Genes controlling grain chalkiness in rice

With rising living standards,there is an increasing demand for high-quality rice.Rice quality is mainly defined by milling quality,appearance quality,cooking and eating quality,and nutrition quality.Among them,chalkiness is a key trait for appearance quality,which adversely affects cooking and eating quality,head rice yield,and commercial value.Therefore,chalkiness is undesirable,and reducing chalkiness is a major goal in rice quality improvement.However,chalkiness is a complex trait jointly influenced by genetic and environmental factors,making its genetic study and precision improvement a huge challenge.With the rapid development of molecular techniques,much knowledge has been gained about the genes and molecular networks involved in chalkiness formation.The present review describes the major environmental factors affecting chalkiness and summarizes the quantitative trait loci(QTL)associated with chalkiness.More than 150 genes related to chalkiness formation have been reported.The functions of the genes regulating chalkiness,primarily those involved in starch synthesis,storage protein synthesis,transcription regulation,organelle development,grain shape regulation,and hightemperature response,are described.Finally,we identify the challenges associated with genetic improvement of chalkiness and suggest potential strategies.Thus,the review offers insight into the molecular dynamics of chalkiness and provides a strong basis for the future breeding of high-quality rice varieties.

GL9 from Oryza glumaepatula controls grain size and chalkiness in rice

Grain size is a key factor influencing grain yield and appearance quality in rice.We identified twelve quantitative trait loci(QTL)for grain length(GL),nine for grain width(GW),and nine for 1000-kernel weight(TKW)using GLU-SSSLs,which are single-segment substitution lines with Oryza glumaepatula as donor parent and Huajingxian 74(HJX74)as recipient parent.Among the QTL,qGL1-2,qGL1-4,qGL9-2,qGW2-2,qGW9-1 and qTKW9-2 contributed to high grain yield.GL9 was identified as a candidate gene for qGL9-2 by map-based cloning and sequencing,and is a novel allele of GS9.The kernel of NIL-gl9was slenderer and longer than that of HJX74,and the TKW and grain yield per plant of NIL-gl9 were higher than those of HJX74.The proportion of grain chalkiness of NIL-gl9 was much lower than that of HJX74.Thus,gl9 increased grain yield and appearance quality simultaneously.Three pyramid lines,NIL-gs3/gl9,NIL-GW7/gl9 and NIL-gw8/gl9,were developed and the kernel of each was longer than that of the corresponding recipient parent lines.The gl9 allele may be beneficial for breeding rice varieties with high grain yield and good appearance quality.

Identification and characterization of the chalkiness endosperm gene CHALK-H in rice(Oryza sativa L.)

Chalkiness is one of the most important agronomic traits in rice breeding,which directly affects the quality of rice seed.In this study,we identified a chalkiness endosperm mutant,chalk-h,from N-methyl-N-nitrosourea(MNU)-induced japonica rice cultivar Hwacheong(HC).Compared with wild type(WT)-HC,chalk-h showed severe chalkiness in the endosperm,yellowish green leaves,as well as reduced plant height.Scanning electron microscopy(SEM)analysis showed that starch grains in the chalk-h mutant were irregular in size and loosely arranged,with large gaps between granules,forming ovoid or orbicular shapes.MutMap analysis revealed that the phenotype of chalk-h is controlled by a single recessive gene LOC_Os11g39670 encoding seryl-tRNA synthetase,which is renamed as CHALK-H.A point mutation occurs in chalk-h on the sixth exon(at nucleotide 791)of CHALK-H,in which adenine(A)is replaced by thymidine(T),resulting in an amino acid codon change from glutamine(Glu)to valine(Val).The chalk-h mutant exhibited a heat-sensitive phenotype from the 3-leaf stage,including yellow-green leaves and reduced pigment content.The transcriptional expression of starch synthesis-related genes was down-regulated in the chalk-h mutants compared to WT-HC at different grain-filling stages.With an increase in temperature,the expression of photosynthesis-related genes was down-regulated in the chalk-h mutant compared to WT-HC.Overexpression of CHALK-H rescued the phenotype of chalk-h,with endosperm and leaf color similar to those of WT-HC.Our findings reveal that CHALK-H is a causative gene controlling chalkiness and leaf color of the chalk-h mutant.CHALK-H is the same gene locus as TSCD11,which was reported to be involved in chloroplast development under high temperature.We suggest that CHALK-H/TSCD11 plays important roles not only in chloroplast development,but also in photosynthesis and starch synthesis during rice growth and development,so it has great application potential in rice breeding for high quality and yield.

Quantitative Trait Loci for Grain Chalkiness and Endosperm Transparency Detected in Three Recombinant Inbred Line Populations of Indica Rice

Quantitative trait loci（QTL） for percentage of chalky grain,degree of chalkiness,and endosperm transparency were detected using 3 recombinant inbred line populations derived from crosses between parental lines of commercial three-line hybrids of indica rice.Two of the populations showed great variations on heading date,and the other had a short range of heading date variation.A total of 40 QTLs were detected and fell into 15 regions of 10 chromosomes,of which 5 regions were detected for 1 or more same traits over different populations,2 were detected for different traits in different populations,3 were detected for 2 or all the 3 traits in a single population,and 5 were detected for a single trait in a single population.Most of these QTLs have been reported previously,but a region located on the long arm of chromosome 10 showing significant effects in all the 3 populations has not been reported before.It was shown that a number of gene cloned,including the Wx and Alk for the physiochemical property of rice grain,and GW2,GS3 and GW5 for grain weight and grain size,could have played important roles for the genetic control of grain chalkiness in rice,but there are many more QTLs exerting stable effects for rice chalkiness over different genetic backgrounds.It is worth paying more attentions to these regions which harbor QTL such as the qPCG5.2/qDC5.2/qET5.2 and qPCG10/qDC10/qET10 detected in our study.Our results also showed that the use of segregating populations having high-uniform heading date could greatly increase the efficiency of the identification of QTL responsible for traits that are subjected to great environmental influence.

Effects of Chalkiness on Cooking,Eating and Nutritional Qualities of Rice in Two indica Varieties

An experiment was conducted to investigate the effects of chalkiness on cooking, eating and nutrition qualities of rice using Gangyou 527 （indica hybrid rice） and Zhaiyeqing 8 （conventional indica rice） as materials. Compared with the milled rice without chalkiness, amylose content, final viscosity, setback and consistence increased significantly, while gel consistence, peak viscosity and breakdown decreased remarkably and other RVA values did not have significant changes in the chalky rice of Gangyou 527. The differences in the above indices between the chalky and non-chalky rice was not remarkable in Zhaiyeqing 8. The crude protein content of chalky rice in Zhaiyeqing 8 was significantly lower than that of non-chalky rice, but there was no remarkable change between the chalky and non-chalky rice in Gangyou 527. Glutelin content fell significantly, and albumin, globulin, prolamine and lysine contents did not change significantly in chalky rice compared with non-chalky rice in the two varieties.

Reduced grain chalkiness and its possible physiological mechanism in transgenic rice overexpressing L-GalLDH

Chalkiness is one of the key factors determining rice quality and price. Ascorbic acid(Asc) is a major plant antioxidant that performs many functions in plants. L-Galactono-1,4-lactone dehydrogenase(L-Gal LDH, EC1.3.2.3) is an enzyme that catalyzes the final step of Asc biosynthesis in plants. Here we show that the L-Gal LDH-overexpressing transgenic rice, GO-2,which has constitutively higher leaf Asc content than wild-type(WT) plants, exhibits significantly reduced grain chalkiness. Higher foliar ascorbate/dehydroascorbate(Asc/DHA)ratios at 40, 60, 80, and 100 days of plant age were observed in GO-2. Further investigation showed that the enhanced level of Asc resulted in a significantly higher ribulose-1,5-bisphosphate(Ru BP) carboxylase/oxygenase(Rubisco) protein level in GO-2 at 80 days. In addition, levels of abscisic acid(ABA) and jasmonic acid(JA) were lower in GO-2 at 60, 80, and100 days. The results we present here indicate that the enhanced level of Asc is likely responsible for changing redox homeostasis in key developmental stages associated with grain filling and alters grain chalkiness in the L-Gal LDH-overexpressing transgenic by maintaining photosynthetic function and affecting phytohormones associated with grain filling.

Rice Grain Chalkiness Is Negatively Correlated with Root Activity During Grain Filling

Field and pot experiments were conducted using indica rice varieties with differential chalkiness. There were significant differences in root activity among the varieties. The percentages of chalky grains and chalky area were both negatively correlated with root activity expressed as α-naphthylamine oxidation ability （RA） per gram of fresh root （RAfw） , RA per spikelet （RAgrn）, or RA per sink capacity （RAsink）. The RAsink was more closely related to chalkiness than RAt, and RAgrn when varieties differed greatly in panicle size and grain weight. Application of NO3^--N fertilizer at heading resulted in higher root activity and reduced chalkiness. Application of 30 mg/L NaN3 （respiration inhibitor） resulted in reduced root activity and increased chalkiness for one variety ＇GD9501＇, but for the other variety ＇Qinluai＇ was in reverse. The percentages of chalky grains and chalky area were negatively correlated with root activity at 10 days after heading under different chemical treatments （r=-0.8567^＊ and r=-0.9211^＊＊, respectively）.

Chalkiness Characters and Scanning Electron Microscope Observation of Rice Grain Endosperm of Japonica Varieties in Southern Henan

Chalkiness characters affect not only the grain appearance,milling,eating and cooking qualities but also the grain nutritional quality in rice,thus it is one of the most important traits in rice. It is very important for us to investigate the relation of the chalkiness formation and the development of endosperm structure and starch granule of different rice varieties. Here,we have investigated the chalkiness characters such as chalkiness rate,chalkiness degree and chalkiness area in 15 japonica rice varieties from southern Henan. Furthermore,the endosperm structure and starch granules of rice grain were also observed with scanning electron microscope. The results showed that the 15 japonica varieties have a significantly linear relationship between the chalkiness rate and chalkiness degree. Among the varieties,the biggest difference is the chalkiness rate,the second is the chalkiness area,and the last is the chalkiness degree. Moreover,there is a certain correlation between the distribution of starch granules,the arrangement of endosperm cells and the occurrence of grain chalkiness in the different rice varieties. For the same variety,the starch granules of chalky and non-chalky grains have obvious difference,while the starch granules from the transparent part of chalky rice and non-chalky rice do not have significant difference. The results would provide useful references for the improvement of grain quality in rice.

Genetic separation of chalkiness by hybrid rice of Huanghuazhan and CS197

The present study focused on the segregation of the percentage of grains with chalkiness(PGWC),using Huanghuazhan as the female parent and CS197 as the male parent to construct the hybrid rice F2 population.Molecular markers were used for genotype analysis among the extremely low and extremely high PGWC individuals from the F2 population.The results revealed that the genotypes of 10 extremely low PGWC individuals were 80.00%and 3.33%,which is identical to Huanghuazhan and CS197,respectively.The heterozygotes accounted for 16.67%.On the contrary,the genotypes of 10 extremely high PGWC individuals were 37.78%and 32.22%,which is identical to Huanghuazhan and CS197,respectively.The heterozygotes accounted for 30.00%.The extremely low and extremely high PGWC individuals had high coincidence with the corresponding F2:3 family lines in phenotype and genotype.Scanning electron microscopy(SEM)images showed that the shape and spacing of starch granules in the extreme low and high PGWC individuals corresponded to Huanghuazhan and CS197,respectively.Correlation analysis showed that the F2 individuals showed a normal distribution trending towards Huanghuazhan.The broad heritability sense was 26.31%,and the narrow heritability sense was 17.21%.The results of cluster analysis showed that the range of PGWC variations of group III based on phenotype varied from 5.66%to 50.00%with an average of 25.46%,and group II results based on genotype varied from 14.34%to 60.67%with an average of 32.33%.Groups II and III shared 28 identical individuals.

A method to measure the rice kernel chalkiness objectively

Rice kernel chalkiness is an impor-tant quality character.Being the un-transparent portions in grain en-dosperm,chalkiness iS always mea-sured by some subjective eye-judgingmethods domestically and interna-tionally.Results measured by suchmethods aye subjective,inaccurate,and unstable.This research is in-

QTL Analysis of Chalkiness in Rice

A population of 150 recombination inbred lines （RILs） derived from the cross between rice varieties V20B and CPSLO17, was applied to locate the QTLs related to chalkiness traits and evaluate their genetic effects. A genetic linkage map was constructed based on 8 602 SLAF （specific-locus amplified fragment） markers, combine with the chatkiness traits of the tested lines. Four QTLs that related to chalkiness were detected using MapQTL 5 software, named qC-5a, qC-5b, qC-5c and qC-5d. The LOD threshold values of qC-5a, qC-5b, qC-5c and qC-5d were 4.02, 4.09, 3.94 and 4.1, respectively, explaining 11.6%, 11.8%, 11.2% and 11.8% of the observed phenotypic variance. All the four detected QTL alleles came from Iow-chalkiness parent V20B.

AMMI analysis of interaction of genotype and environment in chalkiness of indica rice

Chalkiness is an unpleasant trait for rice con-sumer,which is known to be controlled geneti-cally and affected by environment during grainmaturing.We used the model of Additive Main

OsNLP3 enhances grain weight and reduces grain chalkiness in rice

Grain weight,a key determinant of yield in rice(Oryza sativa L.),is governed primarily by genetic factors,whereas grain chalkiness,a detriment to grain quality,is intertwined with environmental factors such as mineral nutrients.Nitrogen(N)is recognized for its effect on grain chalkiness,but the underlying molecular mechanisms remain to be clarified.This study revealed the pivotal role of rice NODULE INCEPTION-LIKE PROTEIN 3(OsNLP3)in simultaneously regulating grain weight and grain chalkiness.Our investigation showed that loss of OsNLP3 leads to a reduction in both grain weight and dimension,in contrast to the enhancement observed with OsNLP3 overexpression.OsNLP3 directly suppresses the expression of OsCEP6.1 and OsNF-YA8,which were identified as negative regulators associated with grain weight.Consequently,two novel regulatory modules,OsNLP3-OsCEP6.1 and OsNLP3-OsNF-YA8,were identified as key players in grain weight regulation.Notably,the OsNLP3-OsNF-YA8 module not only increases grain weight but also mitigates grain chalkiness in response to N.This research clarifies the molecular mechanisms that orchestrate grain weight through the OsNLP3-OsCEP6.1 and OsNLP3-OsNF-YA8 modules,highlighting the pivotal role of the OsNLP3-OsNF-YA8 module in alleviating grain chalkiness.These findings reveal potential targets for simultaneous enhancement of rice yield and quality.

Identification of a Stable Quantitative Trait Locus for Percentage Grains with White Chalkiness in Rice (Oryza sativa)

High chalkiness is a major problem in many rice-producing areas of the world, especially in hybrid rice （Oryza sativa L.） in China. We previously showed a major quantitative trait locus for the percentage of grains with white chalkiness （QTLqPGWC-8） in the interval G1149-R727 on chromosome 8 using a chromosome segment substitution line （CSSL）. Here, we selected the line-CSSL50 harboring the QTLqPGWC-8 allele from the CSSLs derived from a cross between Asominori （as a recurrent parent） and IR24 （as a donor parent）, which had higher percentage chalkiness, markedly different from that of Asominori. There were also significant differences in starch granules, appearance of amylose content （AAC） and milling qualities between Asominori and CSSL50, but not in grain size or thousand grain weight （TGW）. The BC4F2 and BC4F3 populations from a cross between CSSL50 and Asominori were used for fine mapping of qPGWC-8. We narrowed down the location of this QTL to a 142 kb region between Indel markers 8G-7 and 8G-9. QTLqPGWC-8 accounted for 50.9% of the difference in PGWC between the parents. The markers tightly linked to qPGWC-8 should facilitate cloning of the gene underlying this QTL and will be of value for marker-assisted selection in breeding rice varieties with better grain quality.

Dissecting the Genetic Basis of Grain Shape and Chalkiness Traits in Hybrid Rice Using Multiple Collaborative Populations

Dear Editor Through the efficient use of heterosis, hybrid rice varieties generally have higher grain yield potential than inbred varieties. With the significant advantage in grain yield, over the past 30 years approximately half of China＇s total rice-growing area is planted with rice hybrids. However, grain quality has now become one of the most important targets in hybrid rice breeding for meeting consumer demands. Grain shape and chalkiness are two important components of rice grain quality, in which slender grains （typically, grain length-to-width ratio 〉3） with low chatkiness are preferred by most consumers of hybrid rice.

OsbZIP60-mediated unfolded protein response regulates grain chalkiness in rice

Grain chalkiness,an undesirable trait caused by complex factors,has great negative impacts on the quality and economic value of rice.However,little is known about the regulatory mechanism of grain chalkiness,particularly the effect of endoplasmic reticulum(ER)stress.Here,a genome-wide association study(GWAS)reveals that the transcription factor Osb ZIP60 is a vital regulator of rice grain chalkiness.Genetic analysis demonstrates that knockout of Osb ZIP60 results in extremely high grain chalkiness and aberrant structure of storage substances.Notably,the expression of unfolded protein response(UPR)genes,such as OsbZIP50,Os Bi P1,Os Bi P2 and Os Bi P3,is up-regulated in the endosperm cells of osbzip60,and overexpression of all these UPR genes causes various degrees of chalkiness.Furthermore,Osb ZIP60 is found to activate the expression of key genes related to grain chalkiness,such as GPA3,FSE1,FLO7,Chalk5,Os NF-YB1,and Os PK2,whose expression is significantly suppressed in osbzip60 and overexpression lines of Osb ZIP50,Os Bi P1,Os Bi P2,and Os Bi P3.Our study provides novel insights into the function of Osb ZIP60 and the role of the UPR pathway in the formation of grain chalkiness in rice.

利用稻米垩白度分析软件测量叶片相对病斑面积

【目的】提出一种利用稻米垩白度分析软件Chalkiness 1.0测量衡量植物叶片病害综合指标之一叶片相对病斑面积(RLA)的方法。【方法】通过扫描或拍照获得病害叶片的数字图像;然后利用图像处理软件(如Photoshop)对图像进行处理,最重要的是用肉眼识别病斑并用"画笔工具"涂黑,使叶片中的病斑明晰化;最后利用Chalkiness 1.0计算出图像中的RLA。【结果】用本方法对稻瘟病叶上病斑的相对面积进行测量。比较5次重复测量的结果,发现该方法重复性好,误差小。【结论】该方法简单、快速、准确,具有广阔的应用前景。

Structural and Histochemical Characterization of Developing Rice Caryopsis

The development of pericarp, seed coat, starchy endosperm and aleurone of the rice caryopsis was investigated, histochemically and structurally, from the time of flowering to maturity. The results showed that during its growth, the maximum length of the caryopsis was attained first, followed by width and then thickness. Histochemical examination of the caryopsis showed that starch was mainly accumulated in the endosperm, but the endosperm showed no metabolic activity, while embryo and pericarp contained a few starch grains, and embryo and aleurone were strongly active. Aleuronic cells contained many aleurone grains and spherosomes, and aleurone in the dorsal region developed earlier and contained more layers of cells. Amyloplasts in endosperm contained many starch granules and were spherical at early stages but polyhedric at late stages. The protein bodies appeared later than amyloplasts, and the number of protein bodies in subaleurone was greater than those in the starchy endosperm. The white-belly portion of endosperm might be relative to the status of amyloplast development.

Effect of Nitrogen Application Rates on the Nitrogen Utilization, Yield and Quality of Rice

Research on the effect of the nitrogen application rate on the balance of the nitrogen utilization, yield and quality of rice is common in South China but is relatively lacking in Northeast China, especially in the Liaohe Delta. In this study, Yanfeng 47 rice was planted in Panjin city, China, to explore the effect of six nitrogen rates (0, 160, 210, 260, 315 and 420 kg N/ha) on the nitrogen use efficiency, rice quality and grain yield of rice plants. The results showed that the application of an appropriate nitrogen rate (210-260 kg N/ha) remarkably increased the nitrogen use efficiency of rice plants, grain yield, rice milling quality and nutritional quality and resulted in a moderate rice eating quality. Although low nitrogen rates (160 kg N/ha) maintained a high rice eating quality, they decreased grain yield and other rice qualities, and excessive nitrogen (315 kg N/ha) increased rice appearance quality but significantly reduced the nitrogen use efficiency (p < 0.05), yield and eating quality of rice. Therefore, to produce rice in the Liaohe Delta by an environmentally friendly method and guarantee rice with high quality and yield, the recommended nitrogen application rate is 210 kg N/ha.

Revisiting the role of delta-1-pyrroline-5-carboxylate synthetase in drought-tolerant crop breeding

The delta-1-pyrroline-5-carboxylate synthetase(P5CS)gene exercises a protective function in stressed plants.However,the relationship between proline accumulation caused by P5CS and abiotic stress tolerance in plants is not always clear,as P5CS overexpression has been reported to repress plant growth under normal conditions in several reports.We re-evaluated the role of P5CS in drought-tolerant rice breeding by expressing the AtP5CS1 and feedback-inhibition-removed AtP5CS1(AtP5CS1^(F128A))genes under the regulation of an ABA-inducible promoter to avoid the potential side effects of P5CS overexpression under normal conditions.ABA-inducible AtP5CS1 and AtP5CS1^(F128A) increased seedling growth in a nutrient solution(under osmotic stress)and grain yield in pot plants.However,the evidently deleterious effects of AtP5CS1 on grain quality,tiller number,and grain yield in the field indicated the unsuitability of P5CS for drought-tolerance breeding.