Heng Ye

Document Type

Thesis - University Access Only

Award Date

2011

Degree Name

Doctor of Philosophy (PhD)

Department / School

Plant Science

Abstract

Seed dormancy is an adaptive trait for wild and weedy species and also a major domestication-related trait for cereal crops. Domestication has reduced seed dormancy due to selection for rapid, uniform germination, which also causes a problem of pre-harvest sprouting (PHS) in crop production. The goal of this dissertation research was to elucidate genetic, physiological and molecular mechanisms that directly regulate natural variation in seed dormancy in grass species. The previous work in Dr. Gu's Seed Molecular Biology Lab has developed weedy rice as a genetic system to achieve this goal, which includes a detection of seven quantitative trait loci (QTLs) associated with seed dormancy and introduction of QTL alleles from the weedy into the cultivated rice to characterize their genetic and molecular functions. The objectives of this project were: 1) to identify new seed dormancy quantitative trait loci (QTLs) that differentiated between weedy and cultivated of rice; 2) to isolate the QTLs into a near isogenic background to characterize their genetic effects and physiological functions; 3) to clone candidate genes underling the isolated QTLs. To identify new seed dormancy QTLs differentiated between the cultivated rice (EM93-1) and weedy rice (SS18-2), two BC1F2 populations derived from BC1F1 plants, in which the five previously known QTL-containing segments from the weedy rice donor were substituted by the genome of the recurrent parent, were developed to perform QTL mapping for seed dormancy. Three new QTLs named qSD1-2, qSD3, and qSD10 were detected on chromosomes 1, 3, and 10, respectively, which were further confirmed in three BC1 F3 populations. Counting previously identified seven QTLs, the parental lines differentiate a total of 10 dormancy loci that vary in dominance (dominant, partially dominant, co-dominant, and recessive types). The weedy and cultivated parents contribute dormancy alleles with eight and two of the 10 loci, respectively, suggesting that a large proportion (8/10) of seed dormancy genes have been eliminated during domestication and weedy rice could be a rich resource of genes for seed dormancy. During mapping for seed dormancy QTLs in BC1F2 and BC1 F3 populations, seed dormancy and plant height were found be associated with each other at the qSD1-2 and qSD7-2 regions on chromosome 1 and 7. These QTL clusters, which were named as qSD1-2lqPH1 and qSD7-2/qPH7, were selected to be isolated into the cultivated background for further characterization. After three generations of recurrent backcross from a BC1F 3 plant to EM93-1 with marker assisted selection, about 15cM genomic regions containing each individual QTL cluster were isolated into the EM93-1 background. The following QTL analysis on the segregating populations only segregating the two QTL clusters, showed that the QTL cluster (qSD1-2/qPH1) of weedy parent reduced seed dormancy but increased plant height; whereas, the QTL cluster (qSD7-2/qPH7) of cultivated parent enhanced seed dormancy but reduced plant height. Each of the clusters consisted of gene additive (a), dominance (cl), and epistatic (axa and/or axe/) effects on the both traits. These findings indicated that the naturally occurring genes reducing plant height or enhancing seed dormancy distribute in both weedy and cultivated rice and had a similar epistasis pattern between the two loci of the two traits. The subsequent physical mapping of the two loci by progeny tests on recombinants for the qSD1-2/qPH1 or qSD7-2/qPH7 region excluded OsVp1 or Sdr4 to be the candidate genes of the two QTL clusters and revealed the tight linkage (within ~700kbp) between seed dormancy and plant height. These results indicated that seed dormancy and plant height were co-selected during rice domestication. The exogenous gibberrelic acid (GA3) treatment on imbibed seeds and 2-day old seedlings of four near isogenic lines for qSD1-2/qPH1 and qSD7-2/qPH7 at EM93-1 background identified the inhibitory effects of qSD1-2/qPH1, but not qSD7-2/qPH7, on germination and plant height, can be rescued by application of exogenous GA 3, suggesting that the seed dormancy and plant height traits may share some physiological pathways, such as GA biosynthesis or signaling. To clone qSD1-2/qPH1, a population of 2112 plants segregating only for the QTL-containing region was genotyped with molecular DNA markers on the target region to select recombinants within the QTL containing region. The following progeny tests for seed dormancy and plant height on 11 selected recombinants for the qSD1-2/qPH1 region were performed, from which qSD1-2/qPH1 was narrowed into a ~20kbp genomic region containing two predicted genes (LOC_01g66100 and ~2kbp 3'-region of LOC_01g66110). The subsequent sequence analysis and comparison revealed that the ~2kbp 3'-region of LOC_01g66110 had no variation between the two parental lines. LOC_01g66100 was found to be the same locus of semi-dwarf 1 (Sd1) encoding a GA biosynthesis enzyme (GA20 -oxidase) and the allele of EM93-1 had a 382bp deletion across exon 1 to 2 producing a premature un-functional protein. It was suggested that LOC_01g66100 was most likely the candidate gene of qSD1-2/qPH1. The germination test on seeds from a line, which was only heterozygous at qSD1-2 genomic region, showed that the germination rates corresponded with the genotypes of the endosperm. This discovery, which was further confirmed by observing the expression of both qSD1-2 in endosperms during seed development, indicated that qSD1-2 was involved in the endosperm-imposed dormancy. The comparisons on the morphology, water content, and germination rate of the developing seeds between EM93-1 and the near isogenic line (IL 1-2SS) revealed that the allele qSD1-2 8 from SS18-2 accelerated the seed maturation at the early stage of seed development, which offered the evidence that the active biosynthesis of GA in the endosperm of cereal seeds would promote seed maturation and affect seed dormancy level during seed development. To clone qSD7-2/qPH7, a population of 2079 plants segregating only for the QTL-containing region was genotyped with molecular DNA markers on the target region to select recombinants within the QTL containing region. The following progeny tests for seed dormancy and plant height on 9 selected recombinants for the qSD7-2/qPH7 region were performed, from which qSD7-2/qPH7 was narrowed to a genomic region containing a single predicted gene. This candidate gene was predicted to encode a novel protein kinase with an S-receptor, PAN-domain, and kinase domain from the N-terminus to C-terminus. The functional mutation of the allele qSD7-zE from EM93-1 was due to a single nudeotide substitution (T to A) at position -9, which led to a missing DNA transcriptional regulatory TTAAA box and caused low expression level of qSDl-2 in EM93-1 background. The further GA and after-ripening treatments revealed that the expression of qSDl-2 was negatively regulated by GA and the expression level of qSDl-2 in the imbibed seeds was found to be correlated with the dormancy levels altered by different after ripening periods treatments. It was suggested that qSDl-2 played a role in the seed germination stage to inhibit the germination and maintain the dormancy of the seeds via reducing the sensitivity of the imbibed seeds to GA. A new mechanism of dormancy regulation by after-ripening was proposed from this research that after-ripening had a potential to increase the sensitivity to GA of the imbibed seeds by suppressing the expression of qSD7-2 to break dormancy during germination. By comparing the sequences among 36 lines, the allele qSD7-2 5 from SS18-2 was found to be in an ancient category farthest from the allele qSD7-~. whereas qSD7-~ and the functional mutation was found in a small group of Indian landraces, which suggested that qSD7-~ could be introduce into current indica and japonica semi-dwarf varieties for compensation of their negative GA deficient phenotypes. In summary, this project made significant progress in seed dormancy research including: 1) identified three new seed dormancy QTLs, which offered new genetic resources to assist plant breeding in manipulation of seed dormancy level in current cultivars; 2) discovered only two seed dormancy QTLs remained in the cultivated parent, which revealed the eliminative impact of domestication on seed dormancy genes; 3) isolated two QTL clusters controlling both seed dormancy and plant height and their genetic effects at the background of cultivated parent, which offered materials and properties of the two QTL clusters for future research; 4) discovered the tight linkage or pleiotropic effects on both seed dormancy and plant height, which indicated the co- evolutionary trend of the two traits; 5) fine-mapped and cloned the candidate genes for the two QTL clusters and GA treatment revealed the candidate genes for the two QTL clusters involved in different GA pathways (synthesis and signaling) in controlling both seed dormancy and plant height; 6) the further physiological and molecular characterization on near isogenic lines for the two QTL clusters reveal detailed mechanism of how the two loci controlling seed dormancy during seed development, after-ripening, and germination.

Library of Congress Subject Headings

Rice -- Seeds -- Dormancy

Rice -- Genetics

Format

application/pdf

Number of Pages

90

Publisher

South Dakota State University

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