| Sesame (Sesamum indicum L.), one of the most important oil crops in China, is rich in vitamin A, vitamin E, sesamol, and sesamin in oil. It has long been consumed as healthy food for the nourishing and anti-aging effects. Breeding of sesame variety with high yield, good quality and disease resistance has been the prime target for breeders. Many evidents have shown that crop yield can be substantially improved by utilizing heterosis. Currently, genic male sterility (GMS) is the major approach for heterosis utilization in sesame. However, up to now only a few GMS lines that have value of practical application have been reported, and almost all of them are recessive GMS. The unique features of recessive GMS lines include availability of massive restorer lines and easiness of introducing male sterility into other genetic backgrounds. However, such lines can only be used in a ’two-line’system, which requires the removal of50%fertile plants (a time-consuming and labor-intensive process that limits their wide applications). In other crops such as rapeseed, both recessive and dominant GMS lines, as well as their respective maintainer lines and restorer lines, have been identified. Such GMS lines have been widely used in a ’three-line’ system. These progresses have set good examples for sesame research.In this study, two new GMS lines, D248AB and W1098AB, have been developed by means of natural mutation, distant hybridization and backcrossing. Using these two GMS lines as experimental materials, a systematic study have been carried out on the inheritance of male sterility, the characteristics of aborted pollen, cytological process of anther abortion, physiological and biochemical indices variation during anther abortion, molecular makers for male sterility, as well as selection of F1hybrids with strong heterosis. The major results were summarized as follows.1. Inheritance of sesame recessive GMS line D248ABTwo spontaneous male sterile plants were found in the conventional variety ’Zhilzhi4’ during reproduction. By consecutive crossing of these male sterile plants with fertile plants from ’Zhuzhi4’, a new male sterile line (D248AB) was developed, which showed a1:1and3:1fertility segregation ratio in sib-mating and selfing population, respectively. Furthermore, D248AB were crossed with6genotypes, and all resulting F1s were fertile, suggesting that the fertility of D248AB was controlled by a single recessive gene. The anther of D248AB was greenish, flat or curled, without any powder inside when being extruded. The pollen was irregular in shape, stainless (or slightly stained) by aceto-carmine, and not germinative in culture medium. These data indicated that D248AB was completely male sterile. Compared with other GMS line such as95ms-2, blossom duration for D248AB was10-12d longer, and plant yield was42-95%higher. Four Fi hybrids were produced by crossing D248A with5other elite lines respectively. The average yield of these hybrids ranged from931.4kg/ha to1319.1kg/ha, with4.99-48.70%yield advantage over Zhongzhi14, indicating that D248AB has great potential in breeding practice. 2. Development and inheritance of dominant GMS line W1098ABA distant cross was made by pollinating wide sesame (Yezhi2) with a cultivar (Ezhi1). In the resulting generations, complete male sterile plants were selected to backcross with Ezhi1twice and sib-mate with fertile plant from same population for>4times. A new male sterile line (W1098AB) was then developed. For this line,1:1fertility segregation was observed in sib-mating population but no segregation in progonies from selfing of fertile plants. In addition, sterile plants (in a proportion of24-79%) were observed in all test-crossing populations obtained by polinating W1098A with respecitve26genotypes. The above data inferred that fertility of W1098AB was under the control of a single dominant gene. W1098AB has great potential in breeding as its sterility was complete and stable even in different environments. Currently, restorer lines for W1098AB have not yet been identified.3. Cytological observation of abortion process in GMS line W1098ABThe morphology of anthers and pollens from W1098AB was observed by optical microscope and scanning electron microscope (SEM). The sterile anther was white, slightly oval in shape and not so obviously different from fertile anther. The sterile pollen looked like a reduced ball or was concave in shape, without any contents inside.In the view of SEM, sterile pollen looked like a hemisphere or a felt hat, while fertile pollen was plump, spherical or oval in shape, with10-12germinal furrows. These observations indicated that W1098AB was completely sterile.Paraffin section study revealed that anther abortion was initiated at pollen mother cell (PMC) stage. Abortion progressively prevailed at following stages and finally peaked at microspore stage. Such abortion could possibly be attributed to premature disintegration of tapetum at early stage or incomplete disintegration of tapetum at late stage.Transmission electron microscope study also showed that anther abortion was initiated at PMC stage and was caused by the abnormality of anther wall structure. During the process of pollen development, abortion could be aggravated by a number of factors such as reduction of ubisch bodies secreted in tapetum cells, incomplete degradation of tapetum cells, deformation and degradation of middle layer cells, uneven thickening of secondary in anther wall cells, uneven thickening of nuclear membrane in microspore, and reduction and uneven distribution of granule bar. The abortion was peak at microspore stage.4. Variation of physiological and biochemical indices during anther abortion in W1098ABWith the technology of enzyme-linked immunosorbent assay (ELISA), contents of four phytohormones (IAA, ABA, JA and SA) in buds and leaves were measured at different developmental stages. The ratios of different hormones, as well as contents of soluble sugar and starch, were compared between male sterile and fertile plants. The results indicated that:(a) during anther development, the variation trends and contents of four hormones were distinct between male sterile and fertile plants. The significant decrease of IAA and increase of ABA in male sterile plant tended to be connected with occurrence of male sterility;(b) significant increase of JA and ABA and decrease of IAA were observed in leaf in male sterile plants;(c) variation trends and ratios of IAA/ABA, IAA/SA and IAA/JA were quite different from male sterile plants to fertile plants, indicating that imbalance of hormones was also responsible for anther abortion;(d) The soluble sugar and starch contents in buds of male sterile plants were higher than that in fertile plants, which was at least partially responsible for male sterility.Free amino acid contents were measured in buds from sterile and fertile plants. There were no obvious differences between sterile and fertile plants at sporogonium, PMC, tetrad and microspore stages. However, in sterile plants the contents of major free amino acids (such as Thr, Glu, Lys and Arg) raised suddently at mature pollen stage, which was possibly a result rather than a cause of anther abortion, given that abortion mainly occurred at microspore stage.5. Molecular markers for dominant GMSA sib-mating population comprising224plants segregating for fertility was used to identify molecular markers linked to male sterility in W1098AB. Fertile and sterile bulks were constructed and subjected to screening of1500SSR markers. Fifteen markers showing polymorphic bands between two bulks were identified and further investigated in the whole population. Among them,13markers were confirmed to be closely associated with male sterility, with a recombinant rate of0.45-14.73%. A linkage group was constructed by software JoinMap, in which7markers sat on one side of male sterility locus (Ms) and6on the other side. The closest markers on both sides of Ms were SBM298and GB50, with a genetic distance of0.15Cm and0.70Cm, respectively. These markers will be valuable for marker aided breeding of GMS hybrids and map-based cloning of Ms in future. |
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