Cloning And Identification Of Genes Encoding Two Types Of Transcription Factor, GmNAC And GmLFY, And QTL Mapping For Seed Traits In Soybean (Glycine Max (L.) Merr.)

Seed development is one of the vital parts of plant biology and breeding project. Forsoybean it's an especially complexed process regulated and controlled by many diverse genes.During this process the regulation by transcription factors at the transcriptional level playsimportant roles. NAC-like and LFY-like proteins both are identified as two types of plant-specific transcription factors. Genes encoding NAC TFs are involved in many physiologicaland biochemical processes including plant growth and development, responsiveness to stress;while those encoding LFY-like TFs are implicated in the regulation of reproductivedevelopment, such as flowering time control and floral development et al.In this study, we focus on the cloning and identification of genes encoding NAC andLFY-like TFs involved in seed development, and mapping QTLs controlling seed characters insoybean.Firstly, six NAC-like genes from soybean, designated as GmNAC1 to GmNAC6, werecloned and characterized. Sequence analysis showed that six GmNAC proteins uniformlycontained a conserved NAC domain at N-terminus with high homology and a diverged C-terminus. Also, NAC domains in GmNAC protein were found to contain five differentsubdomains A-E. They shared conservative structures of genomic organization: each containedtwo introns and three exons and the first two exons encoded NAC domain. Moreover, insertionsites of introns were much conserved among six proteins. RT-PCR analysis indicated that eachGmNAC gene exhibited a specific expression pattern in tissues examined, revealing thedifferent roles for GmNAC genes in soybean development. GmNAC2, 3, 4, and 6 were detectedto express in most tested tissues while GmNAC1 and GmNAC5 were limited to few tissues. Infloral organs, except for GmNAC3, GmNAC4, and GmNAC6, which were expressedconstitutively, other three GmNAC genes showed unique expression patterns. Furthermore,expression patterns of GmNAC genes were characterized during seed filling and coordinatedexpression was observed between GmNAC genes. Most of them were expressed at a low levelat the early stages of seed development and reached a maximum level between 30-35DAF thendecreased afterward. After treatment of SA, NaCI and wounding, GmNAC genes were regulated differently. Subcellular localization of GmNAC5 in onion epidermic cells suggested GmNAC5was targeted to the nucleus. Based on phylogenetic analysis, six GmNAC proteins were classedinto five subgroups. GmNAC1, GmNAC2, GmNAC5, and GmNAC6 belonged to the NAP,ATAF, NAM, and TERN subgroup, respectively; while GmNAC3 and GmNAC4 wereclassified into the AtNAC3 subgroup.These results provide potent basis for futureinvestigations of NAC-like genes' roles in seed development and other physiological processesin soybean.Secondly, a LEAFY homologue in soybean, GmLFY, was cloned from cDNA preparedfrom young inflorescences by rapid amplification of cDNA ends (RACE) method. A GmLFY-specific CAPS marker was developed and then was mapped on molecular linkage group C2 in amapping population NJ(SP)BN. The marker was located between two SSR markers Sat₂13and Satt322 with the distance of 8.0 and 14.1cM, respectively. Comparative analysis of GmLFYgenomic and cDNA sequences showed that GmLFY gene had three exons and two introns withthe similar spliced sites as other LFY-like genes. RT-PCR analysis indicated that GmLFY waspredominantly expressed in reproductive organs such as inflorescences, pods without seeds, anddeveloping seeds. Moreover, GmLFY was mainly expressed in whorl 1 and whorl 4, the sepalsand pistils, of the floral organs. Expression pattern of GmLFY in developing seeds was alsoinvestigated. GmLFY was expressed at a middle level in seeds at 15 days after flowering (DAF)and decreased in 20 DAF seeds. Thereafter, it increased, reaching its maximum expression levelbetween 35 and 40 DAF and decreased gradually after that time. Result of expression in E.colishowed that GmLFY could be expressd and translated in prokaryote; Subcellular localization ofGmLFY in onion epidermic cells suggested GmLFY was targeted to the nucleus. Phylogeneticanalysis showed that GmLFY belonged to the family of legume together with LjLFY of Lotusjaponicus, MtUNI of Medicago truncatula and UNI of Pisum sativum.In soybean seed traits such as protein content, oil content, and seed size are the majortargets in breeding programs. Using a mapping population NJ(SP)BN derived from Bogao xNG94-156, an SSR-based molecular genetic map with 268 loci was developed. This linkagemap contained 24 molecular linkage groups and spaned 2,854.9cM of the soybean genome withan average interval distance of 10.65cM, the average markers per group of 11.17cM and theaverage length of the LGs of 119.0cM. Using CIM method, QTLs for each seed trait wereanalyzed. A total of 10 QTLs controlling protein content, oil content, and seed size in soybeanwere identified in the years of 2004 and 2005. Analysis and comparison of detected QTLsshowed that one QTL for protein content (qpcD1a₁) and one for oil content (qocC2₁) were stable across two years. Our results will be contributing for genetic improvement for seed traitswith marker-assisted selection in soybean.