QTL Mapping and Candidate Gene Analysis for Starch-Related Traits in Tartary Buckwheat (Fagopyrum tataricum (L.) Gaertn).

Starch is the main component that determines the yield and quality of Tartary buckwheat. As a quantitative trait, using quantitative trait locus (QTL) mapping to excavate genes associated with starch-related traits is crucial for understanding the genetic mechanisms involved in starch synthesis and molecular breeding of Tartary buckwheat varieties with high-quality starch. Employing a recombinant inbred line population as research material, this study used QTL mapping to investigate the amylose, amylopectin, and total starch contents across four distinct environments. The results identified a total of 20 QTLs spanning six chromosomes, which explained 4.07% to 14.41% of the phenotypic variation. One major QTL cluster containing three stable QTLs governing both amylose and amylopectin content, qClu-4-1, was identified and located in the physical interval of 39.85-43.34 Mbp on chromosome Ft4. Within this cluster, we predicted 239 candidate genes and analyzed their SNP/InDel mutations, expression patterns, and enriched KEGG pathways. Ultimately, five key candidate genes, namely FtPinG0004897100.01, FtPinG0002636200.01, FtPinG0009329200.01, FtPinG0007371600.01, and FtPinG0005109900.01, were highlighted, which are potentially involved in starch synthesis and regulation, paving the way for further investigative studies. This study, for the first time, utilized QTL mapping to detect major QTLs controlling amylose, amylopectin, and total starch contents in Tartary buckwheat. The QTLs and candidate genes would provide valuable insights into the genetic mechanisms underlying starch synthesis and improving starch-related traits of Tartary buckwheat.

Molecular insights on the origin and development of waxy genotypes in major crop plants.

Starch is a significant ingredient of the seed endosperm with commercial importance in food and industry. Crop varieties with glutinous (waxy) grain characteristics, i.e. starch with high amylopectin and low amylose, hold longstanding cultural importance in some world regions and unique properties for industrial manufacture. The waxy character in many crop species is regulated by a single gene known as GBSSI (or waxy), which encodes the enzyme Granule Bound Starch Synthase1 with null or reduced activity. Several allelic variants of the waxy gene that contribute to varying levels of amylose content have been reported in different crop plants. Phylogenetic analysis of protein sequences and the genomic DNA encoding GBSSI of major cereals and recently sequenced millets and pseudo-cereals have shown that GBSSI orthologs form distinct clusters, each representing a separate crop lineage. With the rapidly increasing demand for waxy starch in food and non-food applications, conventional crop breeding techniques and modern crop improvement technologies such as gene silencing and genome editing have been deployed to develop new waxy crop cultivars. The advances in research on waxy alleles across different crops have unveiled new possibilities for modifying the synthesis of amylose and amylopectin starch, leading to the potential creation of customized crops in the future. This article presents molecular lines of evidence on the emergence of waxy genes in various crops, including their genesis and evolution, molecular structure, comparative analysis and breeding innovations.

Evaluation of altered starch mutants and identification of candidate genes responsible for starch variation in wheat.

BACKGROUND: Induction of mutation through chemical mutagenesis is a novel approach for preparing diverse germplasm. Introduction of functional alleles in the starch biosynthetic genes help in the improvement of the quality and yield of cereals. RESULTS: In the present study, a set of 350 stable mutant lines were used to evaluate dynamic variation of the total starch contents. A megazyme kits were used for measuring the total starch content, resistant starch, amylose, and amylopectin content. Analysis of variance showed significant variation (p < 0.05) in starch content within the population. Furthermore, two high starch mutants (JE0173 and JE0218) and two low starch mutants (JE0089 and JE0418) were selected for studying different traits. A multiple comparison test showed that significant variation in all physiological and morphological traits, with respect to the parent variety (J411) in 2019-2020 and 2020-2021. The quantitative expression of starch metabolic genes revealed that eleven genes of JE0173 and twelve genes of JE0218 had consistent expression in high starch mutant lines. Similarly, in low starch mutant lines, eleven genes of JE0089 and thirteen genes of JE0418 had consistent expression in all stages of seed development. An additional two candidate genes showed over-expression (PHO1, PUL) in the high starch mutant lines, indicating that other starch metabolic genes may also contribute to the starch biosynthesis. The overexpression of SSII, SSIII and SBEI in JE0173 may be due to presence of missense mutations in these genes and SSI also showed overexpression which may be due to 3-primer_UTR variant. These mutations can affect the other starch related genes and help to increase the starch content in this mutant line (JE0173). CONCLUSIONS: This study screened a large scale of mutant population and identified mutants, could provide useful genetic resources for the study of starch biosynthesis and genetic improvement of wheat in the future. Further study will help to understand new genes which are responsible for the fluctuation of total starch.

Development of novel gene-based markers for waxy1 gene and their validation for exploitation in molecular breeding for enhancement of amylopectin in maize.

Waxy corn possessing high amylopectin is widely employed as an industrial product. Traditional corn contains ~ 70-75% amylopectin, whereas waxy corn with the mutant waxy1 (wx1) gene possesses ~ 95-100% amylopectin. Marker-assisted breeding can greatly hasten the transfer of the wx1 allele into normal corn. However, the available gene-based marker(s) for wx1 are not always polymorphic between recipient and donor parents, thereby causing a considerable delay in the molecular breeding program. Here, a 4800 bp sequence of the wx1 gene was analyzed among seven wild-type and seven mutant inbreds employing 16 overlapping primers. Three polymorphisms viz., 4 bp InDel (at position 2406 bp) in intron-7 and two SNPs (C to A at position 3325 bp in exon-10 and G to T at position 4310 bp in exon-13) differentiated the dominant (Wx1) and recessive (wx1) allele. Three breeder-friendly PCR markers (WxDel4, SNP3325_CT1, and SNP4310_GT2) specific to InDel and SNPs were developed. WxDel4 amplified 94 bp among mutant-type inbreds, while 90 bp was amplified among wild-type inbreds. SNP3325_CT1 and SNP4310_GT2 revealed the presence-absence polymorphisms with an amplification of 185 bp and 189 bp of amplicon, respectively. These newly developed markers showed 1:1 segregation in both BC1F1 and BC2F1 generations, while 1:2:1 segregation was observed in BC2F2. The recessive homozygotes (wx1wx1) of BC2F2 identified by the markers possessed significantly higher amylopectin (97.7%) compared to the original inbreds (Wx1Wx1: 72.7% amylopectin). This is the first report of novel wx1 gene-based markers. The information generated here would help in accelerating the development of waxy maize hybrids.

Mutation of TL1, encoding a novel C2H2 zinc finger protein, improves grains eating and cooking quality in rice.

KEY MESSAGE: The cloning and characterization of a novel C2H2 zinc finger protein that affects rice eating and cooking quality by regulating amylose content and amylopectin chain-length distribution in rice. One of the major objectives in rice breeding aims to increase simultaneously yield and grain quality especially eating and cooking quality (ECQ). Controlling amylose content (AC) and amylopectin chain-length distribution (ACLD) in rice is a major strategy for improving rice ECQ. Previous studies show that some starch synthesis-related genes (SSRGs) are required for normal AC and ACLD, but its underlying regulating network is still unclear. Here, we report the cloning and characterization of a novel C2H2 zinc finger protein TL1 (Translucent endosperm 1) that positively regulates amylose synthesis in rice grains. Loss of TL1 function reduced apparent amylose content (AAC), total starch, gel consistency, and gelatinisation temperature, whereas increased viscosity, total lipid, and ratio of amylopectin A chains with degree of polymerization (DP) 6-12 to B1 chains with DP 13-24, resulting in an enhanced grain ECQ. The improved ECQ was accompanied by altered expression patterns of several tested SSRGs in tl1 mutant grains. Furthermore, knockout of TL1 in the high-yielding rice variety JiaHua NO.1 reduced AAC without obvious side effects on major agronomic traits. These findings expand our understanding of the regulating networks of grain starch metabolism and provide new insights into how rice ECQ quality can be improved via genetic approach.

Potassium Fertilization Stimulates Sucrose-to-Starch Conversion and Root Formation in Sweet Potato (Ipomoea batatas (L.) Lam.).

A field experiment was established to study sweet potato growth, starch dynamic accumulation, key enzymes and gene transcription in the sucrose-to-starch conversion and their relationships under six K2O rates using Ningzishu 1 (sensitive to low-K) and Xushu 32 (tolerant to low-K). The results indicated that K application significantly improved the biomass accumulation of plant and storage root, although treatments at high levels of K, i.e., 300-375 kg K2O ha-1, significantly decreased plant biomass and storage root yield. Compared with the no-K treatment, K application enhanced the biomass accumulation of plant and storage root by 3-47% and 13-45%, respectively, through promoting the biomass accumulation rate. Additionally, K application also enhanced the photosynthetic capacity of sweet potato. In this study, low stomatal conductance and net photosynthetic rate (Pn) accompanied with decreased intercellular CO2 concentration were observed in the no-K treatment at 35 DAT, indicating that Pn was reduced mainly due to stomatal limitation; at 55 DAT, reduced Pn in the no-K treatment was caused by non-stomatal factors. Compared with the no-K treatment, the content of sucrose, amylose and amylopectin decreased by 9-34%, 9-23% and 6-19%, respectively, but starch accumulation increased by 11-21% under K supply. The activities of sucrose synthetase (SuSy), adenosine-diphosphate-glucose pyrophosphorylase (AGPase), starch synthase (SSS) and the transcription of Susy, AGP, SSS34 and SSS67 were enhanced by K application and had positive relationships with starch accumulation. Therefore, K application promoted starch accumulation and storage root yield through regulating the activities and genes transcription of SuSy, AGPase and SSS in the sucrose-to-starch conversion.

Genome-Wide Association Study on Total Starch, Amylose and Amylopectin in Barley Grain Reveals Novel Putative Alleles.

The content and composition of starch in cereal grains are closely related to yield. Few studies have been done on the identification of the genes or loci associated with these traits in barley. This study was conducted to identify the genes or loci controlling starch traits in barley grains, including total starch (TS), amylose (AC) and amylopectin (AP) contents. A large genotypic variation was found in all examined starch traits. GWAS analysis detected 13, 2, 10 QTLs for TS, AC and AP, respectively, and 5 of them were commonly shared by AP and TS content. qTS-3.1, qAC-6.2 and qAP-5.1 may explain the largest variation of TS, AC and AP, respectively. Four putative candidate genes, i.e., HORVU6Hr1G087920, HORVU5Hr1G011230, HORVU5Hr1G011270 and HORVU5Hr1G011280, showed the high expression in the developing barley grains when starch accumulates rapidly. The examined 100 barley accessions could be divided into two groups based on the polymorphism of the marker S5H_29297679, with 93 accessions having allele GG and seven accessions having AA. Moreover, significantly positive correlation was found between the number of favorable alleles of the identified QTLs and TS, AC, AP content. In conclusion, the identified loci or genes in this study could be useful for genetic improvement of grains starch in barley.

Morphology, structure, properties and applications of starch ghost: A review.

Starch ghost, an insoluble structure of gelatinized starch, plays an important role in the applications of starch. In this review, we summarized the preparation, morphology, structure, properties and applications of starch ghost. The preparation steps of starch ghost include gelatinization, purification and preservation, and many factors influence the yield of starch ghost. The morphology and content of starch ghost can be influenced by many factors like starch resource and amylose content. Ghosts from non-waxy starches are composed of amylopectin with long branch-chains and amylose. These molecules cross-link to each other to reinforce the structure, and tend to form B-type double helix in ghosts from high-amylose starches. Some surface proteins that bind tightly to starch granules are also present in starch ghost. Protein and lipid are thought to have limited effects on the structural stability, but they make a big difference in the morphology of starch ghost. Starch ghost shows a different resistance to amylase among various starches, but it can be further digested under the high shear force. The mechanical, enzymatic hydrolysis and electrochemical properties of starch ghost make it widely used as emulsifier, stabilizer, thickener and starch-based films or gels in food and non-food processing industries.

Identification and validation of mutation points associated with waxy phenotype in cassava.

BACKGROUND: The granule-bound starch synthase I (GBSSI) enzyme is responsible for the synthesis of amylose, and therefore, its absence results in individuals with a waxy starch phenotype in various amylaceous crops. The validation of mutation points previously associated with the waxy starch phenotype in cassava, as well as the identification of alternative mutant alleles in the GBSSI gene, can allow the development of molecular-assisted selection to introgress the waxy starch mutation into cassava breeding populations. RESULTS: A waxy cassava allele has been identified previously, associated with several SNPs. A particular SNP (intron 11) was used to develop SNAP markers for screening heterozygote types in cassava germplasm. Although the molecular segregation corresponds to the expected segregation at 3:1 ratio (dominant gene for the presence of amylose), the homozygotes containing the SNP associated with the waxy mutation did not show waxy phenotypes. To identify more markers, we sequenced the GBSS gene from 89 genotypes, including some that were segregated from a cross with a line carrying the known waxy allele. As a result, 17 mutations in the GBSSI gene were identified, in which only the deletion in exon 6 (MeWxEx6-del-C) was correlated with the waxy phenotype. The evaluation of mutation points by discriminant analysis of principal component analysis (DAPC) also did not completely discriminate the waxy individuals. Therefore, we developed Kompetitive Allele Specific PCR (KASP) markers that allowed discrimination between WX and wx alleles. The results demonstrated the non-existence of heterozygous individuals of the MeWxEx6-del-C deletion in the analyzed germplasm. Therefore, the deletion MeWxEx6-del-C should not be used for assisted selection in genetic backgrounds different from the original source of waxy starch. Also, the alternative SNPs identified in this study were not associated with the waxy phenotype when compared to a panel of accessions with high genetic diversity. CONCLUSION: Although the GBSSI gene can exhibit several mutations in cassava, only the deletion in exon 6 (MeWxEx6-del-C) was correlated with the waxy phenotype in the original AM206-5 source.

SSIIIa-RNAi suppression associated changes in rice grain quality and starch biosynthesis metabolism in response to high temperature.

High temperature (HT) is a main environmental restraint that affects rice yield and grain quality. In this study, SSIIIa-RNAi and its wild-type (WT) were used to investigate the effect of HT exposure on the isozyme-specific variation of several key starch biosynthesis enzymes in developing endosperms and its relation to starch properties. SSIIIa-RNAi had minimal impact on grain chalky occurrence under normal temperature growth, but it could up-grade the susceptibility of grain chalky occurrence to HT exposure, due to the relatively sensitive response of AGPase and SSI to HT exposure. Different from WT, SSIIIa-RNAi had the relatively enriched proportion of chains with DP 13-16 under HT, and HT-induced decline in the proportion of DP < 12 became much larger for SSIIIa-RNAi relative to WT. SSIIIa-RNAi significantly enhanced the expression of SSI isozyme and total SS activity, whereas SSI-RNAi deficiency had little impact on the expression of SSIIIa isozyme. In this regard, the compensatory increase in SSI isozyme as a result of SSIIIa deficiency occurred only in a one-way manner. SSIIIa-RNAi caused a striking elevation in BEIIa expression, and the effect of SSIIIa deficiency on the chain length distribution in relation to HT exposure was closely associated with the participation of BEIIa, SSI, and their interaction in amylopectin biosynthesis.

Natural Polymorphisms in Arabidopsis Result in Wide Variation or Loss of the Amylose Component of Starch.

Starch granules contain two Glc polymers, amylopectin and amylose. Amylose makes up approximately 10% to 30% (w/w) of all natural starches thus far examined, but mutants of crop and model plants that produce amylose-free starch are generally indistinguishable from their wild-type counterparts with respect to growth, starch content, and granule morphology. Since the function and adaptive significance of amylose are unknown, we asked whether there is natural genetic variation in amylose synthesis within a wild, uncultivated species. We examined polymorphisms among the 1,135 sequenced accessions of Arabidopsis (Arabidopsis thaliana) in GRANULE-BOUND STARCH SYNTHASE (GBSS), encoding the enzyme responsible for amylose synthesis. We identified 18 accessions that are predicted to have polymorphisms in GBSS that affect protein function, and five of these accessions produced starch with no or extremely low amylose (< 0.5% [w/w]). Eight further accessions had amylose contents that were significantly lower or higher than that of Col-0 (9% [w/w]), ranging from 5% to 12% (w/w). We examined the effect of the polymorphisms on GBSS function and uncovered three mechanisms by which GBSS sequence variation led to different amylose contents: (1) altered GBSS abundance, (2) altered GBSS activity, and (3) altered affinity of GBSS for binding PROTEIN TARGETING TO STARCH1-a protein that targets GBSS to starch granules. These findings demonstrate that amylose in leaves is not essential for the viability of some naturally occurring Arabidopsis genotypes, at least over short timescales and under some environmental conditions and open an opportunity to explore the adaptive significance of amylose.

Amylopectin Chain Length Dynamics and Activity Signatures of Key Carbon Metabolic Enzymes Highlight Early Maturation as Culprit for Yield Reduction of Barley Endosperm Starch after Heat Stress.

Abiotic environmental stresses have a negative impact on the yield and quality of crops. Understanding these stresses is an essential enabler for mitigating breeding strategies and it becomes more important as the frequency of extreme weather conditions increases due to climate change. This study analyses the response of barley (Hordeum vulgare L.) to a heat wave during grain filling in three distinct stages: the heat wave itself, the return to a normal temperature regime, and the process of maturation and desiccation. The properties and structure of the starch produced were followed throughout the maturational stages. Furthermore, the key enzymes involved in the carbohydrate supply to the grain were monitored. We observed differences in starch structure with well-separated effects because of heat stress and during senescence. Heat stress produced marked effects on sucrolytic enzymes in source and sink tissues. Early cessation of plant development as an indirect consequence of the heat wave was identified as the major contributor to final yield loss from the stress, highlighting the importance for functional stay-green traits for the development of heat-resistant cereals.

The Zea mays mutants opaque2 and opaque16 disclose lysine change in waxy maize as revealed by RNA-Seq.

In maize, opaque2 (o2) and opaque16 (o16) alleles can increase lysine content, while the waxy (wx) gene can enhance the amylopectin content of grains. In our study, o2 and o16 alleles were backcrossed into waxy maize line (wxwx). The o2o2o16o16wxwx lines had amylopectin contents similar to those of waxy line. Their nutritional value was better than waxy line, but the mechanism by which the o2 and o16 alleles increased the lysine content of waxy maize remained unclear. The o2o2o16o16wxwx lines and their parents on kernels (18th day after pollination) were subjected to RNA sequencing (RNA-Seq). The RNA-Seq analysis revealed 272 differentially expressed genes (DEGs). Functional analyses revealed that these DEGs were mainly related to biomass metabolism. Among them, in o2o2o16o16wxwx lines, 15 genes encoding α-zein were down-regulated, which resulted in the reduction of α-zein synthesis and increased lysine content; lkr/sdh1 and Zm00001d020984.1 genes involved in the lysine degradation pathway were down-regulated, thereby inhibited lysine degradation; sh2, bt2 and ae1 genes involved in starch metabolism were upregulated, leaded to wrinkling kernel and farinaceous endosperm. Our transcriptional-level identification of key genes responsible for increased grain lysine content and farinaceous endosperm formation following introgression of o2 and o16 alleles should promote molecular breeding for maize quality.

MutMapPlus identified novel mutant alleles of a rice starch branching enzyme IIb gene for fine-tuning of cooked rice texture.

Physicochemical properties of storage starch largely determine rice grain quality and food characteristics. Therefore, modification of starch property is effective to fine-tune cooked rice textures. To obtain new resources with modified starch property as breeding materials, we screened a mutant population of a japonica cultivar Nipponbare and found two independent mutant lines, altered gelatinization (age)1 and age2, with moderate changes in starch gelatinization property. A combination of conventional genetic analyses and the latest mapping method, MutMapPlus, revealed that both of these lines harbour novel independent mutant alleles of starch branching enzyme IIb (BEIIb) gene. In age1, amino acid substitution of Met-723 to Lys completely abolished BEIIb enzyme activity without significant reduction in its protein level. A transposon insertion in an intron of BEIIb gene reduced BEIIb protein level and activity in age2. Production of a series of the mutant lines by combining age alleles and indica-type starch synthase IIa allele established stepwise alteration of the physicochemical properties of starch including apparent amylose content, thermal property, digestibility by α-amylase and branched structures of amylopectin. Consistent with the alteration of starch properties, the results of a sensory evaluation test demonstrated that warm cooked rice of the mutants showed a variety of textures without marked reduction in overall palatability. These results suggest that a series of the mutant lines are capable of manipulation of cooked rice textures.

Toxoplasma gondii Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts.

In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue cysts, leading to encephalitis. A characteristic of T. gondii bradyzoites in tissue cysts is the presence of amylopectin granules. The regulatory mechanisms and role of amylopectin accumulation in this organism are not fully understood. The T. gondii genome encodes a putative glycogen phosphorylase (TgGP), and mutants were constructed to manipulate the activity of TgGP and to evaluate the function of TgGP in amylopectin storage. Both a stop codon mutant (Pru/TgGPS25stop [expressing a Ser-to-stop codon change at position 25 in TgGP]) and a phosphorylation null mutant (Pru/TgGPS25A [expressing a Ser-to-Ala change at position 25 in TgGp]) mutated at Ser25 displayed amylopectin accumulation, while the phosphorylation-mimetic mutant (Pru/TgGPS25E [expressing a Ser-to-Glu change at position 25 in TgGp]) had minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions. The expression of active TgGPS25S or TgGPS25E restored amylopectin catabolism in Pru/TgGPS25A To understand the relation between GP and calcium-dependent protein kinase 2 (CDPK2), which was recently reported to regulate amylopectin consumption, we knocked out CDPK2 in these mutants. PruΔcdpk2/TgGPS25E had minimal amylopectin accumulation, whereas the Δcdpk2 phenotype in the other GP mutants and parental lines displayed amylopectin accumulation. Both the inactive S25A and hyperactive S25E mutant produced brain cysts in infected mice, but the numbers of cysts produced were significantly less than the number produced by the S25S wild-type GP parasite. Complementation that restored amylopectin regulation restored brain cyst production to the control levels seen in infected mice. These data suggest that T. gondii requires tight regulation of amylopectin expression for efficient production of cysts and persistent infections and that GP phosphorylation is a regulatory mechanism involved in amylopectin storage and utilization.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that causes disease in immune-suppressed individuals, as well as a fetopathy in pregnant women who acquire infection for the first time during pregnancy. This parasite can differentiate between tachyzoites (seen in acute infection) and bradyzoites (seen in latent infection), and this differentiation is associated with disease relapse. A characteristic of bradyzoites is that they contain cytoplasmic amylopectin granules. The regulatory mechanisms and the roles of amylopectin granules during latent infection remain to be elucidated. We have identified a role of T. gondii glycogen phosphorylase (TgGP) in the regulation of starch digestion and a role of posttranslational modification of TgGP, i.e., phosphorylation of Ser25, in the regulation of amylopectin digestion. By manipulating TgGP activity in the parasite with genome editing, we found that the digestion and storage of amylopectin due to TgGP activity are both important for latency in the brain.

Critical and speculative review of the roles of multi-protein complexes in starch biosynthesis in cereals.

Starch accounts for the majority of edible carbohydrate resources generated through photosynthesis. Amylopectin is the major component of starch and is one of highest-molecular-weight biopolymers. Rapid and systematic synthesis of frequently branched hydro-insoluble amylopectin and efficient accumulation into amyloplasts of cereal endosperm is crucial. The functions of multiple starch biosynthetic enzymes, including elongation, branching, and debranching enzymes, must be temporally and spatially coordinated. Accordingly, direct evidence of protein-protein interactions of starch biosynthetic enzymes were first discovered in developing wheat endosperm in 2004, and they have since been shown in the developing seeds of other cereals. This review article describes structural characteristics of starches as well as similarities and differences in protein complex formation among different plant species and among mutant plants that are deficient in specific starch biosynthetic enzymes. In addition, evidence for protein complexes that are involved in the initiation stages of starch biosynthesis is summarized. Finally, we discuss the significance of protein complexes and describe new methods that may elucidate the mechanisms and roles of starch biosynthetic enzyme complexes.

Highly phosphorylated functionalized rice starch produced by transgenic rice expressing the potato GWD1 gene.

Starch phosphorylation occurs naturally during starch metabolism in the plant and is catalysed by glucan water dikinases (GWD1) and phosphoglucan water dikinase/glucan water dikinase 3 (PWD/GWD3). We generated six stable individual transgenic lines by over-expressing the potato GWD1 in rice. Transgenic rice grain starch had 9-fold higher 6-phospho (6-P) monoesters and double amounts of 3-phospho (3-P) monoesters, respectively, compared to control grain. The shape and topography of the transgenic starch granules were moderately altered including surface pores and less well defined edges. The gelatinization temperatures of both rice flour and extracted starch were significantly lower than those of the control and hence negatively correlated with the starch phosphate content. The 6-P content was positively correlated with amylose content and relatively long amylopectin chains with DP25-36, and the 3-P content was positively correlated with short chains of DP6-12. The starch pasting temperature, peak viscosity and the breakdown were lower but the setback was higher for transgenic rice flour. The 6-P content was negatively correlated with texture adhesiveness but positively correlated with the cohesiveness of rice flour gels. Our data demonstrate a way forward to employ a starch bioengineering approach for clean modification of starch, opening up completely new applications for rice starch.

Systems Genetics Identifies a Novel Regulatory Domain of Amylose Synthesis.

A deeper understanding of the regulation of starch biosynthesis in rice (Oryza sativa) endosperm is crucial in tailoring digestibility without sacrificing grain quality. In this study, significant association peaks on chromosomes 6 and 7 were identified through a genomewide association study (GWAS) of debranched starch structure from grains of a 320 indica rice diversity panel using genotyping data from the high-density rice array. A systems genetics approach that interrelates starch structure data from GWAS to functional pathways from a gene regulatory network identified known genes with high correlation to the proportion of amylose and amylopectin. An SNP in the promoter region of Granule Bound Starch Synthase I was identified along with seven other SNPs to form haplotypes that discriminate samples into different phenotypic ranges of amylose. A GWAS peak on chromosome 7 between LOC_Os07g11020 and LOC_Os07g11520 indexed by a nonsynonymous SNP mutation on exon 5 of a bHLH transcription factor was found to elevate the proportion of amylose at the expense of reduced short-chain amylopectin. Linking starch structure with starch digestibility by determining the kinetics of cooked grain amylolysis of selected haplotypes revealed strong association of starch structure with estimated digestibility kinetics. Combining all results from grain quality genomics, systems genetics, and digestibility phenotyping, we propose target haplotypes for fine-tuning starch structure in rice through marker-assisted breeding that can be used to alter the digestibility of rice grain, thus offering rice consumers a new diet-based intervention to mitigate the impact of nutrition-related noncommunicable diseases.

Introgression of opaque2 into Waxy Maize Causes Extensive Biochemical and Proteomic Changes in Endosperm.

Waxy maize is prevalently grown in China and other countries due to the excellent characters and economic value. However, its low content of lysine can't meet the nutritional requirements of humans and livestock. In the present study, we introgressed the opaque2 (o2) allele into waxy maize line Zhao OP-6/O2O2 by using marker-assisted selection (MAS) technique and successfully improved the lysine content and quality of waxy maize. Transcript abundance analysis indicated that the wx1 expression levels had no difference between Zhao OP-6/o2o2 and Zhao OP-6/O2O2. However, Zhao OP-6/o2o2 was characterized by a phenotype of hard and vitreous kernels and accumulation of protein bodies at smaller size (one third of that of parents) but in larger numbers. Biochemical analyses showed that Zhao OP-6/o2o2 had 16.7% less free amino acids than Zhao OP-6/O2O2, especially those derived from glycolytic intermediates, but its content of lysine was increased by 51.6% (0.47% vs. 0.31%). The content of amylopectin was 98.5% in Zhao OP-6/o2o2, significantly higher than that in Zhao OP-6/O2O2 (97.7%). Proteomic analyses indicated that o2 introgression not only decreased the accumulation of various zein proteins except for 27-kDa γ-zein, but also affected other endosperm proteins related to amino acid biosynthesis, starch-protein balance, stress response and signal transduction. This study gives us an intriguing insight into the metabolism changes in endosperm of waxy maize introgressed with opaque2.

Increasing lysine content of waxy maize through introgression of opaque-2 and opaque-16 genes using molecular assisted and biochemical development.

The low lysine content of waxy maize cannot meet the nutritional requirements of humans, livestock, or poultry. In the present study, the high-lysine genes o2 and o16 were backcrossed into wx lines using the maize high-lysine inbreds TAIXI19 (o2o2) and QCL3021 (o16o16) as donors and the waxy maize inbred line QCL5019 (wxwx) as a receptor. In the triple-cross F1, backcross, and inbred generations, the SSR markers phi027 and phi112 within the wx and o2 genes and the SSR marker umc1121 linked to the o16 gene were used for foreground selection. Background selection of the whole-genome SSR markers was performed for the selected individuals. The grain lysine content was determined using the dye-binding lysine method. The waxiness of the grain was determined with the I2-KI staining and dual-wavelength spectrophotometric analysis. The BC2F2 generation included 7 plants of genotype wxwxo2o2O16_, 19 plants of genotype wxwxo16o16O2_, and 3 plants of genotype wxwxo2o2o16o16. In these seeds, the average amylopectin content was 96.67%, 96.87%, and 96.62%, respectively, which is similar to that of QCL5019. The average lysine content was 0.555%, 0.380%, and 0.616%, respectively, representing increases of 75.1%, 19.9%, 94.3%, respectively, over QCL5019. The average genetic background recovery rate of the BC2F3 families was 95.3%, 94.3%, 94.2%, respectively. Among these 3 wxwxo2o2O16O16 families, 4 wxwxo2o2O16o16 families, and 3 wxwxo2o2o16o16 families, the longest imported parent donor fragment was 113.35 cM and the shortest fragment was 11.75 cM. No significant differences in lysine content were found between the BC2F4 seeds and the BC2F3 seeds in these 10 families. This allowed us to increase the lysine content of waxy corn and produce seeds with excellent nutritional characteristics suitable for human consumption, animal feed, and food processing. This may be of significance in the breeding of high-quality corn and in improvement of the nutrition of humans, livestock, and poultry.