Fine mapping of wheat Tatyd with pleiotropic roles in plant architecture, leaf yellowish and improved powdery mildew resistance

Background: Photosynthesis is the primary energy for biosphere, and is the foundation for life on earth. Leaf color mutations are widely used to study chloroplast structure and development, biosynthesis and metabolism of chlorophyll and plant photosynthesis, such as in rice and maize, with many genes be mapped and cloned. By comparison, there is few researches on the leaf color mutants in wheat. More and more studies have demonstrated that photosynthesis is involved in plant-pathogen interaction, whether leaf color mutations affect plant-pathogen interactions are rarely reported. Results: In this study, a leaf color mutant, NAU31, which has fewer tillers, dwarfed plant and improved powdery mildew resistance was identified from regenerated plant of common wheat cv. Yangmai158. Inheritance analysis of NAU31 showed that the above mutant traits were significantly correlated and controlled by a dominant gene, named Tatyd. Linkage analysis of a mapping population between NAU31 and a synthesized wheat W7984 using SSR markers, the Tatyd was preliminary mapped to 2DS. By BSA and linkage analysis using a 55K wheat SNP array, the Tatyd was fine mapped to the terminal region of 2DS, flanked by markers ZH47 and gwm296 and corresponding to a physical interval of 0.76 Mb. Genome-wide gene expression analysis indicated that the impairing of photosynthesis may be responsible for plant yellowing in NAU31. Photosynthetic physiological analysis showed varied chloroplast inner structure in the mesophyll cells of the mutant, and this may lead to reduced chlorophyll content (chlorophyll “a”, Chlorophyll “b”, total chlorophyll and carotenoid), as well as decreased maximum quantum efficiency of photosystem II (Fv/Fm) and Net photosynthetic rate. Conclusions: A mutant with few-tiller, plant yellowish, dwarfness and improved powdery mildew resistance was identified, and the mutated locus Tatyd was fine mapped to a 0.76 Mb genome region of wheat chromosome 2D. The yellowing trait of NAU31 may be attributed to its reduced photosynthesis ability due to changes of the chloroplast using the young embryo as explant, we identified a stable mutant with a phenotype of yellowing leaf color, fewer tiller and dwarfness. The mutant line was given a genetic stock number of NAU31, and the mutant gene was named Tatyd. Interestingly, the mutant showed improved powdery mildew resistance compared with wildtype Yangmai158. This provide an ideal material for investigating the relationship of plant development and disease resistance. In the present study, we characterized the photosynthesis characters and inheritance of the mutant traits in NAU31. The gene Tatyd was genetically mapped using an integrated BSA and linkage analysis approach.

powdery mildew resistance was identified from regenerated plant of common wheat cv. Yangmai158.
Inheritance analysis of NAU31 showed that the above mutant traits were significantly correlated and controlled by a dominant gene, named Tatyd. Linkage analysis of a mapping population between NAU31 and a synthesized wheat W7984 using SSR markers, the Tatyd was preliminary mapped to 2DS. By BSA and linkage analysis using a 55K wheat SNP array, the Tatyd was fine mapped to the terminal region of 2DS, flanked by markers ZH47 and gwm296 and corresponding to a physical interval of 0.76 Mb. Genome-wide gene expression analysis indicated that the impairing of photosynthesis may be responsible for plant yellowing in NAU31. Photosynthetic physiological analysis showed varied chloroplast inner structure in the mesophyll cells of the mutant, and this may lead to reduced chlorophyll content (chlorophyll "a", Chlorophyll "b", total chlorophyll and carotenoid), as well as decreased maximum quantum efficiency of photosystem II (Fv/Fm) and Net photosynthetic rate.
Conclusions: A mutant with few-tiller, plant yellowish, dwarfness and improved powdery mildew resistance was identified, and the mutated locus Tatyd was fine mapped to a 0.76 Mb genome region of wheat chromosome 2D. The yellowing trait of NAU31 may be attributed to its reduced photosynthesis ability due to changes of the chloroplast inner structure. The cloning of Tatyd will facilitate dissection of the mechanism involved in chloroplast development of wheat and the regulation of plant development and disease resistance. 3 Background Photosynthesis is the primary energy for biosphere, and is the foundation for life on earth [1]. More than 90% of crop biomass is derived from photosynthetic products, and the improvement of photosynthesis would undoubtedly increase crop yield [2]. Photosynthesis and translocation in relation to crop yield have been reviewed previously [3]. There is a positive correlation between photosynthesis and biomass and yield [4,5]. The relative growth rate of plant was positively correlated with daily integrals of photosynthesis expressed in different species and environments, and 93% of the variation in relative growth rate can be explained by photosynthesis [4], when yield increased by 27%, maximum photosynthetic rate increased by 23% in wheat [5]. Increasing photosynthesis by changing the level of SBPase (Calvin-Benson cycle enzyme sedoheptulose1,7biphosphatase) can increase wheat total biomass and dry seed yield [6], and sped up the adaptation of photosynthesis systems to light changes can also increase plant biomass [7]. Decreased photosynthetic electron transport rate of plants leads to reduced plant height and tiller number [8].
Recently, more and more studies have demonstrated that photosynthesis is involved in plantpathogen interaction [9]. Decreased plant photosynthesis affect nutrient uptake and fungal development of obligate biotrophic fungi [10]. MPK3/MPK6 was reported to coordinate plant growth and disease resistance by regulating decreased photosynthetic capacity and up-regulated expression of disease-related genes [11].
Phenotypic analyses of plants that are mutant for leaf color are widely used to investigate genes associated with plant photosynthesis, such as the biosynthesis and metabolism of chlorophyll (Chl), chloroplast structure and development [12]. The leaf color is mainly determined by Chl and carotenoid contents, in which Chl is the main photosynthetic pigment in plant [13], is responsible for light harvesting in the antenna systems and drives electron transport in the reaction centers [14]. The Chl biosynthetic pathway comprises about 20 different steps [15], and mutations in any of these steps can cause changes in the content and proportion of Chl, which ultimately leads to leaf color phenotypic changes. The Photosynthesis occurs in chloroplasts of eukaryotes [16]. Chloroplast biogenesis and development requires a highly coordination of gene transcription, translation, protein 4 renewal and metabolism. Multiple genes regulate these steps, and defects in any of these steps also lead to impaired chloroplast and leaf-color mutation [17,18]. Leaf color mutants have been reported in rice (Oryza sativa) [19], maize (Zea mays) [20], barley (Hordeum vulgare) [21] and etc. In rice, over 200 leaf color genes have been mapped, and more than 50 have been cloned [19]. Among them, 24 and 19 genes are proved to participate in photosynthetic pigmentation and chloroplast development, directly or indirectly [19,22]. In maize, among the cloned leaf color-related genes, elm1 [23] Among the regenerated progenies from tissue cultured Yangmai158 using the young embryo as explant, we identified a stable mutant with a phenotype of yellowing leaf color, fewer tiller and dwarfness. The mutant line was given a genetic stock number of NAU31, and the mutant gene was named Tatyd. Interestingly, the mutant showed improved powdery mildew resistance compared with wildtype Yangmai158. This provide an ideal material for investigating the relationship of plant development and disease resistance. In the present study, we characterized the photosynthesis characters and inheritance of the mutant traits in NAU31. The gene Tatyd was genetically mapped using an integrated BSA and linkage analysis approach.

5
Characterization of a mutant with yellowish leaf, reduced-tillering and dwarfed plant A yellowing mutant was identified from the regenerated population of Yangmai158, besides the mutant has reduced tillers, dwarfed plant and improved powdery mildew (Pm) resistance. After two generations self-fertilization, these mutant traits could be stably inherited (Fig. 1a). The mutant was named as NAU31. At harvest, the plant heights of Yangmai158 and NAU31 were 84.60 cm and 49.00 cm, respectively (Fig. 1b), their plant height difference exists throughout the growing period.
The tiller numbers of Yangmai158 and NAU31 were 5.40 and 1.67, respectively (Fig. 1c). They both have five internodes on the culm. However, each internode in NAU31 was shortened, with the fifth internode length having the maximum reduction (46.01% of Yangmai158) (Fig. 1b). Meanwhile, NAU31 showed enhanced powdery mildew resistance compared with Yangmai158 (Fig. 1d). FISH karyotypes of NAU31 and Yangmai158 were compared, and no obvious chromosome changes between the two materials (Additional file 1: Fig. S1).This revealed NAU31 did not undergo significant chromosomal structural variation.

Inheritance Of The Mutated Traits In Nau31
The (NAU31 × Yangmai158) F 1 exhibited plant yellowing phenotype and powdery mildew resistance.
Segregation for leaf color in F 2 population was observed, the plants could be classified into two types, yellowing type similar to NAU31 and normal green type similar to Yangmai158. Among the 650 F 2 individuals, 489 were yellowing type and 161 were normal green, and the segregation ratio fits 3:1 (χ 2 = 0.018 χ 2 0.05, 1 = 3.84). Moreover, all yellowing plants were powdery mildew resistant and those green were susceptible. These results showed that the yellowing phenotype was controlled by a dominant gene, and the yellowing trait was closely correlated with powdery mildew resistance.
The F 2 population were investigated for plant height, spike length, total and effective tiller number.
The plant height and spike length showed normal distribution, while the total and effective tiller number showed bimodal distribution (Additional file 1: Fig. S2). Genetic correlations analysis showed that leaf color was significantly and positively correlated with plant height, total and effective tiller number (Table 1). We presume that the mutated locus in NAU31 has pleiotropic effects on plant 6 morphology and Pm resistance.
This confirmed the mutated trait was controlled by a single dominant locus, which has pleiotropic effects. In this study, we only used plant yellowing for further genetic mapping of the muatnt traits.
A total of 1280 SSR markers evenly distributed in wheat genome were selected for polymorphism screening between NAU31 and W7984. Among them, 124 markers were pleiotropic and used for BSA using the "yellow" and "green" bulks (Y-Bulk-1 and G-bulk-1, see method). Five markers (WMC111, Cfd51, gwm296, wmc503 and WMC112) were polymorphic, and they all located on chromosome arm BSA was further performed by the wheat 55 k SNP array using the two DNA bulks (Y-Bulk-2 and Gbulk-2) together with the two parents. A total of 374 polymorphic SNPs were identified between the two parents as well as between the two bulks. Among these SNPS, 146 were located on chromosome 2D. Most of these SNPs located within the interval of 6-24 Mb genome region of 2DS, where the Tatyd were located by SSR marker analysis (Fig. 2c).

Fine mapping of Tatyd
To fine mapping Tatyd, two flanking markers WMC111 and gwm296 were used to genotype 1390 'NAU31 × W7984' F 2 plants and 442 derived F 2:3 plants. Seventeen recombinants were identified. To enrich the marker density of the Tatyd region flanked by WMC111 and gwm296, we developed ten 7 new markers (5 SNP and 5 SSR) (Additional file 1: Fig. S3, Additional file 2: Table S1, Additional file 4).
All were polymorphic and used to genotype the 17 recombinants. By this, we divided them into seven haplotypes, three were yellowing type and four were normal green type. Thus, Tatyd was finely mapped to 2DS flanked by ZH47 and gwm296, representing a physical interval of 0.76 Mb (Fig. 2d).
Leaf yellowing is associated with the photosynthesis ability in NAU31 To help the candidate gene prediction and the cause of mutant morphology, we compared the photosynthesis ability and transcriptional profiling between the Yangmai158 and NAU31. The Fv/Fm and Net photosynthetic rate were compared in flag leaf. The flag leaf Fv/Fm of NAU31 significantly lower (0.7940) than Yangmai158 (0.8153) at adult stage (Fig. 3b, 3d), and most of their growth periods (Fig. 3f). The Net photosynthetic rate of NAU31 (2.51 µmol/m 2 ·s) was also significantly lower than Yangmai158 (11.50 µmol/m 2 ·s) (Fig. 3e). This indicated that yellowing leaves of NAU31 had reduced photosynthetic capacity. The photosynthetic pigment contents and SPAD value at the adult stage were measured. The results showed that Chl a, Chl b, total flag leaf chlorophyll and carotenoid contents in NAU31 were 0.46 mg/g, 0.11 mg/g, 0.57 mg/g and 0.09 mg/g, which decreased by 84%, 89%, 86% and 81%, respectively,compared with those in Yangmai158 (Fig. 3g). The SPAD value decreased from 45.21 in Yangmai158 to 25.05 in NAU31 (Fig. 3h). The Chl-index of NAU31 was also lower than that of Yangmai158, with the difference became more and more apparent from seedling to adult stage (Fig. 3c, 3i).
To investigate whether the compromised photosynthesis ability in NAU31 was attributed to chloroplast development impairing or structural changes, chloroplast ultrastructure of Yangmai158 and the NAU31 at 2-week growth stage were compared using TEM. Yangmai158 had oval shape mesophyll cells (Fig. 4a), while NAU31 had irregular shape (Fig. 4b). Yangmai158 and NAU31 had no significant difference in the chloroplast size or number. However, chloroplasts in the mesophyll cells of Yangmai158 had well-defined grana and regularly shaped starch grains; in contrast, chloroplasts in NAU31 showed abnormal shape, had vague thylakoid boundaries, and compromised normal oval shape starch grains (Fig. 4c, 4d). Moreover, the Yangmai158 displayed well-developed lamellar 8 structures, were equipped with normally stacked grana, thylakoid membranes and regularly shaped plastoglobuli. The thylakoid lamellar structures in the chloroplasts of NAU31 were looser and its plastoglobuli were irregular (Fig. 4e, 4f). We proposed yellowing trait of NAU31 may attributed to reduced photosynthesis ability due to changes of the chloroplast inner structure.
The differentially expressed genes (DEGs) between NAU31 and Yangmai158 at two development stages were compared using RNA-seq of NAU31 and Yangmai158 leaves at two-and four-leaf stages. At two-leaf stage, 65 and 3 DEGs were down-and up-regulated, respectively; at four-leaf stage, 66 and 2 DEGs were down-and up-regulated, respectively (Fig. 5a). Twelve DEGs related to the photosynthesis -antenna proteins pathway were selected for to qRT-PCR. The expression levels of all tested DEGs were significantly lower in NAU31 than those in Yangmai158 at both growth stages ( Fig. 5b), further confirming the down-regulation of the photosynthesis pathway in NAU31. These results showed that compromise of photosynthesis may cause the plant yellowing trait in NAU31.

Discussion
The mutant traits in NAU31 is controlled by a dominant locus Leaf color mutants are ideal materials for the study of photosynthesis, chloroplast development and Chlorophyll biosynthesis [20]. Various leaf color mutants have been identified in crops such as maize and rice [19,20]. The type of leaf color mutants were classified into eight classes: white; yellow; light yield and grain quality significantly and negatively. The plant height, tiller number and grain yield were significantly reduced in the OsPS1-F mutant (Photosystem 1-F subunit was mutated by Ds insertion) [8]. In wheat, the agronomic traits spike length, number of kernels per spike and 1000kernel weight of the yglw-1 mutant were significantly decreased, compared to the wild type [34]. In present study, plant height, tiller number, spike length, effective tiller number, spikelet size, seed size and 1000-grain weight were significantly lower in NAU31 mutant than that of wildtype Yangmai158.
Therefore, similar to the previous reports for the productivity and yield influenced in leaf color mutants, this yellowing NAU31 mutant also negatively impact agronomic traits.
In wheat, leaf color mutations have also been reported and were associated with different genetic regions [30, 31, 39, 40]. In common wheat, the leaf color trait of Ygm mutant was controlled by a incompletely dominant gene, named Y1718, the Y1718 was mapped between SSR marker Xwmc25 and EST-STS marker BE498358, with genetic distances of 1.7 and 4.0 cM, respectively [33]. The leaf color trait of yglw-1 mutant was controlled by a recessive gene, the yglw-1 gene was mapped between SSR markers Xhwm1 and Xhwm43, with genetic distances of 1.9 and 23.9 cM, respectively [34]. In the present study, we identified a yellow leaf mutant, the leaf color trait of NAU31 was controlled by a dominant gene, named Tatyd, the Tatyd was mapped to 2DS between SSR markers ZH47 (16,845,340 bp) and gwm296 (17,609,164 bp). As mentioned above, Y1718 and yglw-1 were mapped to 2B. The yglw-1 is mapped within Xhwm1 and Xhwm43, which corresponds to 109,121,927 to 111,574,556 bp region on chromosome 2D of Chinese Spring reference sequence. The Y1718 is mapped within BE498358 and CJ945085, which corresponds to 8,567,431 to 15,604,263 bp region on chromosome 2D. Therefore, they are not homoeologous alleles because they were not mapped to the same synteny regions among sub-genomes. These results indicate that Tatyd is a new gene related to leaf color.
The NAU31 is likely associated with impairing in photosynthetic pigment pathway The impairing in photosynthetic metabolism or photosynthetic pigments and Chlorophyll development could lead to the mutation in leaf color in crop. More than 50 leaf color genes have been cloned [19], among them, some genes directly participated in metabolism of photosynthetic pigments, for example OsCHLH [41] and YGL1 [42]; and some genes indirectly influenced metabolism of photosynthetic pigments through other biological pathways, for example OsObgc [43] and FdC2 [44], the content and distribution of photosynthetic pigment determined the color of leaf and the capacity of photosynthesis [38,45]. Chlorophyll fluorescence is a useful technique for studying the mechanism of photosynthesis, photosynthetic capacity and heritable variants of plants [46,47]. The changes in leaf color generally lead to changes in photosynthesis capacity and photosynthetic pigment of plant.
In maize, the contents of total Chl s, Chl a, Chl b and Car of ygl-1 mutant were significantly reduced of 24.32%, 20.4%, 40.2%, and 1.96%, respectively, which lowered the capacity of photosynthesis compared with the wild type Lx7226 [20]. In Lotus japonicas, the content of chlorophylls and the ratio of Fv/Fm of C16 mutant showed lower than that of wild type, indicating that the photosynthesis capacity of C16 is lower than that of wild type [48]. In rice, the content of photosynthetic pigment is lower in ygl7 than that in the wild type, but the photoelectron utilization rate, photosynthetic rate, the net photosynthesis and dark respiration rates of ygl7 were higher than WT, maybe caused by genes associated with photosynthesis were mostly up-regulated in ygl7 [22]. In wheat, the pigment of Ygm mutant were reduced compared with wild type Xinong1718, but the Chl a/b ratio in Ygm leaves were 3.81-5.64 times higher compared Xinong1718 [33]. In present study, the photosynthetic pigment (Chl a, Chl b, Chl a + b and Car) and net photosynthetic rate of NAU31 mutant were lower than that of wild type Yangmai158 in flowering stage. Fv/Fm of NAU31 was significantly lower than that of Yangmai158 in most of the growth period. Transcriptome analysis results showed that many DEGs associated with photosynthesis were mostly down-regulated in NAU31, indicating that the lower photosynthetic capacity may be caused by lower content of chlorophylls in NAU31, which is similar to the relationship between photosynthetic pigment content and photosynthetic capacity in papaya plants [49].
The Leaf color mutation increase resistance to powdery mildew of NAU31 Changes in photosynthesis capacity directly affect plant disease resistance [9,50]. In recent years, many studies have found that chloroplasts participate in interaction between plants and pathogens through salicylic acid (SA), jasmonic acid (JA), and reactive oxygen species (ROS) pathway [51]. In Arabidopsis, MPK3/MPK6 can inhibit photosynthesis by down-regulated the expression of photosynthesis-related genes and then increase effector-triggered Immunity (ETI) [11]. In wheat, WKS1 interacts with phosphorylates PsbO, and leading a fast degradation of PsbO, to reduce photosynthesis and regulate leaf chlorosis in conferring Pst resistance [52]. In this study, NAU31 was yellowing and its photosynthesis capacity was reduced, and many photosynthesis -antenna proteins pathway relate genes were significantly down-regulated compared with Yangmai158. Indicating that the resistance of NAU31 to powdery mildew may be increased through reduced photosynthesis.

Conclusions
This study identified a new wheat few-tiller, plant yellowish, dwarfness and improved powdery mildew resistance mutant, named NAU31. Inheritance analysis of NAU31 showed that the above mutant traits were significantly correlated and controlled by a dominant gene, named Tatyd. Tatyd was fine mapped to a 0.76 Mb genome region of wheat chromosome 2D. The photosynthetic capacity of NAU31 was significantly reduced compared to the wild type Yangmai158, which may lead to increased resistance to powdery mildew.

Plant materials
Commercial wheat variety Yangmai158 was cultivated in Institute of Agricultural Sciences in Lixiahe District of Jiangsu Province, Sumai3 was cultivated in Suzhou Academy of Agricultural Sciences, Chinese Spring (CS) is a Chinese wheat landrace. All materials were maintained at the Cytogenetics Institute, Nanjing Agricultural University (CINAU). Yangmai158 is susceptible to wheat powdery mildew. NAU31 is a mutant, which was identified from the regenerated plants of a tissue cultured population of Yangmai158. The protocol of wheat tissue culture followed Xing et al. (2008). [53]. The mutant was self-fertilized for two generations, and its mutant phenotype (few-tiller, dwarfness, yellow leaf and powdery mildew resistance) could be stably inherited (Fig. 1a). Synthetic W7984 is a

Evaluation Of Powdery Mildew Resistance
The single Bgt isolate E26 provided by Prof. Yilin Zhou from Chinese Academy of Agriculture Sciences (CAAS) and mixed Bgt isolates (collected from Nanjing, China) were maintained on two-leaf stage seedlings and fresh spores were used as inoculum to test the Pm resistance. For plants grown in an incubator, the 4th leaves of plants at the 5th leaf growth stage were cut to test the Pm resistance by artificial Bgt inoculation. The plants grown in the field were used for adult stage Pm resistance evaluation by artificial inoculation with mixed Bgt isolates. The resistance level was classified as grades 0-4 as described by Sheng (1988) [54], in which grades "0-2" were regarded as resistant, and grades "3-4" were moderately and highly susceptible, respectively. Transmission Electron Microscopy (tem) 13 TEM analysis was carried out as described previously [56] with minor modifications. The leaves of Yangmai158 and NAU31 were harvested in 2-week-old seedlings stage,fixed in 2.5% glutaraldehyde in 0.1 mM phosphate buffer (pH 7.2) at 4 °C for 24 h. After washed with 0.1 M phosphate buffer (pH 7.2), the leaves were post-fixed with 1% osmium tetroxide. The samples were embedded in Spurr resin, and 0.5-µm sections were cut with a UC7 Ultramicrotome (Leica, Germany). The sections were observed and the pictures were captured using an H-7650 transmission electron microscope (Hitachi Science Systems Ltd, Japan).

Measurement Of Photosynthetic Pigment Contents
Two methods were used to determine the photosynthetic pigment contents. The photosynthetic pigment content (mg g − 1 ) was calculated using the formula: m x =C × V × N/W × 1000, in which m is photosynthetic pigment content, C is photosynthetic pigment concentration, V is volume of extraction solution, W is fresh sample weight and N is the dilution factor.

Measurement Of Chlorophyll Fluorescence And Photosynthetic Parameter
The maximum quantum yield of PSII (Fv/Fm) of NAU31 and Yangmai158 were measured after 10 min 14 of dark acclimation by an in vivo chlorophyll "a" fluorometer (Mini-PAM, Germany) at heading stage.
The net photosynthetic rate of the flag leaves at grain filling stage (25 days after flowering) was measured in the field, using the Li-6400 portable photosynthesis system (LI-Cor, Inc.) at midday, when the light intensity is about 1300 µmol m − 2 s − 1 and CO 2 = 400 µmol mol − 1 . Ten individuals of NAU31 and Yangmai158 were measured and then averaged, respectively.
To investigate the difference of photosynthesis and plant growth parameters between NAU31 and Yangmai158 during their whole growth period. The seeds of NAU31 and Yangmai158 were sown on wet filter paper and incubated at 4 °C for 12-14 days. Three seeds of the same genotype were germinated and planted in pots. The growth conditions were as follows: 14/10 h day/night, 24/20 °C day/night temperature and 70% relative humidity. The plant heights were recorded every day. The photosynthesis parameters (Fv/Fm, Chl-index), chlorophyll fluorescence image and normal image were detected every day at 10 am by PhenoMate (PhenoTrait, Netherlands) during the whole growth period.

Marker Development
Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were developed according to the Chinese Spring reference sequence (https://www.wheatgenome.org/). SSR loci were detected using SSR Hunter software, and primer pairs were designed using the online software Primer3 V0.4.0. The 55K wheat SNP array (Affymetrix® Axiom® Wheat55) was used to genotype the NAU31 and W7984. Based on the SNPs detected by SNP array, tetra-SNP primers (Each set contains two forward primers and two reverser primers) were designed by using the online software Primer3 V0.4.0. All the above primer pairs (Additional file 2: Table S1) were synthesized by Tsingke Biological Technology Company.

Bulk Segregant Analysis (bsa)
The genomic DNA were extracted from young leaves using CTAB method [57]. (1) For BSA using SSR markers, the procedure was performed as described by Qin et al (2015) [21] with the following modifications. Equal amounts of DNA from 10 yellow and 10 normal green individuals from the 15 (NAU31 × W7984) F 2 population were pooled and named as Y-bulk-1 and G-bulk-1, respectively. The SSR markers from public databases distributing on the 21 chromosomes of wheat were screened in the parents and the two bulks. PCR reaction and the polyacrylamide gel analysis were conducted to analyze the genotype of the samples according to Xu et al. (2017) [58]. (2) For BSA using 55K SNP assay, equal amounts of DNA from 7 yellow and 7 normal green individuals from (NAU31 × W7984) F 2:3 lines (derived from the above F 2 ) were pooled and named as Y-bulk-2 and G-bulk-2, respectively.
The SNP array was implemented by CapitalBio Corporation (Beijing). Polymorphic SNPs detected from contrasting pools and corresponding parents were assumed to be linked to Tatyd, and they were localized to chromosomes based on data provided by CapitalBio Corporation.

Linkage Map Construction
The linkage map was constructed using JoinMap 4.0 software at a LOD threshold of 3.0. Linkage groups were assigned to chromosomes by referring to the published genetic maps of wheat (http://wheat.pw.usda.gov/GG2/index.shtml).
All of the samples were immediately frozen in liquid nitrogen and were stored at -80 °C. Total RNA was extracted using the Trizol reagent (Invitrogen, USA), according to the manufacturer's instructions.
RNA concentration was determined using a NanoDrop spectrophotometer and 1.2% agarose gel electrophoresis. RNA-Seq was performed using a BGISEQ-500 sequencing platform by BGI Company, Shenzhen, China. The raw reads were cleaned by removing adapter and low-quality sequences (percentage of low-quality bases with a quality value ≤ 10 in more than 20% of a read). Qrt-pcr Analysis qRT-PCR was to validate the RNA-seq data using the same RNA samples used for RNA-sEq. Chlorophyll-related DGEs were selected and specific primers were designed using Primer 3. The tubulin gene was used as the internal control for normalization (All primers were shown in Additional file 2: Table S1). qRT-PCR was performed with AceQ SYBR Green (Vazyme) using a Louts PCR 480 sequence detection system. The reactions were subjected to the following program: 95 °C for 1 min, 41 cycles of 95 °C for 10 s, and 60 °C for 30 s. The relative value of gene expression were derived from 2 −△△CT [59]. Three independent biological replications were performed for each sample.

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Availability of data and materials:
All data generated in this study are included in the paper and in the supporting information files.

Competing interests:
The authors declare that they have no competing interests