Set Of Near-isogenic Lines Research Articles

A multiple near isogenic line (multi-NIL) RNA-seq approach to identify candidate genes underpinning QTL.

This study demonstrates how identification of genes underpinning disease-resistance QTL based on differential expression and SNPs can be improved by performing transcriptomic analysis on multiple near isogenic lines. Transcriptomic analysis has been widely used to understand the genetic basis of a trait of interest by comparing genotypes with contrasting phenotypes. However, these approaches identify such large sets of differentially expressed genes that it proves difficult to isolate which genes underpin the phenotype of interest. This study tests whether using multiple near isogenic lines (NILs) can improve the resolution of RNA-seq-based approaches to identify genes underpinning disease-resistance QTL. A set of NILs for a major effect Fusarium crown rot-resistance QTL in barley on the 4HL chromosome arm were analysed under Fusarium crown rot using RNA-seq. Differential gene expression and single nucleotide polymorphism detection analyses reduced the number of putative candidates from thousands within individual NIL pairs to only one hundred and two genes, which were differentially expressed or contained SNPs in common across NIL pairs and occurred on 4HL. Our findings support the value of performing RNA-seq analysis using multiple NILs to remove genetic background effects. The enrichment analyses indicated conserved differences in the response to infection between resistant and sensitive isolines suggesting that sensitive isolines are impaired in systemic defence response to Fusarium pseudograminearum.

New Races and Novel Strains of the Spinach Downy Mildew Pathogen Peronospora effusa.

Downy mildew disease, caused by Peronospora effusa (=P. farinosa f. sp. spinaciae [Pfs]), is the most economically important disease of spinach. Current high-density fresh-market spinach production provides conducive conditions for disease development, and downy mildew frequently forces growers to harvest early owing to disease development, to cull symptomatic leaves prior to harvest, or to abandon the field if the disease is too severe. The use of resistant cultivars to manage downy mildew, particularly on increasing acreages of organic spinach production, applies strong selection pressure on the pathogen, and many new races of Pfs have been identified in recent years in spinach production areas worldwide. To monitor the virulence diversity in the Pfs population, downy mildew samples were collected from spinach production areas and tested for race identification based on the disease reactions of a standard set of international spinach differentials. Two new races (designated races 15 and 16) and eight novel strains were identified between 2013 and 2017. The disease reaction of Pfs 15 was similar to race 4, except race 4 could not overcome the resistance imparted by the RPF9 locus. Several resistance loci (RPF1, 2, 4, and 6) were effective in preventing disease caused by Pfs 15. The race Pfs 16 could overcome several resistance loci (RPF2, 4, 5, 9, and 10) but not others (RPF1, 3, 6, and 7). One novel strain (UA1014) could overcome the resistance of spinach resistant loci RPF1 to RPF7 but only infected the cotyledons and not the true leaves of certain cultivars. A new set of near-isogenic lines has been developed and evaluated for disease reactions to the new races and novel strains as differentials. None of the 360 U.S. Department of Agriculture spinach germplasm accessions tested were resistant to Pfs 16 or UA1014. A survey of isolates over several years highlighted the dynamic nature of the virulence diversity of the Pfs population. Identification of virulence diversity and evaluation of the genetics of resistance to Pfs will continue to allow for a more effective disease management strategy through resistance gene deployment.

Alternative functions of Hd1 in repressing or promoting heading are determined by Ghd7 status under long-day conditions

Previous studies suggested that Hd1 promoted heading under short-day conditions (SD) and delayed heading under long-day conditions (LD). However in this study, Hd1 was demonstrated to consistently promote heading date in Zhenshan 97 (ZS97) background by upregulating Ehd1, Hd3a and RFT1 expression under both SD and LD. While the high photoperiod sensitivity of Hd1 was observed in Minghui 63 (MH63) background, with heading being suppressed in LD but promoted in SD. Comparative analysis of two sets of near isogenic lines of Hd1 in MH63 and ZS97 backgrounds indicated that the alternative functions of Hd1 in promoting or suppressing heading under LD are dependent on the previously cloned flowering repressor gene Ghd7. The interaction between proteins Ghd7 and Hd1 occurred through binding of the CCT domain of Ghd7 to the transcription-activating domain of Hd1, resulting in suppression of Ehd1 and florigen gene expression. The involvement of the transcription-activating domain of Hd1 in this protein-protein interaction probably blocked or weakened its transcriptional activity. These findings suggest that Hd1 alone essentially acts as a promoter of heading date, and the protein interaction between Ghd7 and Hd1 determines photoperiod sensitivity and integrated Hd1-mediated and Ehd1-mediated flowering pathways in rice.

Molecular mapping and candidate gene analysis of a new epicuticular wax locus in sorghum (Sorghum bicolor L. Moench).

A new epicuticular wax (bloom) locus has been identified and fine mapped to the 207.89kb genomic region on chromosome 1. A putative candidate gene, Sobic.001G269200, annotated as GDSL-like lipase/acylhydrolase, is proposed as the most probable candidate gene involved in bloom synthesis/deposition. Deposition of epicuticular wax on plant aerial surface is one strategy that plants adapt to reduce non-transpiration water loss. Epicuticular wax (bloom)-less mutants in sorghum with their glossy phenotypes exhibit changes in the accumulation of epicuticular wax on leaf and culm surfaces. We report molecular mapping of a new sorghum locus, bloomless mutant (bm39), involved in epicuticular wax biosynthesis in sorghum. Inheritance studies involving a profusely bloom parent (BTx623) and a spontaneous bloomless mutant (RS647) indicated that the parents differed in a single gene for bloom synthesis. Bloomless was recessive to bloom deposition. Genetic mapping involving F2 and F7 mapping populations in diverse genetic backgrounds (BTx623×RS647; 296A×RS647 and 27A×RS647) identified and validated the map location of bm39 to a region of 207.89kb on chromosome 1. SSR markers, Sblm13 and Sblm16, flanked the bm39 locus to a map interval of 0.3cM on either side. Nine candidate genes were identified, of which Sobic.001G269200 annotated for GDSL-like lipase/acylhydrolase is the most likely gene associated with epicuticular wax deposition. Gene expression analysis in parents, isogenic lines and sets of near isogenic lines also confirmed the reduced expression of the putative candidate gene. The study opens possibilities for a detailed molecular analysis of the gene, its role in epicuticular wax synthesis and deposition, and may help to understand its function in moisture stress tolerance and insect and pathogen resistance in sorghum.

Anthocyanins Participate in Protection of Wheat Seedlings from Osmotic Stress

Plant secondary metabolites anthocyanins are considered to play a protective role. In bread wheat (Triticum aestivum L.), anthocyanins can be observed in both adult plants and seedlings. The aim of the current study was to investigate the putative role of anthocyanins present in grains and shoots with respect to the protection of seedlings against drought. For this purpose a set of near isogenic lines (NILs) differing in pericarp and coleoptile colour was used. Water stress was created by artificial shortage of moisture under laboratory conditions. Differences among the lines were observed in a way that the lines with dark-purple grains and coleoptiles (genotype Pp-D1Pp-D1Pp3Pp3Rc-A1Rc-A1Rc-D1Rc-D1) demonstrated a higher seedling drought tolerance than plants with uncoloured pericarp and light-purple coleoptiles (pp-D1pp-D1pp3pp3Rc-A1Rc-A1rc-D1rc-D1). Furthermore, protection of the root system and the shoot was related with the presence of anthocyanins in grains and coleoptiles, respectively.

GmSALT3, Which Confers Improved Soybean Salt Tolerance in the Field, Increases Leaf Cl- Exclusion Prior to Na+ Exclusion But Does Not Improve Early Vigor under Salinity.

Soil salinity reduces soybean growth and yield. The recently identified GmSALT3 (Glycine max salt Tolerance-associated gene on chromosome 3) has the potential to improve soybean yields in salinized conditions. Here we evaluate the impact of GmSALT3 on soybean performance under saline or non-saline conditions. Three sets of near isogenic lines (NILs), with genetic similarity of 95.6–99.3% between each pair of NIL-T and NIL-S, were generated from a cross between two varieties 85–140 (salt-sensitive, S) and Tiefeng 8 (salt-tolerant, T) by using marker-assisted selection. Each NIL-T; 782-T, 820-T and 860-T, contained a common ~1000 kb fragment on chromosome 3 where GmSALT3 was located. We show that GmSALT3 does not contribute to an improvement in seedling emergence rate or early vigor under salt stress. However, when 12-day-old seedlings were exposed to NaCl stress, the NIL-T lines accumulated significantly less leaf Na+ compared with their corresponding NIL-S, while no significant difference of K+ concentration was observed between NIL-T and NIL-S; the magnitude of Na+ accumulation within each NIL-T set was influenced by the different genetic backgrounds. In addition, NIL-T lines accumulated less Cl- in the leaf and more in the root prior to any difference in Na+; in the field they accumulated less pod wall Cl- than the corresponding NIL-S lines. Under non-saline field conditions, no significant differences were observed for yield related traits within each pair of NIL-T and NIL-S lines, indicating there was no yield penalty for having the GmSALT3 gene. In contrast, under saline field conditions the NIL-T lines had significantly greater plant seed weight and 100-seed weight than the corresponding NIL-S lines, meaning GmSALT3 conferred a yield advantage to soybean plants in salinized fields. Our results indicated that GmSALT3 mediated regulation of both Na+ and Cl- accumulation in soybean, and contributes to improved soybean yield through maintaining a higher seed weight under saline stress.

Dissection of the qTGW1.1 region into two tightly-linked minor QTLs having stable effects for grain weight in rice.

BackgroundMost agronomical traits of crop species are complex traits controlled by multiple genes and affected by environmental factors. While considerable efforts have been made to fine-map and clone major quantitative trait loci (QTLs) for yield-related traits in rice, it is not until recently that the attention has been paid to minor QTLs. Following previous dissection of QTLs for grain weight and grain size in a 12-Mb interval on the long arm of chromosome 1 in rice, this study targeted at one putative QTL region for a more precise mapping and for analyzing the genotype-by-environment interaction of minor QTLs.ResultsFour BC2F10 plants of the indica rice cross ZS97///ZS97//ZS97/MY46 were selected. They carried overlapped heterozygous segments that jointly covered the entire putative region for qTGW1.1 detected previously. Four sets of near isogenic lines (NILs) were developed from selfing progenies of the four plants. Each NIL set consisted of 32 ZS97 homozygous lines and 32 MY46 homozygous lines that differed in the corresponding heterozygous region. They were grown in two locations having distinct ecological conditions and measured for 1000-grain weight, grain length and grain width. Two QTLs were separated in an 835.2-kb interval flanked by DNA markers Wn28447 and RM11569. They both showed consistent effects across the two environments. The qTGW1.1a located within the 120.4-kb interval Wn28447 − RM11543 significantly affect all the three traits with the enhancing allele derived from ZS97, showing a stronger influence on grain weight than on grain length and width. The qTGW1.1b located in the 521.8-kb interval RM11554 − RM11569 significantly affect grain weight and length with the enhancing allele derived from MY46, having a stronger influence on grain length than on grain weight. Consistent performance of the two QTLs was confirmed in a validation experiment using five NIL-F2 populations segregated for either qTGW1.1a or qTGW1.1b.ConclusionSeparation of closely-linked QTLs having small effects is achievable in the absence of major-QTL segregation. Minor QTLs for complex traits could act consistently in diverse environments, offering the potential of pyramiding beneficial alleles of multiple minor QTLs through marker-assisted selection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0410-5) contains supplementary material, which is available to authorized users.

Allelic variations in the soluble starch synthase II gene family result in changes of grain quality and starch properties in rice (Oryza sativa L.)

SUMMARYSoluble starch synthase II (SSII) plays an important role in the biosynthesis of starch and in rice it consists of three isoforms encoded by SSII-1, SSII-2 and SSII-3. However, the genetic effects of various SSII alleles on grain quality have not been systematically characterized. In the present study, the japonica alleles on SSII-1, SSII-2 and SSII-3 (SSIIa) loci from a japonica cultivar, Suyunuo, were respectively introgressed by molecular marker-assisted selection into a typical indica cultivar, Guichao2, through successive backcrossing, generating three sets of near-isogenic lines (NILs). Grain quality and starch property analysis showed that NIL-SSII-3j exhibited significant decreases in the following parameters: amylose content, average granule size, and setback viscosity and consistency; but increases in peak viscosity, hot paste viscosity, gelatinization temperature and relative crystallinity. Moreover, the proportion of short amylopectin chains and branching degree also increased when compared with those of NIL-SSII-3i (Guochao2). Similar effects were observed in NIL-SSII-1j, and certain alterations in the fine structure of starch (granule size) were revealed. However, NIL-SSII-2j did not exert significant effect on grain quality and starch properties. In brief, among the SSII gene family, the functional diversity occurred on SSII-1 and SSII-3, and not on SSII-2. Therefore, it appears that more attention should be directed to SSII-1 and SSII-3 loci for improving the eating and cooking quality of rice.

Magnitude of genotype x environment interaction for bacterial leaf blight resistance in rice growing areas of Uganda

Bacterial leaf blight (BLB) of rice ( Oryzae sativa L.), caused by Xanthomonas oryzae pv. oryzae , is a major constraint in most lowland rice producing areas of Uganda. The disease is widely distributed in all irrigated and rainfed lowland rice ecosytems in the country. The pathogen (Xoo) is highly variable and its control is rather difficult. Development and deployment of host resistance is the only effective means of BLB management. The objective of this study was to determine the magnitude of genotype by environment (G x E) interaction for resistance to bacterial leaf blight in rice in Uganda. A study comprised of two sets of germplasms, a total of 30 rice genotypes comprising of 13 lines with varying levels of BLB resistance, and 17 F4 lines that had been previous generated through crossing 7 parental lines, and then advanced in bulk from F1, was conducted in Namulonge-Wakiso, Olweny-Lira and Kibimba- Bugiri districts in Uganda. The study also included 7 parental lines and 6 popular varieties used in most farmers’ fields. Variety IR 24 had been used as a universal check against BLB in Asian rice populations. Results revealed differential reactions on a set of near isogenic lines in the background of IR24, and some national and regional cultivars. IRBB1 (Xa1), IRBB2 (Xa2) and IRBB14 (Xa14) showed moderate susceptibility to susceptibility towards field pathogen populations in the three locations. Whereas genotype IRBB4 with gene Xa4 differentiated pathotypes of Kibimba and Lira from that of Namulonge, IRBB10 (Xa10) and IRBB11 (Xa11) differentiated pathotypes of Lira from the rest. Genotypes that had been pyramided with BLB genes of resistance, showed similar reactions to the three field populations. Generally, the near isogenic lines IRBB1, IRBB2, IRBB11 and IRBB14, had the highest leaf area damaged by disease attack. The highest was shown by IRBB11 with the Kibimba pathotypes for which disease attack was 43%. Low attack was observed on pyramided genotypes in all locations and two with single gene, i.e. IRBB8 and IRBB21, respectively. Interestingly, IR24 was as resistant as any of the pyramided combinations. Results also revealed different reactions of the tested genotypes in the three locations. The analysis of variance by AMMI partitioned the main effects of treatments into genotype, environment, and genotype x environment (G x E) interactions. Results also revealed that, the mean sum of squares due to treatments, genotypes, environments and genotype x environment interaction were significant, and contributed 48.2, 15.3, 19.3 and 13.3%, respectively, PCA1 accounted for 73.02% of the total G x E sum of squares. Key Words: Oryzae sativa , pyramid, Xanthomonas

Further Mapping and Epistasis Analysis of Two Quantitative Trait Loci of Kernel and Cob Anthocyanin Contents in Maize

ABSTRACTIn our previous study, two major quantitative trait loci (QTLs) for kernel anthocyanin content in maize (Zea mays L.) were mapped on chromosome 6 and chromosome 10 by using two related F2:3 populations derived from crosses of ‘Mu6’ × ‘Super Dark Maize’ (SDM) and ‘Mo17’ × SDM. In this study, two sets of near isogenic lines, MuS‐BC4F3 and MoS‐BC4F3, were developed and used to further map the two QTLs and analyze the epsitasis between the two QTLs. GEN6 was further mapped in the interval S8‐umc1105 on chromosome 6 for MuS‐BC4F3 and S8 to mmc0523 on chromosome 6 for MoS‐BC4F3. GEN10 was further mapped in the interval IDP8526 to S44 on chromosome 10 for MuS‐BC4F3 and bnlg1028 to IDP8526 on chromosome 10 for MoS‐BC4F3. GEN6 and GEN10 controlled both kernel anthocyanin content and cob anthocyanin content. The epistasis between GEN6 and GEN10 was a coadaptive interaction. The anthocyanin content is likely to greatly increase when GEN6 and GEN10 coexist. Additive × dominant (AD) effects were the largest of all the epistatic effects for kernel anthocyanin content; additive × additive (AA) effects were the largest for cob anthocyanin content. These results could provide a new reference for anthocyanin's genetic mechanism and high anthocyanin breeding in maize.

Introgression of virus-resistance genes into traditional Spanish tomato cultivars (Solanum lycopersicum L.): Effects on yield and quality

The objective of this study was to determine and quantify the effects of ToMV, TSWV and TYLCV resistance introgressions (determined by Tm-2a, Sw-5 and Ty-1 genes, respectively) on the yield and quality of fresh market tomatoes. Two sets of near isogenic lines (NILs) containing the eight homozygous combinations for the three resistance genes were developed in the backgrounds of the traditional tomato cultivars ‘De la Pera’ and ‘Muchamiel’. These NILs were grown and evaluated in replicated trials in five locations in south-eastern Spain. Across all the analyses performed, the chromosome fragment that consistently had a greater effect was that containing the Ty-1 gene, which conferred resistance to TYLCV. This gene introgression negatively affected all the studied parameters, except for soluble solids content, and its effect was particularly significant on total and commercial yields, which decreased by 50%. This reduction in agricultural yield would only be acceptable when cultivating under high levels of virus infection. The effects of the other introduced fragments (containing Tm-2a and Sw-5) were minor and more variable, increasing or decreasing depending on the trial and the studied parameter. Advanced breeding lines carrying ToMV and TSWV resistance genes in homozygous conditions can be developed without important losses in agronomic and quality characteristics. However, the Ty-1 gene used for TYLCV resistance should only be used in developing cultivars for highly virus-infected areas.

Fine mapping of a large-effect QTL conferring Fusarium crown rot resistance on the long arm of chromosome 3B in hexaploid wheat.

BackgroundFusarium crown rot (FCR) is a major cereal disease in semi-arid areas worldwide. Of the various QTL reported, the one on chromosome arm 3BL (Qcrs.cpi-3B) has the largest effect that can be consistently detected in different genetic backgrounds. Nine sets of near isogenic lines (NILs) for this locus were made available in a previous study. To identify markers that could be reliably used in tagging the Qcrs.cpi-3B locus, a NIL-derived population consisting of 774 F10 lines were generated and exploited to assess markers selected from the existing linkage map and generated from sequences of the 3B pseudomolecule.ResultsThis is the first report on fine mapping a QTL conferring FCR resistance in wheat. By three rounds of linkage mapping using the NILs and the NIL-derived population, the Qcrs.cpi-3B locus was mapped to an interval of 0.7 cM covering a physical distance of about 1.5 Mb. Seven markers co-segregating with the locus were developed. This interval contains a total of 63 gene-coding sequences based on the 3B pseudomolecule, and six of them were known to encode disease resistance proteins. Several of the genes in this interval were among those responsive to FCR infection detected in an earlier study.ConclusionsThe accurate localization of the Qcrs.cpi-3B locus and the development of the markers co-segregating with it should facilitate the incorporation of this large-effect QTL conferring FCR resistance into breeding programs as well as the cloning of the gene(s) underlying the QTL.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2105-0) contains supplementary material, which is available to authorized users.

The Relationships between Development and Low Temperature Tolerance in Barley Near Isogenic Lines Differing for Flowering Behavior.

Flowering time, vernalization requirement, photoperiod sensitivity and low temperature tolerance are key traits in the Triticeae. We characterized a set of isogenic genetic stocks-representing single and pairwise substitutions of spring alleles at the VRN-H1, VRN-H2 and VRN-H3 loci in a winter barley background-at the structural, functional and phenotypic levels. High density mapping with reference to the barley genome sequence confirmed that in all cases target VRN alleles were present in the near isogenic lines (NILs) and allowed estimates of introgression size (at the genetic and physical levels) and gene content. Expression data corroborated the structural and phenotypic results. The latter confirmed that substitution of a spring allele at any of the VRN loci is sufficient to eliminate vernalization requirement. There was no significant change in low temperature tolerance with substitution of a spring allele at VRN-H2, but there were significant losses in cold tolerance with substitutions at VRN-H1 and VRN-H3. Reductions in cold tolerance are ascribed to an accelerated transition from the vegetative to reproductive state. The set of NILs will be a rich resource for understanding the genetics of vernalization, low temperature tolerance and other traits encoded/regulated by genes within the introgressed intervals.

Identification of a novel male sterile wheat mutant dms conferring dwarf status and multi-pistils

Plant height and fertility are two important traits of wheat (Triticum aestivum L.), whose mutants are ideal materials for studies on molecular mechanisms of stem and floral organ development. In this study, we identified a dwarf, multi-pistil and male sterile (dms hereafter) wheat mutant from Zhoumai 18. Simple sequence repeat (SSR) marker assay with 181 primer pairs showed that only one locus of GWM148-2B was divergent between Zhoumai 18 and dms. There were three typical phenotypes in the progeny of dms, tall (T; ca. 0.8 m), semi-dwarf (M; ca. 0.6 m) and dwarf (D; under 0.3 m) plants. Morphological investigation indicated that the internode length of M was shortened by about 20–50 mm each; the internode number of D was 2 less than that of T and Zhoumai 18, and its internode length was shorter also. The pollen vigor and hybridization test demonstrated that dms mutant was male sterility. Segregated phenotypes in progeny of M suggested that the multi-pistils and sterility were controlled by one recessive gene locus which was designated as dms temporarily, and the plant height was controlled by a semi-dominant gene locus Dms. Therefore, progeny individuals of the dms had three genotypes, DmsDms for tall plants, Dmsdms for semi-dwarf plants and dmsdms for dwarf plants. The mutant progenies were individually selected and propagated for more than 6 generations, thus a set of near isogenic lines of T, M and D for dms were developed. This study provides a set germplasms for studies on molecular mechanisms of wheat stem and spike development.

Effects of Allelic Variation in Glutenin Subunits and Gliadins on Baking-Quality in Near-isogenic Lines of Common Wheat cv. Longmai 19

Two lines, L-19-613 and L-19-626, were produced from the common wheat cultivar Longmai 19 (L-19) by six consecutive backcrosses using biochemical marker-assisted selection. L-19 (Glu-D1a, Glu-A3c/Gli-A1?; Gli-A1? is a gene coding for unnamed gliadin) and L-19-613 (Glu-D1d, Glu-A3c/Gli-A1?) formed a set of near-isogenic lines (NILs) for HMW-GS, while L-19-613 and L-19-626 (Glu-D1d, Glu-A3e/Gli-A1m) constituted another set of NILs for the LMW-GS/gliadins. The three L-19 NILs were grown in the wheat breeding nursery in 2007 and 2008. The field experiments were designed using the three-column contrast arrangement method with four replicates. The three lines were ranked as follows for measurements of gluten strength, which was determined by the gluten index, Zeleny sedimentation, the stability and breakdown time of the farinogram, the maximum resistance and area of the extensogram, and the P andWvalues of the alveogram: L-19-613 > L-19-626 > L-19. The parameters listed above were significantly different between lines at the 0.05 or 0.01 level. The Glu-D1 and Glu-A3/Gli-A1 loci had additive effects on the gluten index, Zeleny sedimentation, stability, breakdown time, maximum resistance, area, P and W values. Although genetic variation at the Glu-A3/Gli-A1 locus had a great influence on wheat quality, the genetic difference between Glu-D1d and Glu-D1a at the Glu-D1 locus was much larger than that of Glu-A3c/Gli-A1? and Glu-A3e/Gli-A1m at the Glu-A3/Gli-A1 locus. Glu-D1d had negative effects on the extensibility and the L value compared with Glu-D1a. In contrast, Glu-A3c/Gli-A1? had a positive effect on these traits compared with Glu-A3e/Gli-A1m.

Development and evaluation of near-isogenic lines for major blast resistance gene(s) in Basmati rice.

A set of NILs carrying major blast resistance genes in a Basmati rice variety has been developed. Also, the efficacy of pyramids over monogenic NILs against rice blast pathogen Magnaporthe oryzae has been demonstrated. Productivity and quality of Basmati rice is severely affected by rice blast disease. Major genes and QTLs conferring resistance to blast have been reported only in non-Basmati rice germplasm. Here, we report incorporation of seven blast resistance genes from the donor lines DHMASQ164-2a (Pi54, Pi1, Pita), IRBLz5-CA (Pi2), IRBLb-B (Pib), IRBL5-M (Pi5) and IRBL9-W (Pi9) into the genetic background of an elite Basmati rice variety Pusa Basmati 1 (PB1). A total of 36 near-isogenic lines (NILs) comprising of 14 monogenic, 16 two-gene pyramids and six three-gene pyramids were developed through marker-assisted backcross breeding (MABB). Foreground, recombinant and background selection was used to identify the plants with target gene(s), minimize the linkage drag and increase the recurrent parent genome (RPG) recovery (93.5-98.6%), respectively, in the NILs. Comparative analysis performed using 50,051 SNPs and 500 SSR markers revealed that the SNPs provided better insight into the RPG recovery. Most of the monogenic NILs showed comparable performance in yield and quality, concomitantly, Pusa1637-18-7-6-20 (Pi9), was significantly superior in yield and stable across four different environments as compared to recurrent parent (RP) PB1. Further, among the pyramids, Pusa1930-12-6 (Pi2+Pi5) showed significantly higher yield and Pusa1633-7-8-53-6-8 (Pi54+Pi1+Pita) was superior in cooking quality as compared to RP PB1. The NILs carrying gene Pi9 were found to be the most effective against the concoction of virulent races predominant in the hotspot locations for blast disease. Conversely, when analyzed under artificial inoculation, three-gene pyramids expressed enhanced resistance as compared to the two-gene and monogenic NILs.

Identification and validation of a major QTL controlling the presence/absence of leaf lobes in Brassica rapa L.

Leaves are vital plant organs, and a high level of variation in leaf shape, including the leaf lobes, is present in Brassica rapa. Based on a previously-constructed linkage map, QTL analysis of the leaf lobes was performed using the scores from an F2 population during the spring of 2010, and scores from an F2:3 family in the autumn of 2010, respectively. A total of nine QTLs were found to affect leaf lobes. Among these, a major QTL, lob 10.1, was mapped to the bottom of linkage group A10. To develop near iso-genic lines (NILs) for the identified major QTL, an F2 plant without leaf lobes that was homozygous in the targeted region for marker alleles from the male parent (08A061), was backcrossed to 09A001, the recurrent parent with leaf lobes. Two InDel marker loci, BrID10909 and BrID10233, which are closely linked to the major QTL peak position, and 166 SSR and InDel markers from non-targeted chromosomal regions, were used for foreground and background selection, respectively. Three sets of NILs were developed through four cycles of backcrossing followed by two cycles of self-pollination. Compared to 09A001, the recurrent parent, all the NILs showed the absence of leaf lobes and had no significant differences in other agriculturally important traits. NILs are the ideal genetic resource for QTL validation and fine-scale linkage mapping, and, ultimately, gene discovery.

Mapping of qTGW1.1, a Quantitative Trait Locus for 1000-Grain Weight in Rice (Oryza sativa L.)

1000-grain weight (TGW) is one of the three component traits of the grain yield in rice (Oryza sativa L). This study was conducted to validate and fine-map qTGW1.1, a minor QTL for TGW which was previously located in a 3.7-Mb region on the long arm of rice chromosome 1. Five sets of near isogenic lines (NILs) were developed from two BC2F4 populations of the indica rice cross Zhenshan 973/Milyang 46. The NIL sets consisted of two homozygous genotypic groups differing in the regions RM11448−RM11522, RM11448−RM11549, RM1232−RM11615, RM11543−RM11554 and RM11569−RM11621, respectively. Four traits, including TGW, grain length, grain width and heading date, were measured. Phenotypic difference between the two genotypic groups in each NIL population was analyzed using SAS procedure GLM. Significant QTL effects were detected on TGW with the Zhenshan 97 allele increasing grain weight by 0.12g to 0.14g and explaining 8.30% to 15.19% of the phenotypic variance. Significant effects were also observed for grain length and width, whereas no significant effect was found for heading date. Based on comparison among the five NILs on the segregating regions and the results of QTL analysis, qTGW1.1 was delimited to a 376.9-kb region flanked by DNA markers Wn28382 and RM11554. Our results indicate that the effects of minor QTLs could be steadily detected in a highly isogenic background and suggest that such QTLs could be utilized in the breeding of high-yielding rice varieties.

Transcriptome and allele specificity associated with a 3BL locus for Fusarium crown rot resistance in bread wheat.

Fusarium pathogens cause two major diseases in cereals, Fusarium crown rot (FCR) and head blight (FHB). A large-effect locus conferring resistance to FCR disease was previously located to chromosome arm 3BL (designated as Qcrs-3B) and several independent sets of near isogenic lines (NILs) have been developed for this locus. In this study, five sets of the NILs were used to examine transcriptional changes associated with the Qcrs-3B locus and to identify genes linked to the resistance locus as a step towards the isolation of the causative gene(s). Of the differentially expressed genes (DEGs) detected between the NILs, 12.7% was located on the single chromosome 3B. Of the expressed genes containing SNP (SNP-EGs) detected, 23.5% was mapped to this chromosome. Several of the DEGs and SNP-EGs are known to be involved in host-pathogen interactions, and a large number of the DEGs were among those detected for FHB in previous studies. Of the DEGs detected, 22 were mapped in the Qcrs-3B interval and they included eight which were detected in the resistant isolines only. The enrichment of DEG, and not necessarily those containing SNPs between the resistant and susceptible isolines, around the Qcrs-3B locus is suggestive of local regulation of this region by the resistance allele. Functions for 13 of these DEGs are known. Of the SNP-EGs, 28 were mapped in the Qcrs-3B interval and biological functions for 16 of them are known. These results provide insights into responses regulated by the 3BL locus and identify a tractable number of target genes for fine mapping and functional testing to identify the causative gene(s) at this QTL.

Expansin Genes Expressed Differentially in Peduncle Elongation of Near-Isogenic Wheat Rht Lines

The introduction of reduced height (Rht) genes Rht-B1b and Rht-D1b led to impressive increases in wheat (Triticum aestivum L.) yields during the Green Revolution. In the present study, the dynamic elongation of peduncle in a set of near-isogenic lines (NILs) carrying different Rht alleles (Rht-B1b, Rht-D1b, Rht-B1c, Rht-D1b+Rht-B1b, and Rht-D1b+Rht-B1c) were investigated. The reduction of the final length of peduncle in NILs was dependent mainly on the elongation rate, which was reduced by Rht genes, during rapid elongation phase. Resin sections showed that Rht genes strongly reduced the cell extension in peduncle. The expression of expansin genes, which mediate cell wall loosening and leading to cell expansion, were analysed by using real-time quantitative PCR (qPCR). Among the 23 possible wheat expansin genes, 17 were expressed in the peduncle. The spatial distribution of expression was further analysed for five expansins that showed high expression levels in the peduncles of Rht lines. Compared to wild type plants, the incorporation of Rht-D1b allele decreased about 37 and 80% of the expression levels of ExpA7 and ExpA3 in elongation zone, respectively. The presence Rht-B1c dwarfing genes, however, produced 53% reduction in the expression level of ExpA7, and seriously decreased about 70% of ExpB9 expression. Although the expression levels of five genes exhibited variability among the lines, an expansin gene, ExpB2, showed its expression level highly associated with the cell elongation rate in peduncle of different Rht lines.