 0.676 0.955 0.926 0.490 0.969 0.862 0.987 0.981 0.776 0.967 0.990 0.905 0.819 0.993 0.963 0.883 0.730 0.993 0.937 0.991 0.977 Protein 0.792 0.749 0.804 0.783 0.470 0.791 0.754 0.790 0.466 0.784 0.660 0.791 0.559 0.760 0.442 0.816 0.750 0.808 0.207

0.646 0.530 0.659 0.701 0.564 0.726 0.711 0.552 0.522 0.712 0.643 0.720 0.482 0.705 0.643 0.682 0.633 0.687 a The diagonal indicates trait correlation coefficient between two years; b Coefficient in 2004; c Coefficient in 2005.

Table 3. Putative QTLs identified for protein content of milled rice from 2004 and 2005.

QTL Flanking marker a LOD A b Var (%) c

2004 qPr1 RM493–RM562 4.5 -3.77 9.2 qPr2 RM154–RM233A 1.9 2.47 4.0 qPr7 RM445–RM418 7.6 6.31 25.9 qPr8 RM149–RM433 3.5 -3.95 10.1

2005 qPr1 RM493–RM562 4.1 -5.26 9.6 qPr4 RM349–MRG113 2.2 3.85 5.1 qPr7 RM445–RM418 3.0 4.57 7.2 qPr9 RM460–RM257 2.7 -3.85 5.1

a The bold format means the QTL were detected in two years; b Positive values of additive effects indicate the alleles from Zhenshan 97B with increasing effects; c Percentage of variation explained.


In 2004, the number of QTLs for each amino acid

ranged from 1 to 4, and the phenotypic variance

explained by each QTL ranged from 5.3% (Thr) to

43.7% (Cys). Of the four QTLs identified for lysine

content, the QTLs at the intervals of MRG141−

MRG6383 and RM443−RM297 on chromosome 1

controlled lysine only, whereas the other two QTLs

controlled the content of other amino acids as well.

Most QTLs co-localized, forming 12 QTL clusters on

the chromosomes, with six controlling only one trait,

and the other six controlling multiple traits. The QTL

clusters at the intervals of MRG186−MRG4499 and

RM493−RM562 included 14 and 11 members,

respectively. At most loci, the alleles from Zhenshan

97B increased the amino acid content, which might

explain a high phenotypic value of Zhenshan 97B.

In 2005, the number of QTLs detected for each

trait ranged from 2 to 5, and the phenotypic variance

explained by each QTL ranged from 4.2% (Lys) to

36.1% (Asp). Of four QTLs identified for lysine

content, two at the intervals of MRG2180−RM16 and

RM328−RM107 controlled lysine only, whereas the

others affected the contents of additional amino acids.

Most QTLs co-localized, forming 17 QTL clusters on

the chromosomes with nine affecting only one amino

acid content and eight affecting the contents of

multiple amino acid. The QTL clusters at the intervals

of MRG186−MRG4499 and RM493−RM562 included

Fig. 1. Putative QTL identified for protein and amino acid contents of milled rice. The bold format means the QTLs were detected in both 2004 and 2005, and the normal and italic format mean the QTLs were detected in 2004

and 2005, respectively. Positive values of additive effects indicate the alleles from Zhenshan 97B with increasing effects while negative values indicate the alleles from Delong 208 with increasing effects.


Zhenshan 97B at most loci contributed to an increase

of the amino acid content.

DISCUSSION

The quantity and quality of protein are the main

attributes that determine the nutritional quality of rice

grains. Protein quality is largely specified by the ratio

of essential amino acid content to protein content. The

present study has characterized the genetic basis of

protein content, 17 amino acid contents and total

amino acid content using a mapping population from

the cross between Zhenshan 97B and Delong 208. In

this study, 48 and 64 QTLs were identified in 2004

and 2005, respectively, for the phenotypes of protein,

17 amino acid and total amino acid contents. Most

QTLs co-localized, forming 29 QTL clusters on the

chromosomes, with three major ones detected in both

years and mapped on chromosomes 1, 7 and 9.

Interestingly, the major QTLs for protein and 17

amino acid contents are located in the same genomic

region, so that the chromosomal segment from Zhenshan

97B increases protein content and at the same time

greatly increases the amino acid content. Free amino

Table 4. Putative QTLs identified for amino acid content of milled rice from 2004 and 2005.

Chr-I a Flanking marker b QTL Trait LOD Additive effect c Var (%) d

2004 1-3 MRG141–MRG6383 Lys 4.1 0.12 9.1 1-12* RM493–RM562 qAa1 Asp/Thr/Glu/Gly/

Ala/Val/Leu/Phe/ Arg/Pro/Taa

9.6/5.3/6.6/5.0/ 6.6/8.6/7.3/8.1/ 6.1/6.9/12.3

-0.49/-0.14/-0.96/-0.17/ -0.24/-0.28/-0.50/-0.23/ -0.32/-0.21/-4.97

21.7/11.3/15.3/12.4/ 16.0/18.1/20.7/17.3/ 13.0/18.1/24.2

1-13 RM562–MRG4638 Ser/Ile/Lys 5.2/10.9/7.5 0.24/0.19/0.17 12.9/22.9/17.5 1-14 MRG4638–RM150A Thr/His 2.7/3.8 -0.09/-0.12 5.3/9.7 1-21 RM443–RM297 Lys 3.0 -0.10 6.3 2-1 RM154–RM233A Arg 3.3 0.25 7.5 7-5 MRG7006–MRG186 Leu 2.9 0.39 13.1 7-6* MRG186–MRG4499 qAa7 Asp/Thr/Ser/Glu/Gly/

Ala/Cys/Val/Met/ Ile/Phe/Arg/Pro/Taa

3.3/6.2/4.6/4.0/3.7/ 3.0/3.4/8.4/3.2/ 5.2/3.7/6.4/4.3/7.6

0.28/0.15/0.23/0.82/0.15/ 0.16/0.07/0.28/0.13/ 0.14/0.18/0.36/0.17/3.83

7.2/13.7/12.4/10.8/9.4/ 7.2/7.4/18.7/10.8/ 11.9/10.0/16.4/12.4/14.4

7-7 MRG4499–RM445 His 3.2 0.16 19.6 9-12 RM553–RM201 Glu/Ala/Cys 3.3/3.0/11.2 -0.71/-0.16/-0.16 7.9/6.9/43.7 9-14* RM328–RM107 qAa9 Asp/Thr/Ser/Gly/Val/

Ile/Phe/Lys/Taa 5.6/4.8/3.8/3.4/4.6/ 4.4/4.0/6/8.1

-0.34/-0.12/-0.19/-0.13/-0.18/ -0.11/-0.16/-0.13/-3.70

10.8/8.4/8.5/7.4/7.6/ 8.0/8.0/10.3/13.2

11-5 RM536–RM287 Tyr 3.2 -0.21 10.1 2005

1-8 RM243–RM577 Val/Ile/Arg 6.3/3.4/3.8 0.19/0.10/0.22 15.7/8.0/7.9 1-9 RM577–RM312 Asp/Thr/Glu/Ala/

Leu/Phe/Pro/Taa 4.8/5.3/4.3/1.8/ 4.4/5.2/3.5/5.7

0.26/0.09/0.56/0.12/ 0.26/0.18/0.10/2.75

10.3/9.8/9.4/4.6/ 11.6/12.2/6.8/9.5

1-11 MRG2412–RM493 Gly/Cys/Met/ Tyr/Lys

2.8/3.8/3.5/ 2.2/2.2

-0.14/-0.06/-0.05/ -0.11/-0.07

7.5/8.6/10.7 6.2/4.9

1-12* RM493–RM562 qAa1 Asp/Thr/Ser/Glu/ Ala/Val/Ile/Leu/Phe/ His/Arg/Pro/Taa

15.2/12.4/2.8/12.4/ 6.4/12.2/9.0/9.8/12.8/3.9/13..8/12.2/16.5

-0.48/-0.16/-0.09/-1.00/ -0.22/-0.27/-0.17-0.40/ -0.31/-0.07/-0.41/-0.19/-5.26

36.1/26.6/5.6/30.1/ 16.4/32.0/22.4/28.1/ 33.5/9.8/28.1/25.9/31.7

2-7 RM475–RM183 Thr 6.6 -0.11 11.4 3-7 MRG2180–RM16 Lys 3.2 0.09 8.0 4-5 MRG388B–RM241 Asp/Thr/Cys/

Arg/Pro 4.4/5.4/5.0/ 4.7/6.4

0.23/0.09/0.07/ 0.23/0.13

8.1/9.1/12.5/ 9.0/11.5

4-6 RM241–RM303 Taa 6.1 2.86 10.3 4-7 RM303–RM349 Ser/Gly 6.0/2.8 0.13/0.13 11.4/6.2 4-8 RM349–MRG113 His 2.3 0.05 5.1/5.2 6-2 RM586–MX21 Ser 2.3 -0.09 5.0 6-6 RM584–RM314 Ala 3.4 -0.15 7.8 7-5 MRG7006–MRG186 Gly/Lys/His 4.3/4.9/5.3 0.24/0.16/0.13 21.9/26.6/28.3 7-6* MRG186–MRG4499 qAa7 Asp/Thr/Ser/Glu/Ala/

Cys/Val/Ile/Leu/Tyr/ Phe/Arg/Pro/Taa

7.7/10.1/7.8/10.3/6.6/7.1/9.6/7.7/7.7/4.3/ 10.7/12.3/10.9/14.7

0.31/0.12/0.18/0.91/0.22/ 0.09/0.21/0.15/0.32/0.16/ 0.25/0.37/0.17/4.68

14.6/16.8/20.5/24.6/16.3/19.7/19.8/17.8/18.0/12.4/22.0/23.6/22.3/27.5

9-7 RM105–RM460 Met 3.4 -0.05 10.4 9-14* RM328–RM107 qAa9 Lys 2.0 -0.06 4.2 11-3 RM4B–RM202 Ile 2.3 -0.08 4.5

a Chr-I, The interval (I) on the chromosome (Chr) where QTL located; *, Detected in both years; b Flanking marker means the interval of QTL;c Positive values of additive effects indicate the alleles from Zhenshan 97B with increasing effects whereas negative values indicate the alleles from Delong 208 with increasing effects; d The percentage of the phenotypic variation explained by QTL.


acid contents were also contributed to the total protein

content in the rice endosperm, but the content of free

amino acid is very low. It is about 0.69% in the brown

rice and 0.1%−0.2% in the milled rice of total protein

(Juliano, 1985; Wang et al, 2008). Our results showed

that both the mapping of protein and amino acids

contents were validated by each other.

Compared with the results of previous studies, it

is likely that qAa1 for protein and amino acid contents

on chromosome 1 detected in our study is the same

locus as the one reported by Aluko et al (2004).

Likewise, qAa7 might be the same as previously

reported locus (Wu et al, 2003; Hu et al, 2004; Wang

et al, 2008). However, the QTL for protein content

located near the Wx locus on chromosome 6 (Tan et al,

2001; Weng et al, 2006; Yu et al, 2006) was not

detected in our study.

Research has shown that cooking and eating

quality is largely controlled by the Wx locus on

chromosome 6 (Tan et al, 1999; Septiningsih et al,

2003; Tian et al, 2005; Fan et al, 2006; Wang et al,

2007), whereas some research suggests that proteins

in rice grains influence cooking properties (Martin and

Fitzgerald, 2002). Wang et al (2007) used the same

population to analyze the genetic basis of the cooking

and eating quality of rice as reflected by 17 traits.

They found that two major clusters, which

corresponded to the Wx and Alk loci on chromosome 6,

controlled the traits. However, in our research, only a

very small effective QTL of serine content in milled

rice is located in the Wx region. With respect to grain

quality improvement, it is clear that grain eating

quality and nutritional quality of hybrid rice can be

independently modified, since the major QTLs for

these two traits are located on different chromosomes.

Wang et al (2001a) identified four significant loci

that accounted for about 46% of the phenotypic

variance of free amino acid (FAA) content in the

endosperm. According to their QTL mapping, one of

these loci on the long arm of chromosome 2 was

coincident with the location of genes encoding a

mono-functional aspartate kinase 2 and a bi-functional

aspartate kinase-homo-serine dehydrogenase-2. They

suggested that an alteration of amino acid and carbon

metabolism led to overproduction and accumulation

of FAA in opaque-2 mutants and that aspartate kinase

2 was a candidate gene for the QTL influencing FAA

content in maize endosperm (Wang et al, 2001b).

They also characterized the mono-functional aspartate

kinase genes in maize and showed that these genes

related to FAA content in the endosperm (Wang et al,

2007). Based on those results, we examined whether

some genes encoding storage proteins or controlling

amino acid biosynthesis, assimilation and transport

were located in QTL regions identified in our study.

According to the BLAST search for all genes known

to encode storage proteins or proteins controlling

amino acid biosynthesis, assimilation or transport,

many of them were located in the QTL regions in our

research (Table 5, Fig 1). A wide coincidence was

Table 5. Coincidence of the loci (genes) involved in amino acid metabolism with the QTL for protein and amino acid profiles.

Chr-I Code Description of the gene function Genomic cloneLocation

(Mb) Marker

Amino acid assimilation and transfer 2-1 Asp AT Aspartate aminotransferase, putative, expressed AC105731 4.90 RM154, RM2420 1-3 CGS1 Oryza sativa cystathionine gamma-synthase (CGS1) mRNA AP004869 5.63 MRG141, RM12671

Amino acid biosynthesis 1-19 Thr synthase Threonine synthase, chloroplast precursor, putative AP003248 29.00 RM297, RM1244 1-19 Glu synthetase Glutamate synthase, chloroplast precursor, putative AP004363 28.40 RM443, RM5625 4-7 Gln synthetase Glutamine synthetase, chloroplast precursor, putative AP004087 33.40 RM349, RM8218 4-7 Shikimate kinase Shikimate kinase, chloroplast precursor, putative AL606649 32.40 RM349, RM17532 3-7 GluA-3 GluA-3 gene for glutelin AC1333982 17.80 RM16, RM2420 1-8 DHDS Oryza sativa dihydrodipicolinate synthase gene AL662952 29.70 RM243, RM3474 7-4 Putative AK Aspartate kinase family protein AP006343 11.87 MRG2224, RM214

Storage protein 1-3 RP5 Prolamin AP004943 5.73 MRG141, RM180 1-3 RP6 Prolamin AP003943 5.72 MRG141, RM180 7-7 LMW globulin Low molecular weight globulin AP004002 6.26 RM445, RM3917 7-7 RA5 Albumin AP003963 6.33 RM445, RM5673 7-7 RA14 Albumin AP004002 6.26 RM445, RM5672 7-7 RAG2(RA17) Albumin AP004002 6.25 RM445, RM3917

Chr-I, The interval (I) on the chromosome (Chr) where QTL located.


found between the QTL detected in our study and the

loci involved in amino acid metabolism pathways for

N assimilation and transport, or protein biosynthesis. For

example, in the interval of seven QTL clusters of our

results, 15 genes including two genes for N

assimilation and transfer, seven genes for amino acid

metabolism, and six genes for protein biosynthesis

were found. There were several genes corresponding

to one QTL. Interestingly, there were lots of genes in

the region of qAa7, such as the gene encoding putative

aspartate kinase, low molecular weight globulin and

albumin. The results might be helpful for gene cloning

by the candidate gene method.

In summary, the results from this study can be

useful to improve nutritional quality of rice grain by

molecular marker-assisted selection. The closely

linked markers that flank the identified QTLs can be

used to aid quality selection in breeding programs.

And the results of the coincidence between the QTL

detected in our study and the loci involved in amino

acid metabolism pathways in N assimilation, transfer,

or protein biosynthesis might be helpful for gene

cloning by the candidate gene method.

ACKNOWLEDGEMENTS

This work was supported by grants from the

National Program on the Development of Basic

Research (Grant No. 2011CB100200), the National

Program of High Technology Development (Grant No.

2010AA101801), the National Program of Plant

Transgenic Breeding (Grant No. 2008ZX08009-003)

and the National Natural Science Foundation of China

(Grant No. 30671114).

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