Population genomic analysis reveals key genetic variations and the driving force for embryonic callus induction capability in maize

Genetic transformation has been an effective technology for improving the agronomic traits of maize.However,it is highly reliant on the use of embryonic callus(EC)and shows a serious genotype dependence.In this study,we performed genomic sequencing for 80 core maize germplasms and constructed a high-density genomic variation map using our newly developed pipeline(MQ2Gpipe).Based on the induction rate of EC(REC),these inbred lines were categorized into three subpopulations.The low-REC germplasms displayed more abundant genetic diversity than the high-REC germplasms.By integrating a genome-wide selective signature screen and region-based association analysis,we revealed 95.23 Mb of selective regions and 43 REC-associated variants.These variants had phenotypic variance explained values ranging between 21.46 and 49.46%.In total,103 candidate genes were identified within the linkage disequilibrium regions of these REC-associated loci.These genes mainly participate in regulation of the cell cycle,regulation of cytokinesis,and other functions,among which MYB15 and EMB2745 were located within the previously reported QTL for EC induction.Numerous leaf area-associated variants with large effects were closely linked to several REC-related loci,implying a potential synergistic selection of REC and leaf size during modern maize breeding.

Genetic Diversity of Maize (Zea mays L.) Landraces from Southwest China Based on SSR Data

Genetic diversity of 54 maize landraces from southwest China was tested using bulk DNA samples and 42 microsatellite （SSR） loci distributed on 10 chromosomes of maize. A total of 256 alleles were detected among the landraces. At each locus, the number of alleles varied from 2 to 9, with an average of 6.1. On the basis of the genetic similarity coefficients, clustering analysis separated the landraces into four groups. The landraces collected from the same region were mostly grouped together. To reveal the genetic structure and genetic diversity within landraces, 165 individuals from 11 landraces were analyzed. Individual DNA samples proved to be superior to bulk DNA samples in identifying genetic diversity of landraces. A total of 330 alleles were detected in the 11 landraces. According to the results of the individual DNA sampling analysis, estimates of the mean number of alleles ‘A’, the effective allelic number ‘Ae’, the observed heterozygosity ‘Ho’, and expected heterozygosity ‘He’ were 7.86, 3.90, 0.69, and 0.37, respectively. An obvious genetic deviation from Hardy-Weinberg expectation was observed both among and within landraces and a considerable genetic variation was revealed within rather than among landraces. In addition, genetic diversity of landraces was greater in Sichuan than in the other three regions. It can be concluded that maize landraces in southwest China were initially introduced to Sichuan and from there to adjacent areas.

Genetic Diversity Analysis of Dominant Middle-late Mature Maize Varieties in Jilin Province

[Objective] The aim was to study the genetic diversity of dominant middle-late mature maize hybrids in Jilin Province by the use of Simple sequence repeats （SSRs） molecular mark.[Method] PCR amplification was carried out based on ten core SSR primers and the amplification products were detected through sodium dodecyl sulfate polyacrylamide gel electrophoresis.[Result] Ten core SSR primers gave clear and stable profiles amplified in samples of 43 maize hybrids.A total of 110 polymorphic amplified fragments were produced by 10 primers.The average number of allele per SSR locus was 11 which varied from 4 to 17.The polymorphism information content （PIC） for the SSR loci ranged from 0.63 to 0.93 with an average of 0.842.Genetic similarities among the 43 maize hybrids varied from 0.664 to 0.918 with an average of 0.774 and those among 30 maize hybrids were greater than 0.85.Combined with the cluster analysis and pedigree origin of 43 maize hybrids,it was found that there were more genetic similarities among the 43 maize hybrids.[Conclusion] The germplasm resources of middle-late mature maize hybrids in Jilin Province were comparatively narrow and waited for innovation.

Studies on Genetic Transformation of NPR1 Gene into Maize by Microprojectile Bombardment

[Objective] This study aimed to explore the conditions of transformation of maize by microprojectile bombardment. [Method] Immature embryo-derived callus of maize inbred line 7239 was used as explants to study the effects of shoot distance, helium pressure, vacuum and bombardment frequency on the transformation efficien- cy in the particle bombardment system of maize. [Result] Considering the transfor- mation efficiency, particle bombardment with 100 μg/P of golden particles, at a shoot distance of 9 cm from the target cells, under helium pressure of 1 350 psi and vac- uum 25 inHg, and bombarding twice could achieve relatively ideal results. After se- lection on media supplemented with different concentration of hygromycin, some re- generated plants were obtained. The results of PCR and Southern blotting analysis demonstrated that the NPR1 gene had been integrated into the genome of trans- genic maize plants, with an average transformation efficiency of 1.76%. [Conclusion] The study laid the foundation for the cultivation and breeding of excellent resistant varieties of maize.

Analysis on Combining Ability and Genetic Parameters of Main Agronomic Characters of 22 Maize Inbred Lines

The general combining ability（GCA）, special combining ability（SCA） and genetic parameter of ten characters of 22 maize inbred lines including plant height and ear height were analyzed using 10×12 through incomplete diallel cross（NC Ⅱ）.The results showed that：（1） Among the 22 maize inbred lines, the yield GCAs of11 HN 097, 11 HN 099, 11 HN 105 and 11 HN 110 were high, which were elite inbred lines to collocate hybridized combinations with strong heterosis. The yield of11 HN110 × 11 HN097, 11 HN110 × 11 HN105, 11 HN112 × 11 HN 097 and 11 HN 106 × 11 HN 104 were in the first four place. The yielding abilities, adaptabilities and yielding stabilities of the four combinations can be further identified by experiment. The heredities of the ten characters were mainly controlled by additive gene effect whereas the influence of non-addictive gene effect was small. The narrow heritabilities of plant height, ear height, ear rows, ear length, kernels per row,100-grain weight and seed-producing percentage were more than 50%. The variances were mainly caused by heredity and early-generation selection should be conducted. The narrow heritabilities of ear diameter, bare tip length and yield was low, which should not be selected in early-generation.

Uncertainty in Measuring Construct-specific Fragments of Genetically Modified Maize MON863 by Real Time Quantitative PCR

[Objective] The study aimed at evaluating the uncertainty in measuring the construct-specific fragments of genetically modified maize MON863 by real time quantitative PCR.[Method] The content of construct-specific fragments in MON863 samples was determined by real time quantitative PCR,and then the uncertainty of measurement result was evaluated according to the sources of uncertainty like the PCR system,the data processing and the micropipette.[Result] Type A evaluation of uncertainty（uA） in the measurement was 1.7×10^-2;Type B evaluation of uncertainty（uB） was 9.0×10^-4;the combined standard uncertainty（uC） was 1.7×10^-2;the expanded uncertainty（U95） was 0.036 and the finally measured result was 1.08%±0.036.[Conclusion] The main uncertainty of the result measured by real time quantitative PCR came from the randomizing effect in the experimental process.

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.

Genetic Diversity of Maize Populations Developed by Two Kinds of Recurrent Selection Methods Investigated with SSR Markers

Two cycles of biparental mass selection （MS） and one cycle of half-sib-S3 family combining selection （HS-S3） for yield were carried out in 2 synthetic maize populations P4C0 and P5C0 synchronously. The genetic diversity of 8 maize populations, including both the basic populations and their developed populations, were evaluated by 30 SSR primers. On the 30 SSR loci, a total of 184 alleles had been detected in these populations. At each locus, the number of alleles varied from 2 to 14, with an average of 6.13. The number and ratio of polymorphic loci in both the basic populations were higher than those of their developed populations, respectively. There was nearly no difference after MS but decreased after HS-S3 in both the basic populations in the mean gene heterozygosity. The mean genetic distance changed slightly after MS but decreased in a bigger degree after HS-S3 in both the basic populations. Analyses on the distribution of genetic distances showed that the ranges of the genetic distance were wider after MS and most of the genetic distances in populations developed by HS-S3 were smaller than those in both the basic populations. The number of genotypes increased after MS but decreased after HS-S3 in both the basic populations. The genetic diversity of intra-population was much more than genetic diversity of inter-population in both the basic populations. All these indexes demonstrated that the genetic diversity of populations after MS was similar to their basic populations, and the genetic diversity was maintained during MS, whereas the genetic diversity of populations decreased after HS-S3. This result indicated that heterogeneity between some of the individuals in the developed populations increased after MS, whereas the populations become more homozygotic after HS-S3.

Genetic Diversity of Chinese Temperate and Exotic Tropical, Subtropical Quality Protein Maize Inbreds by SSR Markers

Information on genetic relationship is of great value to maize (Zea mays L.) breeding. Theobjectives of this study were: 1) to classify 22 quality protein maize (QPM) inbreds intodifferent groups by using simple sequence repeats (SSR) markers, which included exotictropical, subtropical and domestic temperate QPM and normal maize inbreds; 2) to examine theconsistency of grouping results obtained from SSR, specific combining ability (SCA) analysis,and genetic backgrounds of these inbreds. A set of 39 polymorphic SSR primers was selected from70 primer pairs, which detected 136 alleles among the 22 lines. The mean polymorphisminformation content was 0.55. Based on analysis of genetic similarities, five groups wereidentified including Luda Red Cob, Sipingtou, Reid, Lancaster and a miscellaneous group withseveral tropical inbreds which could not be classified into the above four groups. The resultsgenerally agreed with previous results based on analysis of yield combining ability andpedigree data.

Genetic Diversity of Two Important Groups of Maize Landraces with Same Name in China Revealed by M13 Tailed-Primer SSRs

Maize landraces White Dent and Golden Queen played a very important role in the pre-hybrid era of maize production in China. However, dozens of accessions with the same names of White Dent and Golden Queen are preserved in China National Genebank （CNG）. The present study investigated the genetic diversity of these two important groups of maize landraces, as well as the relationships within and among them. Thirty-four landrace accessions with the name of White Dent and 10 with Golden Queen preserved in CNG were fingerprinted with 52 simple sequence repeats with tailed primer M13. Summary statistics including average number of alleles per locus, gene diversity/expected heterozygosity, and observed heterozygosity were carried out using PowerMarker ver. 3.25 software. The test of Hardy-Weinberg equilibrium （HWE） and linkage disequilibrium （LD） of all the 44 maize landrace accessions were also performed by PowerMarker. We observed a significant differentiation in terms of the average number of alleles between White Dent and Golden Queen （6.44 alleles per locus in White Dent, 4.48 in Golden Queen）, while both groups of maize landraces had a relatively high but similar gene diversity （0.61 of White Dent, 0.63 of Golden Queen）. The fixation index （FST） was only 0.0044, while the percentage of loci deviated from Hardy-Weinberg equilibrium within these two groups of White Dent and Golden Queen was 32.69 and 3.92%, respectively. The rather high genetic diversity and average number of alleles per locus confirmed that both groups of landraces had a rather broad germplasm base. The extremely low fixation index showed that there was little genetic variation between White Dent and Golden Queen and the molecular variation within these two groups was remarkably high, indicating no genetic drift between White Dent and Golden Queen and suggesting different improvement approaches to these two important groups of landraces. Hardy-Weinberg equilibrium test revealed that the group of White Dent was deviated from HWE, whereas Golden Queen was under HWE.

Analysis of Genetic Diversity and Population Structure of Maize Landraces from the South Maize Region of China

Understanding genetic diversity and population structure of landraces is important in utilization of these germplasm in breeding programs. In the present study, a total of 143 core maize landraces from the South Maize Region （SR） of China, which can represent the general profile of the genetic diversity in the landraces germplasm of SR, were genotyped by 54 DNA microsatellite markers. Totally, 517 alleles （ranging from 4 to 22） were detected among these landraces, with an average of 9.57 alleles per locus. The total gene diversity of these core landraces was 0.61, suggesting a rather higher level of genetic diversity. Analysis of population structure based on Bayesian method obtained the samilar result as the phylogeny neighbor-joining （N J） method. The results indicated that the whole set of 143 core landraces could be clustered into two distinct groups. All landraces from Guangdong, Hainan, and 15 landraces from Jiangxi were clustered into group 1, while those from the other regions of SR formed the group 2. The results from the analysis of genetic diversity showed that both of groups possessed a similar gene diversity, but group 1 possessed relatively lower mean alleles per locus （6.63） and distinct alleles （91） than group 2 （7.94 and 110, respectively）. The relatively high richness of total alleles and distinct alleles preserved in the core landraces from SR suggested that all these germplasm could be useful resources in germplasm enhancement and maize breeding in China.

QTL mapping of general combining abilities of four traits in maize using a high-density genetic map

General combining abilities （GCAs） are very important in utilization of heterosis in maize breeding. However, its genetic basis is unclear. In the present study, a set of 118 doubled haploid （DH） lines were induced from F1 generations produced from the cross between the inbred line Zheng 58 and the inbred line W499 belonging to the Reid subgroup. Using the MaizeSNP50 BeadChip, a high-density genetic map was constructed based on the DH population which included 1 147 bin markers with an average interval length of 2.00 cM. Meanwhile, the DH population was crossed with three testers including W16-5, HD568, and W556, which belong to the Sipingtou subgroup. The GCAs of the ear height （EH）, the kernel moisture content （KMC）, the kernel ratio （KR）, and the yield per plant （YPP） were estimated using these hybrids in three environments. Combining the high-density genetic map and the GCAs, a total of 14 QTLs were detected for the GCAs of the four traits. Especially, one pleiotropic QTL was identified on chromosome 1 between the SNP SYN16067 and the SNP PZE-101169244 which was simultaneously associated with the GCAs of the EH, the KR, and the YPP. These QTLs pave the way for further dissecting the genetic architecture underlying GCAs of the traits, and they may be used to enhance GCAs of inbred lines under the fixed heterotic pattern ReidxSipingtou in China through a marker-assisted selection approach.

Study of Yield Combining Ability and Genetic Relationship Among Exotic Tropical,Subtropical Maize(Zea mays L.) Inbreds and Temperate Maize Inbreds in China

Information on the genetic relationship between tropical maize (Zea mays L), germplasm and temperate maize germplasm is of great value to maize breeding. The objective of this study was to determine the combining ability and genetic relationship of 25 inbreds extracted from five tropical maize populations and a land race, with four temperate maize inbreds (Huangzaosi, Mol7, B73 and Dan 340). The 25 tropical inbreds were crossed with the four temperate inbreds and evaluated. Lines from Suwanl and POP28 had high general combining ability (GCA) for grain yield. The lines from POP32 (ETO) had the highest special combining ability (SCA) with B73; the average SCA value of the 5 lines was 879 kg/ha. The lines from Suwanl had the second-highest SCA (584 kg/ha) with Huangzaosi. The lines from Suwanl had the greatest relative heterosis (20%) with B73, followed by the lines from POP32 (ETO) with B73 (19%). Five heterotic patterns have been identified from this study: Suwanl × Reid, ETO × Reid, POP28× Reid, POP28× Ludahong-gu, and Suwan1× Lancaster.

Genetic Analysis of the Resistance to Aspergillus flavus Infection in Maize (Zea mays L.)

Maize （Zea mays L.）, one of main crops in the world, is easily susceptible to Aspergillusflavus （Link： fr） infection, resulting huge loses worldwide. Breeding for A. flavus resistance has been proved an efficient way to solve the problem of aflatoxin contamination. Genetic analysis of the sources of resistance to A.flavus in maize is necessary for this purpose. The complete diallel crosses of 6 inbred lines with different resistance to A.flavus infection were implemented. Inoculation categorical data of each cross were analyzed with the additive-dominant and additive-dominant-epitasis genetic models. Results indicated some crosses fitted the 2 major genes with additive-dominant-epitasis genetic model. Others fitted the major gene and polygene mixed model. Moreover, the additive, dominant, and epitasis effects varied in crosses. The A.flavus resistance was controlled by both major gene and polygene.

A Comparative Analysis of B Chromosomes and Genetic Diversity in Maize (Zea mays L.) Landraces from Southwest China

The number of B chromosomes （Bs） in 54 maize landraces from Southwest China was tested by means of cytological observations. Nine landraces with Bs were observed. A map, showing the geographic distribution of the landraces with Bs, was plotted. It was found that southeastern Sichuan Province in China was the main distribution area of the landraces with Bs in Southwest China. In order to obtain information on relationships between Bs and genetic variation, genetic diversity both among and within 11 landraces was evaluated. For each SSR marker, the number of alleles ranged from 3 to 12 with an average of 7.86, which revealed a high level of genetic diversity among maize landraces in Southwest China. Based on SSRs data, higher genetic variation was found in the landraces with 2B, and the genetic distance between the landraces with and without Bs was higher. The results together with the principal component analysis （PCA） supported the hypothesis that maize landraces in Southwest China were first introduced to the middle part of southwest Sichuan, China. At the same time, the effect of Bs on genetic variation was discussed.

Determination of the Number of SSR Alleles Necessary for the Analysis of Genetic Relationships Between Maize Inbred Lines

The amount of molecular marker information has considerable impact on the results of studies of crop germplasm genetic relationships in crop. The number of alleles required to reveal genetic relationship in maize inbred lines is a theoretical issue that needs to be addressed. In this study, 112 pairs of SSR （simple sequence repeat） primers and 97 maize inbred lines were selected to study the relationship between the number of inbred lines and the number of SSR primers and alleles required for a stable cluster. The results showed that the number of SSR primers is not tightly associated with the stability of the cluster analysis results, while an increase in the number of alleles can significantly improve the stability of cluster analysis results. The number of inbred lines （X） is significantly associated with the number of alleles required for stable cluster analysis （Y）, and the regression equation is Y- 600.8xe（-15.9/x）. This equation can be used to calculate the number of SSR alleles required for a genetic relationship study of maize inbred lines. These results provide a reference for determining of SSR alleles number in genetic relationship analysis of maize inbred line and other crop germplasm.

Genetic analysis of maturity and flowering characteristics in maize(Zea mays L.)

Objective:To elucidate the pattern of inheritance and determine the relative magnitude of various genetic effects for maturity and flowering attributes in subtropical maize.Methods: Four white grain maize inbred lines from flint group of corn,two with late maturity and two with early maturity,were used.These contrasting inbred lines were crossed to form four crosses.Six generations(P_1,P_2,F_1,F_2,BC_1,and BC_2) were developed for each individual cross.These were evaluated in triplicate trial for two consecutive years.Results:Roth dominance gene action and epistatic interaction played major role in governing inheritance of days to pollen shedding, 50%silking,anlbesis silking interval and maturity.Conclusions:Preponderance of dominance gene action for these traits indicated their usefulness in hybrid programs of subtropical maize.

Genetic Relationships Among Chinese Maize OPVs Based on SSR Markers

Bulk-SSR method was used to analyze the genetic diversity of 44 open-pollinated varieties collected from Henan, Shandong, Shanxi, and Jilin provinces and Guangxi Zhuang Autonomous Region, China using 70 pairs of SSR primers. The purposes of this study were to （1） compare the genetic diversity among 44 Chinese maize open-pollinated varieties; （2） estimate the minimum number of alleles for construction of a stable dendrogram; and （3） trace the genetic relationships among local germplasm from different regions of China. In total, these 70 SSR primers yielded 292 alleles in 176 samples （4×44） analyzed. The number of alleles per locus was 4.17 on average and ranged from 2 to 8. The highest number of alleles per open-pollinated variety （55.25） was detected in Shanxi germplasm, which indicated that open-pollinated varieties from Shanxi possessed the largest genetic diversity among those from the five locations. The correlation coefficients between different genetic similarity matrices suggested that 200 alleles were sufficient for analysis of the genetic diversity of these 44 open-pollinated varieties. The cluster analysis showed that 44 open-pollinated varieties collected from three growing regions in China were accurately classified into three groups that were highly consistent with their geographic origins, and there is no correlation between GS and geographic distance in this study.

An Analysis on the Relationship between Maize Heterosis and Genetic Distance

13 normal maize inbred lines with quite different quantitative traits were used as experimental materials in this paper. The results showed that there was significantly positive correlation between genetic distance and plot yield,mid-parent heterosis,super heterosis,or SCA of yield. In a certain range,the longer the genetic distance between parental inbred lines,the stronger the heterosis. Therefore,in genetic practice,it was necessary to take the molecular genetic distance between inbred lines as an important reference.

Phylogenetic Analysis among Maize Exserohilum turcicum Isolates from Yunnan Province by RAPD

[ Objective ] This study aimed to investigate the genetic variation of Yunnan Exserohilum turcicum isolates from molecular level, and provide theoretical basis for E. turcicum pathogenicity virulence differentiation and effective control of disease. [Method] A total of 56 E. turc/cum isolates from some areas of Yunnan Province were analyzed by RAPD. Based on the genetic distance, a dendrogram was constructed. [Result] Ten genetic groups were formed in the dendrogram. The RAPD groups had no obvious correlation with geographic origins. Some strains from one area were closely related to some from another area. [ Conclusion] Rich ge- netic variation existed among the tested isolates.