Molecular marker assisted selection for improvement of the eating,cooking and sensory quality of rice (Oryza sativa L.)

II-32B is a key maintainer line used for hybrid rice breeding in China. However, it is of poor quality for most Chinese consumers because of its high apparent amylose content (AAC), high gelatinization temperature (GT) and non-fragrance. It is well known that the AAC, GT and fragrance traits are largely controlled by the Wx, starch synthase IIa (SSIIa), and fragrance (fgr) genes, respectively. With the aid of functional markers, we improved the quality of II-32B by introgressing the Wx, SSIIa, and fgr genes from Yixiang B, a fragrant maintainer line that has low AAC and low GT. The microsatellite allele (CT)₁₇ of Wx, the contiguous single nucleotide polymorphism TT allele of SSIIa and the 8-bp deletion allele of fgr were transferred to II-32B by two backcrosses and three selfings. Molecular marker assisted selection was applied in the series to select for individuals carrying Wx-(CT)₁₇, SSIIa-TT, and fgr-deletion alleles. According to the marker genotypes, seventeen homozygous lines for Wx-(CT)₁₇, SSIIa-TT, and fgr gene markers were finally selected. The improved II-32B lines were fragrant with reduced AAC and GT.     

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-ACCⅠ . 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.     

Inheritance of flour paste viscosity is associated with a rice Waxy gene exon 10 SNP marker

Apparent amylose content is a key element for characterizing a rice (Oryza sativa L.) cultivar for cooking quality. However, cultivars with similar apparent amylose content can have widely varying quality attributes, including major parameters of flour paste viscosity. It has been postulated that the presence of a rice Waxy gene single nucleotide polymorphism (SNP) marker is associated with elevated Rapid Visco Analyser (RVA) properties in specific high amylose rice cultivars. A mapping population derived from a cross between two varieties, Cocodrie and Dixiebelle, having similar high apparent amylose contents, but with different paste viscosity properties and Waxy gene markers was analyzed for the genetic segregation of various pasting properties, measured with RVA instrumentation. Marker inheritance analyses revealed that the Waxy exon 10 SNP marker was associated with the proportion of soluble to insoluble apparent amylose and most RVA pasting measurements. Waxy gene markers can be used to efficiently improve the selection of rice with desirable characteristics, particularly for superior parboiling and canning quality.     

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.     

水稻Wx基因的遗传多态性及其与主要米质指标的相关性分析

利用RM190和484/W2R ACCⅠ标记分析了50个水稻非糯品种的Wx基因的等位性变异。其中,RM190揭示出7种(CT)n纯合变异类型和1种杂合类型,分别为 (CT)20、(CT)19、(CT)18、(CT)17、(CT)14、(CT)11、(CT)10和(CT)11/(CT)18,并以（CT）11和（CT）18两种类型为主;484/W2R ACCⅠ共揭示出2种纯合类型和1 种杂合类型,分别为G/G型、T/T型及G/T型;(CT)11大多为G/G型,(CT)18大多为T/T型,这两个标记可将供试材料划分为10种等位基因变异类型。进一步分析表明,RM190揭示的Wx基因多态性可以解释直链淀粉含量变异的59.3 %;484/W2R ACCⅠ揭示的Wx基因多态性可以分别解释直链淀粉含量及胶稠度变异的56.1%和24.6%;而两标记共同可解释直链淀粉含量变异的72.4%。还对这两个标记在育种实践中的应用前景进行了探讨。     

Comparative susceptibility of Ostrinia furnacalis, Ostrinia nubilalis and Diatraea saccharalis (Lepidoptera: Crambidae) to Bacillus thuringiensis Cry1 toxins

Transgenic corn hybrids that express toxins from Bacillus thuringiensis (Bt) are highly effective against the European corn borer, Ostrinia nubilalis (Hübner), and the closely related Asian corn borer, Ostrinia furnacalis (Guenée). Since the registration of Bt corn hybrids in the U.S. in 1996, there has been a great deal of information generated on O. nubilalis. However, relatively little information exists for O. furnacalis. To help determine whether the information generated for O. nubilalis can be leveraged for decisions regarding the use of transgenic Bt corn against O. furnacalis, experiments were designed to determine whether the pattern of sensitivity to various Bt Cry1 toxins is similar between the two species. Test insects included laboratory-reared O. furnacalis originating from Malaysia, a Bt-susceptible laboratory colony of O. nubilalis maintained at the University of Nebraska-Lincoln (UNL) and an out-group consisting of the sugarcane borer, Diatraea saccharalis (F.), from Louisiana which represents a different genus from the same family. O. furnacalis and O. nubilalis exhibited a similar pattern of susceptibility to all the Cry1 toxins and were highly susceptible to the range of Bt toxins tested including Cry1Aa, Cry1Ab, Cry1Ac and Cry1F. Both of the Ostrinia species were more tolerant to Cry1Ba compared with D. saccharalis, although sensitivity of O. furnacalis was intermediate and did not differ significantly from that of O. nubilalis and D. saccharalis. D. saccharalis was also susceptible to the range of toxins tested but unlike the two Ostrinia species, was more tolerant to Cry1F and more susceptible to Cry1Ba. These results indicate that both of the Ostrinia corn borer species are similar in sensitivity to the Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1F toxins, thus suggesting shared toxin receptors and mechanisms of toxicity for the two species.     

Analysis of QTL for resistance to head smut (Sporisorium reiliana) in maize

Head smut of maize, caused by the fungus Sporisorium reiliana, is an important disease in the temperate maize-growing areas worldwide. In this study, we mapped and characterized quantitative trait loci (QTL) conferring resistance to S. reiliana using a F_2:3 population of 184 families derived from a cross between Mo17 (resistant parent) and Huangzao4 (susceptible). The population was evaluated for resistance in replicated field trials with artificial inoculation of S. reiliana chlamydospores in Gongzhuling of Jilin Province and Harbin of Heilongjiang Province of China, two hot spots of head smut incidence, in 2003 and 2004. Genotypic and G × E variances for disease incidence were highly significant in the population. Heritability estimates for percentage disease incidence in the 2-location and 2-year evaluation ranged from 0.62 to 0.70. Composite interval mapping on a linkage map (1956.1 cM distance; 9.34 cM average interval) constructed with 84 SSR and 135 AFLP markers, identified five QTL, one each on chromosomes 1, 3 and 8 and two on chromosome 2, accounting for 5.0–43.7% of the phenotypic variance across four environments. One major QTL on chromosome 2 explaining up to 43.7% of the phenotypic variance can potentially be used in molecular marker-assisted selection for head smut resistance in maize.     

Analysis of starch swelling power in Australian breeding lines of hexaploid wheat (Triticum aestivum L.)

Starch is the major component of wheat (Triticum aestivum L.) grain and is composed of two large glucan molecules, amylose and amylopectin. The ratio between the two polymers types influences the water absorbing properties of starch upon heating, and thus affects the end-use of grain and purified starch. In this study, we evaluated the starch swelling power (SSP) values in seven wheat populations developed from crosses involving low-SSP lines. Analysis of starch produced by the F₂ generation plants showed that the largest SSP variation (11.4–16.2) and lowest SSP mean (13.9) was obtained for a population derived from doubled haploid lines SM1028 (SSP = 14.5) and VK306 (SSP = 13.6). The population of 360 lines was advanced by single seed descent to the following generations for further studies. Starch analysis of grain produced by F₄ generation lines in two field locations during 2006 and in a greenhouse environment during 2005 showed that SSP values were relatively stably inherited. The average broad-sense heritability was 73% and significant (P < 0.001) genotype × genotype and genotype × environment interactions were seen. Starches with the highest and lowest SSP values were inversely related to amylose concentration determined by high pressure liquid chromatography (HPLC)–size exclusion chromatography (SEC) of debranched starch. Developed lines with the lowest SSP values surpassed 40% in apparent amylose concentration. The study illustrates that screening for SSP in early generations can be used to develop wheat lines with desired starch swelling characteristics.     

Integrated management of Striga hermonthica, stemborers,and declining soil fertility in western Kenya

Striga hermonthica (Delile) Benth., stemborers, and declining soil fertility are serious threats to sustainable food production in the Lake Victoria zone of Kenya. To address these constraints, promising integrated crop management technologies were evaluated, using a multi-locational design in four sub-locations in Siaya and Vihiga district (western Kenya) for six cropping seasons. Technologies evaluated consisted of the traditional maize (Zea mays L.) – bean (Phaseolus vulgaris L.) intercrop, maize – Desmodium (Desmodium uncinatum (Jacq.) DC.) push–pull intercrop, Crotalaria (Crotalaria ochroleuca G. Don) – maize rotation, and soybean (Glycine max (L.) Merr) – maize rotation. Within each of these systems, imazapyr-coated herbicide-resistant maize (IR-maize) and fertilizer were super-imposed as sub-plot factors. The push–pull system was observed to significantly reduce Striga emergence and stemborer damage from the second season onwards. IR-maize reduced and delayed Striga emergence from the first cropping season. Differences in Striga emergence and stemborer damage between the other systems were not significantly different. After five cropping seasons, the Striga seedbank was significantly higher in the maize-bean intercrop system than in the push–pull system under both maize varieties while the rotational systems had intermediate values not different from the day zero values. Under IR-maize, the Striga seedbank was significantly lower than under local maize for all cropping systems. Maize yields varied between seasons, districts, and cropping systems. Yields in the push–pull system were higher than in the maize-bean intercrop after two seasons and in the absence of mid-season drought stress. Both maize and soybean responded significantly to fertilizer application for both districts and for most seasons. The various interventions did not substantially affect various soil fertility-related parameters after five seasons. In the short term, IR-maize integrated in a push–pull system is the most promising option to reduce Striga while the rotational systems may need a longer timeframe to reduce the Striga seedbank. Finally, farmer-led evaluation of the various technologies will determine which of those is really most acceptable under the prevailing farming conditions.     

Development of simple and co-dominant PCR markers to genotype puroindoline a and b alleles for grain hardness in bread wheat (Triticum aestivum L.)

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.). A large deletion in the puroindoline a (Pina) gene or single nucleotide polymorphisms (SNPs) in the puroindoline b (Pinb) gene results in hard grain texture. So far, nine Pina alleles (Pina-D1a–Pina-D1b, Pina-D1k–Pina-D1q) and seventeen Pinb alleles (Pinb-D1a–Pinb-D1g, Pinb-D1p–Pinb-D1ab) have been identified in bread wheat. The major Pina and Pinb alleles identified in hard wheat cultivars are Pina-D1b, Pinb-D1b, Pinb-D1c and Pinb-D1d. In this study, a three-primer PCR system was employed to develop nine co-dominant STS markers for genotyping Pina-D1a and Pina-D1b, whereas temperature-switch (TS) PCR was used to develop six co-dominant SNP markers for genotyping the Pinb-D1a, Pinb-D1b, Pinb-D1c and Pinb-D1d alleles. These STS and TS-PCR markers were used to verify the grain hardness genotype of 100 wheat cultivars. The reliability and genotyping accuracy of TS-PCR markers were confirmed through sequencing of PCR products and a comparison with previously published results. Therefore, STS and TS-PCR markers offer a simple, cost-effective and reliable method for high-throughput genotyping Pina and Pinb alleles to select grain hardness in wheat quality breeding programs and for wheat market classification.     

Waxy gene haplotypes: Associations with apparent amylose content and the effect by the environment in an international rice germplasm collection

Apparent amylose content (AAC), the key determinant of rice end-use quality attributes, is primarily controlled by the Waxy gene which codes for granule bound starch synthase (GBSS). We examined the combination of sequence variation in the Waxy gene and environmental effects, and their associations with AAC using 171 rice accessions originating from 43 countries. The combination of two single-nucleotide-polymorphism (SNP) markers in the Waxy gene allows for the identification of three marker haplotypes in this gene. The first SNP is at the leader intron splice site (In1 SNP), and the second polymorphism is in exon 6. The haplotypes explained 86.7% of the variation in AAC and discriminated the three market classes of low, intermediate and high AAC rice from each other. The environment affected the AAC of all haplotypes. Higher air temperature during grain development associated with a decrease in AAC of low and intermediate AAC-types, but with an increase in AAC of high AAC-type. The association of AAC with several Waxy RM190 microsatellite-(CT_n) alleles in combination with the In1 SNP was also examined. In conclusion, the Waxy haplotypes studied appear to be useful markers for selecting the AAC of breeding lines developed from the world's rice germplasm.     

Discriminant analysis of selected yield components and fatty acid composition of chosen Triticum monococcum, Triticum dicoccum and Triticum spelta accessions

This study analyses the variability of key yield components, the content of protein and crude fat in grain and the fatty acid composition of 50 spring accessions of Triticum monococcum, Triticum dicoccum and Triticum spelta of various origins. The average protein content of the grain of T. monococcum was 20.8%, of T. dicoccum 19.7%, and of spelt 17.0%. The crude fat content of T. monococcum grain (2.7%) was significantly higher compared with T. spelta (2.4%) and T. dicoccum (2.3%). In crude fat, fatty acids C18:2, C18:1 and C16 predominated. T. spelta was characterised by the highest concentrations of C18:2 and C16 (55.89% and 18.77% respectively), while T. monococcum had the highest content of C18:1 (26.35%). The structure of analysed fatty acids proved to be highly desirable in this species. A discriminant analysis performed separately for five variables: protein and fat content and three biometrical characters and separately for fatty acid composition enabled three Triticum species to be distinguished. These species also differed significantly with respect to the C18:1/C16 ratio which was equal to 1.78, 1.06, 1.47 and 0.99 in T. monococcum, T. dicoccum, T. spelta and Triticum aestivum respectively.     

Marker assisted backcross breeding to improve cooking quality traits in Myanmar rice cultivar Manawthukha

Several components contribute to rice grain quality, and among these, fragrance and taste qualities are the most important to consumers. Manawthukha, an elite non-fragrant cultivar with high amylose content (AC), intermediate gel consistency (GC) and gelatinisation temperature (GT), occupies the largest number of acres in Myanmar. To improve fragrance and intermediate AC in Manawthukha, Basmati370 was used as a donor parent to introgress the Basmati alleles of genes conferring fragrance and intermediate AC into Manawthukha by Marker Assisted Backcrossing (MAB). Four backcrosses and one selfing were conducted to transfer positive alleles of badh2 and Wx from Basmati into Manawthukha. Twelve BC₄F₂ selected lines that carried the homozygous Basmati alleles were planted in multi-location trials (four in Myanmar and one in Thailand) and examined for their agronomic performance and cooking quality. The seeds of the improved lines and their parents, harvested from Kamphaeng Saen, Kuaukse, Kyauktada and Latpatan, were investigated for fragrance and AC. All improved lines had fragrance and intermediate AC similar to the donor parent Basmati, whereas their agronomic performances were the same as the original Manawthukha. All improved lines had medium plant heights, high tiller numbers, dense panicles, non-lodging phenotypes and high yielding potentials. Simultaneous improvement of fragrance and intermediate AC indicated that badh2 and Wx genes had major effects on the fragrance and AC, respectively. We show here the success of improving fragrance and intermediate AC by MAB and the high efficiency of selection in early generations. In the present study, MAB accelerated the development of superior qualities in the genetic background of Manawthukha. The improved Manawthukha should be immediately useful for Myanmar farmers and will help farmers to increase their incomes.     

Effects of crude seed extracts of Annona atemoya and Annona squamosa L. against the cabbage looper, Trichoplusia ni in the laboratory and greenhouse

Antifeedant, growth inhibitory and toxic effects of crude seed extracts of Annona squamosa and Annona atemoya from Fazenda Viveiro Bona, Parasisópolis – Minas Gerais, Brazil, were evaluated against the cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) using different bioassays. Crude methanolic seed extracts deterred feeding of third instar T. ni larvae in a leaf disc choice bioassay. A. squamosa was ∼10 times more active as a feeding deterrent than A. atemoya (DC₅₀ = 2.3 mg/ml vs. 20.1 mg/ml). A. squamosa was ∼three times more active as a growth inhibitor than A. atemoya (EC₅₀ = 38.0 ppm vs. 117.0 ppm). Methanolic seed extracts of A. squamosa and A. atemoya were toxic to third instar T. ni larvae both through topical and oral application. A. squamosa was more toxic through feeding (LC₅₀ = 167.5 ppm vs. 382.4 ppm) whereas, A. atemoya exerted greater toxicity via topical application (LC₅₀ = 301.3 μg/larva vs. 197.7 μg/larva). Both A. squamosa and A. atemoya extracts reduced leaf area consumption and larval growth in a greenhouse experiment. Our results indicate that both A. squamosa and A. atemoya have potential for development as botanical insecticides, especially for local use in Brazil.     

Insecticidal activities of crude extracts and phospholipids from Chenopodium ficifolium against melon and cotton aphid, Aphis gossypii

Several organic solvent extracts of Chenopodium ficifolium were tested for their insecticidal activity against melon and cotton aphid, Aphis gossypii, on cucumber plants. Both methanol and ethanol extracts, at 5000 μg ml⁻¹, were highly active giving over 80% control. The other crude extracts displayed moderate or weak insecticidal activity giving control in the range of 16–69%. Two phospholipids were isolated as insecticidal active substances from C. ficifolium. Their chemical structures were identified as 1-palmitoyl-2-(3-trans)-hexadecenoyl-sn-glycero-3-phosphoglycerol and 1-palmitoyl-2-linoleoyl-3-glycerophosphocholine by GC–MS, EDS, mass and NMR spectral analyses. Both compounds displayed a dose-dependent mortality of A. gossypii. Furthermore, the liquid formulation that was obtained by partitioning with n-hexane from the methanol extract of C. ficifolium controlled melon and cotton aphid on cucumber plants effectively. These results indicate that extracts of C. ficifolium have potential for development as botanical insecticides for controlling A. gossypii infesting cucumber plants.     

Reduced height alleles (Rht) and Hagberg falling number of wheat

Near-isogenic lines varying for alleles for reduced height (Rht) and photoperiod insensitivity (Ppd-D1) in cv. Mercia (2005/6–2010/11; rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht8c+Ppd-D1a, Rht-D1c, Rht12) and cvs Maris Huntsman and Maris Widgeon (2007/8–2010/11; rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht-B1b+Rht-D1b, Rht-D1b+Rht-B1c) were compared at one field site, but within different systems (‘organic’, O, 2005/6–2007/8 v. ‘intensive’, I, 2005/6–2010/11). Further experiments at the site (2006/7–2008/9) compared 64 lines of a doubled-haploid (DH) population [Savannah (Rht-D1b) × Renesansa (Rht-8c+Ppd-D1a)]. Gibberellin (GA) insensitive dwarfing alleles (Rht-B1b; Rht-B1c; Rht-D1b; Rht-D1c) could reduce α-amylase activity and/or increase Hagberg falling number (HFN) but effects depended greatly on system, background and season. Only Rht-B1c increased grain dormancy despite producing plants taller than Rht-D1c. The GA-sensitive Rht8c+Ppd-D1a in Mercia was associated with reduced HFN but analysis of the DH population suggested this was more closely linked with Ppd-D1a, rather than Rht8c. The GA-sensitive severe-dwarfing allele Rht12 was associated with reduced HFN. Instability in HFN over season tended to increase with degree of dwarfing. There was a negative association between mean grain weight and HFN that was in addition to effects of Rht and Ppd-D1 allele.     

Agronomic and quality characteristics of triticale (X Triticosecale Wittmack) with HMW glutenin subunits 5+10

Sets of triticale (X Triticosecale Wittmack) lines derived from the cv. Presto with HMW glutenin allele Glu-D1d (subunits 5+10) translocated from bread wheat (Triticum aestivum L.) chromosome 1D to chromosome 1R were evaluated for agronomic and grain quality characteristics in 2002–2005. Two different translocation types were used: (a) single translocation 1R.1D₅₊₁₀-2 where the long arm of 1R carries the wheat segment from 1DL with the Glu-D1d replacing a secalin locus Sec-3, (b) double translocation Valdy where the long arm of 1R has the translocation 1R.1D₅₊₁₀-2 and the short arm has a segment from 1DS carrying wheat loci Gli-D1 and Glu-D3. The presence of Glu-D1d was determined by polyacrylamide gel electrophoresis (PAGE-ISTA) and DNA markers. The tested lines of triticale were compared with the check triticale cv. Presto and with wheat cultivars of different bread making quality (E-C quality classes). Single translocation 1R.1D₅₊₁₀-2 reduced grain yield by 16% and Valdy translocation by 24% as compared with cv. Presto. The Valdy translocation had substantially shortened spike length and reduced specific weight in comparison with check cv. Presto. Wet gluten content (according to the Perten method) was 12% in both translocation types, 8% in check Presto and on average 24% in wheat. Translocations increased the Zeleny sedimentation value (Valdy — 27 ml, 1R.1D₅₊₁₀-2 – 25 ml, cv. Presto — 23 ml). Triticale had a very low Hagberg falling number (FN) of 62–70 s without significant differences, while wheat had on average 301 s. The translocations did not significantly increase loaf volume; however, they improved loaf shape (height/width ratio): Valdy — 0.61, 1R.1D₅₊₁₀-2 – 0.56, cv. Presto 0.44, wheat on average 0.70. The dough was non-sticky in Valdy, slightly sticky in 1R.1D₅₊₁₀-2 and sticky in cv. Presto. Problems with a low FN for improving bread making quality of triticale are discussed. Higher bread making quality can be influenced by appropriate combination with donors of low α-amylase activity.