QTL-seq and transcriptomic integrative analyses reveal two positively regulated genes that control the low-temperature germination ability of MTP-maize introgression lines.

KEY MESSAGE: Two candidate genes (ZmbZIP113 and ZmTSAH1) controlling low-temperature germination ability were identified by QTL-seq and integrative transcriptomic analyses. The functional verification results showed that two candidate genes positively regulated the low-temperature germination ability of IB030. Low-temperature conditions cause slow maize (Zea mays L.) seed metabolism, resulting in slow seedling emergence and irregular seedling emergence, which can cause serious yield loss. Thus, improving a maize cultivar's low-temperature germination ability (LTGA) is vital for increasing yield production. Wild relatives of maize, such as Z. perennis and Tripsacum dactyloides, are strongly tolerant of cold stress and can thus be used to improve the LTGA of maize. In a previous study, the genetic bridge MTP was constructed (from maize, T. dactyloides, and Z. perennis) and used to obtain a highly LTGA maize introgression line (IB030) by backcross breeding. In this study, IB030 (Strong-LTGA) and Mo17 (Weak-LTGA) were selected as parents to construct an F2 offspring. Additionally, two major QTLs (qCS1-1 and qCS10-1) were mapped. Then, RNA-seq was performed using seeds of IB030 and the recurrent parent B73 treated at 10 °C for 27 days and 25 °C for 7 days, respectively, and two candidate genes (ZmbZIP113 and ZmTSAH1) controlling LTGA were located using QTL-seq and integrative transcriptomic analyses. The functional verification results showed that the two candidate genes positively regulated LTGA of IB030. Notably, homologous cloning showed that the source of variation in both candidate genes was the stable inheritance of introgressed alleles from Z. perennis. This study was thus able to analyze the LTGA mechanism of IB030 and identify resistance genes for genetic improvement in maize, and it proved that using MTP genetic bridge confers desirable traits or phenotypes of Z. perennis and tripsacum essential to maize breeding systems.

QTL Mapping and a Transcriptome Integrative Analysis Uncover the Candidate Genes That Control the Cold Tolerance of Maize Introgression Lines at the Seedling Stage.

Chilling injury owing to low temperatures severely affects the growth and development of maize (Zea mays.L) seedlings during the early and late spring seasons. The existing maize germplasm is deficient in the resources required to improve maize's ability to tolerate cold injury. Therefore, it is crucial to introduce and identify excellent gene/QTLs that confer cold tolerance to maize for sustainable crop production. Wild relatives of maize, such as Z. perennis and Tripsacum dactyloides, are strongly tolerant to cold and can be used to improve the cold tolerance of maize. In a previous study, a genetic bridge among maize that utilized Z. perennis and T. dactyloides was created and used to obtain a highly cold-tolerant maize introgression line (MIL)-IB030 by backcross breeding. In this study, two candidate genes that control relative electrical conductivity were located on MIL-IB030 by forward genetics combined with a weighted gene co-expression network analysis. The results of the phenotypic, genotypic, gene expression, and functional verification suggest that two candidate genes positively regulate cold tolerance in MIL-IB030 and could be used to improve the cold tolerance of cultivated maize. This study provides a workable route to introduce and mine excellent genes/QTLs to improve the cold tolerance of maize and also lays a theoretical and practical foundation to improve cultivated maize against low-temperature stress.

[Analysis on breeding potential of eight synthetic populations to improve a Chinese maize hybrid Zhengdan 958].

Maize (Zea mays L.) populations are potential sources of favorable alleles absent in parental inbred lines to improve elite hybrids. The maize hybrid Zhengdan 958 has been hampered by the lack of favorable new alleles for improving yield and commodity quality. In the present study, 16 testcrosses made by using eight synthetic populations as the donors and the two parental lines of Zhengdan 958 as the receptors were evaluated in 2009 and 2010 at Shunyi, Beijing and Xinxiang, Henan Province for grain yield and test weight. Four genetic parameters were used to determine the breeding potential of eight synthetic populations as the donors to improve the target hybrid. Several synthetic populations were identified as the potential sources of favorable alleles absent in the target hybrid for each trait evaluated. The two most promising germplasms, WBMC-4 and Shanxi Syn3, had the potential for simultaneously improving grain yield and test weight of the target hybrid, which could be used to improve the parental lines Zheng 58 and Chang 7-2, respectively, and further broaden the germplasm base of Chinese heterotic groups PA and Sipingtou.

[Combining ability analysis for SP4 lines of maize from space flight].

Three maize (Zea mays L.) inbred lines 08-641, RP125, and 18-599 were carried into cosmic space by recoverable satellite "Shijian 8". Some mutant lines were selected from SP4 and combinations were made according to the NC II genetic design. The materials were planted in Sichuan and Yunnan separately to analyze combining ability based on the incomplete diallel cross design. The results showed that space flight affected the combining ability of mutant lines, and the GCA value of mutant lines were different in two kinds of environmental condition. The GCA of ear length, row per ear, kernel per row, and yield per plant for the mutant line C03 showed substantial increase compared with the control 08-641; the GCA of row per ear, kernel per row, and other yield component traits for the mutant lines C01 and C04 were significantly higher than those of the control. The SCA of yield and yield components for the combinations derived from the mutant lines C06, R18, and S22 were higher than others. These results laid a material foundation for maize breeding and provided some important references for improving and utilizing the mutant lines.

[Genetic analysis of maize cytoplasmic male sterile mutants obtained by space flight].

Three maize male sterile mutants were obtained from the offsprings of two maize inbred lines 18-599 and 08-641, which were carried into space by the Shijian 8 Satellite. The stability of male sterile expression was observed in different locations, years, and seasons. In order to analyze the genetic characteristic of male sterility, testcross, backcross and reciprocal cross were made with these male sterile plants. The results showed that the male sterility character was stable in different locations, years, and seasons, and the sterility was inheritable. Because the maintainer lines and restorer lines for these sterile materials were found, and there was no male sterile plant separated among the reciprocal cross F2. Thus, we concluded that these mutants could be cytoplasmic male sterile. Combining the results of male fertility restoration test and PCR analysis, we could conclude that the three male sterile mutants were classified into the CMS-C type in maize. Owing to their difference in fertility restoration, these mutants may belong to different subgroups of CMS-C type. The discovery of the three male sterile mutants increased the genetic diversity of CMS-C type, improved the tolerance to Bipolaris maydis, and laid a foundation for extensive application of CMS-C in seeds production.

RNA editing of mitochondrial functional genes atp6 and cox2 in maize (Zea mays L.).

RNA editing of two mitochondrial or organs genes, atp6 and cox2, in different tissues were analyzed using homonucleic but alloplasmic, and homoplasmic but heteronucleic maize (zea mays L.) as experimental materials. A total of 18 and 26 editing sites for atp6 conservative region transcript were identified by direct and clone sequencing, respectively. By direct sequencing 23 and 22 editing sites for cox2 transcript were identified in 48-2 and Huangzaosi nuclear backgrounds, respectively. From the direct sequencing results, the occurrence rates of different transcripts generally increase in sterile lines. It is concluded that RNA editing of atp6 and cox2 might have a certain relationship with maize CMS.

[Advance of Agrobaterium-mediated genetic transformation system of maize (Zea mays L.).].

The Agrobacterium-mediated transformation system is more and more attractive to scientists of microbiology, molecular biology and crop genetics and breeding due to its unique advantages, which promote its development rapidly in the past decade. Great success has been achieved in the Agrobacterium-mediated genetic transformation system in maize (Zea mays L.), an important food-supply crop. This article highlights the advances of Agrobacterium-mediated transformation system of maize and the major factors of the transformation process. Then, comments on the problems remained and the prospects of this transformation system are made.

[The editing sites in the transcripts of mitochondrial atp6 gene of maize sterile lines and maintainer line].

Using maize (Zea mays L.) cytoplasmic male-sterile lines T Huang Zao Si, C Huang Zao Si, S Huang Zao Si and maintainer line N Huang Zao Si as the plant materials, editing sites in the conservative area of mitochondrial atp6 gene transcripts of the 4 experimental materials' tassels, of which microspores had developed to uni-nucleate stage, were analyzed. The results showed that DNA sequences of the T, C and S male-sterile cytoplasms were completely unanimous, while being compared with the N-cytoplasm, all the sequences were similar except for the 27th and 28th nucleotides. However, the cDNA sequences of each cytoplasm were not always the same. By comparing DNA and cDNA sequences, we found that within the conservative area of atp6 gene transcripts there were 19, 22, 20 and 19 editing sites in the N, T, C and S cytoplasms, respectively. The 4 cytoplasms also shared 18 sites. The majority of the editings occurred at the 1st or the 2nd position of codons, which might alter the amino acid type. Most the shared editings were fully editing, and the 1st and the 19th sites were partially edited in nearly all cytoplasms, except for the 19th sites editing in the N-cytoplasm. The specific editings in each cytoplasm occurred in the form of partially editing. Thus the editing of atp6 gene in maize was not only sequence specific but also affected by cytoplasmic background. Furthermore, plant RNA editing was inclined to improve the predicted protein's hydrophobicity and enhance the conservation among species.

[Improvement on microwave technology of extracting polysaccharide from yacon leaves].

According to the extraction ratio of polysaccharide in yacon leaves, the comparison between microwave extraction and traditional hot water extraction was conducted, and the two-factor and three-level experiment on the microwave extraction of polysaccharide from yacon leaves was investigated. The result showed that the extraction ratio of polysaccharide by using microwave extraction was better than that by using traditional hot water extraction. Moreover, according to the result of variance analysis and multiple comparison, the optimum conditions for extraction of polysaccharide by using microwave technology from yacon leaves were as follows: 280W microwave power for 2 times and 15 minutes at every time.

[Cytological observation on pollen abortion of genetic male sterile mutant induced by space flight in maize].

In order to understand the cytological mechanism of pollen abortion of genetic male sterile mutant induced by space flight in maize, the sister cross population were used for sterility analysis and cytological observation. Intact anther observation, isolated cells observation and paraffin section were adopted in this research. The results showed that pollen abortion occured mostly in dyad stage of meiosis in genetic male sterile mutant. The dyad were degenerated with abnormal shape. In late anther developing stage, the tapetal cells were giant vacuolated and delayed degeneration. The pollen mother cells (PMC) began to dissolve and degenerate in a few anther before meiosis.

[Initial identification of quantitative trait loci controlling resistance to banded leaf and sheath blight at elongating and heading date in maize].

The genetic linkage map was constructed with 146 SSR markers based on a maize population consisting of 229 F2 individuals from the cross R15 (resistant) x 478 (susceptible), covering 1666cM on a total of ten chromosomes, with an average interval length of 11.4 cM. The disease index from the population of 229 F24 lines were evaluated for BLSB resistance under artificial inoculation at elongating stage and heading date. With the method of composite interval mapping (CIM) described in QTL cartographer v2.0 procedure, 9 QTLs among of 17 QTLs were identified on 1, 2, 3, 4, 5, 6 and 10 chromosomes at Jointing Stage, accounting for 3.72% to 9.26% of the phenotypic variance. The other 10 QTLs were identified on 2, 3, 4, 5, 6, 8 and 9 chromosomes at elongating stage and heading date, accounting for 4.27% to 9.27% of the phenotypic variance. Among of them, two QTLs were detected at two stages, which were located between bnlgl600-umc1818 and umc1006-umc1723 on chromosome 2 and 6, respectively. The result indicated that the significant difference about QTL Controlling Resistance between the two stages had a close connection with Developing Stages, which was showed on the resistance of banded leaf and sheath blight in maize. So this result provided new information to the resistance breeding of maize.

[The discovery of a specific DNA fragment associated with maize cytoplasmic male sterility and its differential display].

Three pairs of PCR primers were designed according to the mitochondrial DNA sequence. PCR amplification was applied to 3 sets of isonuclear alloplasm materials and 3 sets of isoplasm allonuclear materials. Multiplex PCR and general PCR protocol were adopted with total genomic DNA. As for the primers having detected polymorphsim between male sterility and its maintainers, differential display was conducted with mRNA from different development stage of microspore. The results showed as follows: with total genomic DNA template, primer P1-P2 has amplified a specific fragment only in all the male sterile materials, primer P5-P6 has amplified a specific fragment only in maintainer Huangzaosi, primer P3-P4 has no amplification in all the experiment materials. So primer P1-P2 can be used to distinguish male sterile cytoplasm and normal cytoplasm. RT-PCR was conducted with primer P1-P2 in inbred line huangzaosi and 48-2 with male sterile cytoplasm and normal cytoplasm, mRNA was separately isolated from tetrad stage, uninucleate stage and binucleate stage of microspore development, cDNA was obtained with random hexanucleotide primers. With the cDNA template, specific amplified fragments were also detected by primer P1-P2 in the male sterile materials at different development stage of microspore, but there was no amplification by primer P1-P2 in the 2 maintainer lines. This result indicated that primer P1-P2 can be transcripted at 3 development stages of microspore in all male sterile materials, and same transcript was produced by primer P1-P2 among all male sterile materials include 3 sets of isonuclear alloplasm and 3 sets of isoplasm allonuclear. It was suggested from this experiment that the specific DNA sequence detected by primer P1-P2 in all male sterile material total genomic DNA might be related to the cytoplasmic male sterile character.

[Locating maize male sterility gene induced by space flight using microsatellite markers].

Two F2 populations with different kinds of spike derived from maize male sterility materials RP(3)195 (A) x S37 (inbred line) ,which had been sib-bred for many generations,were used for sterility analysis and gene location. There were 138 and 247 plants in the two F2 populations respectively. Among the 326 pairs of microsatellite primers selected,56 were found polymorphic. Linkage analysis of F2 populations with the 56 pairs of primers showed that microsatellite markers bnlg197 and umc1012 were linked with the male sterility gene. The genetic distance between marker bnlgl97 and the male sterility gene in the two different F2 populations were 7 cM and 14.5 cM respectively. The genetic distance between marker umc1012 and the male sterility gene in the 138 plants was 28.5 cM. Thus the male sterility gene was located on chromosome 3L.

[Isolation of the capsid protein gene of maize dwarf mosaic virus and its transformation in maize].

The MDMV (Maize Dwarf Mosaic Virus, MDMV) CP (Coat Protein, CP) gene was cloned by RT-PCR method and introduced into the embryonic calli derived from immature embryos of elite inbred 18-599hong and 18-599bai via particle bombardment. Bombarded calli were selected on selection medium containing 5-10 mg/L (PPT) Bialaphos. From resistant calli, 79 plantlets were regenerated. 18 of 79 were grown and harvested. The results of Southern blotting and PCR analysis demonstrated that MDMV CP have been integrated into the genome of the transgenic plants. PCR-positive progeny plants were artificially inoculated with MDMV strain B, and the average chlorosis of the functional leaves of each plant was investigated. The typical symptoms were observed from the leaves of the control inbreds. while, the presence of the MDMV CP gene provided resistance to inoculation with MDMV strain B.

[The production and multi-color genomic in situ hybridization identification of maize-Z. perennis substituted material].

Genus Zea. L was composed of two sections: sect. Luxuriantes Doebley & Iltis including Z. dipperennis, Z. luxurians and Z. perennis, and sect. Zea. mays consisting only one species, annual Z. mays. To improve the biodiversity of germ-plasm in maize breeding, the study of transferring maize relatives gene into common maize were performed. Firstly, interspecific hybrids between maize (Zea. L) (2n = 20) and Z. perennis (2n = 40) were produced with the aim of transferring desirable horticultural traits from Z. perennis to maize. The F1 of maize x Z. perennis (2n = 30) plant had the most frequent configuration of 5 III + 5 II + 5 I, which were sterile and difficult to produce progeny because of genomic affinities. However,few F2 individuals of maize x Z. perennis could be obtained by some specially treatments, and one maize-like F2 plant were obtained, which were used as a female parent in backcrossing with maize parent. Twelve F2 x P1 ( BC1 F2 ) plants were obtained and then self-crossed to produce self-crossing generation of F2 x P1 (2n = 20) ( BC1 F3). The phenotypic characters of parents, F1 (2n = 30) hybrids, F2 and F2 x P1 were investigated, such as plant height, flowering, leaf shape and tillers. To further verify the genomic organization of maize-Z. perennis material, maize (inbred line 48-2) and BC1 F3 chromosomes, the root tip cells were analyzed by Multi-color GISH. We probed maize and BC1 F3 chromosomes with the probe mixture containing biotin-labeled Z. perennis genomic DNA and digoxigenin-labeled maize genomic DNA, the maize spread exhibited 10 chromosomes with yellow signals and the other 10 chromosomes carried green fluorescing bands. However, although the BC1 F3 was 2n = 20 in all cases. Multi-color GISH images revealed that 17 chromosomes had uniform signals similar to maize chromosomes, but dispersed red signals over the remaining three chromosomes were observed, which indicated that the 3 chromosomes originated from Z. perennis, and they were smaller than maize chromosomes. Data obtained from multi-color GISH images indicated that BC1 F3 was probably a substituted material from maize-Z. perennis.

[Genetic analysis about maize male sterile mutant obtained by space flight].

The seeds of maize single hybrid Chuandan No. 9 were carried into space in 1996 by satellite. After the seeds were planted in field in comparison with travel in space seeds which was not carried into space. Fortunately, male sterile plants were discovered in one of the ear rows. The stability of male sterile expression was observed in different years, different locations and different generations. In order to analysis the genetic characteristic of male sterility, test cross, sister cross, back cross, reciprocal cross and self-pollination were conducted with these male sterile plants. The results showed that the male sterility was stable in different years and different locations, it is inheritable from generation to generation. The sterility is controlled by a single nuclear recessive gene. The appearance of male sterile mutant is the conclusion of gene mutation which happened in nuclear by space flight. This mutant material always accompanies with lower plant height.

[A study of genetic stability of male sterile mutant of maize obtained from space flighted seeds].

OBJECTIVE: To select a male sterile mutant of maize through space flight for production application. METHOD: Air dried seeds of maize (Chuan Dan No. 9) were carried to space for 15 d. After returned to the earth, a male sterile mutant was selected and a sterile line was obtained through direction breeding. RESULT: It was found that the sterile material was thoroughly abortive. The sterile was trail stable and expressed a genetic feature of single recessive gene controlled nucleus sterility. CONCLUSION: The appearance of male sterile mutant was due to gene mutation caused by space conditions.