Yann-Rong Lin

Convergent domestication of cereal crops by independent mutations at corresponding genetic Loci.

Independent domestication of sorghum, rice, and maize involved convergent selection for large seeds, reduced disarticulation of the mature inflorescence, and daylength-insensitive flowering. These similar phenotypes are largely determined by a small number of quantitative trait loci (QTLs) that correspond closely in the three taxa. The correspondence of these QTLs transcends 65 million years of reproductive isolation. This finding supports models of quantitative inheritance that invoke relatively few genes, obviates difficulties in map-based cloning of QTLs, and impels the comparative mapping of complex pheno-types across large evolutionary distances, such as those that separate humans from rodents and domesticated mammals.

Comparative genomics of plant chromosomes

Comparative genomics, the study of similarities and differences in structure and function of the hereditary information in different taxa, uses molecular tools to investigate notions that far preceded the discovery that DNA was the hereditary molecule. Vavilov’s (1922) “law of homologous series in variation” was an early suggestion of the possibility of underlying commonality in the genetic blueprints of different (plant) species. In plants, genetic analysis based upon morphological and isozyme markers provided early hints that the arrangements of genes along the chromosomes of various taxa may have retained parallels since their divergence from common ancestors. DNA-level investigations in diverse taxa point to two broad messages: (1) The small but essential portion of most plant genomes encoding genes evolves relatively slowly, with corresponding genes retaining recognizable DNA sequences and similar order along the chromosomes of taxa that have been reproductively-isolated for millions of years. (2) A wide range of factors, such as DNA sequence mobility, gene deletion, and localized rearrangements, are superimposed on the relatively slow tempo of chromosomal evolution and cause many deviations from co-linearity. (3) Genetic loci that account for common phenotypes in different taxa are often at corresponding genomic locations, and may represent orthologous genes or members of orthologous clusters of genes.

A detailed RFLP map of Sorghum bicolor x S. propinquum, suitable for high-density mapping, suggests ancestral duplication of Sorghum chromosomes or chromosomal segments

The first “complete” genetic linkage map of Sorghum section Sorghum is described, comprised of ten linkage groups putatively corresponding to the ten gametic chromosomes of S. bicolor and S. propinquum. The map includes 276 RFLP loci, predominately detected by PstI-digested S. bicolor genomic probes, segregating in 56 F2 progeny of a cross between S. bicolor and S. propinquum. Although prior cytological evidence suggests that the genomes of these species are largely homosequential, a high level of molecular divergence is evidenced by the abundant RFLP and RAPD polymorphisms, the marked deviations from Mendelian segregation in many regions of the genome, and several species-specific DNA probes. The remarkable level of DNA polymorphism between these species will facilitate development of a high-density genetic map. Further, the high level of DNA polymorphism permitted mapping of multiple loci for 21 (8.2%) DNA probes. Linkage relationships among eight (38%) of these probes suggest ancestral duplication of three genomic regions. Mapping of 13 maize genomic clones in this cross was consistent with prior results. Mapping of heterologous cDNAs from rice and oat suggests that it may be feasible to extend comparative mapping to these distantly-related species, and to ultimately generate a detailed description of chromosome rearrangements among cultivated Gramineae. Limited investigation of a small number of RFLPs showed several alleles common to S. bicolor and S. Halepense (“johnson-grass”), but few alleles common to S. propinquum and S. halepense, raising questions about the origin of S. halepense.

The pineapple genome and the evolution of CAM photosynthesis

Pineapple (Ananas comosus (L.) Merr.) is the most economically valuable crop possessing crassulacean acid metabolism (CAM), a photosynthetic carbon assimilation pathway with high water-use efficiency, and the second most important tropical fruit. We sequenced the genomes of pineapple varieties F153 and MD2 and a wild pineapple relative, Ananas bracteatus accession CB5. The pineapple genome has one fewer ancient whole-genome duplication event than sequenced grass genomes and a conserved karyotype with seven chromosomes from before the ρ duplication event. The pineapple lineage has transitioned from C3 photosynthesis to CAM, with CAM-related genes exhibiting a diel expression pattern in photosynthetic tissues. CAM pathway genes were enriched with cis-regulatory elements associated with the regulation of circadian clock genes, providing the first cis-regulatory link between CAM and circadian clock regulation. Pineapple CAM photosynthesis evolved by the reconfiguration of pathways in C3 plants, through the regulatory neofunctionalization of preexisting genes and not through the acquisition of neofunctionalized genes via whole-genome or tandem gene duplication.

Comparative analyses of linkage maps and segregation distortion of two F2 populations derived from japonica crossed with indica rice

To facilitate genetic research, we constructed two linkage maps by employing two F₂ populations derived from rice inter-subspecific crosses, japonica Tainung 67 (TNG67)/indica Taichung Sen 10 (TCS10) and japonica TNG67/indica Taichung Sen 17 (TCS17). We established linkage map lengths of 1481.6 cM and 1267.4 cM with average intervals of 13.8 cM and 14.4 cM by using 107 and 88 PCR markers for coverage of 88% of the rice genome in TNG67/TCS10 and TNG67/TCS17, respectively. The discrepancy in genetic maps in the two populations could be due to different cross combinations, crossing-over events, progeny numbers and/or markers. The most plausible explanation was segregation distortion; 18 markers (16.8%) distributed at nine regions of seven chromosomes and 10 markers (11.4%) at four regions of four chromosomes displayed severe segregation distortion (p < 0.01)in TNG67/TCS10 and TNG67/TCS17, respectively. All segregation-distorted markers in these two populations corresponded to reported reproductive barriers, either gametophytic or zygotic genes but not to hybrid breakdown genes. The observed recombination frequency, which was higher or lower than the intrinsic frequency, revealed the association of segregation distortion skewed to the same or different genotypes at the consecutive markers. The segregation distortion, possibly caused by reproductive barriers, affects the evaluation recombination frequencies and consequently the linkage analysis of QTLs and positional cloning.

The Evolution of Photoperiod-Insensitive Flowering in Sorghum, A Genomic Model for Panicoid Grasses

Of central importance in adapting plants of tropical origin to temperate cultivation has been selection of daylength-neutral genotypes that flower early in the temperate summer and take full advantage of its long days. A cross between tropical and temperate sorghums [Sorghum propinquum (Kunth) Hitchc.×S. bicolor (L.) Moench], revealed a quantitative trait locus (QTL), FlrAvgD1, accounting for 85.7% of variation in flowering time under long days. Fine-scale genetic mapping placed FlrAvgD1 on chromosome 6 within the physically largest centiMorgan in the genome. Forward genetic data from “converted” sorghums validated the QTL. Association genetic evidence from a diversity panel delineated the QTL to a 10-kb interval containing only one annotated gene, Sb06g012260, that was shown by reverse genetics to complement a recessive allele. Sb06g012260 (SbFT12) contains a phosphatidylethanolamine-binding (PEBP) protein domain characteristic of members of the “FT” family of flowering genes acting as a floral suppressor. Sb06g012260 appears to have evolved ∼40 Ma in a panicoid ancestor after divergence from oryzoid and pooid lineages. A species-specific Sb06g012260 mutation may have contributed to spread to temperate regions by S. halepense (“Johnsongrass”), one of the world’s most widespread invasives. Alternative alleles for another family member, Sb02g029725 (SbFT6), mapping near another flowering QTL, also showed highly significant association with photoperiod response index (P = 1.53×10 − 6). The evolution of Sb06g012260 adds to evidence that single gene duplicates play large roles in important environmental adaptations. Increased knowledge of Sb06g012260 opens new doors to improvement of sorghum and other grain and cellulosic biomass crops.

Three novel alleles of FLOURY ENDOSPERM2 (FLO2) confer dull grains with low amylose content in rice.

Rice is a major food source for much of the world, and expanding our knowledge of genes conferring specific rice grain attributes will benefit both farmer and consumer. Here we present novel dull grain mutants with a low amylose content (AC) derived from mutagenesis of Oryza sativa, ssp. japonica cv. Taikeng 8 (TK8). Positional cloning of the gene conferring the dull grain phenotype revealed a point mutation located at the acceptor splice site of intron 11 of FLOURY ENDOSPERM2 (FLO2), encoding a tetratricopeptide repeat domain (TPR)-containing protein. Three novel flo2 alleles were identified herein, which surprisingly conferred dull rather than floury grains. The allelic diversity of flo2 perturbed the expression of starch synthesis-related genes including OsAGPL2, OsAGPS2b, OsGBSSI, OsBEI, OsBEIIb, OsISA1, and OsPUL. The effect of the flo2 mutations on the physicochemical properties of the grain included a low breakdown, setback, and consistency of rice, indicating a good elasticity and soft texture of cooked rice grains. The effects of FLO2, combined with the genetic background of the germplasm and environmental effects, resulted in a variety of different amylose content levels, grain appearance, and physicochemical properties of rice, providing a host of useful information to future grain-quality research and breeding.

Lack of Genotype and Phenotype Correlation in a Rice T-DNA Tagged Line Is Likely Caused by Introgression in the Seed Source.

Rice (Oryza sativa) is one of the most important crops in the world. Several rice insertional mutant libraries are publicly available for systematic analysis of gene functions. However, the tagging efficiency of these mutant resources–the relationship between genotype and phenotype–is very low. We used whole-genome sequencing to analyze a T-DNA–tagged transformant from the Taiwan Rice Insertional Mutants (TRIM) resource. The phenomics records for M0028590, one of the TRIM lines, revealed three phenotypes–wild type, large grains, and tillering dwarf–in the 12 T1 plants. Using the sequencing data for 7 plants from three generations of this specific line, we demonstrate that introgression from an indica rice variety might occur in one generation before the seed was used for callus generation and transformation of this line. In addition, the large-grain trait came from the GS3 gene of the introgressed region and the tillering dwarf phenotype came from a single nucleotide change in the D17 gene that occurred during the callus induction to regeneration of the transformant. As well, another regenerant showed completely heterozygous single-nucleotide polymorphisms across the whole genome. In addition to the known sequence changes such as T-DNA integration, single nucleotide polymorphism, insertion, deletion, chromosome rearrangement and doubling, spontaneous outcrossing occurred in the rice field may also explain some mutated traits in a tagged mutant population. Thus, the co-segregation of an integration event and the phenotype should be checked when using these mutant populations.

The Green Fence of Chinese Hibiscus (Hibiscus rosa-sinensis L.) Prevents Pollen Dispersal of Transgenic Rice (Oryza sativa)

Abstract Transgene escape mediated by pollen dispersal is one of the preeminent concerns about genetically modified crops, including rice. In this study, the rice pollen donor of non-glutinous Oryza sativa ssp. japonica cv. ‘Tainung 67’ [TNG 67] had a greater potential of pollen flow, which was shown by the greater quantity, germination rate, and viability of pollen, than the other rice pollen donor of transgenic AAN. The pollen-mediated gene flow was detected by the frequency of outcrossed seeds in a “checker-board pattern” and alternating row arrangement of rice pollen donor (TNG 67 or AAN) and pollen recipient (glutinous rice ‘TNG 73’) in the fields. We conducted field experiments to assess rice pollen dispersal with or without a “green” fence of Chinese hibiscus, Hibiscus rosa-sinensis L., of approximately 2 m in height and 0.6 m in thickness. Without a green fence, the outcrossing rate of TNG 73 seeds decreased with increasing distance from the pollen donor, from 1.68% at 1 m to 0.01% at 35 m, with no outcrossing beyond 40 m. The outcrossing rate varied with the direction of pollen donor, from 0.05% in the northeastern direction to 0.78% in the southern direction, which was caused by prevailing wind direction. With the green fence, no seeds of TNG 73 were outcrossed. Since a buffer zone of at least 40 m is needed to prevent outcrossing of rice by pollen dispersal in an open field, planting of Chinese hibiscus around the rice field as a green fence would be an effective measure for preventing transgene escape mediated by pollen flow.

Subtropical adaptation of a temperate plant (Brassica oleracea var. italica) utilizes non-vernalization-responsive QTLs

While many tropical plants have been adapted to temperate cultivation, few temperate plants have been adapted to the tropics. Originating in Western Europe, Brassica oleracea vernalization requires a period of low temperature and BoFLC2 regulates the transition to floral development. In B. oleracea germplasm selected in Taiwan, a non-vernalization pathway involving BoFLC3 rather than BoFLC2 regulates curd induction. In 112 subtropical breeding lines, specific haplotype combinations of BoFLC3 and PAN (involved in floral organ identity and a positional candidate for additional curd induction variation) adapt B. oleracea to high ambient temperature and short daylength. Duplicated genes permitted evolution of alternative pathways for control of flowering in temperate and tropical environments, a principle that might be utilized via natural or engineered approaches in other plants. New insight into regulation of Brassica flowering exemplifies translational agriculture, tapping knowledge of botanical models to improve food security under projected climate change scenarios.