Map-based Cloning Of Leaf Color Regulating Genes YL1 And YL2 And Gene Mapping Of Extreme Short-petiole In Soybean [Glycine Max L.)Merr.]

As the main organ of photosynthesis,leaf is the main source of carbohydrate.Leaf position is also an important factor determining plant type.Both leaf morphology and petiole length can affect plant architecture and light energy utilization efficiency of soybean.Therefore,it is of great theoretical and application value to explore and identify leaf related mutants,cloning genes related and study their working mechanism for soybean breeding.During the early stage,a batch of leaf morphology mutants were obtained via physical,chemical mutagenesis or natural mutation by National Soybean Improvement Center.Among them,yl（yellow leaf）is a yellow leaf soybean recombinant which derived from F2 progenies of green seed coat varieties and yellow seed coat varieties,defined as yl yellow leaf mutant.dsp（derived short petiole）is a recombinant with extreme short petiole that derived from progenies of wild and cultivated soybean,defined as dsp extreme short petiole mutant.In this study,genetic studies were conducted on yl（yellow leaf）and dsp（derived short petiole）mutants.For yl yellow leaf mutant,we carried out a series of phenotype identification experiments including leaf morphology,chloroplast development,photosynthetic physiological characteristics.Furthermore,crosses were made between yl mutant and wild type for genetic analyses,gene mapping,screening and cloning candidate genes.Then we verify the function of candidate genes by VIGS interference analysis in tobacco.What’s more,a transcriptome sequencing was conducted between near isogenic line.At the same time,genetic analysis and gene mapping were carried out on dsp extreme short petiole mutant.The main results are as follo ws:1.yl mutant characterized with unstable chloroplast structure and the increased photosynthetic activity in young leaves.Physiological characteristics analysis revealed that the photosynthetic pigment content of yl was significantly decreased,while,the Chl a/b was increased significantly.The chloroplast ultrastructure of young leaves of yl was almost the same as that in wild-type leaves.However,the grana thylakoid disappeared in the yellowing leaves,only linearized lamellar structure left.These results showed that the chloroplast of yl mutant is unstable and the chloroplast structure is easily destroyed with the development of leaves.What’s more,potential maximum photochemical efficiency Fv/Fm of PSII was significantly improved in yl,the yl showed similar photosynthetic characteristics under different genetic backgrounds,showing that the net photosynthetic rate（Pn）of top leaves reached the maximum,then followed by a gradual decline in adjacent leaves.Pn of top leaves and second leaves from the top in yl were higher than in wild type.Therefore,mutation in yl improves the potential light energy capture and utilization ability of yl,but with the development of leaves,the photosynthetic structure is destroyed,and the photosynthetic rate decreases.2.Simultaneously mutation of YL1 and YL2 results in a yellow leaf phenotype of soybean.yl mutant was derived from F2 populations of the crosses between normal greenish soybeans with yellow seed coat and normal greenish soybeans with green seed coat.Genetic analysis revealed that the yellow trait of yl mutant was controlled by two recessive nuclear genes,defined as yll and yl2.Map-based cloning of yl1 based on 3 F2 segregation populations revealed that there was a single base G deletion（G383-）in first exon of Glymallg04660,which lead to a premature stop codon and results in a truncated protein containing only 169 amino acids.The homologous gene of Glyma11g04660 on chromosome 01,Glyma01g40650,had a base substitution of guanine to adenine（G3000A）in intron region in yl resulted in alternative splicing of the coding protein and a premature stop codon,forming a truncated protein with C-terminal variant,the latter was the recently reported soybean green seed coat gene G.Therefore,we regarded Glyma11g04660 and Glyma01g40650 as candidates for yll and yl2 respectively.qRT-PCR analysis showed that YL1 and YL2 were expressed in various tissues,including leaves,stems,flowers,inflorescence and seed coat,especially in leaves.The tobacco YL homologous gene silencing（VIGS）plants had similar physiological characteristics with yl,showing that the leaf loss green,chlorophyll a,chlorophyll b and carotenoid content decreased significantly,and the Chl a/b increased.Sequencing analysis of different materials revealed three allelic variations of YL1,named as YL1,yl1373 and yl1383;two allelic variations of YL2,named as YL2 and yl2 respectively.In combination with the results of field breeding experiments,only crosses between soybean materials with the genotypes as coupling series were able to produce yl mutants,for example,plants with genotype as yl1373yl1373YL2YL2 or yl1383yl1383 YL2YL2 cross to YL1YL1 yl2yl2,which further proved that Glyma11g04660 and Glyma01g40650 were YL1 and YL1 genes.3.Combination of subcellular localization and transcriptome analysis briefly revealed a working mechanism of YL1 and YL2.YL1 and YL2 are two homologous proteins containing Abi（CPBP）domain,subcellular localization revealed that both YL1 and YL2 were located to the chloroplast.The Abi family protein belongs to Type Ⅱ CAAX protease,which binds to metal ions and has proteolytic enzyme activity.Protein cluster analysis showed that YL1 and YL2 were far away from each member of Type Ⅱ CAAX protease family.Homologous protein sequence alignment found that YL protein only existed in eukaryotes,and YL1 and YL2 proteins in different species were relatively conserved,both containing EExxxR and HxxxB motifs.Therefore,we speculated that YL1 and YL2 proteins are new members of Type Ⅱ CAAX protease family,and they contain Abi（CPBP）domain,but whether they have proteolytic enzyme activity needs to be further verified.The transcriptome analysis found that the expression level of genes related to chlorophyll synthesis pathways were raised in yl,but the expression of chlorophyll a oxygenase（CAO）gene which catalyze the convert chl a to chl b displayed no difference between yl and wild type,suggesting that mutants in yl promote the synthesis of chl a and change chlorophyll constitution.On the other hand,genes related to photosynthesis pathway,light harvesting protein complexes and carboxylation and reduction of ribulose 1,5-diphosphate（RUBP）were increased,indicating that the photoenergy capture and electron transfer in yl were enhanced.The down-regulated expression of RUBP regeneration related genes in yl indicated that the supply of RUBP was limited in the dark reaction process,suggesting that the energy utilization of light reaction and dark reaction process was unbalanced in the mutants,and excessive accumulation of reactive oxygen species subsequent probably induced programmed cell death in yl mutant.Therefore,YL1 and YL2 control the absorption of light energy and avoid the oxidative damage of photosynthetic structure by inhibiting chlorophyll synthesis and the expression of photosynthetic related genes during photosynthesis.4.Two novel short petiole genes dspl and dsp2 were located on chromosomes 7 and 11.The soybean short petiole mutant dsp has very short petiole,which is different from those short petiole lines reported before.Genetic analysis showed that dsp was controlled by 2 pairs of recessive nuclear genes,named as dspl and dsp2.Gene mapping showed that dsp1 was located to a 550.5-Kb interval near to centromere of chromosome 7,with BARCSOYSSR₀7₀787 and BARCSOYSSR₀7₀808 as flanking markers,containing 36 genes.dsp2 located to a 263.3-Kb interval of the front part of chromosome 11,between BARCSOYSSR—11₀037 and BARCSOYSSR₁1₀043,containing 33 genes.Further analysis found that both were in two non-homogenous segments.Further candidate gene mining and cloning have certain guiding significance for clarifying the development process of soybean petiole development.In summary,YL1 and YL2 encoded Type Ⅱ CAAX proteases which locate to chloroplast.During photosynthesis process,YL1 and YL2 modulate light energy absorption by inhibiting the expression of genes related to chlorophyll synthesis and photosynthesis.The exist of them avoid photodamage and maintain the stability of photosynthetic apparatus.They showed functional redundancy on the regulation of photosynthesis,but functional differentiation in the determination of seed coat color.The mechanism of YL2 results in chlorophyll retention on seed coat remains to be further studied.dsp is a new soybean short petiole system with an extremely short petiole and well-developed leaf and leaf pillow.We identified 2 novel loci which controlled soybean leaf petiole length based on dsp mutant,laying a foundation for map-based cloning of short petiole genes.