Modification of plant height via RNAi suppression of OsGA20ox2 gene in rice

GA 20-oxidase (GA20ox) is a regulatory enzyme for the syntheses of biologically active GAs in plants. The loss-of-function mutations in OsGA20ox2 of rice (Oryza sativa L.) generate the well-known Green Revolution gene sd-1, which cause the semi-dwarfism phenotype. In our present investigation, semi-dwarf plants were generated from a taller rice variety QX1 by RNAi suppression on the expression of OsGA20ox2. The 531bp-fragment of OsGA20ox2 was amplified by PCR from genomic DNA of QX1 and used to construct the hairpin RNAi vector pCQK2. The wild type QX1 was transformed with pCQK2 by Agrobacterium-mediated transformation and some independent transgenic RNAi lines exhibited semi-dwarfism. RT-PCR and Northern blot analyses showed that the expression of OsGA20ox2 was specifically suppressed in the RNAi semi-dwarf lines. Endogenous GA assays revealed that the contents of the GA20ox2-catalyzed products GA₁₉, GA₂₀ and the down-stream biologically active GA₁ were drastically reduced in the RNAi semi-dwarf lines. We further showed that the RNAi semi-dwarf lines could be restored to normal plant height by applying exogenous GA₃. The results indicated that the semi-dwarfism of the RNAi semi-dwarf lines was associated with the decreased expression of OsGA20ox2 gene and the reduced content of endogenous biologically active GA₁. Analyses of panicle length, seeds per panicle and 1000-grain weight suggested that the RNAi semi-dwarf lines showed stable grain yield compared with the wild type plants. It is demonstrated that the RNAi approach could be useful for plant breeding purposes in the future. Qing Yang, Chun-Lian Wang have contributed equally to this work.     

Segregation of 12 isozyme genes among doubled haploid lines derived from a japonica x indica cross of rice (Oryza sativa L.)

Summary The segregation of 12 heterozygous isozyme markers was analyzed among F₂ plants and 51 anther culture (AC)-derived lines obtained from the japonica × indica cross of rice, IRAT 177 × Apura. All the lines except two were homozygous products of recombination of the two parental phenotypes. Doubled haploid (DH) lines derived from plants regenerated from the same callus were identical, confirming previously obtained results in rice. Surprisingly, some lines derived from different calli were also identical, suggesting a phenomenon of early callus fragmentation. All these observations at the isozyme level were confirmed by field evaluation. Deviations of segregations from the expected 1 : 1 ratio were observed at 4 loci among the DH lines. Among these, two were also noted among the F₂ plants. The two other distortions, both in favor of the japonica allele, were observed specifically in the AC-derived materials.Although this concerns a small proportion of the genes under study, it suggests that the embryogenic microsporal population does not represent a random gametic array. On the other hand, evaluation of recombination between isozyme genes located on chromosome 6 appears consistent with F₂ data and data previously recorded on the other japonica × indica crosses. The potential use of isozymes in breeding doubled haploids derived from remote crosses in rice is discussed.Abbreviations MCPA = 2-methyl-4-chlorophenoxyacetic acid - IAA = indolacetic acid - AC plant or line = anther culture-derived plant or line - DH line = doubled haploid line     

水稻显性半矮秆基因的SCAR标记及初步定位

粳稻品系Y98149是从离子束诱变的后代中获得的显性半矮秆突变体，与野生型Y98148是一对株高近等基因系。将已经获得的3个与水稻显性半矮秆基因紧密连锁的RAPD标记分别克隆、测序，根据测序结果设计了3对特异性PCR引物，成功地将RAPD标记S1041₅₂₅、S1076₅₄₉和S1272₄₀₃转化成更稳定的SCAR标记SCS1041₄₉₈、SCS1076₅₁₀和SCS1272₃₈₈。通过Y98148×Y98149的F₂代分离群体的分析，这3个SCAR标记与显性半矮秆基因的遗传距离分别为12.6 cM、7.5 cM和16.3 cM, 且位于基因的同一侧。序列同源性比较表明，标记S1272₄₀₃为单拷贝，其核苷酸序列与水稻第7染色体上两个BAC克隆B1249D05(AP006451)和OJ1212-C12(AP005604)同源性为99%，B1249D05与OJ1212-C12有23 kb的重叠区域，标记S1272₄₀₃位于这个重叠区域，据此初步将显性半矮秆基因定位，为进一步精确定位和图位克隆奠定了基础。     

New efforts to overcome apomixis in Poa pratensis L.

Summary By means of a new method, plants of Poa pratensis can be classified rapidly and reliably as to whether they are capable or incapable of parthenogenesis. Parthenogenesis was found to be under strong genetic control, dominant over obligatory fertilization. The selected sexual plants lack all genes/alleles responsible for parthenogenesis, while the polyploid apomictic varieties investigated were heterozygous with one or more dominant alleles. Also dosage effects and/or modifying genes are probably involved. Crosses of sexual individuals with various apomictic varieties resulted in sexual as well as highly apomictic F₁ hyrids. A scheme of recurrent hybridization for breeding of Kentucky bluegrass is proposed.Two other experimental ways to overcome apomixis in Poa pratensis were studied in addition. By application of growth regulators temporary sexuality could not be induced. Attempts of in vitro regeneration of plants from endosperm resulted only in callus and root formation.     

Photosynthetic ability,grain yield and an esterase band in rice genotypes

Summary Four Indica and one Japonica (Tainung 67) of rice (Oryza sativa L.) varieties had an esterase band (tentatively designated as E₁), and also had higher photosynthetic ability than other five Japonica varieties without E₁ band. The F₁ plants of Tainung 67 × Mineyutaka (low photosynthetic ability, no E₁ band) showed E₁ band and a low photosynthetic ability. Of 34 F₂ plants, 28 had E₁ band, but 6 had no E₁ band of which 5 plants showed a low photosynthetic ability. These results suggest that an esterase gene and one of the photosynthesis gene are linked, and the gene for low photosynthetic ability is dominant.Among 42 new Japonica strains and 2 control varieties bred in Taiwan, most genotypes with E₁ band showed higher grain yield potential (grain field/growth days) in local test.Esterase band may be used as a marker for high photosynthesis and grain yield ability in breeding.     

Inheritance of plant elongation ability at seedling stage in rice

Summary In a comparison of methods to study inheritance of plant elongation ability, 15-, 20-, 25-, 30-, and 35-day-old F₂ populations of a cross between Baisbish (floating variety) and IR42 (nonelongating semidwarf modern variety) of rice, (Oryza sativa L.) were subjected to 65 cm water depth for 7 days. Frequency distribution of plant height before and after submergence was obtained. Bimodal curves in 15-, 20- and 25-day-old populations gave good fits to 9:7 elongating: nonelongating plants, suggesting that elongation was due to two dominant complementary genes. Segregation in the 30-day-old population was not clear-cut. A seedling age of 20 days was subsequently chosen for further studies.Two F₂'s involving floating rice and a nonelongating semidwarf; four F₂'s involving floating rice and an elongating semidwarf; and two F₂'s involving elongating and nonelongating semidwarf parents were studied with 20-day-old seedlings in the same way. Floating rice combinations with nonelongating semidwarf parents as well as with elongating semidwarf parents segregated into 9:7 elongating: nonelongating ratio. It is possible that because elongating and nonelongating dwarf parents did not differ much in elongation ability at seedling age, their combination with floating rice parents provided similar segregation. The F₂ distributions for height in elongating and nonelongating dwarf cross combinations were continuous with one peak.Genetic constitution of parents proposed are Sd1 Sd1 El El for floating parents, sd1 sd1 El El for semidwarf elongating, and sd1 sd1 el el for dwarf nonelongating.     

Anther culture of recalcitrant indica × Basmati rice hybrids

Fertile, green, di-haploid plants were obtained at high frequencies from several indica × Basmati rice F₁ hybrids and/or F₂ plant populations using an improved anther culture procedure. Anthers from cold-pretreated (10 ^°C for 10 d) panicles of six indica (HKR120, HKR86-3, HKR86-217, PR106, Gobind andCH2 double dwarf) and two Basmati rice (Basmati 370,Taraori Basmati) varieties and 14 heterotic indica ×Basmati F₁/F₂ hybrids were cultured in modified agarose-solidified N6M, Heh5M and RZM media. Best callus induction frequencies (2.6–78%) were obtained in RZM medium containing 4% (w/v) maltose,2,4-D, NAA and kinetin. F₂ plants compared to F₁ hybrids and parental rice varieties, were more responsive to anther culture. Androgenesis frequencies of 31–78% were obtained for indica × Basmati F₂ plants in RZM medium in just 30 d which are comparable to or higher than that reported for japonica rice varieties and hybrids involving japonica rice parent(s). Agarose (1.0% w/v)-solidified MS medium containing 3.0% maltose, kinetin, BAP, and NAA, induced green shoot regeneration in 0–51% of the anther-derived callide pending upon the genotype. High plant regeneration frequencies (67–337 green plants per 1000 anthers)were obtained from anther calli of several F₁hybrids (Gobind × Basmati 370 and HKR120 ×Taraori Basmati) and F₂ plants (Gobind × Basmati370, Gobind × Taraori Basmati, HKR86-3 × TaraoriBasmati). A sample of 498 plants obtained from the above hybrids, were transferred to pots with>90% survival; 8–78% of these plants had >5%spikelet fertility and were diploid. In addition,18% of the haploid plants could be diploidized by submerging in 0.1% colchicine solution for 16–18 h. The improved anther culture procedure reported here, resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice hybrids compared to previous published procedures. The study may accelerate the introgression of desirable genes from indica into Basmati rice using anther culture as a breeding tool. This revised version was published online in July 2006 with corrections to the Cover Date.     

On the inheritance of the dwarf character in Polyantha × Rosa chinensis minima (Sims) Voss F1-populations

Summary The inheritance of the dwarf character was studied in F1-seedling populations arisen from crosses between diploid Polyantha cultivars and the diploid dwarf species R. chinensis minima (Sims) Voss. Dwarfness is controlled by a single dominant gene D. R. chinensis minima and its dwarf descendants are heterozygous for D, while polyanthas are homozygous for d. The origin of R. chinensis minima and its potential for breeding new pot roses are discussed.     

Genetic analysis and fine mapping of a semi-dwarf gene in a centromeric region in rice (Oryza sativa L.)

Superior plant architecture is a key means of enhancing yield potential in high yielding varieties. A newly identified recessive gene, named sd-c, controls plant height and tiller number. Genetic analysis of an F₂ population from a cross between the semi-dwarf mutant and japonica cv. Houshengheng showed that the sd-c locus was flanked by SSR markers RM27877 and RM277 on chromosome 12. Thirty nine InDel markers were developed in the region and the sd-c gene was further mapped to a 1 cM centromeric region between InDel markers C11 and C12. These sequenced markers can be used to distinguish wild type and mutants and thus can be used in marker-assisted selection. The sd-c mutant decreases culm length by about 26% and doubles the tiller number without changing seed weight. Until now only sd-1 has been used in indica rice breeding programs. The sd-c mutant seems to have no undesirable pleiotropic effects and is therefore a potential genetic resource for breeding semi-dwarf indica rice cultivars.     

Trisomic genetic analysis of aroma in three Japanese native rice varieties (Oryza sativa L.)

Information on the genetics of aroma in rice facilitates breeding and selection of new aromatic varieties with high yield and good quality. Objective of the present study was to make clear the number of genes controlling aroma, and the allelism of aroma genes and the location of aroma gene(s) on the chromosome in three Japanese native aromatic rice varieties (Kabashiko, Shiroikichi and Henroyori). Lack of leaf aroma in all F₁ plants of non-aromatic/aromatic crosses indicated the recessive nature of aroma, and the segregation ratios (3:1) of non-aromatic to aromatic plants in its F₂ populations from Nipponbare/aromatic varieties crosses revealed that each of the three aromatic varieties contains a single recessive gene for aroma. Through trisomic analysis, the segregation of non-aromatic and aromatic plants in all F₂ populations from the crosses between trisomics lines NT8, with an extra chromosome 8, and aromatic varieties deviated significantly from disomic segregation of 3:1 ratios, and fitted to trisomic segregation, however, in other F₂ populations derived from other 7 types of trisomic F₁ plants, the segregation ratios of non-aromatic to aromatic were 3:1, indicating that the single recessive aroma gene was located on chromosome 8 in three aromatic varieties. This revised version was published online in July 2006 with corrections to the Cover Date.     

NORIN 10 semi-dwarfism in tetraploid wheat and associated effects on yield

Summary A genetic study of a range of NORIN 10 based semi-dwarf durum wheats showed that only Gai/Rht 1, located on chromosome 4A, was present. No varieties carrying a second Gai/Rht allele were identified and deliberate attempts to introduce Gai/Rht2 into tetraploid wheats have so far been unsuccessful.In a spaced plant trial of homozygous random F₃ lines from two tall x semi-dwarf crosses, the semi-dwarfs has lower ear yields, due mainly to reduced kernel weight, but had higher tiller numbers than the tall genotypes. Although there was no difference in overall plant yield between talls and semi-dwarfs, an analysis of character associations within the semi-dwarf F₃'s showed positive height-yield and height-kernel weight correlations indicating that selection for tall dwarfs may be a useful breeding strategy in tetraploid wheats.     

Resistance to the cabbage root fly, Delia radicum, within Brassica fruticulosa

Two transgenic Bt rice lines, KMD1 and KMD2, both containing a synthetic cry1Ab gene from Bt, were crossed with conventional rice varieties. The inheritance of resistance to SSB of KMD1 and KMD2was investigated through LSB and field examination of their progenies, e.g. F₁, BC₁ and F₂ populations. In LSBs, 100.0% of newly hatched SSB larvae died on the second day after feeding on leaf tissues of F₁ and GUS positive BC₁ plants, of which the area of leaf tissues consumed by SSB is also similar to that of transgenic parents. These results imply that the resistance of Bt rice to SSB is dominantly controlled and could be easily exploited in hybrid rice production. Field evaluation showed that segregation ratios for SSB resistance to susceptibility in BC₁ populations fit the ratio of 1:1, which was also confirmed by LSBs. However, in F₂ populations, the ratio was significantly smaller than 3:1 for resistant to susceptible plants in all 6 indica × japonica (KMD1 and KMD2) crosses, though it fitted 3:1 in all 4 japonica × japonica crosses. The results implied that the resistance of Bt rice to SSB was controlled by a dominant gene which was present in a homozygous condition in both KMD1 and KMD2, but the inheritance could be affected by other factors. Assays for Cry1Ab protein showed that, in most crosses, the content of Cry1Ab is significantly higher in leaves of GUS positive F₁, BC₁ and F₂ plants than that in transgenic Bt parent plants, which accounts for the high resistance observed in these plants to SSB. This revised version was published online in July 2006 with corrections to the Cover Date.     

The genetical control of the grass-dwarf phenotype in Triticum aestivum L.

Summary Crosses between certain normal varieties of wheat (T. aestivum) produce grass-dwarfs in the F₁ or F₂ generations. Grass-dwarfs are defined as short grass-like plants which do not become reproductive when grown in an 8-hour photoperiod at a maximum temperature of 21°C. This phenotype is not related to either the winter or semi-dwarf habits of wheat. At least three major dominant genes D₁ D₂ and D₃ interact to produce the grass-dwarf phenotype.The interaction of these genes has been studied using monosomic lines. The results indicate that D ₁ and D ₂ interact by complementation, D ₂ being effective only in the homo- and hemizygous condition, but not when heterozygous. D ₃ probably has an additive interaction with D ₁ and D ₂. Evidence for the existence of multiple alleles at these three loci is discussed.Data from F₂ and F₃ segregation studies is presented and details of procedure which must be observed if accurate segregation ratios are to be obtained. The data obtained support the hypothesis proposed for the inheritance of the grass-dwarf phenotype.     

Hybrid dwarfness in wheat

Hybrid dwarfness is the phenomenon that after crossing of normal genotypes dwarfs are obtained in the F₁ or not before the F₂-generation. The literature on hybrid dwarfness in wheat is critically discussed. A new hypothesis on its genetic basis is given, taking McMillan's (1937) as a starting point. Dwarfness is assumed to be determined by the additive interaction of three genes D ₁, D ₂ and D ₃, differing in dominance relations and in quantitative contribution to the dwarf phenotype.Three dwarf types are described. Type 1-dwarfs are dwarf during their whole life cycle and normally do not produce seeds. Type 2-dwarfs start as normal seedlings, become dwarfs while tillering and die dwarfs; some produce seeds, others do not. Type 3-dwarfs emerge as normal seedlings, become dwarfs during the tillering stage, but after some time they start to shoot and develop into nearly or even completely normal plants; in the F₂ the proportion of dwarfs decreases during the growing season. The occurrence and genetic basis of the three dwarf types is discussed.On the basis of their genotype 315 varieties and lines are divided into six genotypeclasses. Over 1000 intra- and inter-class crosses were made and F₁, F₂, F₃, and BC studied. Also some triple crosses and crosses with pure breeding dwarfs were investigated. In general the results obtained fit the hypothesis. Unstable ratios involving type 3-dwarfs are discussed separately.Linkage of the dwarf gene D ₂ and the necrosis gene Ne ₂ (both on chromosome 2B (XIII) was apparent from F₂-data and from results of a triple cross in which both forms of hybrid weakness occurred. Crossing-over between D ₂ and Ne ₂ is calculated to be 34%.Methods are outlined to use hybrid dwarfness in a wheat breeding programme. The possible incompleteness of the three-gene hypothesis and the variability of dwarfness are discussed and finally some suggestions are made for future research.     

Isolation of a Spontaneous Chromosome Translocation in Common Wheat

The genes Ms2 for male sterility and Rht10 for dominant dwarfing located on the short arm of chromosome 4D in common wheat arc closely linked. Male sterile, dwarf F₁ plants from the cross of male sterile‘Chinese Spring’× dwarf‘Ai-bian’were backcrossed with the variety‘Chinese Spring, From this offspring a spontaneous chromosome translocation was isolated resulting in a recombinant male sterile and dwarf genotype.     

A dwarfing mutant caused by deactivation function of alpha subunit of the heterotrimeric G-protein in rice

Dwarf mutants in plants are crucial for elucidating regulatory mechanisms for plant growth and development. Previous studies suggested that the heterotrimeric G-protein alpha subunit known as D1/RGA1 in rice was involved in deactivation function of the G protein. However, so far no partner has been analyzed the spatial structure change acting with D1. In this study, a dwarf mutant designated Mu101 was obtained in M₂ population of rice indica cultivar M804 treated with ⁶⁰Co γ-ray. Genetic analysis of Mu101 indicated that the dwarf phenotype was controlled by a single dwarf gene, and the dwarf mutant was insensitive to gibberellin (GA), which was named dwarf 89 (d89). Using a large F₂ population derived from a cross between the d89 and a japonica rice variety, Taigeng16, the D89 gene was fine mapped into a 62.13 kb physical distance on chromosome 5, where eight open reading frames were predicted. Sequence analysis indicated that only one bp substitution (A-G) was found in LOC_Os05g26890 between M804 and the d89 mutant. The rice GA insensitive dwarf mutant DWARF1 gene was in this locus. The modeling analysis showed amino acid threonine to alanine mutation was likely to make the alpha helix short, and led to the G protein deactivation.     

Seedling emergence,coleoptile length,and plant height relationships in crosses of dwarf and standard-height wheats

Summary Seedling emergence was closely correlated with coleoptile length and plant height among parents, F₂ and F₃, populations of crosses involving dwarf wheats Olesen Dwarf (CI 14497), Norin 10 derivative D6301, Tom Thumb derivative D6899, and the standard-height varieties Ramona 50 and Nainari 60. Genetic mechanisms that governed plant height also influenced coleoptile length, but the relative effects of genes showing dominant or epistatic effects appeared to be different. With respect to the two parents involved in each of 15 crosses, mean F₂ coleoptile lengths were consistently closer to the low parent value than were corresponding mean F₂ plant heights. A slight curvilinear relationship was also found between coleoptile length and plant height of F₃ lines. The results suggest that selection of semidwarf wheats with long coleoptiles and improved emergence properties from crosses involving the dwarf wheats of this study would be unlikely.     

Inheritance of seed colour in Brassica campestris L. and breeding for yellow-seeded B. napus L.

Summary Seed colour inheritance was studied in five yellow-seeded and one black-seeded B. campestris accessions. Diallel crosses between the yellow-seeded types indicated that the four var. yellow sarson accessions of Indian origin had the same genotype for seed colour but were different from the Swedish yellow-seeded breeding line. Black seed colour was dominant over yellow. The segregation patterns for seed colour in F₂ (Including reciprocals) and BC₁ (backcross of F₁ to the yellow-seeded parent) indicated that the black seed colour was conditioned by a single dominant gene. Seed colour was mainly controlled by the maternal genotype but influenced by the interplay between the maternal and endosperm and/or embryonic genotypes. For developing yellow-seeded B. napus genotypes, resynthesized B. napus lines containing genes for yellow seed (Chen et al., 1988) were crossed with B. napus of yellow/brown seeds, or with yellow-seeded B. carinata. Yellow-seeded F₂ plants were found in the crosses that involved the B. napus breeding line. However, this yellow-seeded character did not breed true up to F₄. Crosses between a yellow-seeded F₃ plant and a monogenomically controlled black-seeded B. napus line of resynthesized origin revealed that the black-seeded trait in the B. alboglabra genome was possibly governed by two independently dominant genes with duplicated effect. Crossability between the resynthesized B. napus lines as female and B. carinata as male was fairly high. The sterility of the F₁ plants prevented further breeding progress for developing yellow-seeded B. napus by this strategy.     

Molecular tagging of a gene for resistance to brown planthopper in rice (Oryza sativa L.)

An introgression line derived from an interspecific cross between Oryzasativa and Oryza officinalis, IR54741-3-21-22 was found to beresistant to an Indian biotype of brown planthopper (BPH). Genetic analysisof 95 F₃ progeny rows of a cross between the resistant lineIR54741-3-21-22 and a BPH susceptible line revealed that resistance wascontrolled by a single dominant gene. A comprehensive RAPD analysisusing 275 decamer primers revealed a low level of (7.1%) polymorphismbetween the parents.RAPD polymorphisms were either co-dominant (6.9%), dominant forresistant parental fragments (9.1%) or dominant for susceptible parentalfragments (11.6%). Of the 19 co-dominant markers, one primer,OPA16, amplified a resistant parental band in the resistant bulk and asusceptible parental band in the susceptible bulk by bulked segregantanalysis. RAPD analysis of individual F₂ plants with the primerOPA16 showed marker-phenotype co-segregation for all, with only onerecombinant being identified. The linkage between the RAPD markerOPA16₉₃₈ and the BPH resistance gene was 0.52 cM in couplingphase. The 938 bp RAPD amplicon was cloned and used as a probe on122 Cla I digested doubled haploid (DH) plants from aIR64xAzucena mapping population for RFLP inheritance analysis and wasmapped onto rice chromosome 11. The OPA16₉₃₈ RAPD markercould be used in a cost effective way for marker-assisted selection of BPHresistant rice genotypes in rice breeding programs.     

Genetic analysis of methionine content in indica-japonica hybrid rice (Oryza sativa L.) at different grain developmental stages

Genetics of methionine content in indica-japonica hybrid rice (Oryza sativa L.) was studied in 35 F₁ hybrids derived from crossing 7 male-sterile indica rice cultivars with 5 restorer japonica rice cultivars. Two genetic models and their corresponding statistical methods for quantitative traits of triploid endosperm in cereal crops were used for the analysis. One is the unconditional model, which refers to the analysis of cumulative measurements along the developmental stages, while another is the conditional model, which relates to analysis throughout the developmental stages. Results showed that methionine content of indica-japonica hybrid rice was controlled by expression of triploid endosperm nuclear genes, cytoplasm genes, diploid maternal plant nuclear genes, and their genotype-environment interaction effects. Dominant effects were important at the first three developmental stages, while additive effects were important at the next two stages of grain development under both unconditional and conditional analyses. With regard to the components of heritability, maternal and cytoplasm general heritabilities and their environmental interaction heritabilities were important. It was suggested that enhancing methionine content could be more efficient when selection is based on maternal plants in early breeding generations.