{'fraction_non_alphanumeric': 0.03643402813785475, 'fraction_numerical': 0.024128037609319367, 'mean_word_length': 4.787157431486297, 'pattern_counts': {'":': 0, '<': 14, '<?xml version=': 0, '>': 0, 'https://': 1, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 1, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Flag leaf vein traits and their correlation with photosynthesis and grain yield in wheat genotypes of differing ploidy

Leaf venation and coupled physiological function of wild plants co-evolve during the natural selection. How artificial selection affects leaf vein traits and coordinated physiological functions of main crops are largely unknown. This study examined the changes of leaf vein traits and their correlation with gas exchange of flag leaves and yield in eight wheat genotypes of differing ploidy under the same growing conditions. The results indicate that flag leaf vein density (VLA), major-vein density (VLAmajor), and minor-vein density (VLAminor) decreased whereas the proportion of minor-vein length and interveinal distance between small longitudinal veins (IVD) increased during the polyploidization process, and the major advance occurred from the period from diploids to tetraploids. The VLA, VLAmajor, and VLAminor were closely coordinated with maximum net photosynthetic rate (PN) and photosynthetic N use efficiency (PNUE), but not with stomatal conductance. The proportion of minor-vein length and IVD were negatively related with PN and PNUE but positively related with N content per area (Narea) during wheat evolution. A higher proportion of minor-vein length and IVD, and a lower VLAmajor in flag leaves along with a larger Narea were largely responsible for the increased yield in modern cultivars. The decreased redundancy of leaf vein density and increased minor-vein proportion in modern cultivars can confer a yield advantage during wheat evolution. Additional key words: evolution,⋅gas exchange, leaf N content, stomatal conductance,⋅Triticum aestivum. Submitted 23 April 2020, last revision 4 June 2020, accepted 9 June 2020. Abbreviations: gs stomatal conductance; IVD interveinal distance between small longitudial veins; LMA leaf mass per area; Narea N content per area; Nmass N content per mass; NRE N remobilization efficiency; PN net photosynthetic rate; PNUE photosynthetic N use efficiency; VLA vein density, VLAmajor major-vein density; VLAminor minor-vein density; WUEi intrinsic water use efficiency. Acknowledgements: This work was funded by the National Science and Technology Supporting Program (2015BAD22B01). * Corresponding author; e-mail: yyli@ms.iswc.ac.cn Introduction Leaf veins are an important constituents of leaf structure. They provide mechanical support, transport nutrients and water for photosynthesis and transpiration through the xylem, and transport photosynthates and signal molecules from the mesophyll to the rest of the plant through the phloem (Niklas 1999, Roth-Nebelsick et al. 2001). Depending on climatic and environmental conditions during leaf development, leaf vein traits are highly diverse across and within species (Uhl and Mosbrugger 1999, Boyce et al. 2008, Sack et al. 2012, Sack and Scoffoni 2013). Leaf vein traits have shown repeated evolutionary trajectories across major plant groups. Throughout 380 million years of evolution, angiosperm vein densities (vein length per leaf area, VLA) have reached 8 ~ 25 mm mm-2 (about 2 mm mm-2 in non-angiosperms) with increasing number of vein orders. High value of leaf VLA enables unparalleled photosynthetic rate and transpiration rate in the angiosperms than their competitors, thus dominate in a range of habitats than other plant lineages (Boyce et al. 2009). In the evolution of C4 within eudicot genera Cleome and Flaveria, leaf VLA increased from C3 to C3/C4 intermediate and C4 species (Marshall et al. 2007, McKown and Dengler 2007). C4 grasses have evolved a higher VLA (2.1 times of C3 grasses), denser transverse veins and denser small longitudinal veins than the C3 grasses without significant difference in large longitudinal veins (Ueno et al. 2006). Increasing VLA may initially have little effect on the performance of an effective CO2 concentration mechanism, but may enhance the water status of C4 leaves in hot environments (Sage 2004). Compared to the natural evolution, crop evolution is mainly made by artificial selection. Wheat, as one of the oldest and most important of cereal crops worldwide, began to domesticate ~10 000 years ago (Tanno and Willcox 2006). Modern cultivars of the Triticum aestivum hexaploids with large grain yield were domesticated from the more primitive diploids and


Introduction
Leaf veins are an important constituents of leaf structure. They provide mechanical support, transport nutrients and water for photosynthesis and transpiration through the xylem, and transport photosynthates and signal molecules from the mesophyll to the rest of the plant through the phloem (Niklas 1999, Roth-Nebelsick et al. 2001. Depending on climatic and environmental conditions during leaf development, leaf vein traits are highly diverse across and within species (Uhl and Mosbrugger 1999, Boyce et al. 2008. Leaf vein traits have shown repeated evolutionary trajectories across major plant groups. Throughout 380 million years of evolution, angiosperm vein densities (vein length per leaf area, VLA) have reached 8 ~ 25 mm mm -2 (about 2 mm mm -2 in non-angiosperms) with increasing number of vein orders. High value of leaf VLA enables unparalleled photosynthetic rate and transpiration rate in the angiosperms than their competitors, thus dominate in a range of habitats than other plant lineages (Boyce et al. 2009). In the evolution of C4 within eudicot genera Cleome and Flaveria, leaf VLA increased from C3 to C3/C4 intermediate and C4 species (Marshall et al. 2007, McKown andDengler 2007). C4 grasses have evolved a higher VLA (2.1 times of C3 grasses), denser transverse veins and denser small longitudinal veins than the C 3 grasses without significant difference in large longitudinal veins . Increasing VLA may initially have little effect on the performance of an effective CO2 concentration mechanism, but may enhance the water status of C4 leaves in hot environments (Sage 2004). Compared to the natural evolution, crop evolution is mainly made by artificial selection. Wheat, as one of the oldest and most important of cereal crops worldwide, began to domesticate ~10 000 years ago (Tanno and Willcox 2006). Modern cultivars of the Triticum aestivum hexaploids with large grain yield were domesticated from the more primitive diploids and tetraploids, and polyploidization played an important role during the cultivation and breeding of wheat. How the leaf vein traits changed during this polyploidization process was largely not clear.
Leaf vein traits have been reported to closely associate with leaf physiological function. Generally, high VLA values are positively correlated with higher leaf hydraulic conductance, greater stomatal density and stomatal conductance, and higher rate of gas exchange per unit leaf area (Sack and Frole 2006, Brodribb et al. 2007, Brodribb and Jordan 2011, Zhang et al. 2012. But this is not so under all cases. Negative (Nardini andSalleo 2000, Sack and and no correlations (Flexas et al. 2013, Xiong et al. 2015, Gleason et al. 2016 have also been reported between leaf VLA and leaf hydraulic conductance. Leaf VLA is negatively correlated with leaf net photosynthetic rate (PN) and stomatal conductance (gs) in rice cultivars under different water status (Tabassum et al. 2016). Leaf VLA shows no apparent association with stomatal conductance and maximal net CO2 assimilation across a diverse group of 35 evergreen Australian angiosperms (Gleason et al. 2016). These inconsistent results indicate that the evolutionary drivers linking leaf vein traits and hydraulic efficiency or gas exchange are not fully understood. Moreover, leaf VLA was reported to be positively related with N and P resorption efficiencies across 17 dipterocarp tree species . Another study found that leaf VLA is not correlated with leaf N content per area (Narea), but interveinal distance is positively correlated with leaf Narea among 11 rice cultivars (Xiong et al. 2015). How leaf vein traits are coordinated with these leaf physiological functions during wheat evolutionary process is yet to be known. Knowledge on such comprehensive correlations between leaf vein traits and leaf physiological traits is critical for enhancing our understanding of leaf structural attributes to crop water use and photosynthesis.
Despite the potential importance of leaf vein traits, few studies have examined associations between these traits and whole plant performance. Leaf vein traits are weakly, but significantly related to tree demographic performance (relative growth rate and mortality) along soil environments for 54 co-occurring species in a subtropical forest (Iida et al. 2016). Increasing leaf vein vensity via mutagenesis in rice results in an enhanced photosynthetic efficiency without increased transpiration (Feldman et al. 2017). Leaf VLA is positively correlated with the grain yield in 292 rice landraces (Nawarathna et al. 2017), suggesting that the higher VLA can be used in future rice breeding programs. Whether leaf vein traits in wheat have the similar associations with yield potential as in rice remains unknown. Identification of the presence of such leaf vein traits will be helpful to future wheat breeding.
Some morphological and physiological changes have been reported among wheat species differing in ploidy (Evans and Dunstone 1970, Austin et al. 1982, Zhang et al. 2002, Xiong et al. 2006, Huang et al. 2007, Li et al. 2014, Wang et al. 2017. The diploids generally have smaller leaf area, higher stomatal frequencies, and smaller mesophyll cells than the hexaploids, net photosynthetic rate is the highest in diploids, intermediate in tetraploids, and the lowest in hexaploids (Austin et al. 1982), but the hexaploids have higher root water uptake capacity, higher yield and water use efficiency, as well as higher drought resistance (longer survival duration and higher yield stability under drought) (Zhang et al. 2002, Xiong et al. 2006, Wang et al. 2017. However, little is known about variation of leaf vein traits and their potential importance in leaf physiological function and yield increment during the evolutionary process. Therefore, the aim of this study was to examine the variation of leaf vein traits, physiological function, yield, and their relationship in eight wheat species of three different ploidy. We assumed that during wheat evolution from diploids to tetraploids and to hexaploids, leaf VLA increased as that directed by natural selection, and leaf VLA is closely coordinated with gas exchange and yield.

Materials and methods
Plants and treatments: Eight species, including two diploids, two tetraploids, and four hexaploids, were used in a field experiment in Yangling, Shaanxi Province in China (Table 1 Suppl.). Soil was silty clay with pH 8.36, bulk density 1.26 g cm -3 , organic matter 13.6 g kg -1 , total N 0.82 g kg -1 , alkaline N 78.4 mg kg -1 , Olsen-P 14.2 mg kg -1 , and available K 210.2 mg kg -1 . Seeds were sown on 11 October 2018 at a spacing of 5 × 15 cm in plots 2 × 3 m 2 . There were three replicate plots of each species arranged in randomized blocks. Each plot received 0.022 5 kg(N) m -2 and 0.012 kg(P2O5) m -2 , 70 % of N fertilizer and all P fertilizer were applied before sowing, the left 30 % of N fertilizer was applied with precipitation during the jointing stage. The precipitation during the whole growth season (11. 10. 2018 -16. 6. 2019) was 252 mm, and 30 mm of irrigation was added in the winter of 2018. During the end of May 2019, the plots were sprayed with chemicals at the manufacturer's recommended rate to prevent aphid. Weeds were pulled out manually.
The flowering date of each species was labeled with tags, and 13 main stems in each plot of each species were labeled. Two tagged flag leaves were used for the measurements of leaf area and leaf vein traits on the fourth day after flowering, two tagged flag leaves were used for the measurement of gas exchange parameters and photosynthetic N use efficiency (PNUE) at the same period, and the left nine flag leaves was used for determination of leaf mass per area (LMA) and N remobilization efficiency.

Measurement of leaf vein traits:
Two flag leaves per plot per species were scanned with a scanner at first, and the leaf images were analyzed for leaf area using imageJ software (https://imagej.en.softonic.com/), then the two leaves were used to determined leaf vein traits, totally six leaves per species. Two 0.5 cm 2 pieces were quickly excised from the middle portion of each leaf, and preserved in FAA [37 % (m/v) aqueous formaldehyde, 50 % (v/v) ethanol, and 13 % (m/v) glacial acetic acid] until use. The fixed pieces were boiled in 70 % ethanol for about 10~20 min and washed several times in distilled water, the leaf pieces were transferred to boiling 85 % (m/v) lactic acid for 20 min, and then stored in chloral hydrate-saturated ethanol before analysis (Ueno 1995). Four fields per pieces was randomly taken using a light microscope (CX31, Olympus, Tokyo, Japan) and attached digital camera system (M-shot, Sci-Tech Cor., Guangzhou, China), and above mentioned imageJ software was used to analyzed vein traits. Wheat vascular bundles can be categorized into four types: midvein (MV), large longitudinal veins (LLVs), small longitudinal veins (SLVs), and transverse veins (TVs) (Nelson and Dengler 1997). In the present study, the major-vein density (VLAmajor, mm mm -2 ) was the sum of the densities of the MV and LLVs, and the minor-vein density (VLAminor, mm mm -2 ) was the sum of the densities of the SLVs and TVs. Vein density and the proportion of minor-vein length [%] were also calculated, and interveinal distance between two neighboring SLVs (IVD) were also measured.

Measurement of gas exchange, intrinsic water use efficiency (WUEi), and photosynthetic N use efficiency (PNUE):
Gas exchange was measured using a portable gas-exchange system (LI-6400, Li-Cor, Lincoln, NE, USA) from 9:30 to 11:30 on sunny days, and the radiation source was set to a photosynthetic photon flux density (PPFD) of 1 200 µmol m -2 s -1 . The ambient temperature, relative humidity, and CO2 concentration were 28.4 -33.2 °C, 35.5 -46.8 % and 400 -412 µmol mol -1 , respectively. The leaf enclosed in the chamber of the portable gas-exchange system was marked for identifying the projected areas after each measurement. Then the leaf was cut and taken to the lab for determination of N content per area (Narea). Leaf N content was determined using an Auto-Kjeldahl method (Kjeltec system 2300 Distilling Unit, Foss, Hoganas, Sweden). The WUEi was calculated as PN/gs, and the PNUE was calculated as PN/Narea.

Measurement of N remobilization efficiency (NRE) and leaf mass per area (LMA) in flag leaves:
The NRE was used to represent the phloem transport capacity. N content based on leaf area and dry mass was determined on three dates. Flag leaf N content reached the maximum on the date of flowering for modern wheat cultivars (Cai et al. 2008), but we are not sure if this was true in primitive wheat species. So, we sampled on the date of flowering, the fourth day after flowering, and at physiological maturity, and three flag leaves were taken away at each date per plot per species. These leaves were scanned first, then killed at 105 °C for 30 min, later oven-dried at 80 °C to a constant mass. The scanned pictures were used for determining leaf area with imageJ software, and leaf mass per area (LMA) was calculated as leaf dry mass/leaf area. The dried leaves were grounded for determination of N content. N content on the fourth day after flower were always larger than those on the day of flowering, so we take N content on the fourth day as the maximum N content (Nmax). N content based on leaf area and leaf dry matter are strongly correlated (R 2 = 0.87, P < 0.01, n = 72), so we use only N content per dry mass to calculate N remobilization efficiency (NRE) (Zhang et al. 2014) as NRE [%] = (Nmax -Nmaturity) × 100 / Nmax, where Nmaturity is the N content per dry matter at maturity.
Grain yield: At physiological maturity of each species, 1 m 2 of each plot was harvested to determine aboveground biomass and grain yield; biomass and grain were weighted after oven-drying at 80 °C for 3 d.

Statistical analyses:
Two-level nested analysis of variance (ANOVA) was used to assess the significance of leaf vein and physiological parameters across different wheat ploidy. A post-hoc multiple comparison was conducted using the Tukey-Kramer method if the effect of ploidy was significant. A principal component analysis (PCA) was analyzed to examine multivariate associations of leaf traits. The allometric relationships between leaf vein traits and leaf area, LMA were modeled by power functions, and the regressions between leaf vein traits and leaf physiological parameters, biomass and yield were fitted by linear models. The nested ANOVA was conducted by a "two-level nested anova" EXCEL file provided by Dr. J.H. McDonald on the webpage (http://www.biostathandbook. com/nestedanova.html), all other statistics were conducted using SPSS Statistics 20.0 software (IBM Corporate, Armonk, USA).
VLA, VLAmajor, and VLAminor across wheat genotypes of differing ploidy decreased with leaf area (pseudo R 2 = 0.74, P < 0.01 for VLA; pseudo R 2 = 0.66, P < 0.05 for VLAmajor, and pseudo R 2 = 0.76, P < 0.05 for VLAminor) (Fig. 1A), while proportion of minor-vein length and IVD increased with leaf area (pseudo R 2 = 0.46, P = 0.06 for proportion of minor-vein length; pseudo R 2 = 0.53, P < 0.05 for the IVD) (Fig. 1B,C). Due to the strong correlation between flag leaf area and LMA (R 2 = 0.81, P < 0.01), the variation of leaf vein traits with LMA followed the same trend as with leaf area (Fig. 1 Suppl.). The allometric relationship may partly explain the trend of leaf vein traits during the wheat polyploidization.

Discussion
During the evolution of wheat, VLA, VLAmajor, and VLAminor all show the trend to decrease, while proportion of minor-vein length and IVD increased (Table 1). This trend differs from that exhibited in angiosperm and C4 grasses evolution (Boyce et al. 2009. The variation of wheat leaf vein traits across differing ploidy can be partly explained by the developmental algorithm for vein formation during leaf expansion, showing declined major-vein density with increasing leaf size, and minor-vein density independent of leaf size . Generally, major veins include the first three orders of veins, and vein density of these three orders declines with leaf area ). The first two orders of veins (midrib and large longitudinal veins) of wheat leaves are directly connected with leaf sheath, and participate in water transport to leaves, photosynthate export, etc. (Altus and Canny 1985). The VLAmajor in the present study only covered the first two orders of veins and it decreased due to the increased flag leaf area during wheat evolution process. Vein density of the third order of veins (small longitudinal veins) also decreased with increased leaf area and it contributed more than 60 % to VLA and 75 % to VLAminor (data not shown), resulting in decreased VLA and VLAminor during wheat evolutionary process. Meanwhile, we found that proportion of minor-vein length and IVD increased with the increase in leaf area (Fig. 1). Although there is no mechanic association between LMA and vein traits , the changes of vein traits with LMA also showed the same trend as those with flag leaf area during the study since LMA and leaf area are closely related.
Higher VLA in diploid than in tetraploid and hexaploid species may favour their photosynthesis. The earlier studies Dunstone 1970, Austin et al. 1982) and present study all supported that the more primitive diploids have higher net photosynthetic rate than modern hexaploid cultivars. Except the role of mechanical support, majorvein density has been found to play a role in determining the water transport capacity, in damage tolerance of the vein system, and in leaf drought tolerance (Sack et al. 2008, Scoffoni et al. 2011. Thus larger major-vein density in diploids provide better water transport and so enable their better survival in harsh environments than tetraploids and hexaploids although some research do not support this (Xiong et al. 2006, Wang et al. 2017. The minor veins Means ± SEs, n = 6. The curves are fitted by a power function; Rp 2 -pseudo R 2 of modeling curves. * -P < 0.05, ** -P < 0.01. (transverse veins and small longitudinal veins) of wheat leaves play a vital role in lateral transport of photosynthates from the small to the large longitudinal veins (Altus and Canny 1982) because they have large surface area for uploading photosynthates from the surrounding mesophyll. Thus, diploids have acquired a better photosynthate translocation system than the tetraploids and hexaploids to match with their higher photosynthetic capacity. The decreased VLAmajor in tetraploid and hexaploid species may reduce the carbon cost for construction, and lower vein projected area might potentially enhance mesophyll radiation capture , and larger IVD may allow more mesophyll cells occupy (Austin et al. 1982).
Past studies showed VLA (especially VLAminor) is closely correlated with gs, and PN at intraspecific and interspecific levels (Sack and Frole 2006, Brodribb et al. 2007, Brodribb and Jordan 2011, Zhang et al. 2012. This study also supported the strong positive association between VLA and PN (Fig. 2), but leaf vein density and gs was decoupled (Fig. 3 Suppl.). The PN and gs also decoupled across wheat genotypes of differing ploidy (Fig. 3 Suppl.). In an early study by Austin et al. (1982), the mean gs of the diploids was only 5 % greater than that of genotypes in this study; gs in diploids was 17.1 % higher than that of hexaploid cultivars with no statistic difference, indicating leaf internal anatomical features rather than the difference of gs mainly drive the variation in photosynthetic rate. Anatomically, small leaf size, small mesophyll cell size, and close spacing of veins are correlated with high photosynthetic rate in primitive genotypes (Dunstone and Evans 1974). Water supply capacity of leaves did not constrain water loss and CO2 diffusion under present ample soil water condition. Although there are reports of increased water and nutrient use efficiency during the wheat evolution process (Zhang et al. 2002, Xiong et al. 2006, Huang et al. 2007), we did not find this at leaf level. Leaf WUEi did not change and PNUE decreased (Table 2), which were possibly related with the selected genotypes and different expressions in water and nutrient use efficiency at different organ levels. Leaf veins traits were closely associated with PNUE but not related with WUEi since WUEi was mainly determined by gs, and PNUE was mainly determined by PN (Fig. 3 Suppl.).
Theoretically, leaves acclimated to higher nutrient supplies would have vein traits associated with higher leaf hydraulic conductance and gas exchange rate . VLA across rice cultivars was independent of Narea, but IVD was positively correlated with Narea (Xiong et al. 2015). In this study, we found Narea was negatively correlated with VLA, VLAmajor, and VLAminor, Fig. 2. Relationships between net photosynthetic rate (PN) or photosynthetic N use efficiency (PNUE), and leaf vein density (A,D, respectively), proportion of minor-veins (B,E, respectively) and interveinal distance(C,F, respectively) nin wheat genotypes with differing ploidy. Means ± SEs, n = 6. * -P < 0.05, ** -P < 0.01, *** -P < 0.001. and positively correlated with proportion of minor-vein length as well as IVD (Fig. 3). This can be explained by the developmental algorithm for vein formation  because leaf higher N content is accompanied with larger leaf area (R 2 = 0.88, P < 0.01). Although higher N content stimulated proportion of minor-vein length, N remobilization efficiency did not change during wheat evolution, the reason may be related to enlarged interveinal distance at higher N content, which increased distance for photosynthate transport to phloem. Leaf phloem transport was a complex process, and N remobilization efficiency may be affected by many factors, such as the distribution of phloem in the vein network, and on species' loading strategies, post-flowering leaf duration period, grain sink size, etc. Even tetraploids and hexaploids had higher Narea, their PN was lower than that of diploids, showing high N content in the tetraploids and hexaploids did not increase photosynthesis, contrasted with the studies claiming that leaf N content was closely linked to maximum assimilation across species (Field and Mooney 1986, Harrington et al. 1989, Wright et al. 2004. The reason may be related to lower N content allocation to the photosynthetic apparatus or high N demand in tetraploids and haxaploids. The biggest evolution advance of leaf vein and physiological traits occurred during the period from diploids to tetraploids, thereafter they changed little. Tetraploid Triticum dicoccoides (AABB) hybridized with Aegilops squarrosa (DD) to give rise to the cultivated hexaploid wheats. So, the increased D genome changed the leaf vein traits and physiological function a little and did not increase the photosynthesis. Watanabe et al. (1997) found that the increased doses of the D genome reduces photosynthesis, but the depression is dependent on the source of the D genome. This study partly supports their finding. Although there were not much difference in vein traits and physiological function between tetraploids and hexaploids, hexaploids achieved a higher yield than tetraploids, the relevant mechanism needs further study.
We found at leaf level, that larger biomass mainly attributed to the increase in leaf area, proportion of minor-vein length and Narea, while high yield was mainly attributed to increased proportion of minor-vein length, IVD, and Narea during the process of wheat evolution. It was not possible to determine the relative importance of each trait, but both VLAmajor and proportion of minor-vein length showed closer association with yield than other leaf traits, and also linked with leaf physiological function like Narea, PN, and PNUE, so they are important leaf attributes for yield increase in modern cultivars. Modern wheat genotypes may improve the carbon allocation proportion to grain production due to artificial selection by lowering carbon construction cost in leaf main veins (decreased VLAmajor) and/or by optimizing carbon distribution pattern among different orders of leaf vein system (increased proportion of minor-vein length). In contrast to the study in rice, where VLA was positively correlated with the grain yield (Feldman et al. 2017, Nawarathna et al. 2017, the potential of vein traits in future wheat breeding needs further testing.
Modern cultivars of the hexaploid Triticum aeativum produce larger grain yield than the more primitive diploid and tetraploid species despite they have lower PN Dunstone 1970, Austin et al. 1982). So, PN is not the reason of yield increase during wheat domestication, the increase in harvest index, grain, and leaf size, aboveground biomass, the proportion of dry mass mobilized to the grain and water and nutrient use efficiency may be the other major Fig. 3. Relationships between leaf vein density (A), proportion of minor-veins (B) and interveinal distance (C), and N content per area (Narea) in wheat genotypes with differing ploidy. Means ± SEs, n = 6. * -P < 0.05, ** -P < 0.01. factors in the selection of wheat varieties for increasing yield (Dunstone et al. 1973, Zhang et al. 2002, Xiong et al. 2006, Huang et al. 2007, Wang et al. 2017. This study showed decreased VLAmajor and increased proportion of minor-vein length were also closely associated with the yield improvement during wheat evolution. These traits are worthy to be further assessed in future wheat breeding.

Conclusions
The VLA, VLAmajor, and VLAminor decreased while proportion of minor-vein length and interveinal distance increased during wheat evolution, and the major advance occurred from diploids to tetraploids. VLA, VLAmajor, and VLAminor were closely correlated with PN and PNUE but not with gs. Proportion of minor-vein length and interveinal distance was positively related with Narea and negatively with PN and PNUE, respectively, during wheat evolution. Higher proportion of minor-vein length and interveinal distance, lower VLAmajor along with increased Narea in flag leaves were largely responsible for the increased yield in modern cultivars. This study demonstrates that evolution of leaf vein density in wheat is different from those in wild plants, but a high coordination still exists between leaf vein density and PN.The study highlights the proportion of minor-vein length, IVD, and VLAmajor as the potential indexes for future wheat high yield breeding.