Analysis of transgene(s) (psy+crtI) inheritance and its stability over generations in the genetic background of indica rice cultivar Swarna

Rice the major staple food crop which feeds more than half of the world’s population but, lacks pathway to synthesize and accumulate provitamin A in endosperm therefore rice eaters particularly children, and pregnant women suffer due to vitamin A deficiency. The pathway for provitamin A synthesis in rice endosperm has been engineered and transgenic rice lines have yellow endosperm, called ‘Golden Rice’. The present study aimed at studying the inheritance of transgene(s) in six transgenic events of ‘Golden Rice’ and transfer of provitamin A trait from transgenic lines to a widely grown mega rice variety Swarna. The events E1, R1 and W1 showed normal Mendelian inheritance in F₂, BC₁F₁ and BC₁F₂ generations. The event W1 was studied in BC₁F₃ as well and showed normal Mendelian inheritance of 3:1. The inheritance pattern in L1 event in BC₁F₁ and BC₁F₂ showed normal Mendelian inheritance following expected ratio 1:1 and 3:1 respectively. The two events G1 and T1 showed distorted segregation in BC₁F₂ and BC₂F₂ respectively in Swarna genetic background. In G1 event, transgene inheritance showed segregation distortion in BC₁F₂ in favour of transgene negative plants. In T1 event, inheritance followed expected Mendelian segregation in BC₁F₁, BC₂F₁ and BC₂F₂, generations. However, when tested against co-dominant inheritance 1:2:1 pattern in BC₂F₂, segregation distortion was observed with less than the expected transgene homozygotes. While against 3:1 ratio, it showed the expected segregation pattern in BC₂F₂ generation. Segregation distortion probably due to differential transmission of transgene positive/negative gametes through either/both parents which needs further study.     

QTLs for Tolerance of Drought and Breeding for Tolerance of Abiotic and Biotic Stress: An Integrated Approach

Background

The coupling of biotic and abiotic stresses leads to high yield losses in rainfed rice (Oryza sativa L.) growing areas. While several studies target these stresses independently, breeding strategies to combat multiple stresses seldom exist. This study reports an integrated strategy that combines QTL mapping and phenotypic selection to develop rice lines with high grain yield (GY) under drought stress and non-stress conditions, and tolerance of rice blast.

Methodology

A blast-tolerant BC₂F₃-derived population was developed from the cross of tropical japonica cultivar Moroberekan (blast- and drought-tolerant) and high-yielding indica variety Swarna (blast- and drought-susceptible) through phenotypic selection for blast tolerance at the BC₂F₂ generation. The population was studied for segregation distortion patterns and QTLs for GY under drought were identified along with study of epistatic interactions for the trait.

Results

Segregation distortion, in favour of Moroberekan, was observed at 50 of the 59 loci. Majority of these marker loci co-localized with known QTLs for blast tolerance or NBS-LRR disease resistance genes. Despite the presence of segregation distortion, high variation for DTF, PH and GY was observed and several QTLs were identified under drought stress and non-stress conditions for the three traits. Epistatic interactions were also detected for GY which explained a large proportion of phenotypic variance observed in the population.

Conclusions

This strategy allowed us to identify QTLs for GY along with rapid development of high-yielding purelines tolerant to blast and drought with considerably reduced efforts. Apart from this, it also allowed us to study the effects of the selection cycle for blast tolerance. The developed lines were screened at IRRI and in the target environment, and drought and blast tolerant lines with high yield were identified. With tolerance to two major stresses and high yield potential, these lines may provide yield stability in rainfed rice areas.     

QTLs for hybrid fertility and their association with female and male sterility in rice

Hybrid sterility is one of the major barriers to the application of wide crosses in plant breeding and is commonly encountered in crosses between indica and japonica rice varieties. Ten mapping populations comprised of two reciprocal F2 and eight BC₁F₁ populations generated from the cross between Ilpumbyeo (japonica) and Dasanbyeo (indica) were used to identify QTLs and to interpret the gametophytic factors involved in hybrid fertility or sterility between two subspecies. Frame maps were constructed using a total of 107 and 144 STS markers covering 12 rice chromosomes in two reciprocal F2 and eight BC₁F₁ populations, respectively. A total of 15 main-effect QTLs and 17 significant digenic-epistatic interactions controlling spikelet fertility (SF) were resolved in the entire genome map of F₂ and BC₁F₁ populations. Among detected QTLs responsible for hybrid fertility, four QTLs, qSF5.1 and qSF5.2 on chromosome 5, qSF6.2 on chromosome 6, and qSF12.2 on chromosome 12, were identified as major QTLs since they were located at corresponding positions in at least three mapping populations. Loci qSF5.1, qSF6.1 and qSF6.2 were responsible for both female and male sterility, whereas qSF3.1, qSF7 and qSF12.2 affected the spikelet fertility only through embryosac factors, and qSF9.1 did through pollen factors. Five new QTLs identified in this study will be helpful for understanding the hybrid sterility and for breeding programs via inter-subspecific hybridization.     

A single genetic locus in chromosome 1 controls conditional browning during the induction of calli from mature seeds of Oryza sativa ssp. indica

Being the crucial step for rice transgenic manipulation, callus culture from mature seeds is severely restricted by browning of induced calli, especially in the case of indica (Oryza Sativa L.) rice. Once this browning occurs, the callus will die and no embryonic calli can be obtained for regeneration. Here we report an induction procedure that overcomes callus browning was found. To clarify the inheritance pattern of callus browning, two reciprocal crosses F₂ and two backcrosses BC₁ were made between indica cultivar inbred lines 93-11 and YueTaiB (YTB) which produced normal and browning respectively in the same induction medium. The ratio of browning to normal in the reciprocal F₂ and backcross (BC₁) populations tested was approximately 1:3 and 1:1, respectively, these results indicate that callus browning is controlled by one single chromosomal locus which is tentatively named Ic1 (Induced callus 1). The genetic mapping of this locus was carried out using microsatellite markers (SSR) in a 216 extremely browning F₂ seed callus. The analysis of genetic linkage indicated that one single locus that mapped to chromosome 1 was correlated to callus browning, and the closest marker in this study was mapped within 1.9 cM from the target locus.     

Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop × weed hybrid generations

The level of transgene expression in crop × weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T₁ single-locus insert GFP/Bacillus thuringiensis (Bt) transgenic canola (Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC₁F₁, BC₂F₁) were produced by backcrossing various GFP/Bt transgenic canola (B. napus, cv Westar) and birdseed rape (Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC₂F₂ Bulk) were generated by crossing BC₂F₁ individuals in the presence of a pollinating insect (Musca domestica L.). The ploidy of plants in the BC₂F₂ Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F₁ hybrid generations contained 95–97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15–29% presence in the BC₂F₂ Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC₂F₂ Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F₁, BC₁F₁ and BC₂F₁). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.Communicated by J. Dvorak     

The QTL analysis on maternal and endosperm genome and their environmental interactions for characters of cooking quality in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Investigations to identify quantitative trait loci (QTLs) governing cooking quality traits including amylose content, gel consistency and gelatinization temperature (expressed by the alkali spread value) were conducted using a set of 241 RIL populations derived from an elite hybrid cross of “Zhenshan 97” × “Minghui 63” and their reciprocal backcrosses BC₁F₁ and BC₂F₁ populations in two environments. QTLs and QTL × environment interactions were analyzed by using the genetic model with endosperm and maternal effects and environmental interaction effects on quantitative traits of seed in cereal crops. The results suggested that a total of seven QTLs were associated with cooking quality of rice, which were subsequently mapped to chromosomes 1, 4 and 6. Six of these QTLs were also found to have environmental interaction effects.     

Pyramided rice lines harbouring Allium sativum (asal) and Galanthus nivalis (gna) lectin genes impart enhanced resistance against major sap-sucking pests

We have developed transgene pyramided rice lines, endowed with enhanced resistance to major sap-sucking insects, through sexual crosses made between two stable transgenic rice lines containing Allium sativum (asal) and Galanthus nivalis (gna) lectin genes. Presence and expression of asal and gna genes in pyramided lines were confirmed by PCR and western blot analyses. Segregation analysis of F₂ progenies disclosed digenic (9:3:3:1) inheritance of the transgenes. Homozygous F₃ plants carrying asal and gna genes were identified employing genetic and molecular methods besides insect bioassays. Pyramided lines, infested with brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH), proved more effective in reducing insect survival, fecundity, feeding ability besides delayed development of insects as compared to the parental transgenics. Under infested conditions, pyramided lines were found superior to the parental transgenics in their seed yield potential. This study represents first report on pyramiding of two lectin genes into rice exhibiting enhanced resistance against major sucking pests. The pyramided lines appear promising and might serve as a novel genetic resource in rice breeding aimed at durable and broad based resistance against hoppers.     

A marker-assisted backcross approach for developing submergence-tolerant rice cultivars

Submergence stress regularly affects 15 million hectares or more of rainfed lowland rice areas in South and Southeast Asia. A major QTL on chromosome 9, Sub1, has provided the opportunity to apply marker assisted backcrossing (MAB) to develop submergence tolerant versions of rice cultivars that are widely grown in the region. In the present study, molecular markers that were tightly linked with Sub1, flanking Sub1, and unlinked to Sub1 were used to apply foreground, recombinant, and background selection, respectively, in backcrosses between a submergence-tolerant donor and the widely grown recurrent parent Swarna. By the BC₂F₂ generation a submergence tolerant plant was identified that possessed Swarna type simple sequence repeat (SSR) alleles on all fragments analyzed except the tip segment of rice chromosome 9 that possessed the Sub1 locus. A BC₃F₂ double recombinant plant was identified that was homozygous for all Swarna type alleles except for an approximately 2.3–3.4 Mb region surrounding the Sub1 locus. The results showed that the mega variety Swarna could be efficiently converted to a submergence tolerant variety in three backcross generations, involving a time of two to three years. Polymorphic markers for foreground and recombinant selection were identified for four other mega varieties to develop a wider range of submergence tolerant varieties to meet the needs of farmers in the flood-prone regions. This approach demonstrates the effective use of marker assisted selection for a major QTL in a molecular breeding program. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dynamic QTL analysis of the Na+ content,K+ content,and Na+/K+ ratio in rice roots during the field growth under salt stress

Rice (Oryza sativa L.) is seriously impacted by global soil salinization. To determine the quantitative trait loci (QTLs) related to salt tolerance in rice roots, F_2:3 and BC₁F_2:3 populations derived from a cross between the cv. Dongnong 425 of high quality and yield and the salt-tolerant cv. Changbai 10, were studied at different development stages. Two genetic linkage maps of F_2:3 and BC1F_2:3 populations were constructed. A 66 mM NaCl solution was used to irrigate the field and to analyze the dynamic QTL of some rice root traits. Using unconditional and conditional QTL mapping methods, 30 unconditional QTLs and 16 conditional QTLs related to the 6 root traits were detected on the 9 rice chromosomes during different developmental stages. Fourteen pairs of unconditional and conditional QTLs were detected at the identical developmental stage in the identical population. A number of QTLs were detected at different developmental stages, however, many did not appear at the last stage. Remarkably, qRKC1 appeared continuously at multiple stages in both the populations suggesting its key role in regulating the salt tolerance of rice roots.     

Fine mapping of qHd1, a minor heading date QTL with pleiotropism for yield traits in rice (Oryza sativa L.)

Key message

A minor QTL for heading date located on the long arm of rice chromosome 1 was delimitated to a 95.0-kb region using near isogenic lines with sequential segregating regions.

Abstract

Heading date and grain yield are two key factors determining the commercial potential of a rice variety. In this study, rice populations with sequential segregating regions were developed and used for mapping a minor QTL for heading date, qHd1. A total of 18 populations in six advanced generations through BC₂F₆ to BC₂F₁₁ were derived from a single BC₂F₃ plant of the indica rice cross Zhenshan 97 (ZS97)///ZS97//ZS97/Milyang 46. The QTL was delimitated to a 95.0-kb region flanked by RM12102 and RM12108 in the terminal region of the long arm of chromosome 1. Results also showed that qHd1 was not involved in the photoperiodic response, having an additive effect ranging from 2.4 d to 2.9 d observed in near isogenic lines grown in the paddy field and under the controlled conditions of either short day or long day. The QTL had pleiotropic effects on yield traits, with the ZS97 allele delaying heading and increasing the number of spikelets per panicle, the number of grains per panicle and grain yield per plant. The candidate region contains ten annotated genes including two genes with functional information related to the control of heading date. These results lay a foundation for the cloning of qHd1. In addition, this kind of minor QTLs could be of great significance in rice breeding for allowing minor adjustment of heading date and yield traits.     

A QTL for rice grain yield in aerobic environments with large effects in three genetic backgrounds

A large-effect QTL associated with grain yield in aerobic environments was identified in three genetic backgrounds, Apo/²*Swarna, Apo/²*IR72, and Vandana/²*IR72, using bulk-segregant analysis (BSA). Apo and Vandana are drought-tolerant aerobic-adapted varieties, while Swarna and IR72 are important lowland rice varieties grown on millions of hectares in Asia but perform poorly in aerobic conditions. Two closely linked rice microsatellite (RM) markers, RM510 and RM19367, located on chromosome 6, were found to be associated with yield under aerobic soil conditions in all three backgrounds. The QTL linked to this marker, qDTY6.1 (DTY, grain yield under drought), was mapped to a 2.2 cM region between RM19367 and RM3805 at a peak LOD score of 32 in the Apo/²*Swarna population. The effect of qDTY6.1 was tested in a total of 20 hydrological environments over a period of five seasons and in five populations in the three genetic backgrounds. In the Apo/²*Swarna population, qDTY6.1 had a large effect on grain yield under favorable aerobic (R ² ≤ 66%) and irrigated lowland (R ² < 39%) conditions but not under drought stress; Apo contributed the favorable allele in all the conditions where an effect was observed. In the Apo/IR72 cross, Apo contributed the favorable allele in almost all the aerobic environments in RIL and BC₁-derived populations. In the Vandana/IR72 RIL and BC₁-derived populations, qDTY6.1 had a strong effect on yield in aerobic drought stress, aerobic non-stress, and irrigated lowland conditions; the Vandana allele was favorable in aerobic environments and the IR72 allele was favorable in irrigated lowland environments. We conclude that qDTY6.1 is a large-effect QTL for rice grain yield under aerobic environments and could potentially be used in molecular breeding of rice for aerobic environments.     

Genetic control of linolenic acid concentration in seed oil of rapeseed (Brassica napus L.)

Summary Results from a diallel mating of two rapeseed lines with distinctly different linolenic acid concentration show that this trait is mainly under control of nuclear genes of the embryo. However, significant differences in reciprocal F₁, BC₁ and BC₂ indicate maternal control, which is realized by interaction between maternal genotype and nuclear genes of the embryo. Additionally, temperature exerts considerable influence on the degree of maternal control. Since no reciprocal differences are detectable in F₂, cytoplasmic factors seem not to be involved in the inheritance of linolenic acid concentration. Hypotheses on the physiological nature of maternal control of this trait are discussed.     

The TaDREB3 transgene transferred by conventional crossings to different genetic backgrounds of bread wheat improves drought tolerance

Drought tolerance of the wheat cultivar Bobwhite was previously enhanced by transformation with a construct containing the wheat DREB3 gene driven by the stress‐inducible maize Rab17 promoter. Progeny of a single T₂ transgenic line were used as pollinators in crosses with four elite bread wheat cultivars from Western Australia: Bonnie Rock, IGW‐2971, Magenta and Wyalkatchem, with the aim of evaluating transgene performance in different genetic backgrounds. The selected pollinator line, BW8‐9‐10‐3, contained multiple transgene copies, had significantly improved drought tolerance compared with wild‐type plants and showed no growth and development penalties or abnormalities. A single hybrid plant was selected from each cross‐combination for three rounds of backcrossing with the corresponding maternal wheat cultivar. The transgene was detected in all four F₁BC₃ combinations, but stress‐inducible transgene expression was found in only three of the four combinations. Under well‐watered conditions, the phenotypes and grain yield components of the F₂BC₃ transgene‐expressing lines were similar to those of corresponding recurrent parents and null‐segregants. Under severe drought conditions, the backcross lines demonstrated 12–18% higher survival rates than the corresponding control plants. Two from four F₃BC₃ transgenic lines showed significantly higher yield (18.9% and 21.5%) than control plants under limited water conditions. There was no induction of transgene expression under cold stress, and therefore, no improvement of frost tolerance observed in the progenies of drought‐tolerant F₃BC₃ lines.     

Dynamic expression of green fluorescent protein and Bacillus thuringiensis Cry1Ac endotaxin in interspecific hybrids and successive backcross generations (BC1 and BC2) between transgenic Brassica napus crop and wild Brassica juncea

The persistence and stability of a transgene encoding a Bacillus thuringiensis (Bt) Cry1Ac insecticidal protein was investigated in hybrids between crop Brassica napus and a recurrent wild Brassica juncea population. Interspecific hybrids (F₁) and backcross progenies (BC₁, BC₂) containing green fluorescent protein (GFP) and Bt genes were successfully produced in the greenhouse. Stable Bt toxin levels were found in hybrid and advanced backcross progenies formed in wild B. juncea. Bt Cry1Ac concentration was significantly lower in BC₂ plants than in transgenic B. napus, F₁, BC₁, while no significant differences were detected among the latter three plant genotypes. A GFP marker gene was used as a scorable marker and indicator of Bt transgene expression. GFP fluorescence intensity was significantly correlated with Bt Cry1Ac concentration at the flowering stage and the pod formation stage in both transgenic oilseed rape hybrids and backcrossed progenies (BC₁, BC₂). It was demonstrated that GFP was a suitable marker for Bt protein in the backcross of B. juncea, which could facilitate the detection of gene flow and is useful in biosafety management.     

Fine mapping of quantitative trait loci Hd-1, Hd-2 and Hd-3, controlling heading date of rice, as single Mendelian factors

 Fine mapping was carried out on three putative QTLs (tentatively designated as Hd-1 to Hd-3) of five such QTLs controlling heading date in rice that had been earlier identified using an F₂ population derived from a cross between a japonica variety, ‘Nipponbare’, and an indica variety, ‘Kasalath’, using progeny backcrossed with ‘Nipponbare’ as the recurrent parent. One BC₃F₂ and two BC₃F₁ plants, in which the target QTL regions were heterozygous and most other chromosomal regions were homozygous for the ‘Nipponbare’ allele, were selected as the experimental material. Self-pollinated progeny (BC₃F₂ and BC₃F₃) of the BC₃F₁ or BC₃F₂ showed continuous variation in days to heading. By means of progeny testing based on BC₃F₃ or BC₃F₄ lines, we determined the genotypes of each BC₃F₂ or BC₃F₃ individual at target QTLs. Their segregation patterns fitted Mendelian inheritance ratios. When the results obtained by RFLP analysis and progeny tests were combined, Hd-1, Hd-2 and Hd-3 were mapped precisely on chromosomes 6, 7 and 6, respectively, of a rice RFLP linkage map. The results demonstrated that QTLs can be treated as Mendelian factors. Moreover, these precise locations were in good agreement with the regions estimated by QTL analysis of the initial F₂ population, demonstrating the high reliability of QTL mapping using a high-density linkage map. Received: 5 November 1997 / Accepted: 10 February 1998     

Breeding lines of the Indian mega-rice variety,MTU 1010, possessing protein kinase <Emphasis Type="Italic">OsPSTOL</Emphasis> (<Emphasis Type="Italic">Pup1</Emphasis>), show better root system architecture and higher yield in soils with low phosphorus

MTU 1010 is a high-yielding mega-variety of rice grown extensively in India. However, it does not perform well in soils with low phosphorus (P) levels. With an objective to improve MTU 1010 for tolerance to low soil P, we have transferred Pup1, a major quantitative trait locus (QTL) associated with tolerance from another mega-variety, Swarna, through marker-assisted backcross breeding (MABB). Foreground selection of the F₁ and backcross plants was performed with the co-dominant, closely linked CAPS marker, K20-2, while two flanking markers RM28011 and RM28157 were utilized for recombinant selection. At each backcross generation, positive plants were also analyzed with a set of 85 parental polymorphic SSR markers to identify the QTL-positive plants possessing maximum introgression of MTU 1010 genome. At BC₂F₁, the best backcross plant was selfed to generate BC₂F₂s. Among them, the plants homozygous for Pup1 (n?=?22) were reconfirmed using the functional marker for Pup1, viz., K46-1, and they were advanced through pedigree method of selection until BC₂F₆ generation. A total of five elite BC₂F₆ lines, possessing Pup1 and phenotypically similar to MTU 1010, were screened in the low soil P plot and normal plot (with optimum soil P levels) during wet season, 2016. All the selected lines showed better performance under low P soil with more number of productive tillers, better root system architecture, and significantly higher yield (>?390%) as compared to MTU 1010. Further, under normal soil, the lines were observed to be similar to or better than MTU 1010 for most of the agro-morphological traits and yield. This study represents the successful application of marker-assisted selection for improvement of tolerance to low soil P in a high-yielding Indian rice variety.     

Identification and Validation of Quantitative Trait Loci for Agronomic Traits in Advanced Backcross Breeding Lines Derived from Oryza rufipogon × Oryza sativa Cultivar MR219

A backcross breeding strategy was used to identify quantitative trait loci (QTLs) associated with 14 traits in a BC₂F₂ population derived from a cross between MR219, an indica rice cultivar and an accession of Oryza rufipogon (IRGC 105491). A total of 261 lines were genotyped with 96 microsatellite markers and evaluated for plant morphology, yield components and growth period. The genetic linkage map generated for this population with an average interval size of 16.2?cM, spanning 1,553.4?cM (Kosambi) of the rice genome. Thirty-eight QTLs were identified with composite interval mapping (CIM), whereas simple interval mapping (SIM) resulted in 47 QTLs (LOD >3.0). The O. rufipogon allele was favourable for 59% of QTLs detected through CIM. Of 261 BC₂F₂ families, 26 advanced backcross breeding lines (BC₂F₅) were used for QTL validation. These lines were selected on the basis of the yield traits potentiality in BC₂F₃ and BC₂F₄ generations. The field trial was conducted at three different locations in Malaysia using randomized complete block design with three replications. Trait based marker analysis was done for QTL determination. Twenty-five QTLs were detected in BC₂F₅ generation whereas 29 QTLs were detected in BC₂F₂ generation of the same population. Two QTLs (qPL-1 and qSPL-7) were not considered for validation due to their low R ² values and two QTLs (qPSS-3-2 and qGW-3-2) were not detected in the BC₂F₅ population. Fifteen QTLs showed the beneficial effect to enhance the trait value of the breeding lines. QTL validation aided to select the promising lines for further utilization.     

Improving blast resistance of maintainer line DRR 9B by transferring broad spectrum resistance gene Pi2 by marker assisted selection in rice

Hybrid rice technology offers great promise to further enhance rice production and productivity for global food security. Improving hybrid rice parental lines is the first step in developing heterotic rice hybrids. To improve resistance against blast disease, a maintainer line DRR 9B was fortified with a major broad-spectrum blast resistance gene Pi2 through marker-assisted selection. The rice blast caused by Magnaporthe oryzae is a major disease and can cause severe yield losses upto 100%. The NILs of Samba Mahsuri namely BA-23-11-89-12-168 possessing Pi2 was utilized as a donor parent. The PCR-based molecular marker tightly linked to Pi2 gene was used for the foreground selection at BC₁F₁ generation. The molecular marker tightly linked to the major fertility restorer gene Rf4 was used for negative selection (i.e., selection of plants possessing non fertility restoring alleles) at BC₁F₁ generation to identify maintainer lines. The positive plants with Rf4 gene were added to the restorer pool for restorer line development. At each stage, MAS for Pi2 coupled with stringent phenotypic selection for agro-morphological and grain quality traits were exercised. At BC₁F₃ generation, one hundred families were screened against blast disease at uniform blast nursery (UBN) and selected resistant lines were advanced to next generations. In the BC₁F₅ generation plants were subjected to agro-morphological evaluation for yield and yield-contributing traits. The selected plants at BC₁F₅ generation were crossed with DRR 9A to assess the maintainer ability of blast resistance lines and for further CMS line conversion for hybrid rice breeding for developing blast resistance rice hybrids.     

Mapping and Marker-assisted Selection of a Brown Planthopper Resistance Gene bph2 in Rice (Oryza sativa L.)

Nilaparvata lugens Stål (brown planthopper, BPH), is one of the major insect pests of rice (Oryza sativa L.) in the temperate rice-growing region. In this study, ASD7 harboring a BPH resistance gene bph2 was crossed to a susceptible cultivar C418, a japonica restorer line. BPH resistance was evaluated using 134 F_2:3 lines derived from the cross between “ASD7” and “C418”. SSR assay and linkage analysis were carried out to detect bph2. As a result, the resistant gene bph2 in ASD7 was successfully mapped between RM7102 and RM463 on the long arm of chromosome 12, with distances of 7.6 cM and 7.2 cM, respectively. Meanwhile, both phenotypic selection and marker-assisted selection (MAS) were conducted in the BC₁F₁ and BC₂F₁ populations. Selection efficiencies of RM7102 and RM463 were determined to be 89.9% and 91.2%, respectively. It would be very beneficial for BPH resistance improvement by using MAS of this gene.