The transformation of the photo-thermo sensitive genic male-sterile line 261S of rice via an expression vector containing the anti-Waxy gene

Transgenic photo-thermo sensitive genic male sterility Oryza sativa L. cv. “261S” plants with the anti-Waxy gene were successfully obtained using an Agrobacterium tumefaciens-mediated co-transformation method. Marker-free homozygous transgenic lines with the anti-Waxy gene were obtained. The setting seed rates of the transgenic plants via self-pollination or via crossing with the restorer line WX99075 rice and the 1000-grain weight of the transgenic plants and the F₂ hybrid seeds obtained by crossing the transgenic or non-transgenic plants with the restorer line WX99075 rice, and the number of panicles of the transgenic plants and yields of the F₂ hybrid rice, were analysed. Quality indexes of the transgenic plants and of the F₂ hybrid seeds were analysed. Our researches results indicate that hybrid female and hybrid descendant edibility could be improved via the introduction of the anti-Waxy gene, but the grain yields of the reserve seeds via self-pollination of the transgenic photo-thermo sensitive genic sterile lines and of the hybrid rice were not affected.     

Identification of qRL7, a major quantitative trait locus associated with rice root length in hydroponic conditions

Root system development is an important target for improving yield in rice. Active roots that can take up nutrients more efficiently are essential for improving grain yield. In this study, we performed quantitative trait locus (QTL) analyses using 215 recombinant inbred lines derived from a cross between Xieqingzao B (XB), a maintainer line with short roots and R9308, a restorer line with long roots. Only a QTLs associated with root length were mapped on chromosomes 7. The QTL, named qRL7, was located between markers RM3859 and RM214 on chromosome 7 and explained 18.14–18.36% of the total phenotypic variance evaluated across two years. Fine mapping of qRL7 using eight BC₃F₃ recombinant lines mapped the QTL to between markers InDel11 and InDel17, which delimit a 657.35 kb interval in the reference cultivar Nipponbare. To determine the genotype classes for the target QTL in these BC₃F₃ recombinants, the root lengths of their BC₃F₄ progeny were investigated, and the result showed that qRL7 plays a crucial role in root length. The results of this study will increase our understanding of the genetic factors controlling root architecture, which will help rice breeders to breed varieties with deep, strong and vigorous root systems.     

Mapping three new interspecific hybrid sterile loci between Oryza sativa and O. glaberrima

Hybrid sterility hinders the transfer of useful traits between Oryza sativa and O. glaberrima. In order to further understand the nature of interspecific hybrid sterility between these two species, a strategy of multi-donors was used to elucidate the range of interspecific hybrid sterility in this study. Fifty-nine accessions of O. glaberrima were used as female parents for hybridization with japonica cultivar Dianjingyou 1, after several backcrossings using Dianjingyou 1 as the recurrent parent and 135 BC₆F₁ sterile plants were selected for genotyping and deducing hybrid sterility QTLs. BC₆F₁ plants containing heterozygous target markers were selected and used to raise BC₇F₁ mapping populations for QTL confirmation and as a result, one locus for gamete elimination on chromosome 1 and two loci for pollen sterility on chromosome 4 and 12, which were distinguished from previous reports, were confirmed and designated as S37(t), S38(t) and S39(t), respectively. These results will be valuable for understanding the range of interspecific hybrid sterility, cloning these genes and improving rice breeding through gene introgression.     

Rice tungro spherical virus resistance into photoperiod-insensitive japonica rice by marker-assisted selection

Rice tungro disease (RTD) is one of the destructive and prevalent diseases in the tropical region. RTD is caused by Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. Cultivation of japonica rice (Oryza sativa L. ssp japonica) in tropical Asia has often been restricted because most japonica cultivars are sensitive to short photoperiod, which is characteristic of tropical conditions. Japonica1, a rice variety bred for tropical conditions, is photoperiod-insensitive, has a high yield potential, but is susceptible to RTD and has poor grain quality. To transfer RTD resistance into Japonica1, we made two backcrosses (BC) and 8 three-way crosses (3-WC) among Japonica1 and RTSV-resistant cultivars. Among 8,876 BC₁F₂ and 3-WCF₂ plants, 342 were selected for photoperiod-insensitivity and good grain quality. Photoperiod-insensitive progenies were evaluated for RTSV resistance by a bioassay and marker-assisted selection (MAS), and 22 BC₁F₇ and 3-WCF₇ lines were selected based on the results of an observational yield trial. The results demonstrated that conventional selection for photoperiod-insensitivity and MAS for RTSV resistance can greatly facilitate the development of japonica rice that is suitable for cultivation in tropical Asia.     

Comparison of phenotypic versus marker-assisted background selection for the SUB1 QTL during backcrossing in rice

Marker assisted backcrossing has been used effectively to transfer the submergence tolerance gene SUB1 into popular rice varieties, but the approach can be costly. The selection strategy comprising foreground marker and phenotypic selection was investigated as an alternative. The non-significant correlation coefficients between ranking of phenotypic selection and ranking of background marker selection in BC₂F₁, BC₃F₁ and BC₃F₂ generations indicated inefficiency of phenotypic selection compared to marker-assisted background selection with respect to recovery of the recipient genome. In addition, the introgression size of the chromosome fragment containing SUB1 was approximately 17 Mb, showing the effects of linkage drag. The significant correlation coefficient between rankings of phenotypic selection with the percentage of recipient alleles in the BC₁F₁ generation suggested that background selection could be avoided in this generation to minimize the genotyping cost. The phenotypically selected best plant of the BC₃F₁ generation was selfed and backcross recombinant lines were selected in the resulting BC₃F₄ generation. The selection strategy could be appropriate for the introgression of SUB1 QTL in countries that lack access to high-throughput genotyping facilities.     

QTL analysis on rice grain appearance quality,as exemplifying the typical events of transgenic or backcrossing breeding

Rice grain shape and yield are usually controlled by multiple quantitative trait loci (QTL). This study used a set of F_9–10 recombinant inbred lines (RILs) derived from a cross of Huahui 3 (Bt/Xa21) and Zhongguoxiangdao, and detected 27 QTLs on ten rice chromosomes. Among them, twelve QTLs responsive for grain shape/ or yield were mostly reproducibly detected and had not yet been reported before. Interestingly, the two known genes involved in the materials, with one insect-resistant Bt gene, and the other disease-resistant Xa21 gene, were found to closely link the QTLs responsive for grain shape and weight. The Bt fragment insertion was firstly mapped on the chromosome 10 in Huahui 3 and may disrupt grain-related QTLs resulting in weaker yield performance in transgenic plants. The introgression of Xa21 gene by backcrossing from donor material into receptor Minghui 63 may also contain a donor linkage drag which included minor-effect QTL alleles positively affecting grain shape and yield. The QTL analysis on rice grain appearance quality exemplified the typical events of transgenic or backcrossing breeding. The QTL findings in this study will in the future facilitate the gene isolation and breeding application for improvement of rice grain shape and yield.     

Quantitative trait loci for whiteness of cooked rice detected in improved rice cultivars in Hokkaido

Improving the eating quality of cooked rice has been one of the most important objectives in rice breeding programs. Eating quality of cooked rice is a complex trait including several components, such as external appearance, taste, aroma, and texture. Therefore, dissection of these components followed by marker-assisted selection of detected QTL(s) may be a useful approach for achieving desirable eating quality in rice breeding. Whiteness of cooked rice (WCR) is an important factor related to the external appearance of cooked rice. WCR is known to be associated with the amylose and protein contents of the endosperm. However, the genetic basis of WCR remains unclear. In this study, we evaluated phenotypic variation in WCR among recently developed rice cultivars from Hokkaido, Japan. Then, we developed doubled haploid lines (DHLs) derived from a cross between two cultivars from Hokkaido, Joiku No. 462 (high WCR) and Jokei06214 (low WCR). Using the DHLs, we detected two QTLs for WCR, qWCR3 and qWCR11, on chromosomes 3 and 11, respectively. We also examined the dosage effect of the two QTLs based on both the categorized segregants in the DHLs and the relationship between the WCR phenotype and inheritance around the QTL regions in cultivars from Hokkaido.     

一份新的水稻斑点叶突变体spl32的鉴定和基因定位

从F2(粤晶丝苗2号/H4)群体中,鉴定出一份显性斑点叶突变体spl32(spotted leaf 32)。其叶片褐色斑点受自然光诱导,在幼穗分化期从叶尖逐渐扩散至叶鞘,台盼蓝染色表明斑点并非由细胞死亡引起。以从F5杂合个体分离出的正常叶色植株为对照,斑点叶植株的穗粒数、结实率显著降低。斑点出现后,spl32的POD活性和MDA含量均显著高于对照;同时,spl32叶片光合色素含量降低,但荧光动力学参数并无显著变化。抽穗期人工接菌表明,spl32对水稻白叶枯病菌抗性较对照显著提高。遗传分析表明spl32斑点性状由一个显性基因Spl32(t)控制,利用F2(02428/spl32)群体将其定位在第11染色体Ind-c和RM206之间,推测该基因为一个新的水稻斑点叶基因。     

Differential contribution of two Ppd-1 homoeoalleles to early-flowering phenotype in Nepalese and Japanese varieties of common wheat

Wheat landraces carry abundant genetic variation in heading and flowering times. Here, we studied flowering-related traits of two Nepalese varieties, KU-4770 and KU-180 and a Japanese wheat cultivar, Shiroganekomugi (SGK). These three wheat varieties showed similar flowering time in a common garden experiment. In total, five significant quantitative trait loci (QTLs) for three examined traits, the heading, flowering and maturation times, were detected using an F₂ population of SGK/KU-4770. The QTLs were found at the Ppd-1 loci on chromosomes 2B and 2D and the 2B QTL was also confirmed in another F₂ population of SGK/KU-180. The Ppd-D1 allele from SGK and the Ppd-B1 alleles from the two Nepalese varieties might be causal for early-flowering phenotype. The SGK Ppd-D1 allele contained a 2-kb deletion in the 5′ upstream region, indicating a photoperiod-insensitive Ppd-D1a allele. Real-time PCR analysis estimating the Ppd-B1 copy number revealed that the two Nepalese varieties included two intact Ppd-B1 copies, putatively resulting in photoperiod insensitivity and an early-flowering phenotype. The two photoperiod-insensitive Ppd-1 homoeoalleles could independently contribute to segregation of early-flowering individuals in the two F₂ populations. Therefore, wheat landraces are genetic resources for discovery of alleles useful for improving wheat heading or flowering times.     

Persistent C genome chromosome regions identified by SSR analysis in backcross progenies between Brassica juncea and B. napus

Given that feral transgenic canola (Brassica napus) from spilled seeds has been found outside of farmer’s fields and that B. juncea is distributed worldwide, it is possible that introgression to B. juncea from B. napus has occurred. To investigate such introgression, we characterized the persistence of B. napus C genome chromosome (C-chromosome) regions in backcross progenies by B. napus C-chromosome specific simple sequence repeat (SSR) markers. We produced backcross progenies from B. juncea and F₁ hybrid of B. juncea × B. napus to evaluate persistence of C-chromosome region, and screened 83 markers from a set of reported C-chromosome specific SSR markers. Eighty-five percent of the SSR markers were deleted in the BC₁ obtained from B. juncea × F₁ hybrid, and this BC₁ exhibited a plant type like that of B. juncea. Most markers were deleted in BC₂ and BC₃ plants, with only two markers persisting in the BC₃. These results indicate a small possibility of persistence of C-chromosome regions in our backcross progenies. Knowledge about the persistence of B. napus C-chromosome regions in backcross progenies may contribute to shed light on gene introgression.     

Two linked genes on rice chromosome 2 for F1 pollen sterility in a hybrid between Oryza sativa and O. glumaepatula

Hybrid incompatibility plays an important role in establishment of post-zygotic reproductive isolation. To unveil genetic basis of hybrid incompatibilities between diverged species of genus Oryza AA genome species, we conducted genetic dissection of hybrid sterility loci, S22(t), which had been identified in backcross progeny derived from Oryza sativa ssp. japonica (recurrent parent) and South American wild rice O. glumaepatula near the end of the short arm of chromosome 2. The S22(t) region was found to be composed of two loci, designated S22A and S22B, that independently induce F₁ pollen sterility. Pollen grains containing either of the sterile alleles (S22A-glum^s or S22B-glum^s) were sterile if produced on a heterozygous plant. No transmission of the S22A-glum^s allele via pollen was observed, whereas a low frequency of transmission of S22B-glum^s was observed. Cytological analysis showed that the sterile pollen grains caused by S22A could reach the bicellular or tricellular stage, and the nearly-sterile pollen grains caused by S22B could reach the tricellular stage. Our genetic analysis showed repulsion linkage effect is possible to induce strong reproductive barrier by high pollen sterility based on recombination value and transmission ratio of hybrid sterility gene to the progeny was influenced by frequency of competitors on fertilization.     

Cytoplasmic male sterility in Brassicaceae crops

Brassicaceae crops display strong hybrid vigor, and have long been subject to F₁ hybrid breeding. Because the most reliable system of F₁ seed production is based on cytoplasmic male sterility (CMS), various types of CMS have been developed and adopted in practice to breed Brassicaceae oil seed and vegetable crops. CMS is a maternally inherited trait encoded in the mitochondrial genome, and the male sterile phenotype arises as a result of interaction of a mitochondrial CMS gene and a nuclear fertility restoring (Rf) gene. Therefore, CMS has been intensively investigated for gaining basic insights into molecular aspects of nuclear-mitochondrial genome interactions and for practical applications in plant breeding. Several CMS genes have been identified by molecular genetic studies, including Ogura CMS from Japanese radish, which is the most extensively studied and most widely used. In this review, we discuss Ogura CMS, and other CMS systems, and the causal mitochondrial genes for CMS. Studies on nuclear Rf genes and the cytoplasmic effects of alien cytoplasm on general crop performance are also reviewed. Finally, some of the unresolved questions about CMS are highlighted.     

陆地棉Li1纯合显性不致死重组体的遗传分析

极短纤维突变体Li₁自国外引入本实验室后,出现了纯合致死现象。而在杂交组合(Li₁×XZ142 FLM)的后代中意外发现了一些Li₁基因纯合的突变体株系,其自交后代均为极短纤维。这种Li₁基因显性纯合不致死突变体被命名为Li-R重组体。本实验利用Li-R重组体分别与TM-1、海7124及突变体Li₁新组配了3个F₂群体,对Li-R重组体进行遗传分析。组合Li-R×XZ142 FLM、Li-R×TM-1、Li-R×海7124及Li-R×Li₁的F₂后代的分离结果均表明, Li-R重组体的纯合显性不致死表型是由2对基因控制的,一个是显性基因Li₁,另一个是来自XZ142 FLM的隐性基因li_a。其中的li_a基因是本实验室新提出的一个基因。因而Li-R的基因型就是li_ali_aLi₁Li₁;并由此推断,Li₁纯合致死突变体的基因型是Li_aLi_aLi₁Li₁,XZ142 FLM的基因型为li_ali_ali₁li₁,1929年发现的Li₁显性杂合突变体的基因型为Li_aLi_aLi₁li₁。利用(Li-R×TM-1)F_2:3进一步分析Li-R中的新基因li_a的等位性,发现li_a与控制纤维起始发育的基因li₃、n₂均不等位。新基因li_a的提出,进一步丰富了纤维发育基因资源。     

Distorted genetic segregation of the transposon mPing at the long arm of chromosome 12 in rice

A class II transposable element, mPing exists in the rice genome ubiquitously and can transpose even in ordinary cultivation conditions. A copy of mPing was identified at the long arm of chromosome 12. In reciprocal backcrossed F₁s between a heterozygote and a homozygote without mPing, the male gametes with this mPing from heterozygotes were transmitted to the next generation at a lower frequency than those without mPing, resulting in distorted genetic segregation in self-fertilized progenies, as well as in F₁s after backcrossing. Pollens with mPing tended to germinate on stigma less than those without mPing. These results, however, could not explain the lower transmission of male gametes with mPing. In addition, no excision of mPing was observed in a homozygote. Thus, it was suggested that male gametes with mPing were eliminated partly from pollination to fertilization by negative competition against male gametes without mPing. Less formation of microspores with mPing in meiosis could also be a cause for the distorted segregation, although this could not be examined. At least two ORFs, whose functions have not been identified, are located near this mPing. It is plausible that either of these ORFs or both are necessary for the normal functioning of male gametes.     

Production and characterization of an alloplasmic and monosomic addition line of Brassica rapa carrying the cytoplasm and one chromosome of Moricandia arvensis

Intergeneric hybridization was performed between Moricandia arvensis and four inbred lines of Brassica rapa following embryo rescue. Three F₁ hybrid plants were developed from three cross combinations of M. arvensis × B. rapa, and amphidiploids were synthesized by colchicine treatment. Six BC₁ plants were generated from a single cross combination of amphidipolid × B. rapa ‘Ko1-303’ through embryo rescue. One BC₂ and three BC₃ plants were obtained from successive backcrossing with B. rapa ‘Ko1-303’ employing embryo rescue. Alloplasmic and monosomic addition lines of B. rapa (Allo-MALs, 2n = 21) were obtained from backcrossed progeny of three BC₃ plants (2n = 21, 22 and 23) without embryo rescue. An alloplasmic line of B. rapa (2n = 20) degenerated before floliation on 1/2 MS medium due to severe chlorosis. Allo-MALs of B. rapa (2n = 21) showed stable male sterility without any abnormal traits in vegetative growth and female fertility. Molecular analyses revealed that the same chromosome and cytoplasm of M. arvensis had been added to each Allo-MAL of B. rapa. This Allo-MAL of B. rapa may be useful material for producing cytoplasmic male sterile lines of B. rapa.     

Effect of quantitative trait loci for seed shattering on abscission layer formation in Asian wild rice Oryza rufipogon

Asian cultivated rice Oryza sativa L. was domesticated from its wild ancestor, O. rufipogon. During domestication, the cultivated rice lost its seed-shattering behaviour. Previous studies have shown that two major quantitative trait loci (QTLs; qSH1 and sh4) are responsible for the seed-shattering degree. Here, we produced introgression lines carrying non-functional alleles from O. sativa ‘Nipponbare’ at the two major QTLs in the genetic background of wild rice O. rufipogon W630, and examined the effects of the two QTLs on seed shattering and abscission layer formation. The introgression lines, with Nipponbare alleles at either or both loci, showed complete or partial abscission layer formation, respectively, indicating that other unknown loci might be involved in enhancing seed shattering in wild rice. We detected a single QTL named qSH3 regulating seed-shattering degree using an F₂ population between Nipponbare and the introgression line carrying Nipponbare alleles at the two QTLs. Although we generated an introgression line for qSH3 alone, no effects on seed shattering were observed. However, a significant effect on seed-shattering degree was observed for the introgression line carrying Nipponbare alleles at qSH3 and the two QTLs, suggesting an important role of qSH3 on seed shattering in coordination with the two QTLs.     

Genetic analysis of a novel dominant rice dwarf mutant 986083D

This study was to determine the agronomic and genetic characteristics of a novel rice dominant dwarf mutant 986083D (japonica) and its potential in breeding. 986083D derived from the anther culture of an autotetraploid indica/japonica hybrid and its progeny segregated into normal and dwarf plants. Homozygous and heterozygous 986083D plants looked similar phenotypically, showing shortened stature, erect leaves, more tillers and poor fertility. The segregation ratio of dwarf to normal plants fit the expected 3:1 by χ²-test in 77 out of 88 tested lines. Crosses between homozygous 986083D and eight other rice varieties had uniform semi-dwarf F₁ plants. The F₁ plants from crosses between heterozygous 986083D and five other varieties had normal and semi-dwarf plants close to the expected ratio of 1:1. The reduction of plant height in F₁ plants ranged from 40.0 to 53.5% in a subtropical environment and from 37.5 to 48.2% in a temperate environment. 986083D showed moderate sensitivity to exogenously applied GA₃ in terms of elongation of shoots and induction of α-amylase activity in the endosperm. Linkage analysis showed that the dominant dwarf gene (designated as Dx) in 986083D was not allelic to D53. Dx was roughly mapped to the short arm of chromosome 8. All results showed that 986083D was a novel mutant controlled by single dominant gene, providing a valuable material in rice breeding. Ruizhen Qin, Yang Qiu contributed equally to this work.     

Fertility improvement in tetraploid pearl millet

Summary Autotetraploid (2n=4x=28) pearl millet inbred lines, Tift 23BE and Tift 239DB, have been developed for use in crosses with other polyploid Pennisetum species. Each line set less than 1% and 3% selfed and open-pollinated seed, respectively. Seed germination was usually less than 17%. First generation (F₁) hybrids between the two inbreds set up to 61% seed while up to 40% of the seed from hybrids germinated.Seed weight per inflorescence for two planting dates averaged 0.32 g and 3.06 g for the two inbreds and the second generation F₂ progeny, respectively. One hundred seed weight was also significantly higher (0.78 g vs 0.48 g) in the F₂ progeny, probably due better endosperm development. Chromosome behavior and pollen stainability were similar in the inbred parents and hybrids. However, the hybrids shed more pollen than the inbred parents.Heterosis was evident in the F₁ hybrids and F₂ progeny which showed significant increases in plant height, leaf length, leaf width, and inflorescence length in addition to fertility improvement.     

Genetic analysis of low-temperature tolerance during germination in tomato, Lycopersicon esculentum Mill.

The genetic basis of low-temperature tolerance during germination of tomato seed was investigated using two approaches. First, a cold-tolerant (PI 120256) and a cold-sensitive tomato cultivar (UCT5) and their reciprocal F₂, F₃ and BC₁ progeny (total of 10 generations) were evaluated for germination at a low (11 ± 0.5°C) and a high (control) temperature 20 ±0.5° C) Weighted least-square regression analysis indicated that in the low-temperature treatment most of the variation resulted from additive genetic effects, and dominance and epistatic interactions were nonsignificant. Partitioning of the total genetic variance into those attributable to the effects of embryo, endosperm, testa and the cytoplasm indicated that additive effects of endosperm and embryo could individually account for 80% and 77% of the total variance, respectively. In the control treatment, greater than 60% of the variation could be explained by individual additive effects of endosperm or embryo and ? 27% of the variation could be explained by embryo dominance effects. Across generations, there was a positive correlation (r = 0.78, P < 0.01) between germination in the control and low-temperature treatments and there were no significant genotype × temperature interactions. The results indicate the presence of similar or identical genes with predominantly additive effects on germination under both low and high temperatures. In the second approach, the effectiveness of directional phenotypic selection to improve tomato cold tolerance during germination was evaluated by selecting (in an F₂ population of the same cross) the fastest germinating seeds under low temperature and comparing the germination of the selected F₃ progeny with germination of an unselected F₃ population. The results indicated that selection was highly effective and significantly improved germination performance of the progeny; a realized heritability of 0.74 was obtained for low-temperature tolerance during germination. It is concluded that in these tomato lines germination under low temperature is genetically controlled, with additivity being the major genetic component, and thus the trait can be improved by phenotypic selection.