Bulked segregant analysis (BSA) is an efficient method to rapidly and efficiently map genes responsible for mutant phenotypes. BSA requires access to quantitative genetic markers that are polymorphic in the mapping population. We have developed a modification of BSA (BSR-Seq) that makes use of RNA-Seq reads to efficiently map genes even in populations for which no polymorphic markers have been previously identified. Because of the digital nature of next-generation sequencing (NGS) data, it is possible to conduct de novo SNP discovery and quantitatively genotype BSA samples by analyzing the same RNA-Seq data using an empirical Bayesian approach. In addition, analysis of the RNA-Seq data provides information on the effects of the mutant on global patterns of gene expression at no extra cost. In combination these results greatly simplify gene cloning experiments. To demonstrate the utility of this strategy BSR-Seq was used to clone the glossy3 (gl3) gene of maize. Mutants of the glossy loci exhibit altered accumulation of epicuticular waxes on juvenile leaves. By subjecting the reference allele of gl3 to BSR-Seq, we were able to map the gl3 locus to an ∼2 Mb interval. The single gene located in the ∼2 Mb mapping interval whose expression was down-regulated in the mutant pool was subsequently demonstrated to be the gl3 gene via the analysis of multiple independent transposon induced mutant alleles. The gl3 gene encodes a putative myb transcription factor, which directly or indirectly affects the expression of a number of genes involved in the biosynthesis of very-long-chain fatty acids.

/pdf/gene-mapping-via-bulked-segregant-rna-seq-bsr-seq-1q09qtjory.pdf

Gene mapping via bulked segregant RNA-Seq (BSR-Seq).

A dynamically rolled leaf mutant (rl10) was identified from a spontaneous mutation in an Oryza sativa L. subsp. indica line, II-32B. The leaf chlorophyll content of rl10 is higher than that of the ...

Genetic analysis and fine mapping of a dynamic rolled leaf gene, RL10(t), in rice (Oryza sativa L.).

Bacterial leaf steak (BLS) is one of the most destructive diseases in rice. Studies have shown that BLS resistance in rice is quantitatively inherited, controlled by multiple quantitative trait loci (QTLs). A QTL with relatively large effect, qBlsr5a, was previously mapped in a region of ∼380 kb on chromosome 5. To fine map qBlsr5a further, a set of overlapping sub-chromosome segment substitution lines (sub-CSSLs) were developed from a large secondary F2 population (containing more than 7000 plants), in which only the chromosomal region harboring qBlsr5a was segregated. By genotyping the sub-CSSLs with molecular markers covering the target region and phenotyping the sub-CSSLs with artificial inoculation, qBlsr5a was delimited to a 30.0-kb interval, in which only three genes were predicted. qRT-PCR analysis indicated that the three putative genes did not show significant response to the infection of BLS pathogen in both resistant and susceptible parental lines. However, two nucleotide substitutions were found in the coding sequence of gene LOC_Os05g01710, which encodes the gamma chain of transcription initiation factor IIA (TFIIAγ). The nucleotide substitutions resulted in a change of the 39th amino acid from valine (in the susceptible parent) to glutamic acid (in the resistant parent). Interestingly, the resistant parent allele of LOC_Os05g01710 is identical to xa5, a major gene resistant to bacterial leaf blight (another bacterial disease of rice). These results suggest that LOC_Os05g01710 is very possibly the candidate gene of qBlsr5a.

/pdf/toward-the-positional-cloning-of-qblsr5a-a-qtl-underlying-4tfei93tu1.pdf

Toward the Positional Cloning of qBlsr5a, a QTL Underlying Resistance to Bacterial Leaf Streak, Using Overlapping Sub-CSSLs in Rice

During vegetative development, higher plants continuously form new leaves in regular spatial and temporal patterns. Mutants with abnormal leaf developmental patterns not only provide a great insight into understanding the regulatory mechanism of plant architecture, but also enrich the ways to its modification by which crop yield could be improved. Here, we reported the characterization of the rice leafy-head2 (lhd2) mutant that exhibits shortened plastochron, dwarfism, reduced tiller number, and failure of phase transition from vegetative to reproductive growth. Anatomical and histological study revealed that the rapid emergence of leaves in lhd2 was resulted from the rapid initiation of leaf primordia whereas the reduced tiller number was a consequence of the suppression of the tiller bud outgrowth. The molecular and genetic analysis showed that LHD2 encodes a putative RNA binding protein with 67% similarity to maize TE1. Comparison of genome-scale expression profiles between wild-type and lhd2 plants suggested that LHD2 may regulate rice shoot development through KNOX and hormone-related genes. The similar phenotypes caused by LHD2 mutation and the conserved expression pattern of LHD2 indicated a conserved mechanism in controlling the temporal leaf initiation in grass.

/pdf/leafy-head2-which-encodes-a-putative-rna-binding-protein-2cpeusimk8.pdf

LEAFY HEAD2 , which encodes a putative RNA-binding protein, regulates shoot development of rice

In this article, we report an independent work of positional cloning and functional characterization of OsJAG gene in rice The merit of our work is that we used a genuine null mutant, in which the wild-type allele was completely deleted This allowed us to identify the mutant phenotypes accurately without the interference of residual function of the target gene OsJAG is an important gene with pleiotropy, expressing almost throughout the plant and acting in both vegetative phase and reproductive phase But its main and crucial roles are in regulating the development of all floral organs, especially in specifying the identity of stamens Interestingly, OsJAG does not affect the number of floral organ primordial and so of floral organs in each whorl, suggesting that OsJAG does not influence the initiation of floral organ primordia, but affect the developmental fate of all floral organs after their primordia have initiated Loss of OsJAG function results in maldevelopment of all floral organs, such as degenerated lemma and palea, elongated lodicules and deformed and sterile pistil The stamen appears to be more sensitive to the mutation All the six stamens in a mutant floret were thoroughly transformed into six pistil-like organs developed at the presumptive positions of the stamens in whorl 3

https://link.springer.com/content/pdf/10.1007%2Fs11103-010-9703-7.pdf

Molecular cloning and functional characterization of OsJAG gene based on a complete-deletion mutant in rice (Oryza sativa L.).

A mutant of spikelet differentiation in rice called frizzle panicle (fzp) was discovered in the progeny of a cross between Oryza sativa ssp. indica cv. V20B and cv. Hua1B. The mutant exhibits normal plant morphology but has apparently fewer tillers. The most striking change in fzp is that its spikelet differentiation is completely blocked, with unlimited subsequent rachis branches generated from the positions where spikelets normally develop in wild-type plants. Genetic analysis suggests that fzp is controlled by a single recessive gene, which is temporarily named fzp(t). Based on its mutant phenotype, fzp(t) represents a key gene controlling spikelet differentiation. Some F2 mutant plants derived from various genetic background appeared as the “middle type”, suggesting that the action of fzp(t) is influenced by the presence of redundant, modifier or interactive genes. By using simple sequence repeat (SSR) markers and bulked segregant analysis (BSA) method, fzp(t) gene was mapped in the terminal region of the long arm of chromosome 7, with RM172 and RM248 on one side, 3.2 cM and 6.4 cM from fzp(t), and RM18 and RM234 on the other side, 23.1 cM and 26.3 cM from fzp(t), respectively. These results will facilitate the positional cloning and function studies of the gene.

Genetic analysis and mapping of gene fzp(t) controlling spikelet differentiation in rice.

A rice mutant calledleafy head (lhd), in which the differentiation of rachis branches is blocked, was identified in a doubled haploid (DH) population derived through F1 anther culture from a cross between rice (Oryza sativa L.) indica cultivar Gui-630 and japonica cultivar Taiwanjing. The mutant is shorter in plant height, possessing smaller and clumpy leaves, and always stays at the vegetative growth stage. Genetic analysis suggests thatlhd is controlled by a single recessive gene, which is temporarily namedlhd(t). The phenotype of the mutant suggests thatLHD(t) is a key gene controlling the differentiation of rachis branches. In order to map the gene, two F2 populations were constructed by crossing thelhd heterozygote with varieties Minghui-77 (indica) and Jinghua-8 (japonica). In the F2 oflhd heterozygote × Jinghua-8, some mutant plants appeared as the “medium type”, suggesting that the lhd phenotype could be influenced by genetic backgrounds. With the published SSR markers of RM series and additional SSR markers developed by ourselves and using the methods of bulked segregant analysis (BSA) and mutant analysis (with 498 mutant plants in total),LHD(t) gene was mapped onto the distal region of the long arm of chromosome 10. Markers SSR1, RM269, RM258, RM304 and RM171 were located on one side with distances of 6.4, 16.6, 18.4, 22.2 and 26.3 cM toLHD(t); whereas markers SSR4 and SSR5 were on the other side with distances of 0.6 and 2.2 cM toLHD(t). The results will facilitate the positional cloning and functional study of theLHD(t) gene.

https://link.springer.com/content/pdf/10.1007/BF03182852.pdf

Genetic analysis and gene mapping ofleafy head (lhd), a mutant blocking the differentiation of rachis branches in rice (Oryza sativa L.)

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Genetic analysis and mapping of gene fzp(t) controlling spikelet differentiation in rice.

Genetic analysis and gene mapping ofleafy head (lhd), a mutant blocking the differentiation of rachis branches in rice (Oryza sativa L.)