Introgression of bacterial leaf blight (BLB) resistant gene, Xa7 into MARDI elite variety, MR219 by marker assisted backcrossing (MABC) approach

Bacterial leaf blight (BLB) is one of the major rice diseases in Malaysia. This disease causes substantial yield loss as high as 70%. Development of rice varieties which inherited BLB resistant traits is a crucial approach to promote and sustain rice industry in Malaysia. Hence, this study aims were to enhance BLB disease resistant characters of high yielding commercial variety MR219 through backcross breeding approach with supporting tool of marker-assisted selection (MAS). Broad spectrum BLB resistance gene, Xa7 from donor parent IRBB7 were introgressed into the susceptible MR219 (recurrent parent) using two flanking markers ID7 and ID15. At BC3F4, we managed to generate 19 introgressed lines with homozygous Xa7 gene and showed resistant characteristics as donor parent when it was challenged with Xanthomonas oryzae pv. oryzae through artificial inoculation. Recurrent parent MR219 and control variety, MR263 were found to be severely infected by the disease. The improved lines exhibited similar morphological and yield performance characters as to the elite variety, MR219. Two lines, PB-2-107 and PB-2-34 were chosen to be potential lines because of their outstanding performances compared to parent, MR219. This study demonstrates a success story of MAS application in development of improved disease resistance lines of rice against BLB disease.

Whole-Genome Resequencing of Near-Isogenic Lines Reveals a Genomic Region Associated with High Trans-Lycopene Contents in Watermelon

Trans-lycopene is a functional phytochemical abundant in red-fleshed watermelons, and its contents vary among cultivars. In this study, the genetic basis of high trans-lycopene contents in scarlet red flesh was evaluated. Three near-isogenic lines (NILs) with high trans-lycopene contents were derived from the scarlet red-fleshed donor parent DRD and three coral red-fleshed (low trans-lycopene contents) recurrent parents. The lycopene contents of DRD (589.4 ± 71.8 µg/g) were two times higher than that of the recurrent parents, and values for NILs were intermediate between those of the parents. Coral red-fleshed lines and F1 cultivars showed low trans-lycopene contents (135.7 ± 18.0 µg/g to 213.7 ± 39.5 µg/g). Whole-genome resequencing of two NILs and their parents and an analysis of genome-wide single-nucleotide polymorphisms revealed three common introgressed regions (CIRs) on chromosomes 6, 9, and 10. Twenty-eight gene-based cleaved amplified polymorphic sequence (CAPS) markers were developed from the CIRs. The CAPS markers derived from CIR6 on chromosome 6, spanning approximately 1 Mb, were associated (R2 = 0.45–0.72) with the trans-lycopene contents, particularly CIR6-M1 and CIR6-M4. Our results imply that CIR6 is a major genomic region associated with variation in the trans-lycopene contents in red-fleshed watermelon, and CIR6-M1 and CIR6-M4 may be useful for marker-assisted selection.

Identification of Finely Mapped Quantitative Trait Locus and Candidate Gene Mining for the Three-Seeded Pod Trait in Soybean

The three-seeded pod number is an important trait that positively influences soybean yield. Soybean variety with increased three-seeded pod number contributes to the seed number/plant and higher yield. The candidate genes of the three-seeded pod may be the key for improving soybean yield. In this study, identification and validation of candidate genes for three-seeded pod has been carried out. First, a total of 36 quantitative trait locus (QTL) were detected from the investigation of recombinant inbred lines including 147 individuals derived from a cross between Charleston and Dongning 594 cultivars. Five consensus QTLs were integrated. Second, an introgressed line CSSL-182 carrying the target segment for the trait from the donor parent was selected to verify the consensus QTL based on its phenotype. Third, a secondary group was constructed by backcrossing with CSSL-182, and two QTLs were confirmed. There were a total of 162 genes in the two QTLs. The mining of candidate genes resulted in the annotation of eight genes with functions related to pod and seed sets. Finally, haplotype analysis and quantitative reverse transcriptase real-time PCR were carried to verify the candidate genes. Four of these genes had different haplotypes in the resource group, and the differences in the phenotype were highly significant. Moreover, the differences in the expression of the four genes during pod and seed development were also significant. These four genes were probably related to the development process underlying the three-seeded pod in soybean. Herein, we discuss the past and present studies related to the three-seeded pod trait in soybean.

DNA methylome and lncRNAome Analysis Provide Insights Into Mechanism of Genome-dosage Effects in Autotetraploid Cassava

BackgroundDuring newly formed polyploidy, one of the most intriguing aspects is that whole-genome duplication (WGD) increase the dosage of all coding and non-coding genes. However, the molecular implications of genome-dosage effects remain elusive.ResultsWe conducted integrated maps of methylomes and lncRNAomes in autotetraploid cassava (Manihot esculenta Crantz) and its donor parent, both of which were independently clonal propagated for three years. DNA methylation variation of transposable elements (TEs) was observed as widespread in autotetraploid cassava. The hypermethylation of DNA transposons in mCG and mCHH sites may be an effective way to suppress the expression of nearby PCGs in autotetraploid cassava, resulting in similar expression levels for most of PCGs between autotetraploid and diploid cassava. The decreased methylation levels of retrotransposons in mCHG and mCHH sites, which partly attributed to reduction methylation of Cypsy neighboring long intergenic noncoding RNAs in autotetraploid cassava, may be a mechanism that may suppress the expression levels of nearby lncRNA, leading to no significant differences in transcriptome alterations for major of lncRNAs from its diploid parent.ConclusionsThis work highlighted that WGD-induced DNA methylation variation in DNA transposons and retrotransposons may be as direct adaptive responses to dosage of all coding-genes and lncRNAs, respectively.

Development of Submergence-Tolerant, Bacterial Blight-Resistant, and High-Yielding Near Isogenic Lines of Popular Variety, ‘Swarna’ Through Marker-Assisted Breeding Approach

The rice variety ‘Swarna’ is highly popular in the eastern region of India. The farmers of eastern India cultivate mainly rainfed rice and face the adverse effects of climate change very frequently. Rice production in this region is not stable. Swarna variety is highly susceptible to bacterial blight (BB) disease and flash floods, which cause a heavy reduction in the yield. Transfer of five target genes/QTLs was targeted into the variety, Swarna by adopting marker-assisted backcross breeding approach. Direct markers for Sub1A, OsSPL14, and SCM2 QTLs and closely linked markers for xa5 and Xa21 BB resistance genes were screened in the backcross progenies. Swarna-Sub1, CR Dhan 800, and Swarna–Habataki near-isogenic lines (NILs) were used as donor parents in the breeding program. True multiple F1 plants were identified for backcrossing, and 796 BC1F1 seeds were generated. Foreground selection detected all the five target genes in six progenies in BC1F1 generation. The progeny containing all the target genes and more similar with the recipient parent was backcrossed, and a total of 446 BC2F1 seeds were produced. Foreground screening detected four BC2F1 plants carrying the five target genes. A total of 2,145 BC2F2 seeds were obtained from the best BC2F1 progeny. Screening of the progenies resulted in one plant with all five desirable genes, three plants with four, and another three progenies carrying three genes in homozygous conditions. The pyramided lines showed higher BB resistance and submergence tolerance than the recipient parent, Swarna. Culm strength of the pyramided lines showed higher breaking force than the recipient parent, Swarna. The pyramided line, SSBY-16-68-69 yielded the highest grain yield of 7.52 t/ha followed by the lines SSBY-16-68-511 (7.34 t/ha) and SSBY-16-68-1633 (7.02 t/ha). The best-pyramided line showed a yield advantage of 18% over the recipient parent and 6.8% over the yield component donor parent. Seven pyramided lines showed higher yield than the recipient parent, while five lines were better yielders than the yield component donor parent. The pyramided line SSBY-16-68-69 produced 365 grains/panicle, while the recipient had 152. The main morphologic and grain quality features of the recipient parent were retained in the pyramided lines.

GW10, a Member of P450 Subfamily Regulates Grain Size and Grain Number in Rice

Grain size and grain number play extremely important roles in rice grain yield. Here, we identify GW10 , which encodes a P450 subfamily protein and controlls grain size and grain number by using Lemont ( tropical japonica ) as donor parent and HJX74 ( indica ) as recipient parent. The GW10 locus was mapped into a 20.1 kb region on the long arm of Chromosome 10. Lower expression of the gw10 in panicle is contributed to the shorter and narrower rice grain, and the increased number of grains per panicle. Furthermore, the higher expression levels of some of the brassinosteroid (BR) biosynthesis and response genes are associated with the NIL- GW10 , which strongly suggests that the GW10 is a key node in the brassinosteroid-mediated regulation of rice grain shape and grain number.

Enhancing backcross programs through increased recombination

Background Introgression of a quantitative trait locus (QTL) by successive backcrosses is used to improve elite lines (recurrent parent) by introducing alleles from exotic material (donor parent). In the absence of selection, the proportion of the donor genome decreases by half at each generation. However, since selection is for the donor allele at the QTL, elimination of the donor genome around that QTL will be much slower than in the rest of the genome (i.e. linkage drag). Using markers to monitor the genome around the QTL and in the genetic background can accelerate the return to the recurrent parent genome. Successful introgression of a locus depends partly on the occurrence of crossovers at favorable positions. However, the number of crossovers per generation is limited and their distribution along the genome is heterogeneous. Recently, techniques have been developed to modify these two recombination parameters. Results In this paper, we assess, by simulations in the context of Brassicaceae, the effect of increased recombination on the efficiency of introgression programs by studying the decrease in linkage drag and the recovery of the recurrent genome. The simulated selection schemes begin by two generations of foreground selection and continue with one or more generations of background selection. Our results show that, when the QTL is in a region that initially lacked crossovers, an increase in recombination rate can decrease linkage drag by nearly ten-fold after the foreground selection and improves the return to the recurrent parent. However, if the QTL is in a region that is already rich in crossovers, an increase in recombination rate is detrimental. Conclusions Depending on the recombination rate in the region targeted for introgression, increasing it can be beneficial or detrimental. Thus, the simulations analysed in this paper help us understand how an increase in recombination rate can be beneficial. They also highlight the best methods that can be used to increase recombination rate, depending on the situation.

Evaluation of Xa7 Introgressed Lines Against Bacterial Leaf Blight (BLB) Disease and Their Agronomic Performances

Bacterial leaf blight (BLB) is one of the major rice diseases in Malaysia. The disease can potentially cause a 30 to 50% yield loss to the rice crop. Hence, the utilization of resistant rice varieties to BLB is a crucial approach to prevent potential yield loss and to ensure the sustainability of the rice industry in Malaysia. This study aims to incorporate BLB resistant trait into a susceptible, high yielding, popular commercial variety MR219. The identified donor parent is IRBB7, harboring Xa7, a broad-spectrum BLB resistant gene which closely linked to two STS markers, the ID7 and ID15. Backcrossing was initiated, using IRBB7 as the donor and MR219 as the recurrent parents. Marker-assisted selection (MAS) was applied to monitor and confirm the introgression of Xa7 in the progenies of the cross based on alleles produced by the markers. We managed to generate 19 BC3F4 lines harboring homozygous Xa7 gene that were phenotypically resistant when challenged with Xanthomonas oryzae pv. oryzae through an artificial inoculation. The improved lines exhibited similar morphological and yield performance of the recurrent parent. Two lines, PB-2-107 and PB-2-34 were identified for their outstanding performances which are comparable to MR219. This study demonstrates the advantage of MAS application in the improvement of disease resistance traits in the elite rice cultivars.

Mechanism of anoxic tolerance in backcross lines developed through Jyothi x Swarna-Sub 1 under submergence stress.

Rice varieties adapted to flash flood exhibit submergence tolerance by maintaining reduced shoot elongation under submerged conditions. This study focuses on the two main physiological traits leading to flash flood tolerance- high survival percentage with reduced shoot elongation under submergence and increased rate of alcoholic fermentation. Sub1introgressed BC3F2 lines exhibited higher survival percentage with moderate shoot elongation under submergence stress for fourteen days similar to the donor parent Swarna- Sub1. Enzymatic activities of alcohol dehydrogenase and pyruvate decarboxylase were monitored for fourteen days under submergence stress. The developed Sub1 introgressed lines and the donor parent Swarna-Sub1 showed higher rates of alcohol dehydrogenase and pyruvate decarboxylase activities under submergence stress thereby maintaining optimum rates of alcoholic fermentation. The findings of this study confirmed the expression of tolerance mechanism in the Sub1introgressedJyothi Backcross Inbred lines under submergence stress.

QTL Analysis and Fine Mapping of a Major QTL Conferring Kernel Size in Maize (Zea mays)

Kernel size is an important agronomic trait for grain yield in maize. The purpose of this study is to map QTLs and predict candidate genes for kernel size in maize. A total of 199 F2 and its F2:3 lines from the cross between SG5/SG7 were developed. A composite interval mapping (CIM) method was used to detect QTLs in three environments of F2 and F2:3 populations. The result showed that a total of 10 QTLs for kernel size were detected, among which were five QTLs for kernel length (KL) and five QTLs for kernel width (KW). Two stable QTLs, qKW-1, and qKL-2, were mapped in all three environments. Three QTLs, qKL-1, qKW-1, and qKW-2, were overlapped with the QTLs identified from previous studies. In order to validate and fine map qKL-2, near-isogenic lines (NILs) were developed by continuous backcrossing between SG5 as the donor parent and SG7 as the recurrent parent. Marker-assisted selection was conducted from BC2F1 generation with molecular markers near qKL-2. A secondary linkage map with six markers around the qKL-2 region was developed and used for fine mapping of qKL-2. Finally, qKL-2 was confirmed in a 1.95 Mb physical interval with selected overlapping recombinant chromosomes on maize chromosome 9 by blasting with the Zea_Mays_B73 v4 genome. Transcriptome analysis showed that a total of 11 out of 40 protein-coding genes differently expressed between the two parents were detected in the identified qKL-2 interval. GRMZM2G006080 encoding a receptor-like protein kinase FERONIA, was predicted as a candidate gene to control kernel size. The work will not only help to understand the genetic mechanisms of kernel size of maize but also lay a foundation for further fine mapping and even cloning of the promising loci.