Comparative Analysis of Genetic Diversity in Landraces of Waxy Maize from Yunnan and Guizhou Using SSR Markers

Waxy maize landraces are abundant inYunnan and Guizhou of China. Genetic diversity of waxy maize landraces from Yunnan and Guizhou were analyzed using SSR markers. We screened 38 landraces with 50 primers that generated 3 to 6 polymorphic bands, with an average of 4.13 bands. Shannon＇s information indices for genetic diversity of the 14 waxy maize landraces from Yunnan varied from 4.9571 to 42.1138 and averaged 26.5252; Shannon＇s information indices for genetic diversity of the 24 waxy maize landraces from Guizhou varied from 22.0066 to 40.6320 and averaged 32.3156. For the 14 waxy maize landraces from Yunnan, the within-landrace genetic diversity accounted for 45.40% and the among-landrace genetic diversity accounted for 54.60% of the total genetic diversity observed. For the 24 waxy maize landraces from Guizhou, the within-landrace genetic diversity accounted for 50.76% and the among-landrace genetic diversity accounted for 49.24% of the total observed. Some individual landraces possessed as much as 96.86% of the total genetic diversity occurring among landraces within origins. Differentiation between geographic origins accounted for only 3.14% of the total genetic diversity. Both Yunnan and Guizhou would be the diversity centers and the original centers of waxy maize.

Weakened carbon and nitrogen metabolisms under post-silking heat stress reduce the yield and dry matter accumulation in waxy maize

Post-silking high temperature is one of the abiotic factors that affects waxy maize(Zea mays L. sinensis Kulesh) growth in southern China. We conducted a pot trial in 2016–2017 to study the effects of post-silking daytime heat stress(35°C) on the activities of enzymes involved in leaf carbon and nitrogen metabolisms and leaf reactive oxygen species(ROS) and water contents. This study could improve our understanding on dry matter accumulation and translocation and grain yield production. Results indicated that decreased grain number and weight under heat stress led to yield loss, which decreased by 20.8 and 20.0% in 2016 and 2017, respectively. High temperature reduced post-silking dry matter accumulation(16.1 and 29.5% in 2016 and 2017, respectively) and promoted translocation of pre-silking photoassimilates stored in vegetative organs, especially in leaf. The lower leaf water content and chlorophyll SPAD value, and higher ROS(H2O2 and O2^-·) content under heat stress conditions indicated accelerated senescent rate. The weak activities of phosphoenolpyruvate carboxylase(PEPCase), Ribulose-1,5-bisphosphate carboxylase(Ru BPCase), nitrate reductase(NR), and glutamine synthase(GS) indicated that leaf carbon and nitrogen metabolisms were suppressed when the plants suffered from a high temperature during grain filling. Correlation analysis results indicated that the reduced grain yield was mainly caused by the decreased leaf water content, weakened NR activity, and increased H2O2 content. The increased accumulation of grain weight and post-silking dry matter and the reduced translocation amount in leaf was mainly due to the increased chlorophyll SPAD value and NR activity. Reduced PEPCase and Ru BPCase activities did not affect dry matter accumulation and translocation and grain yield. In conclusion, post-silking heat stress down-regulated the leaf NR and GS activities, increased the leafwater loss rate, increased ROS generation, and induced pre-silking carbohydrate translocation. However, it reduced the post-silking direct photoassimilate deposition, ultimately, leading to grain yield loss.

Application of moderate nitrogen levels alleviates yield loss and grain quality deterioration caused by post-silking heat stress in fresh waxy maize

High temperature(HT)during grain filling is one of the most important environmental factors limiting maize yield and grain quality.Nitrogen(N)fertilizer is essential for maintaining normal plant growth and defense against environmental stresses.The effects of three N rates and two temperature regimes on the grain yield and quality of fresh waxy maize were studied using the hybrids Suyunuo 5(SYN5)and Yunuo 7(YN7)as materials.N application rates were 1.5,4.5,and 7.5 g plant-1,representing low,moderate,and high N levels(LN,MN,and HN,respectively).Mean day/night temperatures during the grain filling of spring-and summer-sown plants were 27.6/21.0°C and 28.6/20.0°C for ambient temperature(AT)and 35/21.0°C and 35/20.0°C for HT,respectively.On average,HT reduced kernel number,weight,yield,and moisture content by 29.8%,17.9%,38.7%,and 3.3%,respectively.Kernel number,weight,yield,moisture,and starch contents were highest under MN among the three N rates under both temperature regimes.HT reduced grain starch content at all N levels.HT increased grain protein content,which gradually increased with N rate.Mean starch granule size under MN was larger(10.9μm)than that under LN and HN(both 10.4μm)at AT.However,the mean size of starch granules was higher under LN(11.7μm)and lower under MN(11.2μm)at HT.Iodine binding capacity(IBC)was lowest under MN and highest under HN among the three N levels under both temperature regimes.In general,IBC at all N rates was increased by HT.Peak viscosity(PV)was gradually reduced with increasing N rate at AT.In comparison with LN,PV was increased by MN and decreased by HN at HT.Retrogradation percentage gradually increased with N rate at AT,but was lowest under MN among the three N rates at HT.LN+AT and MN+HT produced grain with high pasting viscosity and low retrogradation tendency.MN application could alleviate the negative effects of HT on the grain yield and quality of fresh waxy maize.

Effect of Shading During Grain Filling on the Physicochemical Properties of Fresh Waxy Maize

Grain physicochemical properties determine the table quality of fresh waxy maize. Two waxy maize varieties, Suyunuo 5 （shading tolerant） and FHN003 （shading sensitive）, were used to estimate the effect of shading （plants received 30% less radiation than control） during grain filling （from 0 d to 23 d after pollination） on physicochemical properties of fresh waxy maize grain. Shading decreased the grain fresh weight of Suyunuo 5 and FHN003 by 8.4 and 19.1%, respectively. Shading increased the grain water content of FHN003, whereas that of Suyunuo 5 was not affected. In both varieties for shading treatment, soluble sugar, starch and protein contents were decreased, whereas zein content was increased. The changes in globulin, albumin and glutenin contents under shading were variety dependent. In both varieties, shading decreased λmax, iodine binding capacity and the percentage of large starch granules （diameter 〉17 μm） but increased crystallinity. The results of rapid visco analysis showed that the viscosity characteristics （except for pasting temperature） of both varieties were decreased by shading; however, FHN003 was more severely affected than Suyunuo 5. Under shading, Antet and %R were decreased in both varieties, whereas the changes in △Hgol and transition temperatures were variety dependent. Hardness, cohesiveness and chewiness were decreased in both varieties. Significant differences in physicochemical characteristics were observed between the two varieties.

Effects of post-silking water deficit on the leaf photosynthesis and senescence of waxy maize

Waxy maize is widely cultivated under rainfed conditions and frequently suffers water shortage during the late growth stage.In this study,a pot trial was conducted to examine the effects of post-silking drought on leaf photosynthesis and senescence and its influence on grain yield.Two waxy maize hybrids,Suyunuo 5(SYN5)and Yunuo 7(YN7),were grown under the control and drought(soil moisture content was 70–80%and 50–60%,respectively)conditions after silking in 2016 and 2017.The decrease in yield was 11.1 and 15.4%for YN7 and SYN5,respectively,owing to the decreased grain weight and number.Post-silking dry matter accumulation was reduced by 27.2%in YN7 and 26.3%in SYN5.The contribution rate of pre-silking photoassimilates transferred to grain yield was increased by 15.6%in YN7 and 10.2%in SYN5,respectively.Post-silking drought increased the malondialdehyde content,but decreased the contents of water,soluble protein,chlorophyll,and carotenoid in the leaves.The weakened activities of enzymes involved in photosynthesis(ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase)and antioxidant system(catalase,superoxide dismutase and peroxidase)reduced the photosynthetic rate(Pn)and accelerated leaf senescence.The correlation results indicated that reduced Pn and catalase activity and increased malondialdehyde content under drought conditions induced the decrease of post-silking photoassimilates deposition,ultimately resulted in the grain yield loss.

Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines

Immature embryos from three elite Guizhou waxy maize inbred lines （W21019, B7, and QCL5036） were evaluated for their ability of forming callus and regeneration into plants. Immature embryos harvested at different physiological stages were used as explants to initiate callus on N6 basal medium with 0-3.5 mg L-1 of 2,4-dichlorophenoxy acetic acid （2,4-D）. The concentration of 2,4-D, physiological age of immature embryos and genotype had a significant effect （P0.05） on the percentage of embryogenic callus formed. The optimum 2,4-D concentration for the initiation of embryogenic callus was varied among the waxy maize genotypes from 2.0 mg L-1 （B7 and QCL5036） to 3.0 mg L-1 （W21019）. The shoots were generated from embryogenic callus which were transferred into the regeneration medium supplemented with 0-2.5 mg L-1 of 6-benzylaminopurine （6-BA）. 6-BA in the medium significantly promoted the regeneration of embryogenic callus. Embryogenic size was also an important factor that affected regeneration capacity. 0.6-0.7 cm was proved to be the best size for regeneration from embryogenic callus and the mean number of shoots per primary callus in all genotypes achieved the highest number. The ability of the plant regeneration was also affected by genotype. W21019 had the highest number of shoots formed per primary embryogenic callus. With the optimum condition, the highest mean number of shoots per primary callus was up to 12.13, 5.73, and 3.33 in line W21019, B7, and QCL5036, respectively. The successful regeneration of the two inbred lines provides a basis for development of genetic transformation to improve priority traits such as enhanced insects and drought tolerance.

Effects of high temperature during grain filling on physicochemical properties of waxy maize starch

Understanding the waxy maize starch physicochemical properties response to heat stress during grain filling could improve starch quality.The effects of heat stress during early（1-15 days after pollination,DAP） and late（16-30 DAP） grain filling stages on the starch physicochemical properties of four waxy maize varieties were evaluated.Crystallinity only increased in Suyunuo 5 after exposure to high temperature at late grain filling stage.The effects of heat stress on digestibility and swelling power were dependent on varieties and stages.Generally,swelling power was increased by heat stress at early grain development stage and digestibility was increased by high temperature at late grain filling stage,respectively.The results of correlation analysis indicated the starch with large granule size could swell well and easy digest.Peak,trough,final,and breakdown viscosities in response to heat stress were dependent on stages and varieties.In general,peak,trough and final viscosities were decreased and increased by heat stress at early grain formation and late grain filling stages,respectively;whereas the breakdown and setback viscosities were similar among the three treatments.Heat stress increased the gelatinization temperatures and retrogradation percentage.Gelatinization range decreased under heat stress at 1-15 DAP but remained constant under heat stress at 16-30 DAP in all varieties.The starch exposed to high temperature at 16-30 DAP presented higher digestibility and peak viscosity and lower retrogradation percentage than those at 1-15 DAP.Therefore,heat stress at early grain formation stage severely affects the physicochemical properties of starch.

Waxy allele diversity in waxy maize landraces of Yunnan Province, China

Waxy maize is one of the main fresh-eating maize types,and a mutation of the waxy gene causes the waxy character of maize grains.China is rich in waxy maize landraces,and Yunnan and its surrounding areas,are the place of origin and genetic diversity center of Chinese waxy maize.The six known waxy alleles of Chinese waxy maize are wx-D7,wx-D10,wx-Cin4,wx-124,wx-Reina,and wx-Xuanwei.The mutation sites of these alleles all occur in the coding region of the waxy gene,however,the mechanism by which the waxy characteristic is caused by the mutation in the regulatory region has only been reported rarely in maize.In this study,405 waxy maize landraces from Yunnan were used as materials to identify the insertion and deletion of a large sequence fragment in the upstream~3.5 kb regulatory region of the waxy gene by molecular marker detection.Three different waxy alleles were identifed in this study:wx-PIF/Harbinger,wx-hAT and wxElote2.These three types of mutations all represented transposons inserted into the regulatory region of the waxy gene.Wx-PIF/Harbinger was a 304-bp MITE class transposon insertion belonging to the PIF/Harbinger family,while wx-hAT was a 560-bp MITE class transposon insertion belonging to the hAT family,and wx-Elote2 was a 6560-bp LTR-like transposon insertion.In this study,the alleles were identifed for more than 70%of the waxy maize landraces in Yunnan,which provids a basis for the utilization of these waxy maize landraces.

Effect of Foliar Application of Selenium Fertilizer on Yield,Selenium Content and Heavy Metal Contents of Waxy Maize

[Objectives]This study aimed to explore the effect of different concentrations of selenium fertilizer on the yield,selenium content and heavy metal contents of waxy maize,thereby providing reference for the scientific application of selenium fertilizer on waxy maize.[Methods]Different varieties of waxy maize(Guitiannuo 611,Guinuo 615 and Guiheinuo 609)were used as test materials.They were sprayed with different concentrations(0,0.5,1.0,1.5 and 2.0 g/L)of water-soluble selenium fertilizer(Xinxibao)at the big flare stage,and the effect on the yield,selenium content and heavy metal contents of the waxy maize was analyzed.[Results]Foliar application of different concentrations of selenium fertilizer showed no significant effect on the yield and yield components of waxy maize.As the concentration of selenium fertilizer increased,the grain selenium content of different varieties of waxy maize increased.Among them,the increase of Guinuo 615 was the largest,of which the selenium content reached 0.265 mg/kg,2.82 times that of the control(water).The cadmium and arsenic contents of different varieties of waxy maize decreased with the increase of the concentration of selenium fertilizer sprayed.Among the waxy maize varieties,the cadmium and arsenic contents of Guitiannuo 611 dropped the most significantly,up to 52.94%and 77.78%.In waxy maize,the correlation coefficients between selenium content and cadmium,arsenic contents were-0.5509(P<0.05)and-0.8530(P<0.01),respectively.[Conclusions]Spraying exogenous selenium fertilizer had no obvious effect on the yield of waxy maize.However,the increase of the concentration of exogenous selenium fertilizer could significantly increase the selenium content and reduce the cadmium and arsenic contents in grains of different varieties of waxy maize.

Composition of Starch and Protein in the Endosperm of Newly Generated Double Recessive Waxy and Opaque 2 Maize （Zea mays L.） Genotypes

Waxy maize with its pure amylopectin starch is the staple food of many ethnic minorities in hilly regions of Southeast Asia （SEA）. A combination of waxy and quality protein maize （QPM） traits would improve the quality of protein of waxy maize for human consumption. Double recessive waxy-QPM （wx-o2） genotypes had been generated from Southern Chinese material by haploid induction of crosses heterozygous for the two quality traits with an absolutely conserved waxy type and an improved amino acid profile. The vitreous kernel trait （due to the additional modifier genes present in QPM） was lost in the wx-o2 plant material; this may be due to the waxy mutation, this is anyhow desirable for acceptance as waxy maize is preferred due to its soft grains. The content of the quality limiting amino acid lysine was greatly increased in double recessive wx-o2 genotypes compared to standard waxy maize, but still with a high variation among genotypes for future improvement. Conclusively, it was indeed possible to combine two grain quality mutations successfully within one genotype and prototypes of double quality wx-o2 are available now to contribute to meet human requirements in essential amino acids and thus reduce malnutrition in various regions of Asia.

Conversion of a normal maize hybrid into a waxy version using in vivo CRISPR/Cas9 targeted mutation activity

Waxy maize is a specialty maize that produces mainly amylopectin starch with special food or industrial values. The objective of this study was to overcome the limitations of wx mutant allele acquisition and breeding efficiency by conversion of parental lines from normal to waxy maize. The intended mutation activity was achieved by in vivo CRISPR/Cas9 machinery involving desired-target mutation of the Wx locus in the ZC01 background,abbreviated as ZC01-DTM^(wx). Triple selection was applied to segregants to obtain high genome background recovery with transgene-free wx mutations. The targeted mutation was identified, yielding six types of mutations among progeny crossed with ZC01-DTM^(wx).The amylopectin contents of the endosperm starch in mutant lines and hybrids averaged94.9%, while those of the wild-type controls were significantly(P < 0.01) lower, with an average of 76.9%. Double selection in transgene-free lines was applied using the Bar strip test and Cas9 PCR screening. The genome background recovery ratios of the lines were determined using genome-wide SNP data. That of lines used as male parents was as high as98.19% and that of lines used as female parents was as high as 86.78%. Conversion hybrids and both parental lines showed agronomic performance similar to that of their wild-type counterparts. This study provides a practical example of the efficient extension of CRISPR/Cas9 targeted mutation to industrial hybrids for transformation of a recalcitrant species.