Waxy Gene Research Articles

Hybrid origins of cultivated potatoes

Solanum section Petota is taxonomically difficult, partly because of interspecific hybridization at both the diploid and polyploid levels. The taxonomy of cultivated potatoes is particularly controversial. Using DNA sequence data of the waxy gene, we here infer relationships among the four species of cultivated potatoes accepted in the latest taxonomic treatment (S. ajanhuiri, S. curtilobum, S. juzepczukii and S. tuberosum, the latter divided into the Andigenum and Chilotanum Cultivar Groups). The data support prior ideas of hybrid origins of S. ajanhuiri from the S. tuberosum Andigenum Group (2x = S. stenotomum) × S. megistacrolobum; S. juzepczukii from the S. tuberosum Andigenum Group (2x = S. stenotomum) × S. acaule; and S. curtilobum from the S. tuberosum Andigenum Group (4x = S. tuberosum subsp. andigenum) × S. juzepczukii. For the tetraploid cultivar-groups of S. tuberosum, hybrid origins are suggested entirely within much more closely related species, except for two of three examined accessions of the S. tuberosum Chilotanum Group that appear to have hybridized with the wild species S. maglia. Hybrid origins of the crop/weed species S. sucrense are more difficult to support and S. vernei is not supported as a wild species progenitor of the S. tuberosum Andigenum Group.

Development of genome-specific primers for homoeologous genes in allopolyploid species: the waxy and starch synthase II genes in allohexaploid wheat (Triticum aestivum L.) as examples

BackgroundIn allopolypoid crops, homoeologous genes in different genomes exhibit a very high sequence similarity, especially in the coding regions of genes. This makes it difficult to design genome-specific primers to amplify individual genes from different genomes. Development of genome-specific primers for agronomically important genes in allopolypoid crops is very important and useful not only for the study of sequence diversity and association mapping of genes in natural populations, but also for the development of gene-based functional markers for marker-assisted breeding. Here we report on a useful approach for the development of genome-specific primers in allohexaploid wheat.FindingsIn the present study, three genome-specific primer sets for the waxy (Wx) genes and four genome-specific primer sets for the starch synthase II (SSII) genes were developed mainly from single nucleotide polymorphisms (SNPs) and/or insertions or deletions (Indels) in introns and intron-exon junctions. The size of a single PCR product ranged from 750 bp to 1657 bp. The total length of amplified PCR products by these genome-specific primer sets accounted for 72.6%-87.0% of the Wx genes and 59.5%-61.6% of the SSII genes. Five genome-specific primer sets for the Wx genes (one for Wx-7A, three for Wx-4A and one for Wx-7D) could distinguish the wild type wheat and partial waxy wheat lines. These genome-specific primer sets for the Wx and SSII genes produced amplifications in hexaploid wheat, cultivated durum wheat, and Aegilops tauschii accessions, but failed to generate amplification in the majority of wild diploid and tetraploid accessions.ConclusionsFor the first time, we report on the development of genome-specific primers from three homoeologous Wx and SSII genes covering the majority of the genes in allohexaploid wheat. These genome-specific primers are being used for the study of sequence diversity and association mapping of the three homoeologous Wx and SSII genes in natural populations of both hexaploid wheat and cultivated tetraploid wheat. The strategies used in this paper can be used to develop genome-specific primers for homoeologous genes in any allopolypoid species. They may be also suitable for (i) the development of gene-specific primers for duplicated paralogous genes in any diploid species, and (ii) the development of allele-specific primers at the same gene locus.

Development of three allele-specific codominant rice Waxy gene PCR markers suitable for marker-assisted selection of amylose content and paste viscosity

Four Waxy haplotypes, previously identified as each having a different combination of three single nucleotide polymorphisms (SNPs) in the Waxy gene, were highly correlated with apparent amylose content and pasting properties, which are important grain quality traits for predicting cooked rice (Oryza sativa L.) texture and processing properties (Chen et al. in J Cereal Sci 47:536–545, 2008a; Chen et al. in J Cereal Sci 48:781–788, 2008b). Three allele-specific PCR markers were developed to genotype the three aforementioned functional SNPs in a single PCR amplification. Each marker contained two allele-specific primers and one common primer. For each marker, the two allele-specific primers differed by one base at the 3′-end to provide discrimination of SNP alleles, and were labeled with unique fluorescence probes. An additional mismatched base, the third base from the 3′-end, was inserted in some allele-specific primers to increase selectivity. The amplification step of the PCR thermal cycling program was initially set for 20× touch-down cycles with the annealing temperature of the first cycle approximately 6°C above the thermal melting temperature of all three primers at a touch-down rate of −0.3°C per cycle, and followed by 25× regular thermal cycles with the annealing temperature at their thermal melting temperature. The allelic genotypes for each SNP were distinguished from each other by both their differential primer-allele fluorescences and their amplification product lengths. The simplicity of these assays makes it easy to utilize these markers as part of a marker-assisted selection strategy in rice breeding programs selecting for these important grain quality traits.

Molecular basis of the waxy endosperm starch phenotype in broomcorn millet (Panicum miliaceum L.).

Waxy varieties of the tetraploid cereal broomcorn millet (Panicum miliaceum L.) have endosperm starch granules lacking detectable amylose. This study investigated the basis of this phenotype using molecular and biochemical methods. Iodine staining of starch granules in 72 plants from 38 landrace accessions found 58 nonwaxy and 14 waxy phenotype plants. All waxy types were in plants from Chinese and Korean accessions, a distribution similar to that of the waxy phenotype in other cereals. Granule-bound starch synthase I (GBSSI) protein was present in the endosperm of both nonwaxy and waxy individuals, but waxy types had little or no granule-bound starch synthase activity compared with the wild types. Sequencing of the GBSSI (Waxy) gene showed that this gene is present in two different forms (L and S) in P. miliaceum, which probably represent homeologues derived from two distinct diploid ancestors. Protein products of both these forms are present in starch granules. We identified three polymorphisms in the exon sequence coding for mature GBSSI peptides. A 15-bp deletion has occurred in the S type GBSSI, resulting in the loss of five amino acids from glucosyl transferase domain 1 (GTD1). The second GBSSI type (L) shows two sequence polymorphisms. One is the insertion of an adenine residue that causes a reading frameshift, and the second causes a cysteine–tyrosine amino acid polymorphism. These mutations appear to have occurred in parallel from the ancestral allele, resulting in three GBSSI-L alleles in total. Five of the six possible genotype combinations of the S and L alleles were observed. The deletion in the GBSSI-S gene causes loss of protein activity, and there was 100% correspondence between this deletion and the waxy phenotype. The frameshift mutation in the L gene results in the loss of L-type protein from starch granules. The L isoform with the tyrosine residue is present in starch granules but is nonfunctional. This loss of function may result from the substitution of tyrosine for cysteine, although it could not be determined whether the cysteine isoform of L represents the functional type. This is the first characterization of mutations that occur in combination in a functionally polyploid species to give a fully waxy phenotype.

DNA Markers for Identifying waxy Mutations and Improving Noodle Quality in Wheat

Approximately 70% of wheat endosperm consists of starch, and variations in the quality and quantity of starch affect the processing characteristics of wheat flour. Amylose content in particular has a major effect on Asian noodle quality, and selection of wheat lines with slightly lower amylose levels is an important goal in Japanese wheat breeding programs. Accurately measuring amylose content by direct methods such as colorimetric assays was found to be problematic, suggesting there was a need for a more efficient and accurate method of screening for reduced amylose content. Therefore, we characterized mutations in the wheat waxy genes, which control amylose synthesis, and developed DNA markers for the identification of null waxy alleles. In this review, we describe the development of these markers and outline their utility for wheat breeding programs.

Waxy strains of three amaranth grains raised by different mutations in the coding region

Waxy strains raised by waxy mutation have been found for three amaranth grains. Three genes encoding waxy protein were isolated from Amaranthus caudatus (Wx-ca), A. cruentus (Wx-cr), and A. hypochondriacus (Wx-hy). Sequence analysis indicated that the Wx-ca, Wx-cr, and Wx-hy genes contained the same exon (13 exons) and intron (12 introns) structure. The lengths of the Wx-ca, Wx-cr, and Wx-hy genes were 3,236, 3,237, and 3,225 bp, respectively. The alignment of the coding sequence of the three Waxy genes showed 12 polymorphic sites, including 11 single nucleotide polymorphisms (SNPs) (in exons 10 and 12 and introns 1, 3, 4, 9, and 11) and 5 deletions or insertions (indels) (in introns 4, 9, and 11). In particular, major polymorphism was detected in 8- and 3-bp indels in intron 4. Moreover, the mutation in the waxy alleles (wx-ca, wx-cr, and wx-hy) of all three species was also isolated and characterized. Comparison of coding sequences of the three Waxy genes and their waxy alleles indicated one base insertion (wx-ca: insert of T base in exon 8) and a base substitution (wx-cr: a G-to-T base substitution in exon 10; wx-hy: a G-to-A base substitution in exon 6), which occurred as internal termination codon in the three Waxy genes, suggesting the involvement of a nonsense or frameshift mutation. Therefore, these different mutations in coding regions were considered to be the cause of the waxy (amylose-free) phenotype.

Physicochemical and Genetic Analysis of an Endemic Rice Variety, Njavara (Oryza sativaL.), in Comparison to Two Popular South Indian Cultivars, Jyothi (PTB 39) and IR 64

Njavara is a medicinal rice strain, endemic to Kerala, South India, bestowed with medicinal qualities. Genetic variations and some of the physicochemical properties were studied using standard molecular protocols and compared with those of nonmedicinal rice varieties: Jyothi and IR 64. Njavara showed 11 unique positive and 36 unique negative markers to differentiate it from Jyothi and IR 64. Genetic similarity coefficient studies showed two well-defined clusters separating Njavara from Jyothi and IR 64. All the three varieties had waxy gene Wx(a) allele. Njavara had (CT)(n) repeats at (CT)(10), while Jyothi and IR 64 had repeats at (CT)(11) in the 5'-untranslated region of waxy gene. Njavara showed a CGTG sequence, while Jyothi and IR 64 had a CGCG sequence at the 14th exon of Sbe 1 gene. Njavara, Jyothi, and IR 64 have similar amylose equivalent (AE), which was confirmed by microsatellite markers. The SSR primers for protein content and setback viscosity primer (RM 4608) were observed to be polymorphic in case of Njavara. Njavara rice, with a distinct gene pool and medicinal properties, can be exploited as a nutraceutical rice.

Characterization of CIMMYT bread wheats for high- and low-molecular weight glutenin subunits and other quality-related genes with SDS-PAGE, RP-HPLC and molecular markers

Two hundred and seventy-three CIMMYT bread wheat cultivars and advanced lines grown under irrigated conditions in Mexico during the 2005-06 Yaqui crop cycle were characterized for quality-related genetic traits using gene-specific markers for some high- and low-molecular-weight glutenin subunit (HMW-GS and LMW-GS) genes, polyphenol oxidase (PPO), phytoene synthase (PSY), and waxy genes. Of them, 142 were analyzed for quality parameters including SDS sedimentation volume (SDS-SV), dough mixing time, and Alveograph parameters, and for HMW-GS and LMW-GS compositions using sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and reversed-phase high-performance liquid chromatography (RP-HPLC). For the Ppo-A1 locus tested with the marker PPO18, the frequencies of alleles Ppo-A1a and Ppo-A1b were 79.1 and 20.2%, respectively, and no PCR fragment was amplified in 2 lines (0.73%), whereas 227 lines (83.2%) contained the allele Ppo-D1a and 46 lines (16.8%) had Ppo-D1b detected by markers PPO16 and PPO29. For the marker YP7A, 142 lines (52.0%) were assumed to have the allele Psy-A1a and 131 lines (48.0%) contained the allele Psy-A1b. In the case of the marker YP7B for the gene Psy-B1, the alleles Psy-B1a and Psy-B1b were detected in 155 (56.8%) and 43 (15.8%) lines, respectively, and 75 (27.4%) lines possessed the allele Psy-B1d detected by the marker YP7B-3. All 273 lines contained the alleles Wx-A1a and Wx-D1a as determined by markers MAG264 and MAG269, respectively. Using the marker Wx-B1, 204 lines (74.7%) were presumed to have the Wx-B1a allele and 69 (25.3%) possessed Wx-B1b. The over-expressing allele of Bx7 OE and subunit By8*, not clearly seen with SDS-PAGE, were detected by RP-HPLC. The numbers of lines with subunits Ax2*, By8, By9, Bx17, Bx20, Dx5, and Glu-B3j were 90, 16, 57, 5, 46, 118, and 33, respectively, in the 142 lines analyzed with molecular markers, and were consistent with the results obtained by SDS-PAGE, except for one line with the 1A.1R translocation. Subunits Ax1 and Ax2* at the Glu-A1 locus showed significantly better effects on all quality parameters than subunit Null. Subunits 5 + 10 gave significantly better effects for all parameters. Subunit Glu-A3b showed more positive effects than its alternative alleles on SDS-SV and SDS-sedimentation volume/protein content index (SPI). The allele Glu-B3g showed the best effect on SDS-SV and Alveograph W, whereas Glu-B3j, associated with the 1B.1R translocation, exhibited a strongly negative effect on all quality parameters.

Molecular Characterization of the Waxy Gene in Einkorn Wheat

Molecular Characterization of the Waxy Gene in Einkorn Wheat

Quality characteristics and field performance of selectable marker-free transgenic rice with antisense Wx gene and improved quality derived from the elite parents of hybrid indica rice

In rice grains, high amylose content (AC) is correlated with poor grain quality, particularly in indica hybrid rice. To obtain indica hybrid rice with improved cooking and eating qualities, we introduced the antisense Waxy ( Wx) gene into 2 elite parental lines of indica hybrid rice by using co-transformation methods. Subsequently, we selected several elite homozygous transgenic lines that did not contain the selectable marker. The expression of the endogenous Wx gene of the selected transgenic lines was significantly downregulated, resulting in low AC in the mature seeds; moreover, the AC in some lines reduced to the level observed in glutinous rice. With the decrease in AC, the gel consistency of the transgenic rice became softer, and the gelatinization temperature tended to be higher than those of the wild types, especially in the case of the Longtefu-derived transformants. We also analyzed the pasting properties of the selected transgenic low-AC lines, and we noted an improvement in the pasting properties of the transgenic rice lines. The results from a field trial indicated that the grain weights of the transgenic lines with lower AC exhibit remarkable reduction compared with those of the wild types.

Breeding and Field Performance of Novel Soft and Waxy Transgenic Rice Lines without Selectable Markers

Amylose content is one of the important factors affecting rice cooking and eating quality, and controlled by the Waxy (Wx) gene which encoding a granule-bound starch synthase. High amylose levels are usually associated with poor cooking and eating quality. In our prevrious studies, in order to generate transgenic rice varieties with improved quality and without of the selectable marker, the antisense Wx gene was co-transformed together with a selectable marker gene, the hygromycin-resistant gene (HPT), to a high-yielding japonica rice cultivar, Wuxiangjing 9, mediated with Agrobacterium tumeficien. And some select-able marker-free (SMF) transgenic lines had been subsequently selected. In present study, several homozygous transgenic lines with soft or waxy grain property were selected from the offspring of these SMF transformants containing the antisense Wx gene. The results from Northern blot analysis indicated that the levels of both the Wx mature mRNA and unspliced transcripts were reduced differently in the endosperm of different transgenic lines. The Wx protein level in mature seeds of the transgenic lines was also reduced differently. The amylose content of two transgenic lines was reduced to that of soft rice (about 10%) and one line was reduced to that of waxy rice (lower than 2%). The gel consistency of these transgenic lines was softer than that of the wild type. The variance of gelatination temperature between the transgenic lines and the wild type did not reach to significant level. The most of the main agronomic traits of these transgenic lines were similar to that of the wild type. Therefore, three SMF trans-genic homozygous lines with improved quality were successfully bred.

Isolation and characterization of the ryeWaxygene

Complete genomic and cDNA sequences of the Waxy gene encoding granule-bound starch synthase I (GBSSI) were isolated from the rye genome and characterized. The full-length rye Waxy genomic DNA and cDNA are 2767 bp and 1815 bp, respectively. The genomic sequence has 11 exons interrupted by 10 introns. The rye Waxy gene is GC-rich, with a higher GC frequency in the coding region, especially in the third position of the codons. Exon regions of the rye Waxy gene are more conserved than intron regions when compared with the homologous sequences of other cereals. The mature rye GBSSI proteins share more than 95% sequence identity with their homologs in wheat and barley. A phylogenetic tree based on sequence comparisons of available plant GBSSI proteins shows the evolutionary relationship among Waxy genes from rye and other plant genomes. The identification of the rye Waxy gene will enable the manipulation of starch metabolism in rye and triticale.

Breeding and Field Performance of Novel Soft and Waxy Transgenic Rice Lines Without Selectable Marker

Amylose content is one of the important factors affecting rice cooking and eating quality, and is controlled by Waxy ( Wx) gene encoding a granule-bound starch synthase. High amylose level is usually associated with poor cooking and eating quality. Several homozygous transgenic lines with soft or waxy grain property were selected from the offspring of selectable marker-free (SMF) transformants containing the antisense Wx gene. The results from Northern blotting analysis indicated that the levels of both Wx mature mRNA and pre-mRNA were reduced in the endosperm of different transgenic lines. The Wx protein content in mature seeds of the transgenic lines also decreased. The amylose contents of 2 transgenic lines were reduced to that of the soft rice (about 10%), and one of them was even reduced to the level of waxy rice (lower than 2%). The gel consistency of these transgenic lines was softer than that of the wild type. The variance of gelatinization temperature between the transgenic lines and the wild type was significant. Most of the main agronomic traits of these transgenic lines were similar to those of the wild type. Therefore, the 3 SMF transgenic homozygous lines with improved quality were successfully developed.

Production of a fully waxy line and analysis ofwaxygenes in the allohexaploid crop, Japanese barnyard millet

With 4 figures and 2 tables Abstract Foxtail millet, common millet and Japanese barnyard millet have traditionally been important food sources in East Asian countries. Although waxy types of foxtail and common millet have been identified, a waxy mutant of the allohexaploid crop Japanese barnyard millet has not yet been reported. However, several Japanese landraces have been identified that have approximately half the level of amylose found in other varieties. We employed one of the low amylose landraces, `Noge-Hie', to produce waxy Japanese barnyard millet using a γ-radiation treatment. The seeds from a single M2 plant stained red-brown with iodine solution, indicating the starch in the seeds lacked amylose. Colorimetric tests indicated that amylose was not present in endosperm tissue of the mutant, and analysis of starch granule bound proteins showed that waxy (Wx) protein was absent from the starch granules. The waxy trait was stably inherited in subsequent generations. Additionally, a PCR-based analysis demonstrated the presence of three separate waxy genes in the millet, and indicated that the low amylose landraces carry a deletion in one of these three genes.

A Modified TILLING Method for Wheat Breeding

The large genome and polyploidy of wheat (Triticum aestivum L.) makes it difficult to identify desirable genetic changes based on phenotypic screening due to gene redundancy. Forward genetics is, therefore, more difficult in wheat than in diploid plants. A modified TILLING (Targeting Induced Local Lesions IN Genomes) method including the harvest of five heads per M1 plant, storage of M2 seeds, using unlabeled primers and agarose gels for mutation detection, and crossing of useful mutants for desired grain quality was explored in this report. A soft wheat cultivar, QAL2000, and a hard wheat cultivar, Ventura, were mutagenized with ethyl methanesulfonate (EMS). Screening of the waxy genes Wx‐A1 and Wx‐D1 in 2348 EMS‐treated M2 plants allowed identification of 121 mutants, including silent, missense, and knockout (truncation) mutations. A complete waxy wheat was successfully bred in 18 mo by crossing two truncation mutants (Wx‐A1‐truncation and Wx‐D1‐truncation; Wx‐B1 is naturally null in both mutants). Screening of two puroindoline genes (Pina and Pinb) in QAL2000 identified 19 mutants. A hard grain variant of a soft cultivar was identified due to a mutation in Pinb caused by a premature stop codon. Background mutations were observed and further self‐fertilization or crossing with a wild type was performed to eliminate deleterious mutations. With the rapid accumulation of wheat genomics information, many potential target genes of interest can be screened for mutations in these TILLING populations.

Analysis on the characters of progenies of the rice transformed with anti-sense <I>waxy</I> gene

The segregation of exogenous genes was studied by hygromycin-resistant and PCR experiments in the transgenic rice (Oryza sativa L. subsp. japonica and indica) with anti-sense waxy gene, meanwhile the change of amylose and waxy protein contents in progenies of transgenic rice was analyzed. The results showed no matter the rice Guangjing No.1 (O. Sativa L. subsp. japonica) were transformed by p13w4 plasmid carrying anti-sense waxy gene and hygromycin-resistant gene, or in the rice 01Z5202 (O. sativa L. subsp. indica) were co-transformed by p13w8 plasmid carrying anti-sense waxy gene and p1300 plasmid carrying hygromycin-resistant gene, the target gene(s) had been segregated in the progenies; the content of amylose of the transgenic plants was lower than those in non-transgenic ones, and the content of amylose in some of transgenic plants was less than 10.0% (of the weight of grain), which was much lower than those in the control (about 22.04%); and the analysis with SDS-PAGE showed the content of the waxy protein are positively correlated with the con-tent of amylose in the tested transgenic rice materials.

QTL detection of rice grain quality traits by microsatellite markers using an indica rice (Oryza sativa L.) combination

The gelatinization temperature (GT), gel consistency (GC) and amylose content (AC) are the three major rice traits that are directly related to cooking and eating quality (Little et al. 1958). GT is a physical trait responsible for cooking time and the capacity to absorb water during the processes cooking, and the temperature at which starch irreversibly loses its crystalline order during cooking. The GC is responsible for softness, and the AC is responsible for texture and appearance in rice. Hence, regulating AC in rice has been a major concern of rice breeders. To facilitate the development of new varieties with high cooking and eating qualities, it is necessary to understand the genetic bases of such traits. Lanceras et al. (2000) found four QTLs for AC on chromosomes three, four, six and seven. These QTLs accounted for 80% of phenotypic variation observeded in AC. Two QTLs on chromosome six, and one on chromosome seven were detected for GC, which accounted for 57% of phenotypic variation. Umemoto et al. (2002) confirmed this and demonstrated that the alk locus encodes the enzyme soluble starch synthase IIa. Bao et al. (2002) and Lanceras et al. (2000) found the effect of the wx region on GC. However, He et al. (1999) and Bao et al. (2002) showed that GC is controlled by two QTLs with minor effects. Zhou et al. (2003) improved the eating and cooking quality of Zhenshan 97 by introgressing the waxy gene region from Minghui 63 (wx-MH), a restorer line that had medium AC, soft GC and high GT. Li et al. (2004) identified four QTLs for AC, three for GT and five for GC using backcross-inbred lines. The four QTLs for AC were located on chromosomes three, four, five and six. The QTL on chromosome six covered the wx gene region and mainly contributed to the variance between

Genetic variability for waxy genes in Argentinean bread wheat germplasm

Amylose and amylopectin are the two polysaccharides that constitute starch in bread wheat and the enzyme GBSSI ( Granule-bound starch synthase I), also known as waxy protein, is responsible for amylose synthesis in storage tissues. Decrease of the amylose content in starch has been associated with the lack of waxy protein(s). In this work, different sets of PCR markers were used to characterize the genetic variability of waxy loci from 103 Argentinean bread wheat cultivars. For the Wx-A1 locus, Wx-A1a and a novel molecular allele designed Wx-A1g were detected. Wx-B1 locus showed three alleles ( Wx-B1a , Wx-B1b, Wx-B1e ), and Wx-D1 locus showed only the Wx-D1a allele. Novel single-locus allele specific markers for Wx-A1b, Wx-B1b and Wx-D1b null alleles were also described. To our best knowledge this is the first study focused to characterize the genetic variability for waxy genes in bread wheat cultivars from South America.

Molecular cloning and characterization of granule bound starch synthase I cDNA from a grain amaranth (Amaranthus cruentus L.)

A full-length cDNA clone encoding granule-bound starch synthase I (GBSSI = Waxy gene) from grain amaranth (Amaranthus cruentus L.) perisperm was isolated and characterized. Segregation of amylose content in F2 population suggested that the amylose content of A cruentus is controlled by a single gene, Waxy (GBSSI). cDNA clone of this gene is 2076 bp in length and contains an open reading frame of 1821 bp corresponding to a polypeptide of 606 amino acids residues, including a transit peptide of 77 amino acids. Comparison of the cDNA and genomic sequences (3492 bp) suggested that the amaranth GBSSI gene has 12 introns, of which exons 1–13 contributed to the coding sequence. The mature protein shares 70.2–75.3% sequence identity with GBSSI of dicots and about 64.0–67.8% identity with those of monocots. This protein contains the conserved motif KTGGL found in other GBSSI proteins, which has been implicated as the active site in glycogen synthase. Sequence analysis predicted that GBSSI of amaranth has a transit peptide of 77 amino acids including FIR↓S, which is different cleavage site that of the other dicot species. These results will provide more useful information for understanding the structure/function relationship of this protein from amaranths perisperm.

Breeding elite japonica-type soft rice with high protein content through the introduction of the anti- Waxy gene

Low amylose rice ( rice grains. In an attempt to analyze whether the anti-Waxy gene could increase protein content while reducing amylose content in the rice grains, and develop good soft rice varieties with high protein content, an anti-Waxy gene was introduced into the high-yield japonica rice strain, Shangshida No. 2. The amylose contents in the T1 grains were analyzed. The amylose content in the T1 grains derived from the T0 plant with two integrated copies of the anti-Waxy gene was only 6.8%, just belonging to the range of soft rice. The T5 transgenic rice seeds were developed, in which amylose content and protein content were 4.3 and 11.2%, respectively, and the corresponding indexes in the wild type were 12.7 and 9.5%, respectively. In conclusion, it was verified that the anti-Waxy gene could increase protein contents while reducing amylose contents of the rice grains; a kind of elite japonica transgenic rice with low amylose content and high protein content was obtained through the introduction of an anti-Waxy gene. In addition, a new rationale and method for breeding marker-free soft rice by Agrobacterium tumefaciens-mediated co-transformation is presented.