Identification and fine mapping of a fertility restorer gene for wild abortive cytoplasmic male sterility in the elite indica rice non-restorer line 9311

The wild abortive(WA)-type cytoplasmic male sterility(CMS)derived from the wild rice species Oryza rufipogon Griff.is used widely in three-line indica hybrids.The identification and mapping of restorer of fertility(Rf)genes aided in the development of WA-type hybrids.Here we report that testcross F1 plants from the WA-type CMS line and 9311 exhibited stainable pollen grains with no seed set,indicating that 9311 carries minor-effect Rfs for WA-type CMS.We developed an advanced backcross population consisting of plants harboring small regions of donor chromosomal segments from 9311 in the WATianfeng A genetic background with moderate seed setting rates.Genetic analysis showed that the pollen fertility levels of the backcross individuals are governed by a single gene from 9311 that we named Rf19(t).By use of the RICE 40 K gene chip,three introduced segments were identified in the fertile lines,and a candidate region spanning 4.37–8.29 Mb on chromosome 1 was identified for Rf19(t).Finally,Rf19(t)was fine-mapped to a region of 90 kb between the DNA marker loci STS1-163 and STS1-183,in which eight ORFs were predicted.Also,using relative expression analyses,comparative sequence analyses and functional domain analyses,we identified LOC_Os01g10530 as the most likely candidate gene for Rf19(t).Furthermore,Rf19(t)was found to function in fertility restoration,most probably by regulating the degradation of m RNA transcribed from the mitochondrial gene WA352.These results increase our knowledge of fertility restoration in WA-type CMS lines and will facilitate the development of high-quality pairs of WAtype CMS and maintainer lines.

Genetic Analysis and Mapping of Genes Involved in Fertility of Pingxiang Dominant Genic Male Sterile Rice

Pingxiang-dominant genic male sterile rice （PDGMSR） was the first dominant genic male sterile mutant identified in rice （Oryza sativa L.）, and the corresponding dominant genic male sterile gene was designated as Ms-p. The fertility of PDGMSR can be restored by introduction of a dominant epistatic fertility restoring gene in some rice varieties. In the present study, E823, an indica inbred rice variety, restored the fertility of PDGMSR, and the genetic pattern was found to be consistent with a dominant epistatic model, therefore, the dominant epistatic fertility restorer gene was designated as Rfe. The F2 population from the cross of PDGMSR/E823 was developed to map gene Rfe. The F2 plants with the genotypes Ms-pMs-pRferfe or Ms-pms-pRferfe were used to construct a fertile pool, and the corresponding sterile plants with genotypes Ms-pMs-prferfe or Ms-pms-prferfe were used to con- struct a sterile pool. The fertility restoring gene Rfe was mapped to one side of the microsatellite markers RM311 and RM3152 on rice chromosome 10, with genetic distances of 7.9 cM and 3.6 cM, respectively. The microsatellite markers around the location of the Ms-p gene were used to finely map the Ms-p gene. The findings of this study indicated that the microsatellite markers RM171 and RM6745 flanked the Ms-p gene, and the distances were 0.3 cM and 3.0 cM, respectively. On the basis of the sequence of rice chromosome 10, the physical distance between the two markers is approximately 730 kb. These findings facilitates molecular marker-assisted selection （MAS） of genes Ms-p and Rfe in rice breeding programs, and cloning them in the future.

Inheritance of Fertility Restoration for Cytoplasmic Male Sterility in a New Gossypium barbadense Restorer

In order to clarify inheritance mechanism of fertility restoration for cytoplasmic male sterility （CMS） in a new Gossypium barbadense restorer line Hai R which was found in the fertility test crossing of G. hirsutum CMS lines with G. barbadense germplasms. 23 fertility segregation populations of F2 and backcross were used to analyze the inheritance of fertility restoring gene（s） of Hai R. The result showed that Hai R had one major dominant gene （RfB） to control the CMS fertility restoration and this fertility restoration gene functioned at the sporophytic level. The sterile cytoplasm background might not only influence the transmission rate of male gamete but also that of female gamete when the restorer gene was recessive. It could be deduced that this fertility restoration gene might come from G. harknessii cotton, Hai R is of value in the application of cotton interspecific hybrid breeding.

Identification and Genetic Analysis of Fertility Restoration Ability in Dongxiang Wild Rice (Oryza rufipogon)

Two populations of Dongxiang wild rice （Oryza rufipogon）, Shuitaoshu and Dongtangshang, were crossed with five male sterile rice lines with different cytoplasmic backgrounds （B06S, Zhenshan 97A, Xieqingzao A, Zhong 9A, and Yuetai A）, and the seed setting rate of the F1 was used to judge the fertility restoration ability in the Dongxiang wild rice. With P1, F1, P2, and F2 populations as materials, the Akaike＇s Information Criterion （AIC） was used to identify the major genes affecting quantitative traits, and when the major genes existed, the genetic effects of the major gene and the polygene and their genetic variance were estimated through segregation analysis. The seed setting rates of the F1 generation varied from 45.98% to 76.57%, suggesting that the Dongxiang wild rice had the fertility restoration ability. One major gene plus polygene mixed inheritance model was the most fitted genetic model for this trait in all the F2 populations. The heritability values of the major genes varied from 56.63% to 88.29% and those of the polygenes from 2.74% to 30.97%, and the total heritability values were from 63.17% to 94.01%. The major gene inheritance of the combination Zhong 9A/Dongtangshang was controlled by the additive effect without dominant effect, and the other nine combinatfons were by the completely dominant inheritance.

Genetics of Fertility Restoration in Cytoplasmic Male Sterile Pepper

Pepper hybrid seeds production using male sterility could lower cost by reducing time and labour, and increase the genetic purity of the F1 seeds. To investigate the genetics of fertility restoration of the Peterson cytoplasmic sterility in pepper, a doubled haploid population of 115 pepper lines obtained from anther culture of the F1 hybrid between Yolo Wonder （sterility maintainer line） and Perennial （fertility restorer line） and the parental lines were test-crossed by 77013A （a strict cytoplasmic-genic male sterile line）. The fertility of the test-crossed lines was assessed in greenhouse and open field with the following three criteria： pollen index （PI, visual estimation of pollen amount per flower）, pollen number （PN, pollen counting under microscope）, and seed number （SN, the number of seeds per fruit in open pollination）. Correlations between the each couple of criteria within, as well as between the cultivation methods ranged from 0.55 to 0.84. Analysis of variance showed that the genotype （DH line） and environment were the significant sources of variation of the fertility. Narrow sense of heritance of fertility restoration ranged from 0.38 to 0.92, depending on the criteria and environment. The distribution of the progeny was continuous between the parental genotypes indicating the quantitative inheritance of fertility restoration. Inferred from segregation according to Snape et al.（1984）, the number of segregating genes was estimated to be that three to four genetic factors were involved in pollen traits （PI and PN） and five to eight genetic factors in seed production （SN）. The heredity analysis of the CMS will be helpful for understanding of the genetic mechanism of the fertility restoration and the exploitation of the CMS in hybrid seed production.

Soil Fertility Self-development Under Ecological Restoration in the Zhuxi Watershed in the Red Soil Hilly Region of China

Current methods that utilize simple data or models to judge whether soil fertility can selfdevelop are not sufficiently rigorous. A new framework has been set up using catastrophe theory, laboratory experiment, field work, and 3S(Geographic information system, Global positioning system, and Remote sensing) to explore soil fertility catastrophe under ecological restoration, discriminate whether soil fertility can self-develop, and propose adjustment of ecological restoration measures in the Zhuxi watershed of Changting County, Fujian Province, China, which is a typical representative of the red soil hilly region of China. The results show that: 1) the soil fertility is obviously improved through the four ecological restoration measures, which impels soil fertility catastrophe. Among 89 soil samples, catastrophic soil samples and stable soil samples account for 26(29.21%) and 63(70.79%) of the samples, respectively. The four ecological restoration measures are listed in the order lowquality forest improvement > arbor–bush–herb mixed plantation > orchard improvement > closing measures according to the proportions of catastrophic soil samples. A typical soil sample in Bashilihe that can self-develop is selected as the criterion to judge the upper lobe and lower lobe of soil fertility in the process surface of the Cusp catastrophe model. Twenty-six(29.21%) were in the middle lobe, 10(11.24%) were in the upper lobe, and 53(70.79%) were in the lower lobe. The catastrophic direction of 26 catastrophic soil samples is to the upper lobe according to soil and water loss change as well as fieldwork. There is a significant positive correlation of Δ with soil and water loss change, and the lower soil and water loss relates to higher catastrophic probability. 2) Soil fertility self-development could be regionalized as "Soil fertility can self-develop" whose area was 12.74 km2(28.33%) distributed mainly in the leftmost and rightmost parts, "Soil fertility tends to self-develop" whose area was 11.63 km2(25.89%) distributed mainly in the middle part, and "Soil fertility cannot self-develop" whose area was 20.58 km2(45.78%) distributed mainly between the above two types. 3) There is no need to take ecological restoration measures and excessive human interference should be avoided in the future in regions of "Soil fertility can self-develop" and "Soil fertility tends to self-develop," and ecological restoration measures should be taken in region of "Soil fertility cannot self-develop." 4) We suggest withdrawal and implementation of ecological restoration measures should be incorporated into the evaluation criteria of ecological restoration to avoid misuse of funds.

Geographic patterns of genetic diversity and fertility restoration ability of Asian and African origin pearl millet populations

A set of 45 diverse Asian and African origin/bred populations of pearl millet were investigated multi-locationally for morphological traits including grain yield and assessed for their fertility restoration ability on three cytoplasmic male sterility(CMS) systems in two contrasting seasons. Significant genetic variation was found for all the yield linked traits. Multivariate analysis for grain yield and its component traits grouped these populations into five clusters. Most of the Asian origin populations grouped into single cluster(Cluster III) and separated from the rest of the African origin populations indicating the presence of correspondence between clustering pattern and geographical origin of the populations. Clusters dominated with Asian populations(As-As) had linkages with early flowering, short plant height, more number of tillers, small and thin panicles, small seeded and low grain yield compared to the clusters dominated by African populations(Af-Af) or African origin and Asian bred(Af-As) populations.Genetically related populations having common parentage were found grouped in same clusters.Fertility restoration/maintainer frequency of 45 populations on three diverse CMS systems revealed that overall fertility restoration frequency was highest for A1(86%) followed by A4(37%) and for A5(7%) CMS system. Five populations were identified as potential sources for developing maintainer lines for all three CMS systems and eight populations were identified specifically for A;and A;CMS systems. A set of 11 and four populations were identified for restorer line development exclusively for A;and A;CMS system,respectively. Six populations were identified for the development of dual restorers for both A;and A;CMS system.

Development of New InDel Marker to Detect Genotypes of Rf-1a Conferring Fertility Restoration of BT-Type Cytoplasmic Male Sterility in Rice

Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility （CMS） in japonica rice, a functional marker InDeI-Rf-la based on the difference of nucleotide sequence in Rf-la locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-larf-la showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-la locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F2 segregation population, and three genotypes in Rf-la locus could be distinguished distinctly. Therefore, the marker InDeI-Rf-la could be widely used for genetic id^ntifio.~tinn ~nd m^rkp.r-~.~.~i^fp.d .~.tAr.tinn （MA.~ in hr~=dinn i^nnnir~ r^fnr~=r lin==~

Characterization of Zm26Sub5 and Its Potential Roles in Regulating Pollen Fertility of S-CMS in Maize

Mitochondrial orf355-orf77 and the nuclear fertility restorer locus Rf3/rf3 mutually control the fertility of male gametes in CMS-S in maize （Zea mays L.）. A fragment of gene Zm26Sub5 was identified through cDNA-AFLP from a set of Rf3/rf3 near-isogenic lines in a previous study. In the present study, real-time PCR analysis revealed that Zm26Sub5 expression levels were much higher in pollen of S-Mo17Rf3Rf3 than in pollen of S-Mo17rf3rf3, and also higher than in fresh leaves, mature leaves, and roots of both S-Mo17Rf3Rf3 and S-Mo17rf3rf3. In silico cloning of full-length cDNA was successfully implemented. Gene Zm26Sub5 was 1 451 bp in size, of which 1 329 bp encoded a protein with 443 amino acids. The structure of this gene was analyzed by comparing its full length cDNA to homologous genomic DNA sequence （GenBank accession： Ac209463.3）. Subsequent sequence analysis led to sub cellular localization of protein ZM26SUB5, and construction of a phylogenetic tree. In silico mapping indicated that Zm26Sub5 was located on chromosome 5 and closed to a reported starch-filled pollen ratio QTL. ZM26SUB5, therefore might have potential roles in repressing mitochondrial PCD which is associated with sterile activity in pollen in S-type cytoplasm.

Inducing fertility restoration in the genic male sterile line of rice with chemical regulators

For the first time, the fertility of rice genic male sterile line was partially restored with the application of chemical regulators at Hainan Rice Breeding Nursery on Mar 1993. A single panicle of the rice plant could bear as many as 27 grains. The chemical agent that could modulate the growth of the rice plants of genic male sterile line was developed by Prof ZHOU Guangqia and his colleagues at the Biology Institute, Hunan Teachers University, China.

Genetic analysis of fertility restoration of Maxie cytoplasmic male sterility in rice(Oryza sativa L.)

The genetic behavior of fertility restorationgenes of the cytoplasmic male sterile line Max-ie A was studied to facilitate the use ofMaweizhan male sterile cytoplasm.The F,F,and Fof Maxie A/Minhui 63 were grownin paddy field,1993-1995.Meanwhile,someof the Fplants were testcrossed with themaintainer of Maxie A.The fertilities were

Development of Novel Cytoplasmic Male Sterile Source from Dongxiang Wild Rice(Oryza rufipogon)

This study was conducted to develop and characterize a novel cytoplasmic male sterile （CMS） source which was identified from Dongxiang wild rice （Oryza rufipogon） by crossing Dongxiang wild rice as female with Zhongzao 35, an indica inbred variety, as male and continuous backcrossing with Zhongzao 35. Observation under optical microscope manifested that this novel CMS belonged to typical abortion type with less pollen compared with wild abortive type cytoplasm （CMS-WA）. Sequential planting showed that this novel CMS has complete and stable male sterility. Testcross experiment showed that all the 24 tested materials including maintainer and restorer lines of CMS-WA and Honglian type cytoplasm （CMS-HL） and other indica inbred varieties are the maintainers with complete maintaining ability, suggesting that this novel CMS has fertility restoration totally different from CMS-WA and CMS-HL and belongs to a novel type of CMS. So far, we only discovered a unique fertility restoration source for this novel CMS. Inheritance analysis showed that the fertility restoration of this CMS was governed by three pairs of independent dominant genes. Prospect for application of this novel CMS system in hybrid rice breeding was also discussed.

Significant association of the novel Rf4-targeted SNP marker with the restorer for WA-CMS in different rice backgrounds and its utilization in molecular screening

In the rice cytoplasmic-genetic male sterility （CMS） system, the combination of a CMS line, maintainer line and restorer line carrying the restorer gene to restore fertility, is indispensable for the development of hybrids. However, the process of screening for the trait of fertility restoration is laborious and time-consuming. In the present study, we analyzed the nucleotide sequence of the Rf4 gene, which is the major locus controlling fertility restoration, to identify allele-specific variation. A single nucleotide polymorphism （SNP） A/C at ＋474 in the coding sequence （CDS） was found to be capable of strictly distinguishing groups of alleles Rf4 （A） and rf4 （C）. Using KASP genotyping, this valuable SNP was converted to an allele-specific PCR marker. We evaluated and validated the marker among three-line parents with different backgrounds, and the results revealed a complete correlation between SNP alleles and the fertility restoration phenotype. Molecular screening was subsequently carried out for the presence of alleles of Rf4 and Rf3 among 328 diverse rice cultivars with worldwide distribution. The results demonstrate that this SNP marker could be the optimal choice for the molecular identification of potential restorers.

MALE STERILITY 3 encodes a plant homeodomain-finger protein for male fertility in soybean

Male-sterile plants are used in hybrid breeding to improve yield in soybean(Glycine max(L.)Merr.).Developing the capability to alter fertility under different environmental conditions could broaden germplasm resources and simplify hybrid production.However,molecular mechanisms potentially underlying such a system in soybean were unclear.Here,using positional cloning,we identified a gene,MALE STERILITY 3(MS3),which encodes a nuclear-localized protein containing a plant homeodomain(PHD)-finger domain.A spontaneous mutation in ms3 causing premature termination of MS3 translation and partial loss of the PHD-finger.Transgenetic analysis indicated that MS3 knockout resulted in nonfunctional pollen and no selfpollinated pods,and RNA-seq analysis revealed that MS3 affects the expression of genes associated with carbohydrate metabolism.Strikingly,the fertility of mutant ms3 can restore under longd conditions.The mutant could thus be used to create a new,more stable photoperiod-sensitive genic male sterility line for two-line hybrid seed production,with significant impact on hybrid breeding and production.

Delayed callose degradation restores the fertility of multiple P/TGMS lines in Arabidopsis

Photoperiod/temperature-sensitive genic male sterility(P/TGMS)is widely applied for improving crop production.Previous investigations using the reversible male sterile(rvms)mutant showed that slow development is a general mechanism for restoring fertility to P/TGMS lines in Arabidopsis.In this work,we isolated a restorer of rvms–2(res3),as the male sterility of rvms–2 was rescued by res3.Phenotype analysis and molecular cloning show that a point mutation in UPEX1 l in res3 leads to delayed secretion of callase A6 from the tapetum to the locule and tetrad callose wall degradation.Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis demonstrated that the tapetal transcription factor ABORTED MICROSPORES directly regulates UPEX1 expression,revealing a pathway for tapetum secretory function.Early degradation of the callose wall in the transgenic line eliminated the fertility restoration effect of res3.The fertility of multiple known P/TGMS lines with pollen wall defects was also restored by res3.We propose that the remnant callose wall may broadly compensate for the pollen wall defects of P/TGMS lines by providing protection for pollen formation.A cellular mechanism is proposed to explain how slow development restores the fertility of P/TGMS lines in Arabidopsis.