Marker-assisted Backcrossing Research Articles

Parental polymorphism survey using rice microsatellite markers between VGD 1 and ADT 55 for marker-assisted backcross breeding program

Parental polymorphism survey using rice microsatellite markers between VGD 1 and ADT 55 for marker-assisted backcross breeding program

The ryanodine receptor mutation I4728M confers moderate-level resistance to diamide insecticides in Spodoptera litura.

The common cutworm, Spodoptera litura (Fabricius), is one of the most widespread and destructive polyphagous pests in tropical and subtropical Asia. S. litura has evolved resistance to different insecticides, including diamide insecticides. Here, we identified a ryanodine receptor (RyR) mutation (I4728M) associated with target site resistance to diamides in a field-collected population of S. litura. The contribution of this mutation to diamide resistance was investigated through establishing a near-isogenic resistant strain of S. litura. The ND21 population of S. litura, collected from Ningde, Fujian province of China in 2021, exhibited 130.6-fold resistance to chlorantraniliprole compared to the susceptible NJ-S strain. S. litura RyR mutation I4728M, corresponding to Plutella xylostella RyR I4790M, was identified in the ND21 population. SlRyR I4728M mutation of ND21 was introgressed into a susceptible background strain (NJ-S) with marker-assisted backcrossing. The introgressed strain named ND21-R, which was homozygous for the mutant 4728M allele, shared about 94% of the genetic background with the NJ-S strain. ND21-R strain showed moderate levels of resistance to two anthranilic diamides (19.1-fold to chlorantraniliprole, 19.7-fold to cyantraniliprole) and the phthalic diamide flubendiamide (23.4-fold). Genetic analysis showed that chlorantraniliprole resistance was autosomal, incompletely recessive and tightly linked with SlRyR I4728M mutation in the introgressed ND21-R strain of S. litura. Identification of the I4728M mutation and its contribution to diamide resistance in S. litura will help develop allelic discrimination assays for resistance monitoring and guide resistance management practices for diamides in S. litura. © 2023 Society of Chemical Industry.

Gene expression of near-isogenic lines (NILs) carrying blast resistance genes Pi9 and Pi54 in the background of rice cultivar Mushk Budji.

Kashmir valley, India is a homeland to rice landraces like Zag, Nunbeoul, Qadirbeigh, Kawkadur, Kamad, Mushk Budji, etc., generally characterized by short grains, aroma, earliness and cold tolerance. Mushk Budji is a commercially important speciality rice known for its taste and aroma, nonetheless, is extremely vulnerable to blast disease. Through the use of the marker-assisted backcrossing (MABC) approach, a set of 24 Near-isogenic lines (NILs) was created, and the lines with the highest background genome recovery were chosen. The expression analysis was carried out for the component genes and other eight pathway genes related to blast resistance. The major blast resistance genes Pi9 (from IRBL-9W) and Pi54 (from DHMAS 70Q 164-1b) were incorporated following simultaneous-but-step-wise MABC. The NILs harbouring genes Pi9 + Pi54, Pi9 and Pi54 expressed resistance to isolate (Mo-nwi-kash-32) under controlled and natural field conditions. The loci controlling ETI (effector triggered immunity) included the gene Pi9 and showed 61.18 and 60.27 fold change in relative gene expression in Pi54 + Pi9 and Pi9 carrying NILs against RP Mushk Budji. Pi54 was up regulated and showed 41 and 21 fold change in relative gene expression for NIL-Pi54 + Pi9 and NIL-Pi54, respectively. Among the pathway genes, LOC_Os01g60600 (WRKY 108) recorded 8 and 7.5 fold up regulation in Pi9 and Pi54 NILs. The NILs showed recurrent parent genome recovery (RPG) per cent of 81.67 to 92.54 and were on par in performance to recurrent parent Mushk Budji. The lines were utilized to study the expression of the loci controlling WRKYs, peroxidases and chitinases that confer overall ETI response.

Stacking Multiple Genes Improves Resistance to Chilo suppressalis, Magnaporthe oryzae, and Nilaparvata lugens in Transgenic Rice.

The ability of various pests and diseases to adapt to a single plant resistance gene over time leads to loss of resistance in transgenic rice. Therefore, introduction of different pest and disease resistance genes is critical for successful cultivation of transgenic rice strains with broad-spectrum resistance to multiple pathogens. Here, we produced resistance rice lines with multiple, stacked resistance genes by stacking breeding and comprehensively evaluated their resistance to Chilo suppressalis (striped rice stemborer), Magnaporthe oryzae (rice blast), and Nilaparvata lugens (brown planthopper) in a pesticide-free environment. CRY1C and CRY2A are exogenous genes from Bacillus thuringiensis. Pib, Pikm, and Bph29 are natural genes in rice. CH121TJH was introduced into CRY 1C, Pib, Pikm, and Bph29. CH891TJH and R205XTJH were introduced into CRY 2A, Pib, Pikm, and Bph29. Compared with those observed in their recurrent parents, CH121TJH significantly increased the mortality of borers. The other two lines CH891TJH and R205XTJH are the same result. Three lines introduction of Pib and Pikm significantly reduced the area of rice blast lesions, and introduction of Bph29 significantly reduced seedling mortality from N. lugens. Introduction of the exogenous genes had relatively few effects on agronomic and yield traits of the original parents. These findings suggest that stacking of rice resistance genes through molecular marker-assisted backcross breeding can confer broad spectrum and multiple resistance in differently genetic backgrounds.

Gene Validation for Brown Plant Hopper Resistance in Rice Varieties Using SSR Markers

The Brown Plant Hopper (BPH), Nilaparvata lugens (Homoptera: Delphacidae) is one of the most destructive insect pests causing significant yield loss in most of the rice cultivars of Asia. Marker-assisted backcrossing is used to introgress the genes for BPH resistance in which the selection of donor parents to confer durable resistance and validation of the presence of genes is a prerequisite. Jyothi the most popular, widely cultivated rice variety in Kerala is screened along with four parental lines viz., PTB33 (Variety released from RARS, Pattambi), IRRI introgressed lines- IR65482-7-216-1-2, IR7103-121-15-B, and a breeding line RP2068-13-5-8 for validating the presence of genes Bph3, BPH18, Bph20, and Bph21 using SSR markers. Parental polymorphism was shown for markers specific to Bph3, BPH18, and Bph20. Besides this, cluster analysis using the UPGMA method produced 4 clusters. The variety ‘PTB33’ was obtained in a separate cluster which can be attributed to its resistant character against BPH and can be considered as a potential donor. The IRRI introgressed lines- IR65482-7-216-1-2, IR7103-121-15-B, and the breeding line RP2068-13-5-8 can also be well utilized as donors by proper standardized BPH screening tests for confirming the phenotypic expression of BPH resistance.

LCVD resistance breeding in heat tolerant bell pepper: Combined phenotypic and marker-assisted backcrossing for introgression of LCVD resistance from hot pepper

LCVD resistance breeding in heat tolerant bell pepper: Combined phenotypic and marker-assisted backcrossing for introgression of LCVD resistance from hot pepper

Identification of multi-race Fusarium wilt resistance in chickpea (Cicer arietinum L.) using rapid hydroponic phenotyping

Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceris (Padwick) Matuo and K. Sato is a major cause for low productivity of chickpea. Presence of multiple pathogenic races makes it difficult for the breeder to screen for Fusarium wilt resistance. Twenty-two chickpea genotypes were grown in Hoagland solution and inoculated with five different F. oxysporum races two isolates of each race), including host and pathogens from the major chickpea growing region of India. The resistant chickpea line “WR 315” showed a “highly resistant” reaction, and the susceptible line “JG 62” showed a “highly susceptible” reaction across all pathogen races and isolates. However, the parent lines “Pusa 372” and “JG 11” showed “susceptible” reactions, while the marker-assisted backcrossing (MABC) lines of “Pusa 372” (IL.11,12,14) and “JG 11” (IL.15,16,17) were superior for assessed characters (lengths of roots and shoots, fresh and dry weights), and were highly resistant to most races. This is the first study to use race specific screening of MABC lines using hydroponic host culture in chickpea.

Performance of improved versions of Swarna introgressed with yield enhancing genes in multi-location trials

Swarna (MTU7029), an Indian mega-variety of rice, is cultivated on an estimated 8Mha of land. There is a pressing need to address yield stagnation and wider adaptability to irrigated lowland and rainfed ecologies as a result of the negative consequences of climate change and population growth. The present study was aimed at improving Swarna for two yield-related traits through marker-assisted backcross breeding strategy by introgression of OsSPL14 (panicle branching) and SCM2 (stronger culm). Foreground and background selection was carried out at each generation. Homozygous BC2F2 plants harbouring both yield-enhancing genes were identified and advanced through pedigree selection till BC2F5 and evaluated in station trials. Three promising lines possessing higher yield over recurrent parent were identified, and a single line, IET 27661 exhibited superior yield in multi-location trials of the All India Coordinated Rice Improvement Programme (AICRIP) and was found to be promising.

Development of bread wheat (Triticum aestivum L) variety HD3411 following marker-assisted backcross breeding for drought tolerance.

Marker-assisted backcross breeding enables selective insertion of targeted traits into the genome to improve yield, quality, and stress resistance in wheat. In the current investigation, we transferred four drought tolerance quantitative trait loci (QTLs) controlling traits, viz canopy temperature, normalized difference vegetative index, chlorophyll content, and grain yield from the drought-tolerant donor line, C306, into a popular high-yielding, drought-sensitive variety, HD2733. Marker-assisted selection coupled with stringent phenotypic screening was used to advance each generation. This study resulted in 23 improved lines carrying combinations of four drought tolerance QTLs with a range of 85.35%-95.79% background recovery. The backcross-derived lines gave a higher yield under moisture-deficit stress conditions compared with the recipient parent. They also showed higher phenotypic mean values for physiological traits and stability characteristics of HD2733. A promising genotype, HD3411, derived from this cross was identified for release after national multi-location coordinating trials under the All India Coordinated Wheat Improvement Project. Our study is a prime example of the advantages of precision breeding using integrating markers and phenotypic selection to develop new cultivars with desirable traits like drought tolerance.

Developing Early Morning Flowering Version of Rice Variety CO 51 to Mitigate the Heat-Induced Yield Loss

By 2050, the rice production needs to be increased by at least 50% in order to meet the growing food demands of the global population. Among various yield limiting factors, high temperature is fast becoming a major threat to sustain rice yields due to its increased frequency of occurrence and severity of stress events. The development of heat-resilient rice cultivars has been slow due to the lack of relevant donors for heat tolerance traits and limited information regarding the genetic basis of these component traits. The early morning flowering (EMF) trait, contributing to heat escape by promoting flowering/anthesis during cooler hours in the morning is demonstrated to offer protection against high-temperature-induced failure of pollination and fertilization. In this study, evaluation of CO 51, IR64 and IR64-qEMF3 (NIL of IR64 harboring QTL promoting EMF revealed that qEMF3 promoted early morning flowering in IR64-qEMF3 (1½ to 2 h earlier than IR64) and thereby reduced the sterility by about 8.15%. Attempts through marker-assisted backcross breeding led to development of advanced backcross progenies (NILs) of CO 51, harboring qEMF3. Evaluation of 88 BC3F2 progenies identified 19 progenies harboring qEMF3 under homozygous conditions. Evaluation of NILs of CO 51 harboring qEMF3 during summer 2019 revealed that the NILs exhibited early (7.30 a.m.) onset of anthesis by 1½ h and completed its peak anthesis well around cooler hours (9.30 a.m.) of the day and thereby recorded reduced spikelet sterility (7.8–9.0%) than their recurrent parent CO 51 (19.2%). The current study clearly demonstrated the efficacy of early morning flowering in the mitigation of yield losses under high-temperature conditions in a farmer preferred rice variety.

Molecular Markers in Plant Breeding

Molecular markers are an important tool for plant breeding. Since the 1980s, in response to the technology development, molecular marker approaches have been further diversified. The establishment of new-generation sequencing and high-throughput plant phenotyping has greatly decreased the time to genotype large numbers of individuals. For breeders who are not very familiar with molecular techniques and want to catch up with the advances in the field, this review offers basic knowledge. Each molecular marker technology has specific advantages as well as limitations. Molecular marker types, diversity studies, QTL mapping, associative mapping, marker-assisted backcrossing and genomic selection are explored. Marker application in plant breeding is also described. In the genome, molecular markers can detect the genetic architecture of a trait, but also identify candidate genes with an important role in plant breeding programs.

Marker-Assisted Pyramiding of Blast-Resistance Genes in a japonica Elite Rice Cultivar through Forward and Background Selection

Rice blast, caused by Pyricularia oryzae, is one of the main rice diseases worldwide. The pyramiding of blast-resistance (Pi) genes, coupled to Marker-Assisted BackCrossing (MABC), provides broad-spectrum and potentially durable resistance while limiting the donor genome in the background of an elite cultivar. In this work, MABC coupled to foreground and background selections based on KASP marker assays has been applied to introgress four Pi genes (Piz, Pib, Pita, and Pik) in a renowned japonica Italian rice variety, highly susceptible to blast. Molecular analyses on the backcross (BC) lines highlighted the presence of an additional blast-resistance gene, the Pita-linked Pita2/Ptr gene, therefore increasing the number of blast-resistance introgressed genes to five. The recurrent genome was recovered up to 95.65%. Several lines carrying four (including Pita2) Pi genes with high recovery percentage levels were also obtained. Phenotypic evaluations confirmed the effectiveness of the pyramided lines against multivirulent strains, which also had broad patterns of resistance in comparison to those expected based on the pyramided Pi genes. The developed blast-resistant japonica lines represent useful donors of multiple blast-resistance genes for future rice-breeding programs related to the japonica group.

Backcrossing with Marker Assistance to Introduce Broad-Spectrum Bacterial Leaf Blight Resistance in the Malaysian Elite Variety MR297

A significant part of marker-assisted backcross breeding (MABB) is recurrent parent genome recovery (RPGR). The purpose of this study was to introduce MABB-mediated resistance genes against numerous bacterial leaf blight (BLB) pathogens. Here, we examine the introgression of four Xoo resistance genes to the high-yielding Malaysian MR297 lineage Xa21, xa13, xa5, and Xa4 from the IRBB60 lineage. To accomplish both foreground and background selection, we employed polymorphic functional linked markers to target genes and simple sequence repeat (SSR) markers. We used 83 of the 475 authorized polymorphic microsatellite markers to determine the proportion of RPGR in the chosen lines. According to the data, the RPGR at BC1F1, BC2F1, and BC2F2 are 81.94, 92.30, and 95.04%, respectively. Incorporating the four BLB resistance genes into the newly created lines would result in long-lasting and comprehensive protection against BLB. Therefore, a resistant gene may be introduced into a population more quickly with MABB than through conventional breeding. All ten lines tested showed a significant level of resistance to BLB, with three lines displaying very high levels of resistance. Consequently, it was recommended that new lines should be used to produce commercially viable rice varieties.

Molecular Breeding for Incorporation of Submergence Tolerance and Durable Bacterial Blight Resistance into the Popular Rice Variety 'Ranidhan'.

Ranidhan is a popular late-maturing rice variety of Odisha state, India. The farmers of the state suffer heavy loss in years with flash floods as the variety is sensitive to submergence. Bacterial blight (BB) disease is a major yield-limiting factor, and the variety is susceptible to the disease. BB resistance genes Xa21, xa13, and xa5, along with the Sub1 QTL, for submergence stress tolerance were transferred into the variety using marker-assisted backcross breeding approach. Foreground selection using direct and closely linked markers detected the progenies carrying all four target genes in the BC1F1, BC2F1, and BC3F1 generations, and the positive progenies carrying these genes with maximum similarity to the recipient parent, Ranidhan, were backcrossed into each segregating generation. Foreground selection in the BC1F1 generation progenies detected all target genes in 11 progenies. The progeny carrying all target genes and similar to the recipient parent in terms of phenotype was backcrossed, and a total of 321 BC2F1 seeds were produced. Ten progenies carried all target genes/QTL in the BC2F1 generation. Screening of the BC3F1 progenies using markers detected 12 plants carrying the target genes. A total of 1270 BC3F2 seeds were obtained from the best BC3F1 progeny. Foreground selection in the BC3F2 progenies detected four plants carrying the target genes in the homozygous condition. The bioassay of the pyramided lines conferred very high levels of resistance to the predominant isolates of bacterial blight pathogen. These BB pyramided lines were submergence-tolerant and similar to Ranidhan in 13 agro-morphologic and grain quality traits; hence, they are likely to be adopted by farmers.

The effects of a few important gene families on sorghum agronomic traits

Sorghum (Sorghum bicolor [L.] Moench), a main food for more than 500 million impoverished and food insecure people in arid and semi-arid regions of Sub-Saharan Africa (SSA) and South Asia, is an important crop for food and nutritional security (SA). Sorghum has the most acceptance in these drought-prone areas due to its good tolerance to harsh settings, high yield, and use as a good source of forages. In this review, the objective of this study is to document the production and use Sorghum in improvement programmed through a literature review, we used publications from journals to explore gene families, how they evolved, gene family theories, how gene families influenced agronomic features in sorghum, and in-depth studies of the key ten gene families in sorghum. The future prospects on sorghum enhancement include genomic selections and gene families, as well as comparative genomic selections. Furthermore, understanding the mechanism of these gene families is important for addressing problems that plague sorghum production, including as infections, drought, and heat stress. We can accurately improve traits using modern techniques such as marker-assisted selection, Genomic selections (GS), Marker-assisted backcrossing (MABC), Marker-assisted recurrent selection (MARS), Marker-assisted selections (MAS), and Genome-wide selections (GWAS) if we have the above gene families of interest (GWAS). Sorghum as a desirable breed: future paths and prospects.

Genetic Diversity, Conservation, and Utilization of Plant Genetic Resources.

Plant genetic resources (PGRs) are the total hereditary material, which includes all the alleles of various genes, present in a crop species and its wild relatives. They are a major resource that humans depend on to increase farming resilience and profit. Hence, the demand for genetic resources will increase as the world population increases. There is a need to conserve and maintain the genetic diversity of these valuable resources for sustainable food security. Due to environmental changes and genetic erosion, some valuable genetic resources have already become extinct. The landraces, wild relatives, wild species, genetic stock, advanced breeding material, and modern varieties are some of the important plant genetic resources. These diverse resources have contributed to maintaining sustainable biodiversity. New crop varieties with desirable traits have been developed using these resources. Novel genes/alleles linked to the trait of interest are transferred into the commercially cultivated varieties using biotechnological tools. Diversity should be maintained as a genetic resource for the sustainable development of new crop varieties. Additionally, advances in biotechnological tools, such as next-generation sequencing, molecular markers, in vitro culture technology, cryopreservation, and gene banks, help in the precise characterization and conservation of rare and endangered species. Genomic tools help in the identification of quantitative trait loci (QTLs) and novel genes in plants that can be transferred through marker-assisted selection and marker-assisted backcrossing breeding approaches. This article focuses on the recent development in maintaining the diversity of genetic resources, their conservation, and their sustainable utilization to secure global food security.

Introgression of Drought Tolerance into Elite Basmati Rice Variety through Marker-Assisted Backcrossing

Drought is one of the major abiotic threat to rice production in the context of climate change. Super Basmati is an elite, fine grain basmati rice variety grown in Punjab, Pakistan. Due to drought sensitive in nature, its yield has been facing an alarming situation in production because of gradual decrease in irrigated water for a couple of years. Three reported novel QTLs for drought tolerance were selected for incorporation into Super Basmati by employing marker assisted selection strategy. IR55419-04 with novel QTLs was used as a donor parent. Foreground selection was performed by applying PCR based QTL linked SSR markers followed by recombinant selection by using 2–4 flanking markers. Background selection was exercised by using polymorphic SSR markers for maximum genome recovery of the Super Basmati. The individuals homozygous at the target QTLs and with maximum background of Super Basmati at the rest of the non-target genome was selected for evaluation of drought tolerance. Under drought stress conditions, the yields of all introgressed lines (ILs) were 44.2%–125.7% higher than recurrent parent. Six superior ILs that are drought tolerant and very similar to Super Basmati in terms of agronomic and grain quality traits are marked for release as drought-tolerant varieties in arid regions or for use in breeding programs of high grain quality and drought-tolerant parents.

Development of low-phytate maize inbred lines through marker-assisted introgression of lpa1

Context Phytic acid is the major storage form of phosphorus in cereals and is considered an anti-nutritional factor because it chelates major mineral micronutrient cations, resulting in micronutrient malnutrition in humans. For monogastric animals fed maize (Zea mays L.) grains, the stored phosphorus does not release into the digestive tract, leading to phosphorus deficiency and environmental pollution. Aims The aim of the study was to develop maize lines with a lower level of phytic acid that might substantially enhance the nutritional value of maize. Methods The lpa1 mutant allele conferring low phytic acid was transferred into the parental lines of popular maize hybrid DMH 121 (i.e. BML 6 and BML 45) through marker-assisted backcross breeding. Foreground selection was performed using a co-dominant single nucleotide polymorphism marker through a high-resolution melting approach, and background selection was undertaken using 50–55 polymorphic sequence-tagged microsatellite site markers. Key results Near-isogeneic lines were produced with >90% recurrent parental genome and reduction of phytic acid content by up to 44–56% compared with the original lines. Conclusions The near-isogeneic lines carrying lpa1 can be used to reconstitute DHM 121 with low phytate content. Implications The low-phytate maize hybrids produced can be useful in reducing micronutrient malnutrition in humans, as well as environmental pollution.

Ugradnja Ga1-S gena u roditeljske linije hibrida kukuruza belog zrna pomoću molekularnih markera

Maize Research Institute Zemun Polje has a marker-assisted backcross breeding (MABB) program aimed to incorporate the strong allele of incompatibility gene Ga1 into parental components of the hybrids with specific traits (white kernel). The main objective of this research was marker-assisted development of the white maize hybrid's parental lines with complete cross-incompatibility to foreign pollens. Ga1-S-tightly-linked molecular markers successfully identified heterozygous plants with a percentage of approximately 50% after all three backcrossings. Regarding the background selection, 69% of progenies had recurrent parent's genome (RPG) above the theoretical value for BC3 generation. Also, a few individuals even had 99%, the value theoretically achieved in the BC6 generation, showing that MABB made a genetic gain in RPG recovery. Foreground selection in this generation aimed to identify homozygous dominant individuals. Out of 264 BC3 F2 plants, 70 (26.5%) were dominant homozygotes, which is in accordance with the expected Mendelian ratio. Progenies of the BC3 F2 homozygous dominant plants were planted alternatively with yellow-kernel maize of the same maturity to check cross-incompatibility. Unfortunately, none of the dominant progenies had a 100% white kernel, most likely due to the presence of modifier genes that increase/decrease the effectiveness of pollen exclusion or that, alternatively, modifiers are lost during the backcrossing. It could be expected that successfully employed functional markers (developed from the sequence polymorphisms present within a functional gene(s) associated with phenotypic trait variations) would outbalance the noted impediments and enhance MABB efficiency to transfer the desired gene(s) controlling simple or complex trait(s) into cultivated varieties.

Stacking effective ASR and APR rust genes for multiple disease resistance in bread wheat cultivars

Rusts and powdery mildews pose serious threats to wheat and have caused substantial yield losses worldwide. Host resistance is the most economical and sustainable approach for managing such diseases. In this study, an effective leaf rust resistance gene Lr45-derived from Secale cereale L. and a linked stem rust and powdery mildew resistance gene Sr36/Pm6-derived from Triticum timopheevii were successfully pyramided. They were validated into well-adapted Indian wheat cultivars that were already carrying the APR stem rust gene Sr2/Lr27/Yr30 through marker-assisted backcross selection (MABC) following two parallel backcrossing schemes. Three efficiently linked microsatellite markers, G372185 (Lr45), Stm773-2(Sr36), and Xgwm533 (Sr2), were used to confirm introgression of these genes. Lines with resistance genes in each background showed improved agronomic traits in comparison to their recurrent parents. These lines could be used in wheat improvement programs as potentially resistant stocks for leaf, stem rusts and powdery mildew to develop new wheat cultivars.