Recessive Genic Male Sterility Line Research Articles

Full-length transcriptome analysis reveals the differences between floral buds of recessive genic male-sterile line (RMS3185A) and fertile line (RMS3185B) of cabbage.

Starch and sucrose metabolism and plant-pathogen interaction pathways play a dominate role in recessive genic male sterility (RGMS) of cabbage (Brassica oleracea L. var. capitata). RGMS is common in plants and has been widely applied as an effective and economic system for hybrid seed production in many crops. However, little is known regarding the molecular mechanisms of RGMS in cabbage. Hence, full-length transcriptomic and physiological analysis were performed in the spontaneous RGMS mutant RMS3185A and its near-isogenic fertile line (NIL) RMS3185B of small (< 1.6mm in diameter), medium (~ 2.5mm in diameter), and large floral buds (~ 3.4mm in diameter) to identify the differentially expressed genes (DEGs) associated with RMGS. The pollen abnormalities between RMS3185B and RMS3185A appeared at the large floral bud stage. In contrast with RMS3185B, the mature anthers and stamens of RMS3185A were shorter than those of RMS3185B, and the anthers did not dehiscent. The concentrations of glucose, fructose, trehalose, starch, and cellulose in RMS3185A were all significantly lower than those in large floral buds of RMS3185B. PacBio sequencing results showed that DEGs were mainly concentrated in large floral bud stage. In combination with the KEGG enrichment analysis of DEGs in GO terms related to cell wall, pollen and anther, pentose and glucuronate interconversions (ko00040), starch and sucrose metabolism (ko00500), and plant-pathogen interaction (ko04626) were significantly enriched. Among which, cell-wall/pectin-related genes of eighteen PEI, twenty-two PEL, three PG, and fifteen PGL involved in ko00040, and one UGDH, one SPS, four CWINV, four TPP/TPS, and four EGL involved in ko00500, as well as plant-pathogen interaction genes, including sixteen calcium-dependent protein kinase (CDPK), one cyclic nucleotide-gated ion channel (CNGC), and twenty-three calcium-binding protein CML (CML), were significantly down-regulated in RMS3185A relative to that in RMS3185B. Besides, genes involved in ko04626, including two CML and one transcription factor WRKY33, were up-regulated in RMS3185A relative to that in RMS3185B. In conclusion, we hypothesized that the expression alterations of these genes were responsible for calcium signaling and sugar metabolism, thus affecting the occurrence of RGMS in cabbage.

Cytological characterization and molecular mapping of a novel recessive genic male sterility in sesame (Sesamum indicum L.).

Recessive genic male sterility (RGMS) has great potential for F1 hybrid seeds production in sesame (Sesamum indicum L.). However, it is not yet widely used in practice due to poor understanding of the underlying mechanism in RGMS. Previously, we have developed a novel sesame RGMS line (D248A) controlled by a single recessive gene. To elucidate its cytological mechanism, histological observations were carried out in sterile and fertile buds. The results indicated that abnormality in D248A began at microspore mother cell stage and persisted until microspore stage. The microsporocytes had less cytoplasm and no obvious nucleus. Normal meiosis failed in microspore mother cells. Cytoplasm condensation and vacuolation frequently occurred in tetrads, leading to the production of crumpled and abortive microspores. To develop molecular markers for breeding of hybrid lines, InDel and SSR markers were analyzed in a fertility segregating NIL population constructed by sib-mating D248A with D248B. Five markers were identified for the male sterile gene (Ms), with a respective genetic distance of 1.47 and 5.17 cM for the two closest markers (SB2993 and LG1-170) on both sides. The Ms gene was further anchored into a 108-kb interval in the downstream of chromosome 1, within which 15 genes were predicted and four were likely to be responsible for male sterility. These findings provide a deeper understanding of the mechanism underlying RGMS in sesame.

Identification of a nuclear-recessive gene locus for male sterility on A2 chromosome using the Brassica 60 K SNP array in non-heading Chinese cabbage

WS24-3 is a newly bred recessive genic male sterility line of the non-heading Chinese cabbage (Brassica rapa ssp. chinensis). Here, an F2 population was produced from the cross between WS24-3 and a male-fertile breeding line (WS135). The Illumina Brassica 60 K single nucleotide polymorphism (SNP) array was used for SNPs detecting between sterile and fertile bulks from the F2 population, and 62 SNPs were identified. BLAST analysis of the 62 SNPs revealed that the A2 chromosome of Brassica rapa genome contained 22 SNPs, whereas the other chromosomes did not contain more than 6 SNPs each. These data indicated that the potential target gene locus, named Bra2Ms, might be located on A2. Based on 10 of the 22 SNPs, allele-specific-polymerase chain reaction (AS-PCR) primers and single sequence repeat (SSR) primers were designed, 5 AS-PCR primers and 9 SSR primers showed difference between the bulks in electrophoretic determination. Analysis of these markers in F2 population revealed that Bra2Ms was genetically delimited to a region of 1.2 cM. We also detected two co-segregated markers SSRa2-951 and SSRa2-960 in this region. The markers identified in our study might facilitate the transfer of recessive genic male sterility alleles to other favorable genetic backgrounds. Furthermore, these markers will support a map-based clone of Bra2Ms.

Transcriptome sequencing and de novo analysis of a recessive genic male sterile line in cabbage (Brassica oleracea L. var. capitata)

Recessive genic male sterility (RGMS) is common in plants and has been widely applied as an effective and economic system for hybrid seed production in many crops. However, little is known regarding the molecular mechanisms of RGMS in cabbage (Brassica oleracea L. var. capitata) due to limited transcriptomic and genomic data. Comparative transcriptomic analyses were performed on the sterile (RGMS632-MS) and fertile plants (RGMS632-SF) of a RGMS line (RGMS632) using second-generation Illumina sequencing to identify critical genes and pathways associated with male sterility. A total of approximately 109 million sequencing reads was obtained using RNA-seq. Abundance analysis identified a total of 5107 unigenes that showed significant differences between the two kinds of plants. Among these, 1558 genes were upregulated while 3549 genes were downregulated by more than twofold in RGMS632-MS compared to RGMS632-SF. KEGG pathway enrichment analysis revealed changes in the transcript abundance of genes involved in the metabolism and signal transduction of various phytohormones. The majority of hormone signalling pathways were downregulated in RGMS632-MS. Furthermore, a set of potential candidate genes involved in the formation or abortion of pollen were investigated. These results increased our understanding of the molecular mechanisms and biological processes in RGMS plants.

Recessive Genic Male Sterility in &lt;italic&gt;Brassica napus&lt;/italic&gt; and Its Application

The Brassica napus recessive genic male sterile (RGMS) line was widely used in heterosis utilization because it possesses stable sterility, no negative cytoplasmic effects, easy restoration and multi-restored resources. There are mainly two recessive genic male sterile lines in China. One is controlled by two duplicate recessive genes, and another is controlled by two loci, one recessive mutant locus and one multiple allele locus. With cloning of these sterile genes, some molecular mechanisms of genic male sterility in Brassica napus was deciphered. In this review, we summarize the advances in research on molecular mechanism of genic male sterility in Brassica napus . A feasible way is proposed to breed entire sterile populations of genic male sterile line in Brassica napus , which shows broad prospects in heterosis utilization.

Development, inheritance and breeding potential of a recessive genic male sterile line D248A in Sesame (Sesamum indicum L.)

Genic male sterility (GMS) has great potential for heterosis exploitation in sesame (Sesamum indicum L.). Two spontaneous male-sterile plants were discovered in a Chinese sesame cultivar (Zhuzhi 4) in 2006. By consecutive sib mating with fertile plants from Zhuzhi 4, a new sterile line, D248A, was developed. Anatomy studies showed that D248A has thin, small and greenish anthers on which there are no or little pollen grains. The pollens are irregularly shaped and completely aborted, resulting in no germination and no formation of pollen tubes as revealed by acetocarmine stain or semi-solid suspension culture. Furthermore, D248A has a better performance in growth vigor, bloom duration and yield per plant than the other GMS lines (i.e. 95 ms-2A and 95 ms-5A). To investigate the inheritance mode of fertility, D248A was crossed and backcrossed with six varieties, and a segregating ratio of 3:1 and 1:1 for fertile and sterile plants was observed in F2 and BC1 populations, respectively. These results suggested that D248A is controlled by a recessive GMS gene. The average yield of four D248A-derived F1 hybrids is as high as 1695 kg·ha-1, which is almost twice of that of 95 ms-5A-derived F1 hybrids. These results indicated that this newly developed recessive GMS line has great potential in sesame hybrid breeding.

High temperature induced fertility transition and anther carbohydrate metabolism modification in a canola recessive genic male sterile line

High temperature stress is an important abiotic stress during plant growth and development that can damage canola male reproductive organ development. In this present study, we characterized a canola genic male sterile line that its fertility can be restored after high temperature induction with heterozygous but not homozygous condition. Anther length and weight was elongated and increased 2 and 5.7 times at 9 days after high temperature treatment in heterozygous line. Furthermore, viable pollen grain was produced in the anther. Anther carbohydrate content was assayed. The result showed that fructose and sucrose contents were much more stable under heat stress both in hetero- or homozygous lines. However, glucose and starch content was considerably increased after high temperature treatment in heterozygous line. In particular, anther glucose content was twice as much as that of starch content after 5 days high temperature treatment. Activity of neutral invertase, acid invertase, and sucrose synthase were increased in the anther of heterozygous line under heat stress. But activity of neutral invertase was the highest among these three sucrose cleavage enzymes. Both activity of sucrose phosphate synthase and starch phosphorylase were decreased under heat stress in the anther of heterozygous line. Activity of ADP glucose pyrophosphorylase was greatly increased in the heterozygous line, which was beneficial for starch accumulation. Transcription level of carbohydrate related genes was analyzed. 27 and 34 of the carbohydrate related genes were identified for down- and up-regulated in the anther of heterozygous line, respectively. Their function involved in the regulation of heat tolerance of heterozygous line was also discussed. Key words: Canola, carbohydrate, gene.

Map-based cloning of a recessive genic male sterility locus in Brassica napus L. and development of its functional marker

We previously mapped one male-sterile gene (Bnms3) from an extensively used recessive genic male sterility line (9012AB) in Brassica napus to a 0.14-cM genomic region. In this study, two highly homologous BAC contigs possibly containing the candidate BnMs3 gene were identified using a map-based cloning strategy. A BnMs3-linked SCAR marker (DM1) capable of differentiating the subgenomes between B. rapa and the B. oleracea aided mapping of BnMs3 on the contig derived from the B. napus chromosome C9. One representative BAC clone was sequenced from each of the two contigs and resulted in a larger number of markers according to the sequence difference between the two clones. To isolate BnMs3, these markers were then analyzed in another two BC(1) populations with different genetic backgrounds. This assay allowed for a delimitation of the mutated functional region of BnMs3 to a 9.3-kb DNA fragment. Gene prediction suggested that one complete open reading frame (ORF, ORF2) and partial CDS fragments of ORF1 and ORF3 reside in this fragment. Sequence comparison and genetic transformation eventually indicated that ORF1 (designated as BnaC9.Tic40), an analogue of the Arabidopsis gene AT5G16620 which encodes a translocon of the inner envelope of chloroplasts 40 (Tic40), is the only candidate gene of BnMs3. Furthermore, two distinct mutation types in ORF1 both causing the male-sterile phenotype were individually revealed from 9012A and the temporary maintainer line T45. The molecular mechanism of this male sterility as well as the application of BnMs3-associated functional and cosegregated markers in true breeding programs was also discussed.

Molecular validation of a multiple-allele recessive genic male sterility locus (BnRf) in Brassica napus L.

The recessive genic male sterility (RGMS) line 9012AB has been used successfully for rapeseed hybrid production in China. This male sterility was previously thought to be controlled by three independent genes (Bnms3, Bnms4, and BnRf). Here, we initially attempted to locate the BnMs4 locus and develop feasible molecular markers for application in practical rapeseed breeding. However, we found that three sequence characterized amplified region markers and five simple sequence repeat markers identified as linked to BnMs4 were also genetically associated with BnRf, suggesting the possible co-localization of these two loci. Moreover, we proved that four intron-based polymorphism markers tightly linked or co-segregated with BnRf could also be mapped to BnMs4 with a genetic distance ranging from 0.054 to 0.594 cM. Finally, integration of genetic maps around BnRf and BnMs4 allows for the physical restriction of both loci to a DNA fragment of about 50 kb. Systematic genetic tests also provided evidence that the candidate BnMs4 locus was allelic to the BnRf locus. These results confirmed a major modification of the sterility inheritance model in 9012A: specifically, that this male sterility was essentially controlled by two loci (BnMs3 and BnRf), whereas the previously designated BnMs4 locus (hereafter designated as BnRf a ) was just one allele of BnRf in addition to BnRf b (the allele from 9012A) and BnRf c (the allele from temporary maintainer), with a dominance relationship of BnRf a > BnRf b > BnRf c . This inheritance model will simplify the breeding process involved with this RGMS line, especially with the BnRf allele-specific molecular markers identified here.

Improvement of the recessive genic male sterile lines with a subgenomic background in Brassica napus by molecular marker-assisted selection

Both the pollination control system and genetic distance are major factors in the utilization of crop heterosis. The recessive genic male sterile line (RGMS) 7-7365A (Bnms3ms3ms4ms4) has been widely applied to hybrid seed production because it can generate a completely male sterile population by crossing with the 7-7365C temporary line (Bnms3ms3rfrf). In this study, the sterile genes of 7-7365A were transferred to the new Brassica napus lines 7-749 and 7-750 with a high content of subgenomes by backcross breeding. We used the amplified fragment length polymorphism (AFLP) technique combined with bulk segregant analysis (BSA) to identify markers linked to the BnMs4 gene. Twelve AFLP markers linked to the BnMs4 gene were identified. Of them, SA06MG09 and P08MG16 were the closest makers, which were on either side of the gene at a distance of 0.9 and 0.8 cM, respectively. Twenty AFLP primer combinations were used to screen the F2, BC1F3, and BC2F4 populations from the breeding program, and the markers linked to the BnMs3 and BnMs4 genes were used to screen the BC2F4 populations. As a result, we obtained two types of improved sterile lines, 7-749A and 7-750A, and their indexes of subgenomic components (ISG) were 44.2–49.8 and 20.2–26.6%, respectively. The combining ability analyses of seed yield character were conducted in the crosses from the three sterile lines and ten restorers within a random block design in three environments for two successive years. The general combining ability (GCA) of the two improved sterile lines were significantly higher than the GCA of 7-7365A in every environment tested. The two improved sterile lines had stability in seed yield, and they will be used in the future for hybrid seed production.

Abnormal Vacuolization of the Tapetum During the Tetrad Stage is Associated with Male Sterility in the Recessive Genic Male Sterile Brassica napus L. Line 9012A

In the recessive genic male sterile line 9012A of Brassica napus, pollen development is affected during the tetrad stage. According to the light and electron microscopy analysis of tapetal cells and tetrads, the sterile tapetal cells swelled with expanded vacuoles at the early tetrad stage and finally filled the center of the locules where a majority of tetrads encased with the thick callose wall collapsed and degraded. We suggested that an absence of callase, which is a wall-degrading enzyme stored in the vacuoles of tapetal cells before secretion, resulted in the failure of tetrad separation. Moreover, transmission electron microscopy analysis showed that the secretory tapetal cells were not observed in sterile anthers, which indicated that the transition of the tapetum from the parietal type to the secretory type was probably aberrant. In plants, degeneration of the tapetum is thought to be the result of programmed cell death (PCD). PCD of tapetal cells was investigated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and signals indicative of deoxyribonucleic acid fragmentation were detected much earlier in sterile anther than in fertile anther. This suggests that tapetal breakdown does not occur by the normal procession of PCD and might be following an alternative mechanism of unscheduled apoptosis in line 9012A. This research supports the hypothesis that premature PCD is associated with male sterility in B. napus.

Genetic and histological characterization of a novel recessive genic male sterile line of Brassica napus derived from a cross with Capsella bursa-pastoris

In a previously made cross Brassica napus cv. Oro (2n = 38) × Capsella bursa-pastoris (2n = 4x = 32), one F1 hybrid with 2n = 38 was totally male sterile. The hybrid contained no complete chromosomes from C. bursa-pastoris, but some specific AFLP (amplified fragment length polymorphism) bands of C. bursa-pastoris were detected. The hybrid was morphologically quite similar to ‘Oro’ except for smaller flowers with rudimentary stamens but normal pistils, and showed good seed-set after pollination by ‘Oro’ and other B. napus cultivars. The fertility segregation ratios (3:1, 1:1) in its progenies indicated that the male sterility was controlled by a single recessive gene. In the pollen mother cells of the male sterile hybrid, chromosome pairing and segregation were normal. Histological sectioning of its anthers showed that the tapetum was multiple layers and was hypertrophic from the stage of sporogenic cells, and that the tetrads were compressed by the vacuolated and disaggregated tapetum and no mature pollen grains were formed in anther sacs, thus resulting in male sterility. The possible mechanisms for the production of the male sterile hybrid and its potential in breeding are discussed.

Identification of AFLP fragments linked to one recessive genic male sterility (RGMS) in rapeseed (Brassica napus L.) and conversion to SCAR markers for marker-aided selection

117AB is a recessive genic male sterility (RGMS) line in which the sterility is controlled by a duplicate recessive gene named ms, located at two separate loci. In the RGMS line, the genotype of the sterile plant (117A) is msmsmsms, and that of the fertile plant (117B) is Msmsmsms. The present study was aimed to identify DNA markers linked to the ms locus by amplified fragment length polymorphism (AFLP). From the survey of 512 AFLP primer combinations, 6 AFLP fragments (y1, k1, k2, k3, k4, k5) were identified as being tightly linked to the Ms locus. The genetic distances between the markers and the Ms locus were all less than 8 cM, among which two fragments, designated as k2 and k3, co-segregated with the target gene in the tested population. Fragment k2 was successfully converted into a sequence characterized amplified region (SCAR) marker. The markers detected could be valuable in marker-assisted breeding of RGMS in Brassica napus.