Genetics of microspore embryogenesis in Intervarietal Substitution Lines of rapeseed (Brassica napus L.)

by Muhammad Munem Khan
Doctoral thesis
Date of Examination:2024-03-26
Date of issue:2024-07-23
Advisor:PD Dr. Wolfgang Ecke
Referee:Prof. Dr. Stefan Scholten
Referee:Dr. Christian Möllers
Referee:Prof. Dr. Heiko C. Becker
Persistent Address: http://dx.doi.org/10.53846/goediss-10634

 

 

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Abstract

English

Microspore embryogenesis, a tissue culture-based propagation technology, is of great importance in rapeseed breeding to produce doubled haploids (DHs) in shortest time. Brassicas have become model plant species to study the induction and behavior of microspore-derived embryos (MDEs). In rapeseed, production of MDEs and DHs is limited by three factors such as the embryogenic potential of isolated microspores, the ability of embryos to convert into plantlets and the capacity of microspores to diploidize in the presence of a doubling agent (Möllers & Iqbal, 2009). The present study is aimed to identify the factors behind the phenomena of enhanced embryogenic potential using Intervarietal substitution lines (ISLs). Intervarietal Substitution Lines (ISLs) are developed by crossing a donor and recurrent parent followed by several generations of backcrosses with the help of marker-assisted selection. Ideally, each ISL will carry a distinctive segment of the donor genome in the genetic background of the recurrent parent, and together they represent the whole donor parent genome across all ISLs. By comparing the embryogenic potential of selected ISLs with the embryogenic potential of the recurrent parent, one can fine-map a region involved in the genetic control of embryogenic potential followed by the number of genes lying in that region with their expression profiles to understand the complex phenomena of embryogenic potential. Ecke et al. (2015) had identified seven Intervarietal substitution lines (ISLs) in rapeseed which showed enhanced embryogenic potential to their recurrent parent. They identified 12 genomic regions that may contain genes controlling the embryogenic potential in isolated microspores of rapeseed. For this purpose, four ISLs named ER085, ER0137, ER0228, and ER1321, covering most of the genomic segments, had been selected further for characterization. Ecke et al. had suggested to fine map the genes by backcrossing the ISLs again with their recurrent parent i.e. “Express 617” which will subdivide the donor segments making it easier to fine map the regions of interest. The current study consisted of two experiments. Before characterizing of donor segments in ISLs, a comparison of several genetics and physical maps was conducted which included two available reference sequences (“Bra_napus_2.0; ZS11” and “DH12075”) against four genetic maps (“DH4079 x E”, “SGDH14 x E” and “L16 x E” and DH12075 x PSA12). This comparison led us to identify a physical map “DH12075” which was best suited to characterize the donor segment in ISLs. While observing plots between the “ZS11” physical map and four genetic maps, large inversions and recombinations were observed on various chromosomes of “ZS11” thus, making it un-ideal for characterization of donor segments. The first part of this study comprised of fine mapping the regions involved in the control of enhanced embryogenic potential of isolated microspores. For this purpose, a new set of 262 substitution lines (ISLs) were produced by backcrossing the original four ISLs (ER085, ER0137, ER0228, and ER1321) to their recurrent parent (“Express 617”). To characterize the donor segments in new ISLs using KASP analysis, 58 SNP markers were used with an average distance of 5 centimorgan (cM) on the SGDH14xE genetic map. These markers had been selected to cover the donor segments of the parental lines of new ISLs. The largest donor segment (60.3 cM) was found on the parental ISL ER085 on LG C03 while smallest donor segments (0 cM) arose from parental ISLs ER1321 (LG A02, A05 and C04) and ER0137 (LG C07). Based on the result and comparison from the data of these 262 ISLs obtained by KASP analyses, 37 new ISLs were selected for microspore preparations to determine their embryogenic potential. These selected ISLs carried complementary subsegments of the parental donor segments that together covered these segments as best as was possible. Out of 37 ISLs, only two ISLs named “ERA049” and “ERD035”, showed significantly higher embryogenic potential compared to their recurrent parent “Express617”. These ISLs showed a ratio of 24.1 times and 9.5 times higher than their recurrent parent, respectively. To further characterize and fine map the regions which may be involved in the control of enhanced embryogenic potential, 20 ISLs were further analyzed with 60K SNP chip of rapeseed using the reference sequence DH12075 v3.1 as a physical map. Upon analyzing ISLs with donor segments, blocks of failed scores (BoFS) were observed on several chromosomes named A01, A04, A09, C01, C04, and C08. Apart from that, BoFS were also discovered in a significant ISL “ERA049” where the original parent, ER085, showed recurrent parent score. Several donor segments were identified, however, there was no direct correlation between the amount of donor segment and embryogenic potential which may further strengthen the idea of the involvement of some specific genetic factors which are controlling the embryogenic potential of isolated microspores. The fine-mapping approach was invalidated by the occurrence of new homoeologous exchanges during the development of new ISLs as indicated by BoFS. This phenomenon created genetic differences among the new lines due to the subdivision of the original donor segments and ultimately created a hindrance in fine mapping the region of interest. To overcome this problem, a qualitative analysis was performed based on the presence and absence of donor segments between the two ISLs with statistically significant enhanced embryogenic potential to their recurrent parent and non-significant ISLs. Based on this analysis, four donor segments were identified on chromosomes A04, A05, and C01 using reference sequence “DH12075” as a physical map. These segments were only present in the two significant lines, ERA049 and ERD035. A synteny analysis was performed between four selected segments (A04DS, A05DSa, A05DSb and C01DS) and “ZS11” genome to identify regions in the “ZS11” genome homoeologous to the selected segments. The region on chromosome C04 which was colinear to the donor segment A04DS, contains a small region of 30 kb and 299 kb in “DH12075” and “ZS11” physical maps, respectively, which had already been identified in the work of Ecke et al. (2015) as a region that could be involved in the control of embryogenic potential. Therefore, a total of 117 genes that had copies in both regions were further analyzed. Massive Analysis of cDNA Ends (MACE) analysis was conducted on four parental ISLs with enhanced embryogenic potentials over their recurrent parent. To induce microspore embryogenesis, microspores from these ISLs were incubated at 32C and then harvested at five different time points (0, 6, 12, 24 and 48 hrs). The harvested microspores were stored and frozen at -80oC before RNA extraction. A total of 217.1 million reads across 32 libraries were retrieved with an average of 6.78 million reads per sample. Reads were cleaned from ribosomal RNA (rRNA) and other organelle RNA which left behind 171.7 million reads across all libraries with an average loss of 1.4 million reads per library. While mapping reads to reference genome “Bra_napus_v2.0” based on ZS11, there was a discovery of 20,000 new genes that did not overlap or correspond to any of the annotated genes in the reference genome which consequently resulted in limiting the mapping of reads to only annotated genes of “ZS11” genome. The reads were mapped against the annotated genes of reference sequence with an average alignment rate of 72%. The genes were further quantified followed by differential expression analysis between four ISLs and recurrent parent. A total of 5,811 genes were found to be differentially expressed at FDR<0.05 between paired comparisons (ISLs x Express617) at five time points. Out of 5,811 genes, 21% (1,228) genes were up-regulated, and 79 % (4,583) genes were down-regulated. From all the genes differentially expressed at certain time points, only genes BnZS11gene13329 and BnZS11gene2552 were differentially expressed in all four ISLs with BnZS11gene13329 being differentially expressed at two time points (0h, 12h) and BnZS11gene2552 at three time points (0h, 24h and 48h). A gene ontology annotation was developed for the protein-coding genes of the “ZS11” genome as there was no such annotation included in the “ZS11” reference sequence. A total of 123,465 protein sequences were available and were allowed to blast against the proteins of the Arabidopsis TAIR10.1 reference sequence using Blast2go software to identify homologous Arabidopsis proteins. Out of these, about 110,438 sequences got blast hits, assigned gene ontology (GO) terms, and functionally annotated. A total of 11,127 sequences had no blast hits at all. Furthermore, 1,900 sequences had blast hits but without any assigned GO term and annotation. The parental line (ER1321) carrying the donor segment of interest “C04CRDS” showed four and three enriched GO terms in the BP and MF category, respectively. Out of 117 genes extracted from shared segment between “C04CRDS” and chromosome A04, only thirteen genes were found to be differentially expressed (FDR<0.05) in at least one of the paired comparisons. Out of 13 differentially expressed genes, 11 genes were significantly down-regulated in most of the paired comparisons while only two genes (BnZS11gene77912; B3 domain-containing protein At2g24670-like) and BnZS11gene78078; uncharacterized protein BNAC04G42730D) were up-regulated in ISLs ER1321 and ER0137 at 0 hours and 06 hours, respectively. Upon further analysis, the gene “BnZS11gene77912; B3 domain-containing protein” has a DNA-binding motif and has been named in plant developmental processes. This gene may be involved in the enhanced embryogenic potential other than transcriptional regulation, however, more research is needed to further verify. Apart from analyzing the expression of genes extracted from the donor segment, the expression of several embryogenesis genes reported in the literature was also estimated. A total of five genes responsible for embryogenesis (LEC1, PKL, AtGAox2, WUS1 and CLV1) were differentially expressed. The highest expression values (Fragments per million mapped reads; FPM) were observed for genes PKL; BnZS11gene45820 (259) in ER1321 at 06 hours, LEC1; BnZS11gene33152 (196) in ER1321 at 48 hours and CLV1; BnZS11gene35816 (12.8) in ER0137 at 24 hours. In Arabidopsis, gene PKL encodes for “chromatic-remodeling factor” and it regulates embryo development. PKL has also been reported for repression of LEAFY genes such as LEC1 and LEC2. We also observed repression effect of PKL on LEAFY genes which is consistent with several studies on PKL gene. The second gene with highest expression, LEC1; BnZS11gene33152, encodes for “nuclear factor Y, subunit B1” in Arabidopsis. Nuclear factor subunits are involved in the transcriptional regulation as well as plant growth and development. The third highly expressed but down-regulated gene CLV1 encodes “leucine-rich receptor-like protein kinase family protein” and is involved in plant and anther development. In conclusion, our result and analysis indicate that, for winter and spring rapeseed, the “DH12075” reference sequence is better suited as a physical map as compared to “ZS11” to characterize the donor segment in ISLs as well as for fine mapping. The reference sequence based on Asian cultivar “ZS11” is a good choice for transcriptome analysis since it is well- annotated. In the experiment of fine mapping, the region of interest in newly developed substitution lines was nearly impossible to fine-map as almost all the newly developed substitution lines showed block of failed scores that were not present in the parents, indicating homoeologous exchanges. This further strengthens the claim of increased genomic instability in resynthesized rapeseed. One shall be careful in opting resynthesized rapeseed for fine mapping-related experiments which can cause problems after every new generation. In the transcriptome analysis, the mapping of reads was limited to annotated genes of the reference genome “ZS11”. We identified a possible candidate gene named BnZS11gene77912; B3 domain-containing protein At2g24670-like) which was up-regulated in one of the ISL containing the donor segment of interest. The gene BnZS11gene77912 is a DNA-binding motif and is responsible for plant developmental processes. The B-3 domain containing proteins are also involved in plant growth and seed maturation and may also be involved in the enhanced embryogenic potential other than transcriptional regulation.
Keywords: Microspore embryogenesis; Intervarietal Substitution Lines; Doubled haploids; MACE; Brassica napus L.; ZS11
 
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