Determination of Amylose Content and Its Relationship with RVA Profile Within Genetically Similar Cultivars of Rice(Oryza sativa L.ssp.japonica)

Anther culture pure lines with antisense Wx gene were generated by Agrobacterium tumefaciens-mediated co-transformation followed by anther culture in transgenic rice （Oryza sativa L. ssp. japonica）. The antisense Wx transgenic pure lines were used to analyze the differences and the relationship between RVA eigenvalues among the lines with different amylose contents. 77 pure lines of anther culture having antisense Wx gene were studied for the amylose content of its offspring cultivar, Wuyunjing 7. It was found that levels ofamylose content were similar to the control in 19 lines （16.0%）; 1-5% lower than control in 50 lines, of which, within 11.0-13.9% in 40 lines; and 3.1-4.0% in 8 lines. The effect of the amylose content on RVA eigenvalue was analyzed through the RVA profile graphic comparison and test of significance for RVA eigenvalues. The analysis of RVA profiles with different amylose contents indicated that there were two kinds of RVA profiles within genetically similar cultivars. The lines with 3.1-4.0% of amylose content were distinctly different from other lines and conventional glutinous rice. The comparison of the similarity curve of RVA profile of the lines with different amylose contents showed that the lines with lower amylose content had a distinguished RVA profile with the curve increasing gradually, but not exceeding the first apex. The test of significance for eigenvalues of the RVA profile in the lines with different amylose contents indicated that there were five eigenvalues remarkably different among the lines with 3.1-4.0% of amylose content compared with other two groups. Further, there were three eigenvalues remarkably different among the lines with 11.0-13.5% of amylose content compared with control group. An optimized mathematical model, which characterizes the relationships between the amylose content and RVA eigenvalue, has been established in this study. From this model, it was clearly indicated that the parameters PeT and SBV were more related to amylose content. It was concluded that the differences in amylose content not only affected the eigenvalues of RVA but also caused different RVA profiles within genetically similar cultivars. The introduction of antisense Wx gene could lead to reduce the amylose content and lay theoretical foundation for quality improvement in rice breeding programs.

Application of Near Infrared Transmittance Spectroscopy(NITS)in Determining Amylose Content of Rice Grain

Rice samples were scanned with a Foss Tecator Infratec and their amylose content was determinedusing a standard wet chemical method.Calibration software along with several mathematicaltreatments of the sample spectra and several regression methods was used to optimize a nearinfrared transmittance spectroscopy equation for determining rice amylose content. The standarderror of calibration, standard error of cross-validation and regression squared of thecalibration were 1.489, 1.761 and 0.885,respectively.

Progresses in Researches of the Application of Low-Amylose Content Rice Gene for Breeding

Amylose content is a key determinant of eating quality of rice. With the characteristics offluffy texture, glossy appearance when cooked, remaining soft when cooled and excellentpuffing ability, the low-amylose rice with amylose content 5-15% could be served as not onlycooked rice directly, but also good material for convenience, mixed rice and puffing foods.Current status on characterization, inheritance, molecular mechanism and breeding of low-amylose content rice was reviewed in this paper, strategy of related researches in the era ofglymics was mainly discussed furthermore. The future research should focus on screening andenhancing the germplasm, further elucidating the molecular mechanism on mutation of lowamylose content, utilizing the genes independent of Wx on low-amylose content rice breedingprogram, and developing high quality functional rice cultivars for special usage throughpyramiding low amylose gene and other special quality genes.

Comparison of Near Infrared Spectroscopy Models for Determining Protein and Amylose Contents Between Calibration Samples of Recombinant Inbred Lines and Conventional Varieties of Rice

The near infrared spectra of 178 recombinant inbred lines （RILs） from the cross of Ⅱ-32B/Yuezaoxian 6 （YZX6） and 511 varieties in rice were acquired. A total of 80 RILs and 96 cultivars were selected as modeling samples by comparing the spectra similarity primarily. Three partial least square （PLS） regression models were developed, based on the RILs （RIL-model）, the varieties （Var-model） and their mixture （Mix-model）, for protein content （PC） and amylose content （AC）, respectively. Cross validation and outer prediction showed that the models were largely influenced by the range and distribution of modeling samples. The regression model of PC based on the cultivars and the model of AC based on RILs had higher coefficient of determination （r^2 ≥ 0.9） and lower root mean square error of cross validation （RMSECVs）. The disadvantages of RIL samples for PC model and variety samples for AC model were probably caused by the narrow range of variance. Aberrant predictions were obtained for outer sample with PC or AC outside the range or within the distribution gap of modeling samples. The Mix-models gave more reliable prediction as the distribution of RIL and variety modeling samples were complementary to each other.

Relationship Between the First Base of the Donor Splice Site of Waxy Gene Intron 1 and Amylose Content in Yunnan Indigenous Rice Varieties

There exists a single nucleotide polymorphism, G or T, at the first base of the donor splice site of waxy gene intron 1 in rice. In order to study the relationship between the first base of the donor splice site of waxy gene intron 1 and amylose content in rice, the one-step PCR method was used to determine whether it is G or T in 220 Yunnan indigenous rice varieties from 14 districts, 55 towns/counties of Yunnan Province, and 101 varieties of which were validated by the PCR-Acc I method. According to the G/T polymorphism, 164 rice varieties showed GG-genotype, while the other 56 fell into TT- genotype, accounting for 74.5% and 25.5% of all the test varieties, respectively. When all the rice varieties were divided into indica and japonica subspecies, it was found that 80.5% of indica rice and 67.0% of japonica rice belonged to GG-genotype. The rice varieties with GG-genotype had significantly higher amylose content （18.95% on average） than those with TT- genotype （all below 16%）, but 33 rice varieties with GG-genotype still had low amylose content ranging from 3.91% to 15.93%, and most of them came from the Dai minority area in the Southwest of Yunnan Province. However, there was no significant difference in the mean amylose content of the same GG or TT genotypes between indica and japonica rice, suggesting that different genetic backgrounds, indica or japonica, had no effect on amylose content. The coefficient of correlation between the genotype and amylose content was 0.733 （P〈0.01）.

Amylose Content,Morphology,Crystal Structure,and Thermal Properties of Starch Grains in Main and Ratoon Rice Crops

Rice ratooning,or the production of a second rice crop from stubble after the harvest of the main crop,is considered to be a green and resource-efficient rice production system.The present study was conducted to examine variance in amylose content(AC),grain morphology,crystal structure,and thermal properties of starch between main-and ratoon-season rice of seven varieties.Ratoon-season rice grains had higher ACs and significantly lower transition gelatinization temperatures(To,Tp,and Tc)than did main-season rice grains.The relative crystallinity and lamellar peak intensity of ratoon-season rice starch were 7.89%and 20.38%lower,respectively,than those of main-season rice starch.In addition,smaller granules with smoother surfaces and lower thermal parameters were observed in the starch of ratoon-season rice.The relative crystallinity and lamellar peak intensity of starch correlated negatively with the AC and positively with transition gelatinization temperatures.These results suggest that the superior cooking quality of ratoon-season rice is attributable to the moderate increase of grain AC,which reduces the relative crystallinity,weakens the crystal structure,and lead to a decrease in the gelatinization temperature.

Molecular marker for screening rice cultivars with intermediate amylose content in indica

The waxy(Wx)gene of rice encodes the granule-bound starch synthase(GBSS),which was required in the synthesis of amylose in endosperm.Theamylose content(AC)of rice endosperm played an important role in grain yield,palatability,and processing quality.Our previous researehes showed that en-dosperm AC and GBSS contents were correlated with the ability of excising intron1 from the leader sequence of the Wx transcript.Cultivars with high endosperm

Lowering Grain Amylose Content in Backcross Offsprings of indica Rice Variety 057 by Molecular Marker-Assisted Selection

To lower the amylose content （AC） of the indica rice restorer line 057 with high AC, backcrosses were made respectively by using four indica varieties （R367, 91499, Yanhui 559, Hui 527） as low AC donor parents and 057 as the recurrent parent. A molecular marker （PCR-Acc Ⅰ） was used to identify the genotypes （GG, TT and GT） of the waxy （Wx） gene. Plants with GT genotype were selected and used as female parent and crossed with 057 to advance generation. The ACs of rice grains harvested from plants with different Wx genotypes were measured and compared to analyze the efficiency of marker-assisted selection. The ACs of the rice grain, harvested from the plants of Wx genotypes GG, GT and TT, were higher than 20%, in the range of 17.7-28.5%, and less than 18%, respectively. The PCR-Acc Ⅰ marker could be used for efficiently lowering the AC of 057 through backcrossing, and there were some influence of parental genetic background on the AC of rice grains with the same Wx genotype.

Amylose Content and Starch Granule Size in Rice Grains are Affected By Growing Season

Amylose content and starch granule size in grains influence rice quality,which differs between the early(ES)and late season(LS).The objective of this study was to determine the variation of amylose content and starch granule size between seasons and find the main reasons(e.g.,temperature and solar radiation)for the observed variation.Field experiments with six rice varieties(three high and three low amylose content rice)planted in the ES and LS were conducted in 2016 and 2017,respectively.The mean temperatures during the filling stage were higher in ES,however,the daily temperatures at 7-10 days after flowering(DAF)in 2016,and at 5-10,13-14 DAF in 2017 were higher in LS.The results showed that amylose content in LS was lower than in ES with high amylose content rice varieties(HACV);the opposite trend occurred with low amylose content rice varieties(LACV).The mean starch granule diameter was higher in LS than ES in 2016,but the opposite result occurred in 2017 with all rice.Our results suggest that higher temperatures increased and decreased the amylose content in HACV and LACV,respectively.Temperatures at 5-15 DAF were important for the formation of starch granules:lower temperatures during 10-14 DAF increased the proportion of larger starch granules(d>6.21μm,some with d>13.3μm),and higher temperatures at 5-6 DAF increased the proportion of starch granules with diameter 4.24-6.21μm.

Analysis of genetic effects and heritabilities for amylose content of indica rice in different environments

An understanding of the genetic effects of em-bryo genes for rice quality traits is of impor-tance for rice quality improvement. Analysisof embryo, endosperm, cytoplasmic, and ma-ternal genetic effects and genotype×environ-

The kinase OsSK41/OsGSK5 negatively regulates amylose content in rice endosperm by affecting the interaction between OsEBP89 and OsBP5

Amylose content(AC) is the main factor determining the palatability, viscosity, transparency, and digestibility of rice(Oryza sativa)grains. AC in rice grains is mainly controlled by different alleles of the Waxy(Wx) gene. The AP2/EREBP transcription factor OsEBP89 interacts with the MYC-like protein OsBP5 to synergistically regulate the expression of Wx.Here, we determined that the GLYCOGEN SYNTHASE KINASE 5(OsGSK5, also named SHAGGY-like kinase 41 [OsSK41]) inhibits the transcriptional activation activity of OsEBP89 in rice grains during amylose biosynthesis. The loss of OsSK41 function enhanced Wx expression and increased AC in rice grains. By contrast, the loss of function of OsEBP89 reduced Wx expression and decreased AC in rice grains. OsSK41 interacts with OsEBP89 and phosphorylates four of its sites(Thr-28,Thr-30, Ser-238, and Thr-257), which makes OsEBP89 unstable and attenuates its interaction with OsBP5. Wx promoter activity was relatively weak when regulated by the phosphomimicvariantOsEBP89E–OsBP5but relatively strong when regulated by the nonphosphorylatable variant OsEBP89A–OsBP5.Therefore, OsSK41-mediated phosphorylation of OsEBP89 represents an additional layer of complexity in the regulation of amylose biosynthesis during rice grain development. In addition, our findings provide four possible sites for regulating rice grain AC via precise gene editing.

Effects of Different Nitrogen Fertilizer Levels on Fe,Mn,Cu and Zn Concentrations in Shoot and Grain Quality in Rice (Oryza sativa)

In a pot experiment, effects of N fertilizer application on the concentrations of Fe, Mn, Cu and Zn in shoot of rice and the quality of brown rice were studied. In the treatments with N fertilizer application, the concentrations of Fe, Mn, Cu and Zn in most parts of rice shoot increased compared with control （no N fertilizer application）. This indicated that the transportation ability of microelements from root to shoot in rice was improved with N fertilizer application. Effect of N fertilizer on IR68144 was similar to that of on IR64, but the concentrations of the microelements in plant differed, suggesting that the characteristic expression of the two rice genotypes was not controlled by the amount of N fertilizer supplied. The concentrations of those microelements in brown rice increased at first and then decreased with increasing N fertilizer application, reaching the highest at 160 kg/ha, at which the Fe, Mn, Cu and Zn concentrations in brown rice increased by 28.96%, 41.34%, 58.31% and 16.0% for IR64, and by 22.16%, 13.75%, 8.75% and 20.21% for IR68144 compared with control, respectively. Moreover, N fertilizer promoted the accumulation of protein, decreased the accumulation of amylose in grain, and enhanced gel consistency of brown rice. These results indicate that appropriate N fertilizer management could increase micronutrient contents in grain and improve nutrition quality of rice.

Effects of Soil Salinity and Alkalinity on Grain Quality of Tolerant,Semi-Tolerant and Sensitive Rice Genotypes

Soil salinity and alkalinity adversely affects the productivity and grain quality of rice. The grain quality of 19 rice genotypes characterized as salt tolerant （T）, semi-tolerant （ST） and sensitive （S） was assessed in lysimeters containing saline and highly alkaline soils. Head rice recovery was reduced by salinity stress whereas it was not affected by alkalinity stress. The ratio of length to width （grain dimensions） was significantly reduced in the T genotype even at low electrical conductivity （EC, 4 mS/cm） and alkalinity （pH 9.5）, whereas in the ST genotype, it was significantly reduced at high salinity （EC 8 mS/cm）. There was no significant effect of any levels of salinity or alkalinity on grain dimensions in the S genotype. Amylose content was significantly reduced in T and ST groups even at low EC （4 mS/cm） and alkalinity （pH 9.5） and the effect in the S genotype was only at high salinity. Starch content showed significant reduction at high salinity and alkalinity （EC 8 mS/cm and pH 9.8） in the T and ST genotypes and no significant effect was observed in the S genotype. The effect of both levels of salinity （EC 4 and 8 mS/cm） and high alkalinity （pH 9.8） on gel consistency was observed only in the S genotype. The tolerant genotypes IR36 under high salinity, and CSIR10 and CSR11 under alkali stress showed less reduction in amylose content. The T genotype BR4-10, and ST genotypes CSR30, CSR29 and CSR13 showed better gel consistency under saline and alkali stress. Amylose content was affected even at low salinity stress and thus important to be considered in breeding rice for salt tolerance. Overall, the grain quality of T and ST genotypes was less affected by saline and alkali stress compared to S ones.

A Cleaved Amplified Polymorphic Sequence Marker to Detect Variation in Wx Locus Conditioning Translucent Endosperm in Rice

The translucent endosperm trait in a japonica rice variety ＇Kantou 194＇ is controlled by a Wx-mq gene which is allelic to Wx locus by genetic analysis and allelic test. The amylose content analysis showed that an intermediate amylose content between those of glutinous and non-glutinous rice existed in endosperm of homozygous Wx-mq genotype. The slight changes of amylose content in different varieties and F1 grains with an identical Wx-mq genotype might be influenced by dissimilar genetic background. To identify the Wx-mq genotype simply and rapidly, a cleaved amplified polymorphic sequence （CAPS） marker was designed. The result from the molecular detection indicated that it could be used for marker-assisted selection for low amylose content varieties in rice breeding.

Genetic Polymorphism of Wx Gene and Its Correlation with Main Grain Quality Characteristics in Rice

The allelic variation of the Wx gene in 50 non-glutinous rice varieties （lines） was analyzed by using the microsatellite marker RM190 [for （CT）n simple sequence repeat （SSR）] and cleaved amplified polymorphic sequence（CAPS） marker 484/W2R-ACCⅠ[for G/T single nucleotide polymorphism （SNP）]. Six homozygous （CT）n types, namely （CT）20, （CT）19, （CT）18, （CT）17, （CT）16, （CT）14, （CT）11 and （CT）10, and a heterozygous genotype （CT）11/（CT）18 were detected for RM190, of which （CT）11 and （CT）18 were predominant. Two homozygous Wx genotypes （G/G and T/T） and one heterozygous （G/T） were detected using 484/W2R-ACC Ⅰ. Most of the materials with a RM190 of （CT）11 were G/G for SNP of 484/W2R-ACC Ⅰ, while T/T for SNP was predominantly appeared in materials with （CT）18. The materials tested could be grouped into 10 categories using the two markers together. Results indicated that 59.3% variance of amylose content was attributed to the polymorphism of Wx gene revealed by RM190, while 56.1% and 24.6% of the variances in amylose content and gel consistency were respectively to the polymorphism of Wx gene revealed by 484/W2R-ACC Ⅰ. Furthermore, with both SSR and CAPS markers, 72.4% of the variance in amylose content could be explained. In addition, the application prospects of the two markers in breeding were also discussed.

Comparison of grain yield and quality of different types of japonica rice cultivars in the northern Jiangsu plain,China

In recent years,an increasing number of different types of japonica rice cultivars have been released in the southern rice region of China.The grain yield and quality of these new cultivars showed significant differences in large scale planting.However,the causes of the differences remain little known.Therefore,three typical types of japonica rice cultivars were used in this study to investigate their grain yield and quality.A scanning calorimeter(DSC),X-ray powder diffractometer(XRD),rapid viscosity analyzer(RVA)and taste analyzer were used to evaluate the cooking and eating properties.The results showed that the yield of non-soft hybrid japonica rice cultivars was significantly higher than that of non-soft inbred japonica rice cultivars and soft inbred japonica rice cultivars.Soft inbred japonica rice cultivars had a low amylose content and moderate protein content,which are the main reasons for the superior cooking and eating quality.In addition,the relative crystallinity of soft inbred japonica rice cultivars was significantly higher than that of non-soft inbred and non-soft hybrid japonica rice cultivars,which is considered the major factor resulting in higher transition temperature and gelatinization enthalpy(ΔH_(gel)).Non-soft hybrid japonica rice cultivars had a higher number of large starch granules than soft inbred and non-soft inbred japonica rice cultivars.The setback value(SB)and breakdown value(BD),indirectly reflecting the cooking and eating quality of the three types of japonica rice cultivars,also confirmed that soft inbred japonica rice cultivars with a low SB value and a high BD value had better palatability than the other two types.This study provides guidance for future plantation of different types of japonica rice cultivars in large rice-producing areas.

The Source of Genes Related to Rice Grain Starch Synthesis Among Cultivated Varieties and Its Contribution to Quality

The property of starch in rice grain endosperm is a very important determinant for rice quality, and it is essential to understand the genetic effect of the genes related to starch synthesis in high-yielding rice varieties for rice quality improvement. The physicochemical properties （e.g., amylose content, gel consistency, and RVA profile） were assessed on 53 rice varieties, including certain typical indica/japonica landraces and certain high-yielding modern varieties. And molecular markers for Sbel, Sbe3 developed on the basis of sequence diversities between the rice subspecies indica and japonica, together with PCR-Acc I marker for Wx gene were used to investigate the genotypes of 53 rice cultivars. The result showed that the developed molecular markers for Wx, Sbel, Sbe3 could distinguish indica or japonica alleles at three loci. Among all the 53 rice cultivars, six genotypes were observed when Sbel, Sbe3, and Wx loci were considered together, while the genotypes of WxiSbelJSbe3i and WxiSbelJSbe3J were absent. In order to explore the genetic effects of the three genes, especially for starch branching enzyme genes, ANOVA and multiple comparison analysis were conducted. The results showed that rice cultivars with different genotypes exhibited different phenotypes, including amylose content, gel consistency and certain RVA characteristics, and the significant differences among the six genotypes were observed. It was concluded that these three genes had randomly recombined during the process of the rice variety development. And the genetic effects of indica and japonica alleles at three gene loci were different, of which, Wx gene plays a major role in determining the starch properties, followed by Sbel and Sbe3, and the genetic effects of Sbel and Sbe3 in different backgrounds （Wx~, WxJ） are different. The results have provided a clue for rice good quality variety development, and the molecular markers will benefit to the improvement in quality of rice.

Effects of Dry-Milling and Wet-Milling on Chemical, Physical and Gelatinization Properties of Rice Flour

Rice flour from nine varieties, subjected to dry- and wet-milling processes, was determined for its physical and chemical properties. The results revealed that milling method had an effect on properties of flour. Wet-milling process resulted in flour with significantly lower protein and ash contents and higher carbohydrate content. Wet-milled flour also tended to have lower lipid content and higher amylose content. In addition, wet-milled rice flour contained granules with smaller average size compared to dry-milled samples. Swelling power at 90℃ of wet-milled samples was higher while solubility was significantly lower than those of dry-milled flour. Dry milling process caused the destruction of the crystalline structure and yielded flour with lower crystallinity compared to wet-milling process, which resulted in significantly lower gelatinization enthalpy.

Analysis on Stability of AC,GT and PC in Rice Varieties (Orzya sativa L.)

The investigation of rice varieties stability for amylose content (AC), gelatinization temperature (GT) and protein content (PC) was carried out using additive main effects and multiplicative interaction (AMMI) model in three locations and two years. Eighteen tested varieties were further clustered and evaluated based on the phenotypic values of three quality traits and their Di values from the AMMI analysis. The results showed that the genotype by environment interactions, the differences of phenotype and stability of AC, GT and PC among different genotypes and environments were all significant at 1% level. Also, only one variety, W002, had high stability for all the three quality traits. Additionally, in consideration with the phenotype of AC, GT and PC, and their stability in rice varieties, etc., four rice varieties, W002, Zaofeng 9, Guangling Xiangjing and Nanjing16, could be also applied as breeding parents to improve eating quality and stability of rice.

Characterization of Myanmar Paw San Hmwe Accessions Using Functional Genetic Markers

Paw San Hmwe （PSM） rice has been cultivated in many areas of Myanmar for a long time. Strong aroma, good taste and its elongation during cooking are the key characteristics of PSM rice. Thirty-one PSM accessions were genotypically characterized, and their physical grain and cooking quality traits were studied. We used specific gene markers associated with aroma, apparent amylose content （AAC） and alkali spreading value to determine the alleles carried by different PSM accessions. The results revealed that six PSM accessions （PSM10, PSM12, PSM13, PSM21, PSM22 and PSM30） had a 3-bp insertion in Os2AP gene. Gel consistency （GC） allele was predominant among the PSM accessions for gelatinization temperature （GT）, however, the phenotype observed was between low and intermediate GT because of the combination of the GC allele with the presence of low GT allele at heterozygous state from the other loci of the SSIla gene. Intermediate to high AAC was observed among the PSM accessions corresponding to the haplotype identified for the single nucleotide polymorphism G/T and the （Cm）n repeat in the Wx gene. The characterization and grouping data of PSM accessions posted benefits to Myanmar seed banks, and our results will help in maintaining the integrity of PSM rice variety.