Correlation of Simple Sequence Repeat (SSR) Variants in the Leader Sequence of the waxy Gene with Amylose Content of the Grain in Rice

Variation of two simple sequence repeats (SSRs) in the leader region of the waxy gene was analyzed in a sample of 74 accessions, including Oryza sativa L. ssp. indica, japonica and wild rice (O.rufipogon) representing a wide distribution range of amylose content (AC) in cultivated rice. Eight alleles were detected in the (CT) n motif and two alleles were resolved in the (AATT) n motif. The distribution of the alleles of the two SSRs was quite uneven as detected by the (CT) n motif. The repeat numbers of the two SSR motifs, (CT) n and (AATT) n, appeared to be inversely related such that the total length of this region was maintained. AC of the varieties was highly correlated with the length of SSRs. Differences in AC among the various SSR genotypes were statistically highly significant as analyzed using genotypes of both SSR motifs. Although the SSR variation did not seem to have obvious function in the synthesis of the starch synthase encoded by the waxy gene, the almost perfect correlation between the two SSRs and AC level could be used for quality improvement in rice breeding programs.

Effect of Fast Neutron Irradiation on Amylose Content and Amylopectin Structure of Thailand Rice Cultivar Jao Hom Nin

[Objective]The aim was to research the effect of fast neutron irradiation on amylose content and amylopectin structure.[Method] The amylose content and amylopectin structure of the M4 plants of Thailand rice cultivar Jao Hom Nin irradiated with fast neutron at 13 Gy were evaluated and analyzed in this paper.[Result]The results showed that amylose content of rice could be changed by fast neutron irradiation,many rice mutants with reduced amylose content and many others with enhanced amylose content even some mutants with amylose content near to waxy rice could be isolated,but fast neutron irradiation almost had no effect on amylopectin structure of the samples.[Conclusion]The study provided a basis for breeding rice cultivar with different amylose content in order to meet with the taste hobby of different people and the further processing of diverse rice products via the irradiation of fast neutron.

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）.

Determination of Rice Amylose Content by Near-infrared Spectroscopy

Amylose contents in 298 rice samples were determined by conventional method, and a near-infrared spectral model of rice amylose content was established by partial least squares. The calibration determination coefficient （RC） was 0.95; the standard error of calibration （SEC） was 1.58; and the determination coefficient of cross validation （RP） was 0.91, and the standard error of prediction SEP was 1.92. External validation was performed with 20 samples, the predicted values and the determined values were not significantly different, and the correlation coefficient between them was over 95%. The calibration model has good prediction perfor- mance, and could rapidly determine rice amylose content instead of chemical ana- lytical method.

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.

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.

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.

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.

Correlation Analysis of Characters of Indica Hybrid Rice with High Content of Amylose

[Objective] The aim was to research and analyze characters and related correlation of indica hybrid rice containing high amylose. [Method] In the research, major characters of 16 species of groups of {ndica hybrid rice and the correlation were explored in rice belts in Sichuan in 2011. [Result] The variation of chalkiness degree was the highest at 26.8%, followed by rate of head rice at 22.89%; varia- tions of head rice rate and chalkiness degree were lower at 0.72% and 2.61%. In addition, rate of unpolished rice was of significantly negative correlation with chalki- ness degree; rate of chalky rice was of extremely positive correlation with chalkiness degree and gel consistency; chalkiness degree was of significantly positive correla- tion with gel consistency; gel consistency was of significantly negative correlation with protein content; amylose content was of insignificant correlation with gel consis- tency （r=0.166 9） of the 16 hybrid groups. These indicated that gel consistency would be still high as the content of amylose grows （AC≥25%）. For example, when the content of anylose in Chuanxiang 29A/Luhui 37, Kangfeng AJ6684, and Lefeng A/SR536 exceeded 27%, the gel consistency was over 80 mm in the research. [Conclusion] The research provides references for improvement of amylose content in breeding hybrid rice.

Analysis of the Conditional Correlations from Different Genetic Systems Between the Protein Content and the Appearance Quality Traits of Indica Rice

A factorial mating design in two environments was conducted using 7 cytoplasmic male sterile lines （A） and 5 restorer lines （R） along with their F1 （A × R） and F2 populations. The unconditional and conditional analyses of genetic models and the corresponding statistic methods, including endospermic, cytoplasmic, and maternal plant genetic systems, were used to analyze the genetic relationships between protein content （PC） and the appearance quality traits of indica rice （Oryza sativa L.）. The results from unconditional analysis indicated that PC was significantly correlated with the appearance quality traits of rice, except for the brown rice thickness （BRT）. Only the genetic covariance between PC and the brown rice width （BRW） was positively correlative, whereas all the other pairwise traits were negatively correlative. The results from conditional analysis revealed that the weight of brown rice （WBR） or the amylose content （AC） could significantly affect the relationships between PC and the appearance quality traits of indica rice. The conditional analysis showed that WBR might negatively affect the relationships between PC and the brown rice length （BRL）, BRW, or BRT through the geuotype x environmental （GE） interaction effects, but positively affected the relationships between PC and the ratio of brown rice length to width （RLW） or the ratio of brown rice length to thickness （RLT）. The amylase content could positively affect the relationships between PC and BRL, RLW, RLT through the cytoplasmic effects and maternal additive effects, but negatively affected the relationships between PC and BRW.

Correlation analysis between major nutritional components and resistant starch content in wheat

With 94 spring wheat cultivars as experimental materials, the correlations between the content of resistant starch （RS） in uncooked flour and cooked flour, and the apparent amylose content （AAC）, protein, lipid were investigated. The results showed that RS contents in both the uncooked flour and cooked flour assumed significantly positive correlation with AAC, and significantly negative cor- relation with protein content; and they were proved to be not significantly correlated with lipid content. RS content in uncooked flour was significantly correlated with that in cooked flour. These results provided references for the genetic improvement of wheat cultivars.

Introgression of lac6/tl1/du13 improves the palatability of japonica rice

Amylose content(AC)is a crucial determinant of the eating and cooking quality(ECQ)of rice,with low AC varieties exhibiting a softer texture and greater stickiness-attributes that enhance palatability and are desirable in specific culinary contexts.To harness these traits,significant efforts have been made to manipulate AC to improve rice ECQ.Our research utilized the MutMap+approach to identify LAC6/TL1,a gene that is an allele of Du13,responsible for low AC.LAC6 encodes a C2H2 zinc finger protein,which specifically increases the splicing efficiency of the Wxb allele without affecting the Wxa allele.Functional studies of LAC6 revealed that its proper integration could rectify the undesirable AC phenotype,whereas mutations within this gene led to reduced AC and were associated with shorter grain length and decreased thousand-grain weight.Despite these drawbacks,such mutations positively impact rice palatability,presenting a trade-off between grain size and eating quality.To address the challenges posed by the reduced grain weight associated with LAC6 mutations,we developed a specific molecular marker for LAC6,which has been effectively used in breeding programs to select lac6/tl1/du13 homozygous individuals with larger grain size.Our findings demonstrate that the“small grain”trait associated with lac6/tl1/du13 can be effectively mitigated through combined phenotype-based and marker-assisted selection.This study highlights the potential of lac6/tl1/du13 as a valuable gene for breeding novel,high-quality soft rice varieties through targeted breeding strategies.

Advances in Breeding of High-amylose Indica Hybrid Rice Combinations in Sichuan Province

During 2000-2013, totally ten high-amylose specific hybrid rice combinations approved and five combinations for production test were bred in Sichuan Province. In the paper, rice yield, economic traits, quality indicators and rice blast-resistance identification results of these 15 hybrid combinations were described and analyzed. Results show that Chuanxiangyou37 and Tianlongyou540 have relatively high amylose content, good yielding ability, high resistance and appropriate growth period, which are new hybrid rice varieties with organic combination of high amylase content, high gel consistency, high yield, stable yield and wide adaptability. Therefore, Chuanxiangyou37 and Tianlongyou540 are recommended as the main combination for rice production.

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.