Pyramided Lines Research Articles

Designing rice panicle architecture via developmental regulatory genes.

Rice panicle architecture displays remarkable diversity in branch number, branch length, and grain arrangement; however, much remains unknown about how such diversity in patterns is generated. Although several genes related to panicle branch number and panicle length have been identified, how panicle branch number and panicle length are coordinately regulated is unclear. Here, we show that panicle length and panicle branch number are independently regulated by the genes Prl5/OsGA20ox4, Pbl6/APO1, and Gn1a/OsCKX2. We produced near-isogenic lines (NILs) in the Koshihikari genetic background harboring the elite alleles for Prl5, regulating panicle rachis length; Pbl6, regulating primary branch length; and Gn1a, regulating panicle branching in various combinations. A pyramiding line carrying Prl5, Pbl6, and Gn1a showed increased panicle length and branching without any trade-off relationship between branch length or number. We successfully produced various arrangement patterns of grains by changing the combination of alleles at these three loci. Improvement of panicle architecture raised yield without associated negative effects on yield-related traits except for panicle number. Three-dimensional (3D) analyses by X-ray computed tomography (CT) of panicles revealed that differences in panicle architecture affect grain filling. Importantly, we determined that Prl5 improves grain filling without affecting grain number.

Marker-Assisted Pyramiding of CRa and CRd Genes to Improve the Clubroot Resistance of Brassica rapa.

Clubroot, caused by Plasmodiophora brassicae, is an economically important soil-borne disease that threatens Brassicaceae crops worldwide. In recent years, the incidence area of Chinese cabbage (Brassica rapa ssp. pekinensis) clubroot disease has increased, which severely affects the yield and quality of Chinese cabbage. The resistance of varieties harboring the single clubroot-resistance (CR) gene is easily broken through by P. brassicae pathotypes. CRa and CRd, genetically identified in B. rapa, are CR genes known to be highly resistant to different P. brassicaea pathotypes. In our study, we perform the gene pyramiding of CRa and CRd in Chinese cabbages through marker-assisted selection (MAS), and develop homozygous pyramided lines. The newly generated pyramided lines exhibit greater resistance to six different pathotypes than that of two parental lines carrying a single CR gene. This study provides new CR-gene-pyramided lines for the development of clubroot-resistant Brassica varieties for future breeding programs.

Marker-assisted pyramiding of southern leaf blight resistance QTLs qSLB3.1 and qSLB8.1 in maize (Zea mays)

Southern leaf blight (SLB) is one of the major diseases that cause substantial yield losses in maize (Zea mays L.) worldwide. Stacking broad-spectrum resistance genes/QTLs into prevalent cultivars is the prerequisite for durable disease resistance breeding programme. Therefore, a study was carried at the research farm and molecular biology laboratory of School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, Punjab during 2017–21 to introgress SLB resistance QTLs from two donors, viz. CM139 (qSLB3.1) and LM5 (qSLB8.1) into CM140 inbred using marker-assisted backcross breeding (MABB). Crosses were made between CM139 × CM140 and LM5 × CM140 to generate two separate F1s. Each F1 was backcrossed twice to generate BC2F1 progenies. Foreground selection was performed at each step using linked flanking markers to each QTL and also evaluated for SLB resistance. The selected heterozygous BC2F1 plants from each cross were inter-crossed to combine qSLB3.1 and qSLB8.1 in the same genetic background. The F2 population was also evaluated phenotypically for SLB resistance and other morphological traits. A total of 18 plants were obtained having both the QTLs with homozygous donor alleles. The F2 plants having both QTLs and singly in the homozygous state were advanced to generate F3 progenies. The pyramided lines exhibited 29% lesser disease severity than the lines with either QTL. The present results indicated that additive effects of the QTLs for SLB resistance played an important role among these lines. To our best knowledge, this is the first report for the pyramiding of QTLs associated with SLB resistance. The pyramided lines would serve as potential donors in maize breeding programs.

Engineering Resistance against Sclerotinia sclerotiorum Using a Truncated NLR (TNx) and a Defense-Priming Gene.

The association of both cell-surface PRRs (Pattern Recognition Receptors) and intracellular receptor NLRs (Nucleotide-Binding Leucine-Rich Repeat) in engineered plants have the potential to activate strong defenses against a broad range of pathogens. Here, we describe the identification, characterization, and in planta functional analysis of a novel truncated NLR (TNx) gene from the wild species Arachis stenosperma (AsTIR19), with a protein structure lacking the C-terminal LRR (Leucine Rich Repeat) domain involved in pathogen perception. Overexpression of AsTIR19 in tobacco plants led to a significant reduction in infection caused by Sclerotinia sclerotiorum, with a further reduction in pyramid lines containing an expansin-like B gene (AdEXLB8) potentially involved in defense priming. Transcription analysis of tobacco transgenic lines revealed induction of hormone defense pathways (SA; JA-ET) and PRs (Pathogenesis-Related proteins) production. The strong upregulation of the respiratory burst oxidase homolog D (RbohD) gene in the pyramid lines suggests its central role in mediating immune responses in plants co-expressing the two transgenes, with reactive oxygen species (ROS) production enhanced by AdEXLB8 cues leading to stronger defense response. Here, we demonstrate that the association of potential priming elicitors and truncated NLRs can produce a synergistic effect on fungal resistance, constituting a promising strategy for improved, non-specific resistance to plant pathogens.

Pyramiding Submergence Tolerance and Three Bacterial Blight Resistance Genes in Popular Rice Variety Hasanta through Marker-Assisted Backcross Breeding

Flash flooding (FF) along with bacterial blight (BB) outbreak are very destructive for rice production in the rainfed shallow-lowland (RSL) ecosystem. The presence of dynamic Xoo races with varying levels of genetic diversity and virulence renders their management extremely challenging under RSL. In this context, the marker-assisted improvement of plant resistance/tolerance has been proven as one of the most promising strategies towards the development of sustainable cultivars. The present study demonstrates the marker-assisted introgression of the submergence tolerant (Sub1) and three bacterial blight resistant genes (Xa21 + xa13 + xa5) into the genetic background of Hasanta, a long duration popular rice variety in the eastern coastal region of India. The rice genotypes, Swarna Sub1 (carrying Sub1) and IRBB66 (carrying Xa21 + xa13 + Xa7 + xa5 + Xa4) had maximum genetic similarity (0.84 and 0.73, respectively) with Hasanta; recurrent parent (RP) was used as donor. The forward analysis of target genes in F1s, IC1F1s and backcross (BC) generations was performed by linked/genic markers (Sub1bc2; pTA248, xa13prom and RM122), whereas background recovery of RP in each BC and segregating generations was performed utilizing 108 hypervariable SSR markers. Intervened speed breeding (SB) strategy and intensive phenotyping could lead the development of near isogenic lines (NILs) as to the RP in all basic traits. The performance of the near isogenic lines (NILs, BC2F3 and BC2F4), HS 232-411-391-756-37, HS 232-411-391-809-8, HS 232-411-391-756-18, HS 110-224-197-10-36, HS 232-411-391-809-81, HS 110-224-197-10-41 and HS 232-411-391-809-63 establishes the utility of marker-assisted backcross-breeding (MAB) and SB in accelerated trait introgression. The introgressed lines carrying Sub1 + Xa21 + xa13 + xa5 showed 76% to 91% survival under 14 days of submergence and durable BB resistance (percent disease index-PDI of 2.68 ± 0.26 to 6.22 ± 1.08 and lesion length, LL of 1.29 ± 0.12 to 4.2 ± 0.64 cm). Physiological analysis revealed that improved NILs, carrying Sub1 gene conquered adaptive physiological modulations, had reduced the consumption of soluble sugar and the degradation of total chlorophyll contents (TCC), and an enhanced level of Alcohol Dehydrogenase activity (ADH) and proline accumulation in all submergence regimes. The pyramided lines attained complete product profile of RP, that will contribute to sustainable rice production under RSL, particularly in the coastal region that has substantial acreage under the variety Hasanta.

Pyramiding of gn1a, gs3, and ipa1 Exhibits Complementary and Additive Effects on Rice Yield.

Pyramiding of quantitative trait loci (QTLs) is a powerful approach in breeding super-high-yield varieties. However, the performance of QTLs in improving rice yield varies with specific genetic backgrounds. In a previous study, we employed the CRISPR/Cas9 system to target three yield-related genes, gn1a, gs3, and ipa1 in japonica ‘Zhonghua 11’, mutants of which featured large panicle, big grain, few sterile tillers, and thicker culm, respectively. In this paper, four pyramided lines, including gn1a-gs3, gn1a-ipa1, gs3-ipa1, and gn1a-gs3-ipa1, were further generated by conventional cross-breeding to be tested. Agronomic traits analysis showed that: (1) the stacking lines carried large panicles with an increased spikelet number in the main panicle or panicle; (2) the grain weight of the stacking lines, especially gs3-ipa1 and gn1a-gs3-ipa1, were heavier than those in single mutants; (3) both gn1a-gs3 and gs3-ipa1 produced more grain yield per plant than single mutant lines; (4) pyramided lines were higher than single mutants and transcriptome analysis found improved expression levels of genes related to lipid, amino acid, and carbohydrate transport and metabolism in lines pyramiding three mutant alleles, possibly as a result of complementary and additive effects. Accordingly, the alteration of gene-expression patterns relating to hormone signaling, plant growth, and seed size control was characterized in pyramided lines. The present study not only investigates the effects of pyramiding genes, but also may provide an efficient strategy for breeding super-high-yield rice by reducing the time cost of developing pyramided lines.

Combined Hybridization and Evaluation of High-Lysine Rice: Nutritional and Physicochemical Qualities and Field Performance.

Rice, as a major food crop, provides necessary energy and nutrition for humans and livestock. However, its nutritional value is affected by lysine. Using point mutation, we previously obtained AK2 (aspartokinase) and DHDPS1 (dihydrodipicolinate synthase) genes insensitive to lysine feedback inhibition and constructed transgenic lines AK2-52 and DHDPS1-22, which show increased lysine synthesis, as well as Ri-12, which shows decreased lysine degradation by inhibiting rice lysine ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH) activity. In this study, further transgenic lines were hybridized and evaluated. The lysine content of mature seeds from pyramid lines PRD and PRA increased 32.5- and 29.8-fold, respectively, compared with the wild-type, while the three-gene pyramiding line PRDA had a moderate lysine content. The total lysine, total free lysine, and total protein contents of PRD and PRA also increased and had no obvious impact on the physical and chemical quality, seed appearance, and main agronomic traits. Meanwhile, comparative analysis with polygenic polymeric lines GR containing bacterial AK (lysC) and DHDPS (dapA) genes revealed differences in the way bacterial and endogenous rice AK and DHDPS regulate lysine biosynthesis. These results provide a reference for further evaluation and commercialization of high-lysine transgenic rice.

Genetic improvement of traditional Basmati rice Ranbir Basmati for semi-dwarfism and blast resistance through molecular breeding

The traditional Basmati rices grown in north-western foothills of Himalayas in India are known world-wide for their exquisite aroma and exceptional eating and cooking qualities. These varieties are tall and suffer heavy losses due to their susceptibility to lodging as well as blast disease. In present study, we improved a popular traditional Basmati rice variety ‘Ranbir Basmati’ for semi-dwarfism and resistance to blast by incorporating the semi-dwarfing gene, sd1 and two blast resistance genes, Pi9 and Pi54 through marker-assisted backcross breeding. Combining marker-assisted background selection with stringent phenotypic selection for aroma and seed quality traits during backcrossing enabled faster recovery of recurrent parent genome and reconstitution of the genomic regions associated with Basmati quality traits in the gene pyramided lines. All pyramided lines were significantly short statured with a reduction of 23.46 to 40.26 cm in height, as compared to recurrent parent, with many of them exhibiting higher grain yield and superior quality attributes than Ranbir Basmati. The yield superiority of the pyramided lines primarily resulted from the improvement in panicle number and semi-dwarf stature conferred by sd1 gene. The gene pyramided lines showed complete resistance to five highly virulent blast races that collectively showed compatibility with 16 different resistance genes, suggesting the broad-spectrum resistance in these lines. The semi-dwarf, high yielding and blast resistant lines generated herein are potential candidates for release as cultivars for overcoming losses in Ranbir Basmati due to lodging and blast disease.

Pyramiding of multiple strong-culm genes originating from indica and tropical japonica to the temperate japonica rice

Severe lodging has recurrently occurred at strong typhoon’s hitting in recent climate change. The identification of quantitative trait loci and their responsible genes associated with a strong culm and their pyramiding are important for developing high-yielding varieties with a superior lodging resistance. To evaluate the effects of four strong-culm genes on lodging resistance, the temperate japonica near isogenic line (NIL) with the introgressed SCM1 or SCM2 locus of the indica variety, Habataki and the other NIL with the introgeressed SCM3 or SCM4 locus of the tropical japonica variety, Chugoku 117 were developed. Then, we developed the pyramiding lines with double,triple and quadruple combinations derived from step-by-step crosses among NIL-SCM1–NIL-SCM4. Quadruple pyramiding line (NIL-SCM1 + 2 + 3 + 4) showed the largest culm diameter and the highest culm strength among the combinations and increased spikelet number due to the pleiotropic effects of these genes. Pyramiding of strong culm genes resulted in much increased culm thickness, culm strength and spikelet number due to their additive effect. SCM1 mainly contributed to enhance their pyramiding effect. These results in this study suggest the importance of identifying the combinations of superior alleles of strong culm genes among natural variation and pyramiding these genes for improving high-yielding varieties with a superior lodging resistance.

Incorporation of Two Bacterial Blight Resistance Genes into the Popular Rice Variety, Ranidhan through Marker-Assisted Breeding

The bacterial blight (BB) disease of rice is a major disease that reduces yield heavily in susceptible varieties. Ranidhan is a late maturing popular rice variety but shows high susceptibility to the disease. Two BB resistance genes were transferred into the variety through a marker-assisted backcross breeding approach. Tightly linked molecular markers were deployed to track the BB resistance genes in the plants carrying the target genes in each backcross generation. Foreground screening detected 17, 16 and 15 progenies to carry the 3 BB resistance genes in BC1F1, BC2F1 and BC3F1 generations, respectively. The selected BC3F1 plant was selfed and three different combinations of BB resistance genes were tracked in homozygous state in seven BC3F2 plants. The pyramided lines carrying three resistance genes in homozygous conditions were evaluated for BB disease resistance by inoculating with eight virulent Xoo strains. Five pyramided lines carrying two resistance gene combinations (Xa21+xa13 and Xa21+xa5) exhibited enhanced resistance against the BB pathogens. The disease resistance was in the order of Xa21+xa5 < Xa21+xa13 < xa13+xa5 gene combinations in conferring the resistance. The developed pyramided lines were similar to the recipient parent for the majority of the important agro-morphologic and grain quality traits.

Development of Broad Spectrum and Durable Bacterial Blight Resistant Variety through Pyramiding of Four Resistance Genes in Rice

Bacterial blight (BB) disease caused by Xanthomonas oryzae pv. oryzae is a major biotic constraint on obtaining higher grain yields in rice. Marker-assisted backcross breeding (MABB) was performed by the pyramiding of Xa4, xa5, xa13 and Xa21 resistance genes in the popular variety, Ranidhan. A foreground selection in BC1F1, BC2F1, and BC3F1 progenies detected all the target genes in 12, 7 and 16 progenies by using the closely linked markers from a population size of 446, 410, and 530, respectively. The BB-positive progenies carrying the target genes with a maximal similarity to the recipient parent was backcrossed in each backcross generation. A total of 1784 BC3F2 seeds were obtained from the best BC3F1 progeny. The screening of the BC3F2 progenies for the four target genes resulted in eight plants carrying all the four target genes. A bioassay of the pyramided lines conferred very high levels of resistance to the predominant isolates of bacterial blight disease. In addition, these pyramided lines were similar to Ranidhan in 16 morpho-quality traits, namely, plant height, filled grains/panicle, panicles/plant, grain length, grain breadth, grain weight, milling, head rice recovery, kernel length after cooking, water uptake, the volume expansion ratio, gel consistency, alkali-spreading value, and the amylose content.

Development of High Yielding Fusarium Wilt Resistant Cultivar by Pyramiding of "Genes" Through Marker-Assisted Backcrossing in Chickpea (Cicer arietinum L.).

Pusa 391, a mega desi chickpea variety with medium maturity duration is extensively cultivated in the Central Zone of India. Of late, this variety has become susceptible to Fusarium wilt (FW), which has drastic impact on its yield. Presence of variability in the wilt causing pathogen, Fusarium oxysporum f.sp. ciceri (foc) across geographical locations necessitates the role of pyramiding for FW resistance for different races (foc 1,2,3,4 and 5). Subsequently, the introgression lines developed in Pusa 391 genetic background were subjected to foreground selection using three SSR markers (GA16, TA 27 and TA 96) while 48 SSR markers uniformly distributed on all chromosomes, were used for background selection to observe the recovery of recurrent parent genome (RPG). BC1F1 lines with 75–85% RPG recovery were used to generate BC2F1. The plants that showed more than 90% RPG recovery in BC2F1 were used for generating BC3F1. The plants that showed more than 96% RPG recovery were selected and selfed to generate BC3F3. Multi-location evaluation of advanced introgression lines (BC2F3) in six locations for grain yield (kg/ha), days to fifty percent flowering, days to maturity, 100 seed weight and disease incidence was done. In case of disease incidence, the genotype IL1 (BGM 20211) was highly resistant to FW in Junagarh, Indore, New Delhi, Badnapur and moderately resistant at Sehore and Nandyal. GGE biplot analysis revealed that IL1(BGM20211) was the most stable genotype at Junagadh, Sehore and Nandyal. GGE biplot analysis revealed that IL1(BGM 20211) and IL4(BGM 20212) were the top performers in yield and highly stable across six environments and were nominated for Advanced Varietal Trials (AVT) of AICRP (All India Coordinated Research Project on Chickpea) in 2018–19. BGM20211 and BGM 20212 recorded 29 and 28.5% average yield gain over the recurrent parent Pusa 391, in the AVT-1 and AVT-2 over five environments. Thus, BGM20211 was identified for release and notified as Pusa Manav/Pusa Chickpea 20211 for Madhya Pradesh, Gujarat and Maharashtra, Southern Rajasthan, Bundhelkhand region of Uttar Pradesh states by the Central Sub-Committees on Crop Standards, Notification and Release of Varieties of Agricultural Crops, Ministry of Agriculture and Farmers Welfare, Government of India, for commercial cultivation in India (Gazette notification number S.O.500 (E) dt. 29-1-2021).Such pyramided lines give resilience to multiple races of fusarium wilt with added yield advantage.

GL9 from Oryza glumaepatula controls grain size and chalkiness in rice

Grain size is a key factor influencing grain yield and appearance quality in rice. We identified twelve quantitative trait loci (QTL) for grain length (GL), nine for grain width (GW), and nine for 1000-kernel weight (TKW) using GLU-SSSLs, which are single-segment substitution lines with Oryza glumaepatula as donor parent and Huajingxian 74 (HJX74) as recipient parent. Among the QTL, qGL1-2, qGL1-4, qGL9-2, qGW2-2, qGW9-1 and qTKW9-2 contributed to high grain yield. GL9 was identified as a candidate gene for qGL9-2 by map-based cloning and sequencing, and is a novel allele of GS9. The kernel of NIL-gl9 was slenderer and longer than that of HJX74, and the TKW and grain yield per plant of NIL-gl9 were higher than those of HJX74. The proportion of grain chalkiness of NIL-gl9 was much lower than that of HJX74. Thus, gl9 increased grain yield and appearance quality simultaneously. Three pyramid lines, NIL-gs3/gl9, NIL-GW7/gl9 and NIL-gw8/gl9, were developed and the kernel of each was longer than that of the corresponding recipient parent lines. The gl9 allele may be beneficial for breeding rice varieties with high grain yield and good appearance quality.

Reconstruction of the High Stigma Exsertion Rate Trait in Rice by Pyramiding Multiple QTLs.

Asian cultivated rice is a self-pollinating crop, which has already lost some traits of natural outcrossing in the process of domestication. However, male sterility lines (MSLs) need to have a strong outcrossing ability to produce hybrid seeds by outcrossing with restorer lines of male parents in hybrid rice seed production. Stigma exsertion rate (SER) is a trait related to outcrossing ability. Reconstruction of the high-SER trait is essential in the MSL breeding of rice. In previous studies, we detected eighteen quantitative trait loci (QTLs) for SER from Oryza sativa, Oryza glaberrima, and Oryza glumaepatula using single-segment substitution lines (SSSLs) in the genetic background of Huajingxian 74 (HJX74). In this study, eleven of the QTLs were used to develop pyramiding lines. A total of 29 pyramiding lines with 2–6 QTLs were developed from 10 SSSLs carrying QTLs for SER in the HJX74 genetic background. The results showed that the SER increased with increasing QTLs in the pyramiding lines. The SER in the lines with 5–6 QTLs was as high as wild rice with strong outcrossing ability. The epistasis of additive by additive interaction between QTLs in the pyramiding lines was less-than-additive or negative effect. One QTL, qSER3a-sat, showed minor-effect epistasis and increased higher SER than other QTLs in pyramiding lines. The detection of epistasis of QTLs on SER uncovered the genetic architecture of SER, which provides a basis for using these QTLs to improve SER levels in MSL breeding. The reconstruction of the high-SER trait will help to develop the MSLs with strong outcrossing ability in rice.

Wheat germplasm development by gene pyramiding for resistance to race TTKSK of stem rust

To develop wheat germplasm with resistance to race TTKSK of stem rust, five stem rust resistance genes were used to produce pyramids with up to 4 Sr genes of various combinations by marker-assisted selection (MAS) and doubled haploid (DH) technology. Thirteen desired pyramids with 2 to 4 of the Sr genes were recovered, and wheat cultivar Hoffman was improved by stacking Sr35 and Sr36. TTKSK resistance test showed that pyramided lines with three or four genes plus improved Hoffman with Sr35+Sr36 were all immune to TTKSK. This type of germplasm will be a useful tool for the production of cultivars with long-term sustainability.

Marker-assisted pyramiding of QTLs for heat tolerance and escape upgrades heat resilience in rice (Oryza sativa L.).

This study demonstrated that pyramiding of early morning flowering and heat tolerance QTLs (qEMF3 and qHTSF4.1) in rice is an efficient approach to maintain high spikelet fertility under high-temperature stress at flowering stage. High temperature at flowering stage of rice causes low spikelet fertility and low yield. To cope with high-temperature stress brought by climate change, two strategies were proposed to develop heat-resilient rice varieties. One is to escape the high temperature by flowering early in the morning, another is to enhance tolerance to high-temperature stress per se. Two promising QTLs for early morning flowering (qEMF3) and heat tolerance (qHTSF4.1) were introgressed into IR64 background, and Near isogenic lines (NILs) IR64 + qEMF3 (IR64EMF3) and IR64 + qHTSF4.1 (IR64HT4) were developed in previous studies. In this study, a QTL pyramiding line IR64 + qHTSF4.1 + qEMF3 (IR64HT4EMF3) was developed by marker-assisted selection of the progenies of previous NILs. The NILs were subjected to different high-temperature regimes in the indoor growth chambers and different locations in the field. In the indoor growth chambers, when high temperature starts early (before 11:00 am), IR64HT4 and IR64HT4EMF3 had higher spikelet fertility than IR64EMF3; when high temperature comes later (after 11:00 am), IR64EMF3 and IR64HT4EMF3 had higher spikelet fertility than IR64HT4. The flowering pattern of the IR64HT4EMF3 was earlier than IR64HT4, but similar to IR64EMF3 in the glasshouse, field and indoor growth chambers. IR64HT4EMF3 showed higher spikelet fertility than IR64EMF3 and IR64HT4 in the field in the Philippines. Thus, combination of early morning flowering and heat tolerance QTLs is an elegant breeding strategy to cope with future extreme climate.

Marker-Assisted Pyramiding of Genes for Multilocular Ovaries, Self-Compatibility, and Clubroot Resistance in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Molecular marker-assisted gene pyramiding combined with backcrossing has been widely applied for crop variety improvement. Molecular marker identification could be used in the early stage of breeding to achieve the rapid and effective pyramiding of multiple genes. To create high-quality germplasm for Chinese cabbage breeding, multi-gene pyramiding for self-compatibility, multilocular, and clubroot resistance was performed through molecular marker-assisted selection. The results showed that self-compatibility and multilocular traits were controlled by a pair of recessive genes. Two flanking markers, sau_um190 and cun_246a, and marker Teo-1, based on the gene sequence related to multilocular ovaries, were used for multilocular ovary trait selection. Two flanking markers, SCF-6 and SC-12, and marker Sal-SLGI /PK1+PK4, based on the gene sequence, were used for self-compatibility selection. Two flanking markers, TCR74 and TCR79, closely linked to clubroot resistance gene CRb, were used as foreground selection markers. Based on Chinese cabbage genomic information, 111 SSR markers covering 10 chromosomes were applied for background selection. After multiple generations of selection, a multi-gene pyramided line from a BC4F2 population with self-compatibility, multilocular ovaries, and clubroot resistance was obtained with a high genomic background recovery rate. The improved pyramided line is expected to be utilized as a potential material in further breeding programs.

Pyramiding and evaluation of segregating lines containing lectin and protease inhibitor genes for aphid resistance in Brassica juncea

Aphids are one of the most devastating pests, affecting the potential yield and quality ofBrassica juncea. In the current study, we have attempted to pyramid two transgenic lines containing chickpea lectin (CHPL, P1) and urdbean protease inhibitor (UPI, P2) in each under the phloem specific rolC promoter, through conventional breeding approach. In the derived F2 population, both lectin and protease inhibitor genes were segregating in a 9:3:3:1 ratio (p-value: 0.81), indicative of a single copy of the transgenes in the parents. Furthermore, the parental, as well as pyramided progenies were evaluated for their potential resistance to aphids in terms of mortality and natality. The lines containing both the transgenes were found to be superior over single gene transgenics as a higher mortality rate (96%) was found in F2on the 9th day as compared to single gene transgenics (86% and 80% in P1 and P2 respectively). A significant decrease in the number of nymphs was observed in P1 and P2 but most in F2 plants as almost 43, 32.08, and 107.5 times decrease in the number of nymphs was found in P1, P2, and F2 individuals over control. Expression profiling was done to see if there was any impact of gene pyramiding on the expression pattern of both transgenes before and after aphid treatment, and no significant changes were observed, indicating constitutive expression of transgenes in pyramided lines also. In conclusion, pyramided lines were found to be promising and were superior for aphid resistance.

Improvement of grain yield under moisture and heat stress conditions through marker-assisted pedigree breeding in rice (

Enhancement of rice (Oryza sativa L.) productivity under rainfed ecosystems is important to food security. Efforts to increase yield have had little success, mainly due to the complexity of inheritance of abiotic stress tolerance traits and the technical challenges of phenotyping these traits. The study was conducted to pyramid quantitative trait loci (QTLs) governing tolerance to moisture and heat stress from cv. Nagina22 and QTLs for high yield traits from variety IR64, with the aid of marker-assisted pedigree breeding. From the IR64/N22 cross, we identified 14 pyramided lines with various combinations of targeted QTLs (5–11 QTLs). The three best lines that performed well under moisture stress conditions were PL76 with two water-use efficiency (WUE) QTLs (Mr19a, SLA 9.1), three drought-tolerant yield (DTY) QTLs (qDTY1.2, qDTY3.2, qDTY6.1), one heat-tolerance (HT) QTL (qSSPF10) and four yield QTLs (qFLW4.1, qFLL9, LP1, GW5); PL476 with two WUE QTLs (%N11.1, qWue7a), two DTY QTLs (qDTY6.1, qDTY12.1), two HT QTLs (qSSPF10, qHT6) and three yield QTLs (qFLW4.1, qFLL9, LP1); and PL130 with two WUE QTLs (Mr19a, qWue7a), four DTY QTLs (qDTY1.2, qDTY3.2, qDTY4.1, qDTY12.1), two HT QTLs (qSSPF10, qHT6) and two yield QTLs (qFLW4.1, LP1). These three lines shown a yield advantage of 51.93%, 55.93% and 60.30%, respectively, over high-yielding IR64. Under heat stress, PL457 and PL130 with HT QTLs qSSPF10 and qHT6 showed 85.02% and 61.55% yield advantage, respectively, over IR64. In conclusion, we have demonstrated that the systematic combination of important QTLs from two contrasting parents through marker-assisted pedigree breeding is an outstanding approach to supplement conventional phenotype-based pedigree breeding, especially to improve complex traits such as yield and tolerance to moisture stress and heat.

Carbon-Ion Beam Radiosensitivity Study and Biological Responses of High-Yielding Rice Line, MR219-PL-5

The carbon ion-beam has emerged as a novel physical mutagen for creating genetic variability and crop improvement. In this study, seeds of a high-yielding pyramided rice line MR219-PL-5 were exposed to carbon ion beam irradiation at 10, 20, 40, 60, 80, and 100 Gy. The radiosensitivity test was conducted to determine the optimum dose of carbon ion beam irradiation based on the lethal dose 50% (LD50) using Sandwich Blotter Technique. The biological responses of carbon-ion beam irradiation were also observed in other characteristics such as germination rate (GeR), survival rate (SR), growth rate (GRoR), shoot length (SL), root length (RL), seedling height (SH), days to flowering (DTF), fertility rate (FR) and thousand-grains weight (TGW). Based on the polynomial curve of SR graph, the lethal dose 50% (LD50) value was 86.12 Gy. However, the optimum dose range of carbon ion-beam irradiation was between 40 and 60 Gy as these two doses recorded the highest SR, 63 and 67%, respectively. Furthermore, the shoulder dose in this study was 60 Gy since SR decreased significantly at higher doses. M1 individuals irradiated at 40 and 60 Gy had the best biological responses where significant differences were found for SR, SL, RL, GRoR, SH, DTF and FR at these two doses compared to the other doses. Further studies on M2 and M3 populations could help to identify potential individuals as well as to understand the inheritance of each trait of interest from one generation to the next.