Development Of Near-isogenic Lines Research Articles

Development of near isogenic lines (NILs) for leaf rust resistance utilizing advanced generation segregating lines of RIL population in wheat (Triticum aestivum L.)

Near-isogenic lines (NILs) are useful genetic resources for basic genetic studies and further understanding of associated molecularmechanisms. The NILs can be developed through standard methods like backcross breeding or advanced generation segregatinglines. The present study aimed the development of NILs for leaf rust resistance from the advanced generation segregating lines withresidual heterozygosity. Advanced generations segregating lines/heterogeneous inbred families (HIFs) segregating for contrastinginfection types (IT ;1 and 3) for leaf rust were identified from the recombinant inbred lines (RILs) between cross of T. timopheevii derivedintrogression line Selection G12, a resistant parent and a susceptible parent Agra local. The molecular analysis using polymorphic SSRmarkers between parents indicated a high level of similarity with 97.07 and 96.49% resemblance among the contrasting NIL pairs fromHIF1 and HIF5, respectively. These NILs may serve as valuable resources for conducting fine mapping and expression analysis of leafrust resistance in wheat and, therefore, will help to identify candidate gene(s) for leaf rust resistance in Selection G12

Genetic Enhancement for Biotic Stress Resistance in Basmati Rice through Marker-Assisted Backcross Breeding.

Pusa Basmati 1509 (PB1509) is one of the major foreign-exchange-earning varieties of Basmati rice; it is semi-dwarf and early maturing with exceptional cooking quality and strong aroma. However, it is highly susceptible to various biotic stresses including bacterial blight and blast. Therefore, bacterial blight resistance genes, namely, xa13 + Xa21 and Xa38, and fungal blast resistance genes Pi9 + Pib and Pita were incorporated into the genetic background of recurrent parent (RP) PB1509 using donor parents, namely, Pusa Basmati 1718 (PB1718), Pusa 1927 (P1927), Pusa 1929 (P1929) and Tetep, respectively. Foreground selection was carried out with respective gene-linked markers, stringent phenotypic selection for recurrent parent phenotype, early generation background selection with Simple sequence repeat (SSR) markers, and background analysis at advanced generations with Rice Pan Genome Array comprising 80K SNPs. This has led to the development of Near isogenic lines (NILs), namely, Pusa 3037, Pusa 3054, Pusa 3060 and Pusa 3066 carrying genes xa13 + Xa21, Xa38, Pi9 + Pib and Pita with genomic similarity of 98.25%, 98.92%, 97.38% and 97.69%, respectively, as compared to the RP. Based on GGE-biplot analysis, Pusa 3037-1-44-3-164-20-249-2 carrying xa13 + Xa21, Pusa 3054-2-47-7-166-24-261-3 carrying Xa38, Pusa 3060-3-55-17-157-4-124-1 carrying Pi9 + Pib, and Pusa 3066-4-56-20-159-8-174-1 carrying Pita were identified to be relatively stable and better-performing individuals in the tested environments. Intercrossing between the best BC3F1s has led to the generation of Pusa 3122 (xa13 + Xa21 + Xa38), Pusa 3124 (Xa38 + Pi9 + Pib) and Pusa 3123 (Pi9 + Pib + Pita) with agronomy, grain and cooking quality parameters at par with PB1509. Cultivation of such improved varieties will help farmers reduce the cost of cultivation with decreased pesticide use and improve productivity with ensured safety to consumers.

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.

Sigma factor binding protein 1 (CsSIB1) is a putative candidate of the major-effect QTL dm5.3 for downy mildew resistance in cucumber (Cucumis sativus).

The dm5.3 major-effect QTL in cucumber encodes a homolog of Arabidopsis sigma factor binding protein 1 (CsSIB1). CsSIB1 positively regulates defense responses against downy mildew in cucumber through the salicylic acid (SA) biosynthesis/signaling pathway. Downy mildew (DM) caused by the oomycete pathogen Pseudoperonospora cubensis is an important disease of cucumber and other cucurbits. Our knowledge on molecular mechanisms of DM resistance is still limited. In this study, we reported identification and functional characterization of the candidate gene for the major-effect QTL, dm5.3 for DM resistance originated from PI 197088. The dm5.3 QTL was Modelized through marker-assisted development of near isogenic lines (NILs). NIL-derived segregating populations were used for fine mapping which narrowed the dm5.3 locus down to a 144kb region. Based on multiple lines of evidence, we show that CsSIB1 (CsGy5G027140) that encodes the VQ motif-containing sigma factor binding protein 1 as the most likely candidate for dm5.3. Local association analysis identified a haplotype consisting of 7 SNPs inside the coding and promoter region of CsSIB1 that was associated with DM resistance. Expression of CsSIB1 was up-regulated with P. cubensis infection. Transcriptome profiling of NILs in response to P. cubensis inoculation revealed key players and associated gene networks in which increased expression of CsSIB1 antagonistically promoted salicylic acid (SA) but suppressed jasmonic acid (JA) biosynthesis/signaling pathways. Our work provides novel insights into the function of CsSIB1/dm5.3 as a disease resistance (R) gene. The roles of sigma factor binding protein genes in pathogen defense in cucumber were also discussed.

Characterization of QTLs for grain weight from New Plant Type rice cultivars through the development of near-isogenic lines with an IR 64 background

Characterization of QTLs for grain weight from New Plant Type rice cultivars through the development of near-isogenic lines with an IR 64 background

Development of near isogenic lines for grain softness through marker assisted backcross breeding in wheat

Development of near isogenic lines for grain softness through marker assisted backcross breeding in wheat

Development of near-isogenic lines of durum wheat (Triticum durum Desf.) for glaucousness inhibitor loci Iw1, Iw3 and Iw4 derived from wild relatives

Development of near-isogenic lines of durum wheat (Triticum durum Desf.) for glaucousness inhibitor loci Iw1, Iw3 and Iw4 derived from wild relatives

Detection of a major QTL conditioning trichome length and density on chromosome arm 4BL and development of near isogenic lines targeting this locus in bread wheat.

The online version contains supplementary material available at 10.1007/s11032-021-01201-8.

Genotypic and phenotypic characterization of a large, diverse population of maize near-isogenic lines.

Genome-wide association (GWA) studies can identify quantitative trait loci (QTL) putatively underlying traits of interest, and nested association mapping (NAM) can further assess allelic series. Near-isogenic lines (NILs) can be used to characterize, dissect and validate QTL, but the development of NILs is costly. Previous studies have utilized limited numbers of NILs and introgression donors. We characterized a panel of 1270 maize NILs derived from crosses between 18 diverse inbred lines and the recurrent inbred parent B73, referred to as the nested NILs (nNILs). The nNILs were phenotyped for flowering time, height and resistance to three foliar diseases, and genotyped with genotyping-by-sequencing. Across traits, broad-sense heritability (0.4-0.8) was relatively high. The 896 genotyped nNILs contain 2638 introgressions, which span the entire genome with substantial overlap within and among allele donors. GWA with the whole panel identified 29 QTL for height and disease resistance with allelic variation across donors. To date, this is the largest and most diverse publicly available panel of maize NILs to be phenotypically and genotypically characterized. The nNILs are a valuable resource for the maize community, providing an extensive collection of introgressions from the founders of the maize NAM population in a B73 background combined with data on six agronomically important traits and from genotyping-by-sequencing. We demonstrate that the nNILs can be used for QTL mapping and allelic testing. The majority of nNILs had four or fewer introgressions, and could readily be used for future fine mapping studies.

Accelerated development of rice stripe virus-resistant, near-isogenic rice lines through marker-assisted backcrossing.

The development of new improved varieties is one of the major goals of plant breeding. Concomitantly, the demand for stable, eco-friendly, and high-quality rice production is constantly increasing. However, most farmers prefer to cultivate familiar rice varieties developed more than 10 years ago to minimize economic risk. A strategy is needed to develop rice varieties without the limitations of the preferred old varieties. Here, we tested the rapid development of near isogenic lines (NILs) using a rapid generation advance system together with marker-assisted backcrossing to overcome the shortcomings of parental materials. For this purpose, we chose rice stripe virus (RSV) susceptible variety Unkwang and RSV resistant variety Haedamssal as experimental materials. First, we backcrossed and screened BC1F1 and BC2F1 plants having similar agronomic traits as Unkwang and the heterozygous genotype for RSV resistant specific marker InDel7 from Haedamssal. Secondly, the genetic background of 11 BC2F1 plants was identified with 73 KASP markers; plants of line YR32548-8 showed 84.5% of recovery of the recurrent parent genome. Among 28 BC2F2 plants, YR32548-8-16 was the line that showed maximum recovery of the recurrent parent genome (96.2%) while effectively introgressed with RSV-resistance loci on chromosome 11. Finally, we selected line YR32548-8-16 as an NIL showing an RSV resistant phenotype and similar agronomic traits to Unkwang. This fast breeding approach will be useful in rice breeding programs for the improvement of varieties preferred by farmers for their stress tolerance, yield, or quality.

Marker-assisted development and characterization of near-isogenic lines carrying the Rx4 gene for hypersensitive resistance to Xanthomonas euvesicatoria pv. perforans race T3 in tomato

Bacterial spot caused by several species of Xanthomonas is a devastating disease resulting in severe fruit yield loss and quality decline in tomato production areas worldwide. Lack of resistant commercial varieties is among the most critical factors limiting the application of integrated management to control the disease. Although sources for resistance to all species and races have been discovered over years, breeding for resistance to the disease is slow because the resistance is quantitatively inherited and sometimes linked to poor horticultural traits. Development of near-isogenic lines (NILs) by introgressing the shortest DNA fragment of the resistance gene region from the donor into elite cultivars is a promising approach to eliminating the negative effect on horticultural traits. In the current study, a strategy of combining traditional backcross with molecular marker-assisted foreground and background selection was adopted to generate NILs carrying the Rx4 gene for resistance to Xanthomonas euvesicatoria pv. perforans race T3 strain. The donor parent is a wild species Solanum pimpinellifolium accession PI 128216, and the recurrent parent is an elite processing tomato breeding line OH88119. Disease evaluation was performed in F1, BC4F1, BC6F1, BC6F2, BC6F3, and BC6F4 generations by infiltration of race T3 strain Xv829 to leaves of plants. Meanwhile, fruit traits including fruit weight, fruit color parameters, soluble solid content, pH, and firmness were collected from replicated field trials in BC6F4 and BC6F5. Five NILs with 159–1901 kb DNA fragments in the Rx4 gene region were obtained. They all showed high level of hypersensitive resistance to race T3, and their fruit traits were not significantly different from the recurrent parent OH88119. However, fruit weight of one NIL Rx4–1812 carrying the largest DNA fragment of 1901 kb in the Rx4 gene region was 9.5–11.8% lower than OH8819 though the difference was not statistically significant. This might be due to the linkage drag of FW11.2, a QTL for fruit weight in the same region. Overall, the NILs created here can be used as sources for developing resistant varieties and deciphering the mechanism of resistance to bacterial spot in tomato.

Deciphering signalling network in broad spectrum Near Isogenic Lines of rice resistant to Magnaporthe oryzae

Disease resistance (R) genes like Pi9, Pita, Pi21, Pi54 are playing important role for broad spectrum blast resistance in rice. Development of near isogenic lines (NILs) using these type of broad spectrum genes and understanding their signalling networks is essential to cope up with highly evolving Magnaporthe oryzae strains for longer duration. Here, transcriptional-level changes were studied in three near-isogenic lines (PB1 + Pi1, PB1 + Pi9 and PB1 + Pi54) of rice resistant to blast infection, to find the loci that are unique to resistant lines developed in the background of Pusa Basmati 1 (PB1). The pathway analysis of loci, unique to resistant NILs compared to susceptible control revealed that plant secondary metabolite synthesis was the common mechanism among all NILs to counter against M. oryzae infection. Comparative transcriptome analysis helped to find out common clusters of co-expressed significant differentially expressed loci (SDEL) in both PB1 + Pi9 and PB1 + Pi54 NILs. SDELs from these clusters were involved in the synthesis and degradation of starch; synthesis and elongation of fatty acids; hydrolysis of phospholipids; synthesis of phenylpropanoid; and metabolism of ethylene and jasmonic acid. Through detailed analysis of loci specific to each resistant NIL, we identified a network of signalling pathways mediated by each blast resistance gene. The study also offers insights into transcriptomic dynamics, points to a set of important candidate genes that serve as module to regulate the changes in resistant NILs. We suggest that pyramiding of the blast resistance gene Pi9 with Pi54 will lead to maximum broad spectrum resistance to M. oryzae.

Candidate genes expression profiling during wilting in chickpea caused by Fusarium oxysporum f. sp. ciceris race 5.

Chickpea production may be seriously threatened by Fusarium wilt, a disease caused by the soil-borne fungus Fusarium oxysporum f. sp. ciceris. F. oxysporum race 5 is the most important race in the Mediterranean basin. Recently, the region responsible for resistance race 5 has been delimited within a region on chromosome 2 that spans 820 kb. To gain a better understanding of this genomic region, we used a transcriptomic approach based on quantitative real-time PCR to analyze the expression profiles of 22 selected candidate genes. We used a pair of near-isogenic lines (NILs) differing in their sensitivity to Fusarium race 5 (resistant vs susceptible) to monitor the transcriptional changes over a time-course experiment (24, 48, and 72 hours post inoculation, hpi). Qualitative differences occurred during the timing of regulation. A cluster of 12 genes were induced by the resistant NIL at 24 hpi, whereas a second cluster contained 9 genes induced by the susceptible NIL at 48 hpi. Their possible functions in the molecular defence of chickpea is discussed. Our study provides new insight into the molecular defence against Fusarium race 5 and demonstrates that development of NILs is a rich resource to facilitate the detection of candidate genes. The new genes regulated here may be useful against other Fusarium races.

Development of 25 near-isogenic lines (NILs) with ten BPH resistance genes in rice (Oryza sativa L.): production, resistance spectrum, and molecular analysis

A first set of 25 NILs carrying ten BPH resistance genes and their pyramids was developed in the background of indica variety IR24 for insect resistance breeding in rice. Brown planthopper (Nilaparvata lugens Stal.) is one of the most destructive insect pests in rice. Development of near-isogenic lines (NILs) is an important strategy for genetic analysis of brown planthopper (BPH) resistance (R) genes and their deployment against diverse BPH populations. A set of 25 NILs with 9 single R genes and 16 multiple R gene combinations consisting of 11 two-gene pyramids and 5 three-gene pyramids in the genetic background of the susceptible indica rice cultivar IR24 was developed through marker-assisted selection. The linked DNA markers for each of the R genes were used for foreground selection and confirming the introgressed regions of the BPH R genes. Modified seed box screening and feeding rate of BPH were used to evaluate the spectrum of resistance. BPH reaction of each of the NILs carrying different single genes was variable at the antibiosis level with the four BPH populations of the Philippines. The NILs with two- to three-pyramided genes showed a stronger level of antibiosis (49.3-99.0%) against BPH populations compared with NILs with a single R gene NILs (42.0-83.5%) and IR24 (10.0%). Background genotyping by high-density SNPs markers revealed that most of the chromosome regions of the NILs (BC3F5) had IR24 genome recovery of 82.0-94.2%. Six major agronomic data of the NILs showed a phenotypically comparable agronomic performance with IR24. These newly developed NILs will be useful as new genetic resources for BPH resistance breeding and are valuable sources of genes in monitoring against the emerging BPH biotypes in different rice-growing countries.

Development of Near-Isogenic Lines in a Parthenogenetically Reproduced Thrips Species, Frankliniella occidentalis.

Although near-isogenic lines (NILs) can standardize genetic backgrounds among individuals, it has never been applied in parthenogenetically reproduced animals. Here, through multiple rounds of backcrossing and spinosad screening, we generated spinosad resistant NILs in the western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), with a haplo-diploid reproduction system. The resultant F. occidentalis NIL-R strain maintained a resistance ratio over 30,000-fold, which was comparable to its parental resistant strain, Spin-R. More importantly, F. occidentalis NIL-R shared 98.90% genetic similarity with its susceptible parental strain Ivf03. By developing this toolset, we are able to segregate individual resistance and facilitate the mechanistic study of insecticide resistances in phloem-feeding arthropods, a group of devastating pest species reproducing sexually as well as asexually.

Development and validation of markers for spike density QTL, Qsd.sau-7A from Tibetan semi-wild wheat (Triticum aestivum ssp. tibetanum)

Spike density is one of the few traits that determines the yield potential in wheat. Three high resolution melting (HRM) markers (0C32–715, DCE-464, and C81A-240) were developed and used to map the QTL for spike density Qsdsau-7A. The markers 0C32–715 and DCE-464 were validated in two of the three validation population. DCE-464 and 0C32715 were the most closely linked markers that could be used to introgress QSd.sau-7A through molecular maker assisted breeding and development of near isogenic lines.

日本型イネにおける脱粒性評価のための準同質遺伝子系統群の育成と脱粒程度の遺伝解析

日本型イネにおける脱粒性評価のための準同質遺伝子系統群の育成と脱粒程度の遺伝解析

Development of near isogenic lines (NILS) using backcross method of breeding and simultaneous screening against Karnal bunt disease of wheat

Karnal bunt of wheat caused by Tilletia indica can be a hindrance to wheat trade as the fungal disease is known to affect the quality of the grain. As a breeding strategy, a set of eight near isogenics lines (NILs) and backcross derived recombinant inbred lines (micro-RILs) were developed in the background of PBW 343 and WH542 for Karnal bunt resistance over a period of time. The donor stocks resistant to Karnal bunt used in the study were ALDAN ‘ S’ / IAS 58, CMH 77.308, H567.7I, HD29, HP1531, W485 and their derived lines KBRL 22 and KBRL 57. Effective method for screening to Karnal bunt was standardized and used for identification of resistant lines across many seasons. Some of the identified lines evaluated for yield were found to be equivalent to the high yielding parents and the commercially grown check varieties. These developed lines will serve as a basic material for production of Karnal bunt free wheat.

Identification of the target region including the Foc0 1 /foc0 1 gene and development of near isogenic lines for resistance to Fusarium Wilt race 0 in chickpea

Three recombinant inbred populations (RIP-1, RIP-7 and RIP-10) were used to validate and saturate the target area involved in the resistance to Fusarium oxysporum f.sp. ciceris race 0 (Foc0) in chickpeas. With the aim of physically localizing the genetic area controlling Foc0 resistance, a new genetic map of the linkage group 5 (LG5) and a physical map of the Ca5 pseudomolecule were drawn up and compared. The genetic and physical between CaGM20820 and CaGM20889 (16759541–17501349 bp) within an interval of 2 cM comprising 27 annotated genes. Only two of the annotated genes in this region could mapping results obtained were useful to refine the position of the target area be involved in resistance mechanisms. The three RIPs were used to develop three pairs of near isogenic lines (NILs) for resistance/susceptibility to Foc0. NILs are a valuable tool for fine mapping, refining the target region and selecting candidate genes for resistance to Foc0. The information reported in this paper will help develop diagnostic markers, considered useful tools for MAS in chickpea breeding programs.

Identification of quantitative trait loci associated with oil content and development of near isogenic lines for stableqOC-A10inBrasscia napusL.

Seed oil content is a key seed quality trait determining the economic value of rapeseed (Brassica napus L.). However, it is a complex quantitative trait controlled by multiple genes. To this point, its genetic mechanism in rapeseed remains to be revealed. In the present study, we separately identified the quantitative trait loci (QTL) controlling seed oil content of B. napus using three generations of recombinant inbred line (RIL) populations (F4:5, F5:6, and F6:7) derived from a cross of two contrasting parents (M201, a high-oil parent, and M202, a low-oil parent) in four trials. The results indicated that the additive effects may be the primary factors contributing to the variation in seed oil content in B. napus. A total of 15 QTL for seed oil content were mapped. Two of them, namely qOC-A9-3 and qOC-A10, were consistently detected across two and all four environments, respectively. Meanwhile, qOC-A10 showed a large effect on phenotypic variation in seed oil content. The stability and significance of qOC-A10 was also validated in the near isogenic lines (NILs-qOC-A10) developed from the RIL population (F4:5) using marker-assisted selection. The qOC-A10 is of particular interest for further fine mapping and map-based cloning.