Deep Sequencing of the Pistil Transcriptome in the Cross Incompatible Maize  

Song Li1 , Chunyan Xu1 , Dezhou Cui1 , Huaihua Liu1 , Peng Li1 , Hua Zhang2 , Detao Li1 , Youhui Tian1 , Huabang Chen2 , Xianrong Zhao2
1. The State Key Lab of Crop Biology, College of Agriculture, Shandong Agricultural University, Tai’an, Shandong, P. R. China
2. The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China
Author    Correspondence author
Molecular Plant Breeding, 2014, Vol. 5, No. 10   doi: 10.5376/mpb.2014.05.0010
Received: 14 Apr., 2014    Accepted: 21 Apr., 2014    Published: 11 Jul., 2014
© 2014 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Li et al., 2014, Deep Sequencing of the Pistil Transcriptome in the Cross Incompatible Maize, Molecular Plant Breeding, 2014, Vol.5, No. 10 1-14 (doi: 10.5376/mpb.2014.05.0010)

Abstract

Plant reproductive development is dependent on successful pollen-pistil interactions. The high degree of specificity in pollen-pistil interactions and the precision of directional pollen tube growth suggest that signals are continually being exchanged between pollen tubes and cells of the pistil that line their path. However, with few exceptions, little is known about the genes that control these interactions. In this investigation, we performed genome-wide transcriptional profiling of pistil tissues of Gametophytic factor1-Strong (Ga1-S) genotype maize after pollination 5 hours(5HAP)with Ga1-S pollen and ga1 pollen respectively , in an attempt to isolate genes involved in pollen tube elongation in maize pistils, using the Solexa deep sequencing technology. The results of this study suggest that the regulation of reinforcing, modifying, or remodeling the structure of the transmitting tract and the pollen tube tip play a critical role in pollen-pistil interactions during pollen tube elongation in maize pistils, and the inhibition of these functions maybe the molecular mechanism of how Gametophytic factor1-Strong (Ga1-S) presence in style tissue completely prevents ga1 pollen to accomplish fertilization.

Keywords
Deep sequencing; Transcriptome; Cross incompatibility; Pistil; Maize

Plant reproductive development is dependent on successful pollen-pistil interactions. The male gametophyte has a very short life-span in the life cycle of higher plants and the pollen grain is a rather simple two- or three-celled organism (Mihaela L. Ma´rton, Simone Cordts, Jean Broadhvest, & Dresselhaus, 2005). However, it is able to exist as a free organism, and once on a female receptive surface, to germinate and produce a pollen tube that, through style tissues, reaches the ovary and achieves fertilization. This process represents a peculiar and striking example of cell migration in plant development, largely based on cell surface interactions between the male gametophyte and the female sporophytic tissues. The high degree of specificity in pollen-pistil interactions and the precision of directional pollen tube growth suggest that signals are continually being exchanged between pollen/pollen tubes and cells of the pistil that line their path(Cheung, 1995; Herrero & Hormaza, 1996; Lord, 2003; Wolters-Arts, Lush, & Mariani, 1998). However, with few exceptions, little is known about the genes that control these interactions.

Recently, Ga1-s was reported in 6 of 14 annual teosinte populations(Lausser, Kliwer, Srilunchang, & Dresselhaus, 2010). However, all of the associated landrace maize populations carried the cross-neutral allele Ga1-m, which fertilizes Ga1-s, but accepts ga1pollen. Thus it is not obvious how Ga1-s could serve as a primary barrier to crossing in this circumstance(A. M. Sanchez et al., 2004).
Many popcorn strains cannot be fertilized by pollen of dent and flint strains although the reciprocal crosses are successful. Similarly, plants of some annual teosinte populations can fertilize maize but do not accept its pollen. Single genes or gene complexes govern these two unilateral barriers to crossing. Failure of fertilization could reflect active rejection by the pistil of pollen containing a contrasting allele (incompatibility). Alternatively, the pistil could require presence of a matching allele in pollen (congruity) (Allen, Thorogood, Hegarty, Lexer, & Hiscock, 2011; Ashman, 1975). To distinguish between these possibilities genetically, the receptivity to pollen having both alleles was determined. If there is active rejection, heteroallelic pollen would not be accepted; if presence of a matching allele is required, heteroallelic pollen would be accepted. In both the popcorn and teosinte crossing barrier systems, heteroallelic pollen functioned, consistent with the congruity model (Ashman, 1975; Boavida, Vieira, Becker, & Feijo, 2005; J. L. Kermicle, 2006; Nelson, 1994).
In order to identify and isolate genes involved in pollen-pistil interaction, we are focusing on the maize mutant Gametophytic factor1 (Ga1), one of the several genes shown to be responsible of the phenomenon of cross sterility in maize and other species. In fact, Gametophytic factors strongly affect pollen tube growth: pollen bearing recessive ga alleles are much less competitive as compared to pollen carrying dominant Ga alleles in style tissues carrying the same Ga dominant allele(Geitmann, Snowman, Emons, & Franklin-Tong, 2000; J. L. Kermicle, 2006). In maize, several Gametophytic factors have been described, which show different degrees of competition between the two types of pollen. For the Ga1 gene it has been identified a strong allele, named Ga1-s, whose presence in style tissue completely prevents ga1 pollen to accomplish fertilization(Larios et al., 2008). Onto female tissues from either homozygous Ga-1s/Ga1-s or heterozygous Ga1-s/ga1 plants, ga1 pollen grains germinate and pollen tubes grow, although never reaching the ovules. The feature of the mutant pollen tubes and the mechanism of cross-incompatibility have not been described yet (Suarez et al., 2013; Xie et al., 2010; Yamashita et al., 2004; Yu et al., 2009; Zhang, Tateishi, & Tanabe, 2010). However this mutant is a powerful tool to shed light on the genetic control of pollen function and on the molecular basis of pollen-pistil interaction.
Here, we used the second Illumina Genome Analyzer platform (GA II) to perform a SAGE-derived Digital Gene Expression (DGE) analysis of the pollen tube elongation in maize pistils(Audic & Claverie, 1997). The Solexa/Illumina system can yield millions of short reads (32–40 bp), and is therefore more suitable for tag-based transcriptome sequencing. Both, the avoidance of laborious cloning steps and the higher sequencing depth in Solexa add to its presumably superior accuracy and precision as compared with older methods(Morrissy et al., 2009). In particular, Solexa is a much more practical technique when small changes in gene expression and low-abundance transcripts are considered. Furthermore, it gives an unbiased view of the transcriptome because it does not require prior knowledge of the gene sequences to be investigated(Fan et al., 2014).
In the present study, we compared the difference of the pistil tissues of Gametophytic factor1-Strong (Ga1-S) genotype maize after pollination 5 hours (5HAP)with Ga1-S pollen and ga1 pollen respectively, in an attempt to isolate genes involved in pollen tube elongation in maize pistils using the Solexa deep sequencing technology.
1 Materials d Method
1.1 Plant materials
All experiments were conducted using maize inbred line Ga25 and W22. Ga25 had the dominant Ga genebut the inbred line W22 did not had the Ga gene. The plants were grown in summer at the experimental field of Shandong Agricultural University in Tai’an, China, where daily temperatures were 20-36 in the growing season. During the flowering period in July, the plants silking consistently were selected to be pollinated. Fresh pollens were paper-bagged between 9:00-11:00 am and silks were cut 1 cm above the husks to make silks uniform. The total silks from the upper and middle of the ears were taken down 0.15 h, 0.5 h, 1 h, 2 h, 5 h, 10 h and 20 h after pollination. 10 silks from each ear were selected randomly and stored in FAA solutions (formaldehyde: 95% alcohol: acetic acid (10:85:5, v/v/v)), which were used for pollen tubes observation. The rest were immersed into distilled water and carded with a soft brush to sweep the pollen, which were repeated three times and conducted rapidly. The absorbent paper was used to absorb redundant water. Then the silks were immersed into liquid nitrogen immediately and stored at -80 freezer until used.
1.2 Pollen tube growth observation
Pollen tube growth observation was performed as per the procedure reported by Zhang et al. (Zhang et al. 2012). For each plant, 10 silks that were relatively uniform in length were used for pollen tube growth observation at each time interval. With respect to each time interval, three individual plants were studied(Kho & Baer, 1968).
1.3 RNA isolation
Fish were snap frozen in liquid nitrogen, stored at -80 , and homogenized in liquid nitrogen using a mortar and pestle. Portions of 50–100 g of powdered tissue were used for extraction of total RNA with 1 ml of TRIZOL(R) Reagent (Invitrogen) according to the manufacturer’s instructions. The RNA samples were incubated for 20 min at 37 C with 10 units of DNaseI (Roche Applied Science) to remove residual genomic DNA prior to clean up using RNase columns (Qiagen). The integrity of the RNA was confirmed by Lab on-chip analysis using the 2100 Bioanalyzer (Agilent Technologies). The samples used had an average RIN value of 9.5 and a minimum RIN value of 8.9.
1.4 Digital gene expression-tag profiling
The experimental process includes sample preparation and sequencing. The main reagents and supplies are Illumina Gene Expression Sample Prep Kit and Solexa Sequencing Chip (flowcell), and the main instruments are Illumina Cluster Station and Illumina Genome Analyzer System.
Specified Experimental Process: Extract 6 µg of the total RNA, use Oligo (dT) magnetic beads adsorption to purify mRNA, and then use Oligo (dT) to guide reverse transcription to synthesize double-stranded cDNA. The generation of 5' ends of tags can be recognized with two types of Endonuclease: NlaIII or DpnII (see table 1 for recognition sites). Usually we use NlaIII which recognizes and cuts off the CATG sites on cDNA, then use magnetic beads precipitation to purify cDNA fragments with 3' ends and add Illumina adapter 1 to their 5' ends. The junction of Illumina adapter 1 and CATG site is the recognition site of MmeI, which is a type of Endonuclease with separated recognition sites and digestion sites. It cuts at 17bp downstream of the CATG site, producing tags with adapter 1. After removing 3' fragments with magnetic beads precipitation, Illumina adapter 2 is introduced at 3' ends of tags, acquiring tags with different adapters at both ends to form a tag library. After 15 cycles of linear PCR amplification, 85 base strips are purified by 6% TBE PAGE Gel electrophoresis. These trips are then digested, and the single-chain molecules are fixed onto the Solexa Sequencing Chip (flow cell). Each molecule grows into a single-molecule cluster sequencing template through Situ amplification. Then add in four types of nucleotides which are labeled by four colors, and perform sequencing with the method of sequencing by synthesis (SBS). Each tunnel
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