A breeding method and application of a rice ld-type cytoplasmic male sterile line

Abstract

The present application relates to a kind of rice LD type cytoplasmic male sterile line breeding method and application, belong to rice genetic breeding and hybrid rice seed production technical field.The rice LD type cytoplasmic male sterile line breeding method includes the following steps: with rice LD type cytoplasmic donor as female, with the maintainer line not carrying Rf1 and Rf2 restorer gene as recurrent father, by continuous backcrossing, obtain rice LD type cytoplasmic male sterile line.The rice LD type cytoplasmic male sterile line screened by the present application Xixiang A shows pollen abortion stability, no self-crossing appearance, main agronomic traits approach Jiaxian A, is conducive to keeping the uniformity of seed parent traits and hybrid seed purity.At the same time, the rich hybrid rice sterile cytoplasm type is conducive to the rich sterile cytoplasm resource outside the existing BT type cytoplasmic male sterile line, reduces the genetic fragility risk brought by cytoplasm singleness, improves the risk resistance of hybrid rice industry.

Description

Technical Field

[0001] This invention belongs to the field of rice genetics and breeding and hybrid rice seed production technology, specifically relating to a method for breeding and applying a rice LD-type cytoplasmic male sterile line. Background Technology

[0002] Cytoplasmic male sterility (CMS) is the core foundation of three-line breeding in hybrid rice. The genetic diversity of sterile cytoplasm is one of the important factors affecting the genetic stability and production safety of hybrid rice, and it is of great significance to reduce the genetic vulnerability caused by cytoplasmic homogenization. At present, the mainstream sterile cytoplasm used in hybrid rice production in my country is mainly of the BT type, WA type, and HL type. The cytoplasm source is relatively singular. Long-term large-scale promotion may increase genetic homogenization and production risks, which is not conducive to the long-term stable development of the hybrid rice industry. There is an urgent need to explore new, stable, and compatible sterile cytoplasmic resources that are suitable for existing breeding systems.

[0003] Lead-type (LD) cytoplasm is a type of rice sterile cytoplasm with specific mitochondrial sequence characteristics. Its identification can be based on a combination of mitochondrial ORF79 sequence characteristics and the ORF79 / ATP6 read depth ratio, exhibiting significant specific molecular marker characteristics and possessing the potential to develop novel sterile lines. However, current technologies lack large-scale screening methods for LD-type cytoplasm germplasm, relying heavily on field phenotypic identification, which is inefficient and inaccurate. Existing sterile line conversion methods primarily target known cytoplasm, lacking a complete set of technologies for precisely locating specific LD-type cytoplasm at the whole-genome level and rapidly converting it into stable sterile lines. This has resulted in the ineffective application of this high-quality cytoplasmic resource in hybrid rice breeding. The fertility of LD-type cytoplasmic sterile lines can be restored by the Rf2 restorer gene, and BT-type restorer lines carrying the Rf1 restorer gene can also be used to restore the fertility of LD-type cytoplasmic sterile lines. This LD-type sterile line can be combined and utilized with existing BT-type restorer line resources. This genetic characteristic breaks through the conventional limitation that cytoplasmic type and restorer gene must correspond. LD-type sterile lines can be directly adapted to existing BT-type restorer line resources, making full use of mature restorer line breeding materials and significantly reducing the technical threshold and promotion cost of applying new cytoplasmic types.

[0004] While existing BT-type male-sterile lines are widely used, the problem of cytoplasmic homogeneity is prominent. Replacing the cytoplasm with new types under a homonuclear background and maintaining heterosis and stable yield traits are the core needs to overcome genetic vulnerability. However, there is still a lack of LD-type male-sterile lines that are compatible with existing BT-type restorer lines and have a nuclear background that is highly consistent with the main maintainer line. Summary of the Invention

[0005] The first objective of this invention is to provide a method for breeding rice LD-type cytoplasmic male sterile lines to solve the technical problems of relatively limited sterile cytoplasmic types and low screening efficiency of LD-type cytoplasmic resources in hybrid rice.

[0006] The second objective of this invention is to provide an application of a rice LD-type cytoplasmic male sterile line.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for screening rice LD-type cytoplasmic donors includes the following steps: based on whole-genome resequencing data, comparing the full-length orf79 sequence and analyzing the orf79 / atp6 read depth ratio, screening rice materials with 100% orf79 coverage, orf79 full-length sequence determined to be LD-type, and orf79 / atp6 read depth relatively high and close to 1, as rice LD-type cytoplasmic donors; preferably, the orf79 / atp6 read depth ratio is 0.70 to 1.00.

[0009] The LD-type materials screened in this embodiment of the invention include Jinan Wan, II Xiang and Gui 630, with orf79 / atp6 read depth ratios of 0.73, 0.93 and 0.85, respectively. Among them, II Xiang is preferred as the LD-type cytoplasmic donor for subsequent transfection.

[0010] A method for breeding a rice LD-type cytoplasmic male sterile line includes the following steps: using the rice LD-type cytoplasmic donors screened above as the female parent and the maintainer line that does not carry the Rf1 and Rf2 restoration genes as the recurrent male parent, the rice LD-type cytoplasmic male sterile line is obtained through continuous backcrossing.

[0011] Furthermore, the parent of the cycle is Jiaxi B.

[0012] Further, the continuous backcrossing step is as follows: the rice LD-type cytoplasmic donor is crossed with the recurrent paternal parent to obtain the F1 generation; the F1 generation is backcrossed with the recurrent paternal parent to obtain the BC1F1 generation; from the BC1F1 generation, individual plants that do not carry the functional restorer genes Rf1 and Rf2 and exhibit male sterility or high sterility are selected and backcrossed with the recurrent paternal parent to obtain the BC2F1 generation; from the BC2F1 generation, individual plants that do not carry the functional restorer genes Rf1 and Rf2 and exhibit male sterility or high sterility are selected and backcrossed with the recurrent paternal parent to obtain the BC2F1 generation. n F1 generation yields the LD-type cytoplasmic male sterile line of rice.

[0013] Furthermore, the continuous backcrossing refers to backcrossing for four or more generations.

[0014] The application of rice LD-type cytoplasmic male sterile lines bred using the above-mentioned breeding method in hybrid rice breeding.

[0015] Furthermore, the LD-type cytoplasmic male sterile line was crossbred with the BT-type restorer line DR610 for hybrid rice breeding.

[0016] The beneficial effects of this invention are:

[0017] This invention provides a method for screening LD-type cytoplasmic rice germplasm based on whole-genome resequencing, which can improve the accuracy and efficiency of screening LD-type cytoplasmic materials and provide a new technical approach for expanding the sterile cytoplasmic resources of hybrid rice.

[0018] The rice LD-type cytoplasmic male sterile line Xixiang A obtained by this invention exhibits stable pollen abortion and no self-pollination phenomenon, with its main agronomic traits closely resembling those of Jiaxi A. This is beneficial for maintaining the uniformity of parental traits and the purity of hybrid seed production. Simultaneously, it enriches the types of sterile cytoplasmic lines in hybrid rice, breaks the monopoly of BT-type cytoplasmic lines, reduces the risk of genetic vulnerability, and enhances the resilience of the hybrid rice industry.

[0019] The F1 hybrids of the rice LD-type cytoplasmic male sterile line Xixiang A and the BT-type restorer line DR610, screened by this invention, showed no significant difference in yield compared to combinations bred from the control BT-type homonuclear background male sterile line, demonstrating comparable yield levels. Therefore, Xixiang A can be directly integrated into the existing hybrid rice breeding system, broadening the resources of sterile cytoplasm and enhancing the genetic diversity of hybrid rice. Thus, Xixiang A has the potential to be used as a novel LD-type cytoplasmic male sterile line for hybrid rice combination breeding. Attached Figure Description

[0020] Figure 1 The graph shows the detection rate of the orf79 sequence and the analysis of cytoplasmic types in 148 rice materials in Example 1. (a) shows the detection rate of the orf79 sequence, (b) shows the proportion of different cytoplasmic types in the orf79 positive materials, and (c) shows the bar graph of the orf79 / atp6 ratio in the orf79 positive materials.

[0021] Figure 2 This is a flowchart illustrating the breeding method for rice LD-type cytoplasmic male sterile lines in Example 1. In the diagram, S represents sterile cytoplasm (which can be fertile when a functional restoration gene is present), N represents normal cytoplasm, Rf1 and Rf2 represent fertility restoration function at the corresponding restoration sites, and rf1 and rf2 represent fertility restoration function at the corresponding restoration sites.

[0022] Figure 3 The image shows a microscopic examination of I2-KI stained pollen from Jiaxi A flower in Example 1. The scale bar is 100 μm.

[0023] Figure 4 The image shows a microscopic examination of I2-KI stained pollen from *Pterocarya stenoptera* A in Example 1. The scale bar is 100 μm.

[0024] Figure 5 Images show the pollen fertility and panicle morphology of F1 cells from crosses between Jiaxi A (control) and Xixiang A with different maintainer lines. (a) shows the I2-KI staining results of pollen from Jiaxi A; (b) shows the I2-KI staining results of pollen from the F1 cells from the cross between Xixiang A and Quanjing 1B; (c) shows the I2-KI staining results of pollen from the F1 cells from the cross between Xixiang A and Zaoxiu 34; (d) shows the I2-KI staining results of pollen from the F1 cells from the cross between Xixiang A and Hubble 2B; and (e) shows the I2-KI staining results of pollen from the F1 cells from the cross between Xixiang A and different maintainer lines. The pollen I2-KI staining results of Jinjing 7B hybrid F1 are shown in the following figures: (f) is the mature panicle morphology of Jiaxi A; (g) is the mature panicle morphology of Xixiang A × Quanjing 1B hybrid F1; (h) is the mature panicle morphology of Xixiang A × Zaoxiu 34 hybrid F1; (i) is the mature panicle morphology of Xixiang A × Hubble 2B hybrid F1; and (j) is the mature panicle morphology of Xixiang A × Jinjing 7B hybrid F1. The scale bar of (a)-(e) in the figure is 100 μm, and the scale bar of (f)-(j) is 3 cm.

[0025] Figure 6 The figures show the field phenotypic diagrams of the F1 hybrids of Jiaxi A and DR610, and Xixiang A and DR610 in Experimental Example 2. (a) is a field growth diagram of the F1 hybrid of Jiaxi A and DR610, (b) is a field growth diagram of the F1 hybrid of Xixiang A and DR610, (c) is a mature rice panicle diagram of the F1 hybrid of Jiaxi A and DR610, (d) is a mature rice panicle diagram of the F1 hybrid of Xixiang A and DR610, (e) is a brown rice diagram of the F1 hybrid of Jiaxi A and DR610, and (f) is a brown rice diagram of the F1 hybrid of Xixiang A and DR610. The scale bar of (c)-(d) is 3 cm, and the scale bar of (e)-(f) is 1 cm. Detailed Implementation

[0026] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0027] Example 1

[0028] The breeding method for the rice LD-type cytoplasmic male sterile line in Example 1 includes the following steps:

[0029] 1. Test materials:

[0030] Germplasm tested: 148 rice cultivars, local varieties and intermediate breeding materials;

[0031] Nuclear donor retention system: Jiaxi B;

[0032] Recovery system: DR610;

[0033] Control sterile line: Jiaxi A (a BT-type cytoplasmic male sterile line with a Jiaxi B nuclear background).

[0034] 2. Breeding steps:

[0035] Step 1: Screening of LD-type cytoplasmic germplasm

[0036] Whole-genome resequencing was performed on 148 rice germplasm materials. Based on the whole-genome resequencing data, the presence of the orf79 gene in the mitochondrial genome was analyzed. By comparing the full-length orf79 gene sequence, rice materials containing the orf79 gene were classified into three types of cytoplasmic sterility: HL-CMS, LD-CMS, and BT-CMS. Figure 1 As shown in (a) and (b) above. In a typical LD-type mitochondrial structure, orf79 is located downstream of atp6, forming an atp6-orf79 structure. Therefore, the orf79 / atp6 read depth ratio should theoretically be close to 1. Due to factors such as sequencing depth, sequence alignment, mitochondrial genome copy number, and sample differences, the measured ratio may deviate from 1. In this embodiment, Jinan Wan, II Xiang, and Gui 630 were all determined to be LD-type by orf79 full-length sequence alignment, with orf79 / atp6 read depth ratios of 0.73, 0.93, and 0.85, respectively. In the above LD-type materials, such as Figure 1 As shown in (c), the orf79 coverage of II Xiang was 100%, the orf79 / atp6 read depth ratio was 0.93, and the material source was clear and could be used for subsequent hybridization and breeding. Therefore, II Xiang was selected as the LD-type cytoplasmic donor maternal parent. The sequencing analysis results of atp6 and orf79 in 148 rice materials are summarized in Table 1.

[0037] Table 1. Summary of sequencing analysis results of ATP6 and ORF79 in 148 rice materials

[0038] 1 Osmanthus Yellow 55 Indica rice 1149 120 Missing 0 0 2 Tetepu 54 Indica rice 1396 85 Missing 0 0 3 Zhejiang Fu 802 55 Indica rice 2101 136 Missing 0 0 4 Miracle Rice 52 Indica rice 2101 144 Missing 0 0 5 Huang Huazhan 55 Indica rice 1584 132 Missing 0 0 6 Hua Zhan 63 Indica rice 1135 135 Missing 0 0 7 IR64 58 Indica rice 1950 148 Missing 0 0 8 Minghui 63 55 Indica rice 1678 140 Missing 0 0 9 Zhenshan 97 57 Indica rice 1994 164 Missing 0 0 10 Guanglu Dwarf No. 4 58 Indica rice 1816 127 Missing 0 0 11 Lansheng 60 Japonica rice 1433 163 Missing 0 0 12 C Fortress 60 Japonica rice 2076 186 Missing 0 0 13 Kunming Xiaobai Valley 62 Japonica rice 1206 113 Missing 0 0 14 Psa33 44 Indica rice 1262 109 Missing 0 0 15 IR56 42 Indica rice 817 91 Missing 0 0 16 Taitung upland rice 69 Japonica rice 1059 103 Missing 0 0 17 Longjing No. 21 48 Japonica rice 1023 113 Missing 0 0 18 International No. 24 56 Indica rice 2405 194 Missing 0 0 19 Miryang 42 61 Indica rice 2234 191 Missing 0 0 20 Haobayong No. 1 62 Japonica rice 1190 109 Missing 0 0 21 77-175 59 Indica rice 1795 126 Missing 0 0 22 Hongyun 33 56 Indica rice 1990 160 Missing 0 0 23 Jinan Evening 54 Indica rice 1111 808 LD type 100 0.73 24 Xuanen Changtan Qingzhan 59 Indica rice 2202 171 Missing 0 0 25 Nanbaozao 60 Indica rice 2150 169 Missing 0 0 26 Minke Early No. 1 52 Indica rice 2197 164 Missing 0 0 27 Yellow shell early 20 days 60 Japonica rice 1437 139 Missing 0 0 28 Wuyunjing No. 7 64 Japonica rice 1660 173 Missing 0 0 29 Yunnan 4 Glutinous Rice 52 Japonica rice 1715 147 Missing 0 0 30 Yunjing 37 55 Japonica rice 870 97 Missing 0 0 31 Yanfeng 47 61 Japonica rice 1388 149 Missing 0 0 32 II Fragrance 73 Indica rice 1246 1164 LD type 100 0.93 33 Yuefeng 55 Indica rice 1705 111 Missing 0 0 34 Bobai 54 Indica rice 1028 70 Missing 0 0 35 Longtepu 58 Indica rice 2166 175 Missing 0 0 36 Chujing No. 27 71 Japonica rice 1235 130 Missing 0 0 37 Nanjing 46 46 Japonica rice 850 89 Missing 0 0 38 Wanlox 53 Japonica rice 1156 126 Missing 0 0 39 Lenon 67 Japonica rice 1166 128 Missing 0 0 40 Jiahe 212 56 Japonica rice 1033 110 Missing 0 0 41 Baitian Rice 55 Indica rice 1314 100 Missing 0 0 42 11 AM 54 Indica rice 1416 105 Missing 0 0 43 Zhan Gu 76 Indica rice 1963 168 Missing 0 0 44 IR40 54 Indica rice 1166 98 Missing 0 0 45 DawDam 59 Japonica rice 1028 128 Missing 0 0 46 Multi-series No. 1 63 Indica rice 1585 131 Missing 0 0 47 Ailishu 59 Indica rice 1061 127 Missing 0 0 48 Pitt 55 Indica rice 1670 139 Missing 0 0 49 Kanto 51 63 Japonica rice 2093 226 Missing 0 0 50 Neixiang No.7 62 Indica rice 1148 168 Missing 0 0 51 Longjing No. 46 50 Japonica rice 1507 163 Missing 0 0 52 Suijing No. 4 57 Japonica rice 1224 132 Missing 0 0 53 Three grains 59 Japonica rice 1218 117 Missing 0 0 54 Taichung No. 1 57 Indica rice 2089 138 Missing 0 0 55 Kanto 146 58 Japonica rice 846 76 Missing 0 0 56 cpslo17 55 Japonica rice 860 73 Missing 0 0 57 Wushan Oil Origin 60 Indica rice 1082 111 Missing 0 0 58 Lijiang New Group Heigu 56 Japonica rice 758 93 Missing 0 0 59 Kyu 630 54 Indica rice 855 723 LD type 100 0.85 60 Miryang 46 54 Indica rice 1089 79 Missing 0 0 61 Lamont 65 Japonica rice 912 75 Missing 0 0 62 Wuyujing No. 3 57 Japonica rice 912 75 Missing 0 0 63 replacement card 47 Japonica rice 1153 89 Missing 0 0 64 SWR17 65 Japonica rice 1835 152 Missing 0 0 65 Rethu Heenati 58 Indica rice 790 76 Missing 0 0 66 Tianfeng-Pigm+Xa23 62 Indica rice 2209 173 Missing 0 0 67 Cypress 71 Japonica rice 1467 133 Missing 0 0 68 Tianfeng 56 Indica rice 1382 116 Missing 0 0 69 Xiushui 11 53 Japonica rice 823 86 Missing 0 0 70 Longge 113 53 Indica rice 1714 139 Missing 0 0 71 Creek Selection No. 4 53 Indica rice 1826 131 Missing 0 0 72 One stick of incense 50 Indica rice 1385 69 Missing 0 0 73 White-shelled drought-resistant rice 50 Indica rice 1657 143 Missing 0 0 74 Huajingxian 74 61 Indica rice 906 77 Missing 0 0 75 Xieqing Early 76 Indica rice 1740 150 Missing 0 0 76 Chunjiang06 56 Japonica rice 1436 123 Missing 0 0 77 International No. 26 50 Indica rice 1377 116 Missing 0 0 78 Koshihikari 58 Japonica rice 1356 161 Missing 0 0 79 IR32 60 Indica rice 1570 117 Missing 0 0 80 Early-maturing Nonghu No. 6 67 Japonica rice 1163 105 Missing 0 0 81 Hexi No. 22 52 Japonica rice 901 99 Missing 0 0 82 R308 60 Indica rice 1241 105 Missing 0 0 83 Dongye No. 1 53 Japonica rice 1056 123 Missing 0 0 84 International No. 36 60 Indica rice 1749 156 Missing 0 0 85 Early dedication to the Party 62 Indica rice 882 95 Missing 0 0 86 Aso Ren 62 Japonica rice 1139 113 Missing 0 0 87 Dahanara 2220 55 Indica rice 842 794 BT type 100 0.94 88 IR58025 55 Indica rice 1585 142 Missing 0 0 89 C418 66 Japonica rice 1070 104 Missing 0 0 90 Guangyuan No. 1 64 Indica rice 1044 1021 HL type 100 0.98 91 Green japonica rice accounts for 1 53 Indica rice 1928 134 Missing 0 0 92 Black stockings 57 Indica rice 1185 98 Missing 0 0 93 Late Feng Early 1 57 Indica rice 2190 133 Missing 0 0 94 Yinhua Zhan 70 Indica rice 2272 209 Missing 0 0 95 Beizhen Dwarf No. 8 77 Indica rice 2098 166 Missing 0 0 96 Green and yellow occupy 77 Indica rice 2194 168 Missing 0 0 97 Ba San Zhan 67 Indica rice 2173 164 Missing 0 0 98 Ai Xiuzhan 60 Indica rice 2001 154 Missing 0 0 99 Suijing 62 Indica rice 825 88 Missing 0 0 100 Xianxi Zhan 56 Indica rice 1184 76 Missing 0 0 101 Narrow Leaf Green No. 8 55 Indica rice 928 109 Missing 0 0 102 Guichao No. 2 54 Indica rice 1761 127 Missing 0 0 103 Chuunibyou Soft Occupation 68 Indica rice 1492 149 Missing 0 0 104 Maguko 54 Japonica rice 774 73 Missing 0 0 105 dryland rice 71 Indica rice 1708 123 Missing 0 0 106 Basmati 385 71 Indica rice 1136 615 BT type 100 0.54 107 Rice husk 71 Indica rice 1313 101 Missing 0 0 108 Jingshan Zhan 77 Indica rice 3164 249 Missing 0 0 109 Low-footed black tip 57 Indica rice 1629 123 Missing 0 0 110 Zhongnong No. 4 64 Indica rice 1623 131 Missing 0 0 111 Gu Mei No. 2 64 Indica rice 972 97 Missing 0 0 112 Nan Shuang Ai 64 Indica rice 2897 195 Missing 0 0 113 Special Indica varieties account for 13 64 Indica rice 1692 98 Missing 0 0 114 Feng Bazhan 66 Indica rice 2267 181 Missing 0 0 115 Thermal Research No. 2 92 Japonica rice 1343 264 Missing 0 0 116 Aizhenzhan 63 Indica rice 1353 132 Missing 0 0 117 Xiangsi Miao No. 2 55 Indica rice 1662 129 Missing 0 0 118 Red Radiation Early No. 2 67 Indica rice 2091 145 Missing 0 0 119 Double Dwarf No. 11 61 Indica rice 1459 129 Missing 0 0 120 Sanhuangzhan No. 2 72 Indica rice 2191 172 Missing 0 0 121 Xinqing 92 62 Indica rice 2254 116 Missing 0 0 122 Seven Green Occupations 56 Indica rice 1364 104 Missing 0 0 123 Katsurano No. 2 55 Indica rice 2174 168 Missing 0 0 124 Nancong 3 65 Indica rice 2280 119 Missing 0 0 125 Ke Qingnuo 67 Indica rice 2264 148 Missing 0 0 126 Guangke No. 36 52 Indica rice 1468 110 Missing 0 0 127 Cantonese incense 70 Indica rice 2249 188 Missing 0 0 128 Gui Nong Zhan 67 Indica rice 2693 138 Missing 0 0 129 Special 351 63 Indica rice 1925 186 Missing 0 0 130 The Second Seven Divination 68 Indica rice 1925 153 Missing 0 0 131 Jade Rice 2 66 Indica rice 2887 152 Missing 0 0 132 Lu Xiang Zhan 3 72 Indica rice 2445 194 Missing 0 0 133 Qingzhen No. 1 60 Indica rice 2214 134 Missing 0 0 134 Hua Si Zhan 62 Indica rice 1896 1430 HL type 100 0.75 135 Shanyou 836-1 58 Indica rice 2373 168 Missing 0 0 136 Zhen Gui Dwarf No. 1 58 Indica rice 2001 180 Missing 0 0 137 Qinggui Dwarf No. 5 59 Indica rice 1303 121 Missing 0 0 138 Lijing Red Rice 71 Indica rice 3383 236 Missing 0 0 139 Double-grain rice 71 Indica rice 2191 156 Missing 0 0 140 Silver Shop 54 Japonica rice 1625 198 Missing 0 0 141 32 Dwarf 64 Indica rice 2959 301 Missing 0 0 142 Japonica and Indica No. 89 64 Indica rice 2959 301 Missing 0 0 143 1537 61 Indica rice 2160 192 Missing 0 0 144 Shuanggui 36 50 Indica rice 2414 162 Missing 0 0 145 Early Osmanthus No. 1 50 Indica rice 2414 162 Missing 0 0 146 Xian Xiaozhan 57 Indica rice 1342 156 Missing 0 0 147 Shuanggui No. 1 60 Indica rice 2581 200 Missing 0 0 148 Jiaxi B 30 Japonica rice 385 48 Missing 0 0

[0039] Table 1 classifies the orf79 state into deletion, LD, BT, and HL types based on sequence variation characteristics. The orf79 / atp6 read depth ratio is only meaningful in materials with 100% orf79 coverage; for materials determined to have orf79 deletion, the reads aligned to orf79 originate from other homologous sequences. The atp6 coverage of all 148 rice varieties was 100%. The average sequencing depth of the whole genome is indicated by ×.

[0040] Step 2: Crossbreeding to obtain F1 generation

[0041] Using 'II Xiang' as the female parent and 'Jiaxi B' as the male parent, F1 seeds were obtained through artificial emasculation and pollination. Since 'Jiaxi B' does not carry functional Rf1 and Rf2 restorer genes, while 'II Xiang' carries Rf1 and Rf2 genes, the F1 generation of the hybrid carries functional restorer allelic variants from 'II Xiang'. However, due to the incompatibility between indica and japonica rice, the seed setting rate will decrease, resulting in only some fertile plants. Therefore, the F1 generation plants exhibit fertility restoration or partial restoration when planted in the field.

[0042] Step 3: Continuous backcrossing and breeding

[0043] Using F1 generation as the female parent and Jiaxi B as the recurrent male parent, backcrossing yields BC1F1 generation seeds. More than 200 BC1F1 generation seeds are planted, and pollen from the BC1F1 generation is examined under a microscope using I2-KI staining. Individual plants meeting the following criteria are selected: (1) Pollen I2-KI staining shows male sterility or high sterility, with some deeply stained pollen grains exhibiting LD-type cytoplasmic characteristics allowed; (2) Does not carry Rf1 and Rf2 fertility restoration genes; (3) Major agronomic traits are similar to Jiaxi A. BC1F1 generation plants meeting the above criteria are backcrossed with Jiaxi B to obtain BC2F1 generation seeds. More than 200 plants of BC2F1 generation seeds were planted, and pollen from the BC2F1 generation was examined under a microscope using I2-KI staining. Individual plants meeting all three conditions were backcrossed with Jiaxi B to obtain BC3F1 generation seeds. More than 200 BC3F1 generation seeds were planted, and pollen from the BC3F1 generation was examined under a microscope using I2-KI staining. Individual plants meeting all three conditions were backcrossed with Jiaxi B to obtain BC4F1 generation seeds. The main agronomic traits of BC4F1 generation plants were relatively uniform and showed no significant difference from Jiaxi A, thus obtaining a rice LD-type cytoplasmic male-sterile line, named Xixiang A. Under the LD-type cytoplasmic background, some pollen grains showed deep I2-KI staining, reflecting a certain accumulation of starch within the pollen grains, but not equivalent to normal pollination and fertilization capacity. Combined with the self-pollination failure phenotype at maturity, Xixiang A was determined to exhibit stable male sterility.

[0044] Experimental Example 1

[0045] Tin Fragrance A Infertility Maintenance Verification

[0046] Using Xixiang A as the female parent, it was crossed with four different japonica type maintainer lines: Quanjing 1B, Zaoxiu 34, Hubble 2B, and Jinjing 7B. The homonuclear background BT-type male-sterile line Jiaxi A was used as a control. The F1 generation of the hybrids was planted. Pollen fertility was examined under a microscope using 1% I2-KI staining at flowering time, and panicle morphology and grain number were assessed at maturity. Figure 5As shown in Table 2, the F1 generation pollen from crosses between Xixiang A and maintainers of Quanjing 1B, Zaoxiu 34, Hubble 2B, and Jinjing 7B all exhibited sterility, with no rice grains settling in the panicles. This is consistent with the sterility trait of the control Jiaxi A, confirming that after Xixiang A is crossed with various japonica maintainer lines, the offspring still retain the male sterility phenotype, indicating that its sterility can be stably maintained.

[0047] Table 2. Statistics on the number of filled seeds and total number of seeds in hybrid combinations of Xixiang A and different maintainer lines.

[0048] Tin Fragrance A × Puree 1B 0 1255 Xi Xiang A × Zao Xiu 34 0 1434 Tin Incense A × Hubble 2B 0 1326 Tin Incense A × Golden Rice 7B 0 1261

[0049] Experimental Example 2

[0050] Verification of Tin Fragrance A Combining Ability and Yield

[0051] Using the male-sterile lines Xixiang A and the control BT-type Jiaxi A as female parents, hybrid F1 was obtained by crossing them with the restorer line DR610 carrying the Rf1 fertility restoration gene. The F1 hybrids were planted at a ratio of 2 plants per hole, with 5 replicates and consistent field management. After maturity, yield, number of grains per ear, and number of tillers were measured. The comparison results of yield-related traits are shown in […]. Figure 6 And Table 3, from Figure 6 As shown in Table 3, the two combinations did not differ significantly in yield traits, exhibiting comparable yield levels. The main yield components, such as the number of tillers and the number of grains per panicle, did not differ significantly, and the overall yield performance was stable. This indicates that Xixiang A can be effectively paired with the restorer line DR610 carrying the Rf1 restorer gene. The yield and main yield components of the resulting hybrid F1 were not significantly different from the control combination, demonstrating its potential for hybrid rice combination breeding and application.

[0052] Table 3 Comparison of yield and yield-related traits between the two combinations

[0053] Yield (kg / mu) 5 742.5±55.3 764.7±59.6 0.560 Number of grains per ear 30 275.1±23.9 280.0±26.8 0.457 Number of tillers (ears / hole) 30 11.8±2.7 12.3±2.0 0.482

[0054] The above results indicate that Xixiang A is an LD-type cytoplasmic male sterile line derived from II Xiang, exhibiting stable male sterility. When it is paired with the restorer line DR610 carrying the Rf1 restorer gene, the yield and main yield-related traits of the hybrid F1 are not significantly different from the control combination, indicating its potential for application in hybrid rice combination breeding.

[0055] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice disclosed herein. This invention is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the claims.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention is determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification should also be included within the scope of protection of the present invention.

Claims

1. The application of a rice LD-type cytoplasmic male sterile line in hybrid rice breeding, characterized in that, Hybrid rice breeding was carried out by crossing LD-type cytoplasmic male sterile rice lines with BT-type restorer lines DR610. The breeding method of the LD-type cytoplasmic male sterile rice lines included the following steps: using LD-type cytoplasmic donors as female parents and maintainer lines without Rf1 and Rf2 restorer genes as recurrent male parents, LD-type cytoplasmic male sterile rice lines were obtained through continuous backcrossing. The screening method of LD-type cytoplasmic donors included the following steps: based on whole-genome resequencing data, the full-length orf79 sequence was compared and the orf79 / atp6 read depth ratio was analyzed. Rice materials with 100% orf79 coverage, LD-type full-length orf79 sequence, and orf79 / atp6 read depth ratio of 0.70 to 1.00 were selected as LD-type cytoplasmic donors.

2. The application of the rice LD-type cytoplasmic male sterile line according to claim 1 in hybrid rice breeding, characterized in that, The orf79 / atp6 read segment depth ratio is 0.

93.

3. The application of the rice LD-type cytoplasmic male sterile line according to claim 1 in hybrid rice breeding, characterized in that, The parent of the cycle is Jiaxi B.

4. The application of the rice LD-type cytoplasmic male sterile line according to claim 1 in hybrid rice breeding, characterized in that, The continuous backcrossing steps are as follows: The rice LD-type cytoplasmic donor is crossed with the recurrent paternal parent to obtain the F1 generation; the F1 generation is backcrossed with the recurrent paternal parent to obtain the BC1F1 generation; from the BC1F1 generation, individual plants that do not carry the functional restorer genes Rf1 and Rf2 and exhibit male sterility or high sterility are selected and backcrossed with the recurrent paternal parent to obtain the BC2F1 generation; from the BC2F1 generation, individual plants that do not carry the functional restorer genes Rf1 and Rf2 and exhibit male sterility or high sterility are selected and backcrossed with the recurrent paternal parent to obtain the BC2F1 generation. n F1 generation yields the LD-type cytoplasmic male sterile line of rice.

5. The application of the rice LD-type cytoplasmic male sterile line according to claim 1 in hybrid rice breeding, characterized in that, The term "continuous backcrossing" refers to backcrossing for four or more generations.

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