NL2033533B1 - Breeding method of white-fleshed loquat and method for shortening juvenile phase - Google Patents
Breeding method of white-fleshed loquat and method for shortening juvenile phase Download PDFInfo
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- 238000009395 breeding Methods 0.000 title claims abstract description 23
- 230000000366 juvenile effect Effects 0.000 title claims abstract description 19
- 238000004904 shortening Methods 0.000 title claims abstract description 15
- 241001092070 Eriobotrya Species 0.000 title claims description 90
- 235000009008 Eriobotrya japonica Nutrition 0.000 title claims description 89
- 230000010152 pollination Effects 0.000 claims abstract description 14
- 238000009331 sowing Methods 0.000 claims abstract description 14
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 12
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 238000012408 PCR amplification Methods 0.000 claims description 27
- 239000002689 soil Substances 0.000 claims description 25
- 239000003147 molecular marker Substances 0.000 claims description 15
- 239000003337 fertilizer Substances 0.000 claims description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 238000005452 bending Methods 0.000 claims description 8
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 6
- 229910021538 borax Inorganic materials 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- 239000002362 mulch Substances 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 239000004328 sodium tetraborate Substances 0.000 claims description 5
- 235000010339 sodium tetraborate Nutrition 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000002274 desiccant Substances 0.000 claims description 4
- 230000007935 neutral effect Effects 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 3
- 239000000618 nitrogen fertilizer Substances 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000012364 cultivation method Methods 0.000 claims 3
- 244000052616 bacterial pathogen Species 0.000 claims 1
- 238000012136 culture method Methods 0.000 claims 1
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- 230000001737 promoting effect Effects 0.000 claims 1
- 238000009396 hybridization Methods 0.000 abstract description 8
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- 238000007493 shaping process Methods 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 3
- 239000013615 primer Substances 0.000 description 7
- 239000002987 primer (paints) Substances 0.000 description 7
- 238000004925 denaturation Methods 0.000 description 6
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- 108020004414 DNA Proteins 0.000 description 5
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 5
- 239000005556 hormone Substances 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 229960003390 magnesium sulfate Drugs 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003086 colorant Substances 0.000 description 3
- 230000004883 flower formation Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 235000007575 Calluna vulgaris Nutrition 0.000 description 2
- 241000135550 Eriobotrya prinoides Species 0.000 description 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000036760 body temperature Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
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- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000000855 fungicidal effect Effects 0.000 description 2
- 239000000417 fungicide Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000004745 nonwoven fabric Substances 0.000 description 2
- 239000003895 organic fertilizer Substances 0.000 description 2
- 239000003415 peat Substances 0.000 description 2
- 239000010451 perlite Substances 0.000 description 2
- 235000019362 perlite Nutrition 0.000 description 2
- 239000011574 phosphorus Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000010902 straw Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000010455 vermiculite Substances 0.000 description 2
- 235000019354 vermiculite Nutrition 0.000 description 2
- 229910052902 vermiculite Inorganic materials 0.000 description 2
- 102100036263 Glutamyl-tRNA(Gln) amidotransferase subunit C, mitochondrial Human genes 0.000 description 1
- 101001001786 Homo sapiens Glutamyl-tRNA(Gln) amidotransferase subunit C, mitochondrial Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000044800 Pellenes Species 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- YYRMJZQKEFZXMX-UHFFFAOYSA-N calcium;phosphoric acid Chemical compound [Ca+2].OP(O)(O)=O.OP(O)(O)=O YYRMJZQKEFZXMX-UHFFFAOYSA-N 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000003898 horticulture Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229940036310 program Drugs 0.000 description 1
- 238000013138 pruning Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 239000002426 superphosphate Substances 0.000 description 1
- CPYIZQLXMGRKSW-UHFFFAOYSA-N zinc;iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+3].[Fe+3].[Zn+2] CPYIZQLXMGRKSW-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/08—Fruits
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/74—Rosaceae, e.g. strawberry, apple, almonds, pear, rose, blackberries or raspberries
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Physiology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
The present disclosure provides a breeding method, of a white— fleshed loguat and a method for shortening a juvenile phase, and relates to the technical field of agricultural breeding. The breeding method, of a white—fleshed loguat and the method for 5 shortening a juvenile phase specifically include the following steps: 81 parent selection, 82 preparation of pollen, S3 hybridization and combination, S4 cold protection and fruit retention, 85 sowing and seedling raising, S6 nursery establishment, 87 transplanting and field, planting, 88 sapling lO fertilization, 89 sapling shaping, and 810 comprehensive juvenile phase shortening technology. In the present disclosure, the method improves a screening efficiency of hybrid parents and hybrid progenies of the white—fleshed, loguat, improves a preservation time and a pollination efficiency of loguat pollen, and improves a 15 yield of hybrid seeds. (+ Fig. 2)
Description
P1642 /NL
BREEDING METHOD OF WHITE-FLESHED LOQUAT AND METHOD FOR SHORTENING
JUVENILE PHASE
The present disclosure belongs to the technical field of ag- ricultural breeding, and in particular relates to a breeding meth- od of a white-fleshed loguat and a method for shortening a juve- nile phase.
Loguat is a famous and characteristic tree species native to
China, with extremely rich resources. Loquat varieties can be di- vided into white-fleshed loquat varieties and yellow-fleshed lo- quat varieties according to the flesh color. White-fleshed loguat varieties have milky white or pale yellow flesh, with tender,
Juicy, and sweet properties, resulting in an excellent flavor; and the white-fleshed loquat varieties are most suitable for fresh- eating due to a better quality than that of yellow-fleshed loqguat varieties. Although the white-fleshed loquat varieties have a bet- ter fruit quality than that of the yellow-fleshed loquat varie- ties, the yellow-fleshed logquat varieties are main varieties of loquat cultivated in the world. The white-fleshed loquat varieties mainly occupy a certain proportion in Jiangsu, Zhejiang, and
Shanghai and other places in a northern logquat production area of
China, and are rarely seen in the market of other loquat produc- tion areas. An important reason for this situation is that there is a relatively small number of white-fleshed loquat varieties, and each production area cannot supply suitable and excellent va- rieties from limited white-fleshed loquat resources. Therefore, it is an urgent task to adjust a variety structure of the loguat in- dustry in China and even in the world by cultivating a large num- ber of excellent varieties of the white-fleshed loquat.
In order to cultivate a large number of excellent varieties of the white-fleshed loquat, it is a key by innovation of breeding methods. So far, white-fleshed loquat varieties have mainly come from three methods: I, direct selection from white-fleshed germplasm resources by seedling selection, which serves as a source of the majority of white-fleshed loquat varieties; II, hy- bridization breeding with the white-fleshed loquat varieties as parents, which serves as a source of the minority of white-fleshed loquat varieties; III, bud mutation, which serves as a source of the tiny minority of white-fleshed loguat varieties. Moreover, in these methods, plants obtained from the first two main breeding methods of white-fleshed loguat generally require a juvenile phase of not less than 5 years before flowering and fruiting. Further- more, grafting or multiple applications of high-concentration hor- mones may affect botanical characters of the seedlings to a cer- tain extent.
An objective of the present disclosure is to provide a breed- ing method of a white-fleshed loquat and a method for shortening a juvenile phase. The method improves a screening efficiency of hy- brid parents and hybrid progenies of the white-fleshed loquat, im- proves a preservation time and a pollination efficiency of loquat pellen, and improves a yield of hybrid seeds. The method further avoids influences of grafting or multiple applications of high- concentration hormones on botanical characters of the loquat hy- brid progenies, and shortens the juvenile phase of the loguat hy- brid progenies by not less than one year.
The present disclosure provides a breeding method of a white- fleshed loquat, including the following steps: (1) using a white- fleshed loquat germplasm resource or a yellow-fleshed loguat germplasm resource with an EjPSY2A®° gene as a parent; (2) conducting artificial pollination on collected male par- ent pollen onto an emasculated female parent, followed by fruit bagging; and (3) sowing loguat seeds harvested in the bag the following year, identifying seedlings of white-fleshed loquat germplasms us- ing a molecular marker method after emergence, and conducting nursery establishment and field planting on the seedlings.
Preferably, in step (1), PCR amplification is conducted on the yellow-fleshed loguat germplasm resource using a molecular marker primer pair, when a PCR amplification product is shown as a band of 300 bp to 400 bp, the yellow-fleshed loquat germplasm re- source is determined as a white-fleshed loquat germplasm; and when the PCR amplification product is shown as a band of 300 bp to 400 bp and a band of greater than 500 bp, the yellow-fleshed loquat germplasm resource is determined as a yellow-fleshed loquat germplasm; and the molecular marker primer pair includes PSY2A-2F with a nu- cleotide sequence shown in SEQ ID NO: 1 and PSY2A-2R with a nucle- otide sequence shown in SEQ ID NO: 2.
Preferably, a program of the PCR amplification includes: ini- tial denaturation at 94°C for 4 min; denaturation at 95°C for 1 min, annealing at 55°C for 45 sec, and extension at 72°C for 1 min, conducting 35 cycles; and extension at 72°C for 10 min.
Preferably, in step (2), a collection method of the pollen includes: collecting all petals and stamens of an upper half of non-bloomed flower buds by clipping, filtering to obtain pure sta- mens, and drying the pure stamens in a constant-temperature green- house at 25°C to 28°C for 24 h to 36 h to obtain the pollen.
Preferably, the breeding method further includes mixing the pollen with a neutral desiccant to conduct preservation.
Preferably, in step (3), the molecular marker method includes the following steps: conducting PCR amplification using PSY2A-2F and PSY2A-2R, and when a PCR amplification product is shown as a band of 300 bp to 400 bp, determining the seedlings as the white- fleshed loquat germplasms; and the PSY2A-2F has a nucleotide sequence shown in SEQ ID NO: 1, and the PSY2A-2R has a nucleotide sequence shown in SEQ ID NO: 2.
The present disclosure further provides a method for shorten- ing a juvenile phase of a white-fleshed loquat obtained by the breeding method, including the following steps: conducting field planting on seedlings of the white-fleshed loquat on a high ridge with a height of 50 cm and a bottom width of 2 m in a nursery, and covering a mulch on a ridge surface; conducting topdressing by a nitrogen fertilizer at a sprout- ing stage in a first year of the field planting, and then top-
dressing by an NPK fertilizer in second and third years after the field planting; in the first year of the field planting, conducting heading trunk at 50 cm to 60 cm, selecting and remaining 3 to 4 main branches, selecting and remaining 2 to 3 lateral branches on each main branch, and conducting bending branch in the second year af- ter the field planting; and in the third year after the field planting, conducting gir- dling on the trunk, and in a fourth year after the field planting, spraying a flower-promoting agent including potassium dihydrogen phosphate, borax, and magnesium sulfate.
Preferably, the bending branch is conducted in late July of the second year after the field planting, such that the main branch and a central trunk have an open-branching angle of 60° to 80°.
Preferably, the loquat trunk is subjected to repeated gir- dling for 3 circles in June of the third year after the field planting.
Preferably, the flower-promoting agent is sprayed three times from June to July in the fourth year after the field planting.
Beneficial effects: the breeding method of a white-fleshed loquat and the method for shortening a juvenile phase specifically include the following steps: S1 parent selection, S2 preparation of pollen, S83 hybridization and combination, S4 cold protection and fruit retention, S5 sowing and seedling raising, S6 nursery establishment, S87 transplanting and field planting, S358 sapling fertilization, S9 sapling shaping, and S10 comprehensive juvenile phase shortening technology. In the present disclosure, the method improves a screening efficiency of hybrid parents and hybrid progenies of the white-fleshed loguat, improves a preservation time and a pollination efficiency of loquat pollen, and improves a yield of hybrid seeds. The method further avoids influences of grafting or multiple application of high-concentration hormones on botanical characters of the loguat hybrid progenies, shortens the juvenile phase of the loguat hybrid progenies by not less than one year, and makes the loquat hybrid progenies in a 4th year after sowing have an effective rate of flowers at not less than 77.5%.
To describe the embodiments of the present disclosure or the technical solutions in the related art more clearly, the accompa- 5 nying drawings required in the embodiments are briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present disclosure. A person of ordi- nary skill in the art may further obtain other accompanying draw- ings based on these accompanying drawings without creative labor.
FIG. 1 shows a method for soil improvement and nursery estab- lishment by soil ridging;
FIG. 2 shows a pruning method of sapling after field plant- ing;
FIG. 3 shows an effect of flower-promoting by bending branch, where a lower left corner of the figure indicates a shooting time, a location, and longitude and latitude, which are November 8§, 2021, the third section of Dongshan Avenue, Chengdu, and 30.369826°N and 104.157098°E, the same below; and
FIG. 4 shows an effect of comprehensive flower-promoting, where the following information is marked on each figure from top to bottom and from left to right: 13-2 x Guifei-54 (large amount of flowers); Huangfeng x Dawuxing-6 (extremely large amount of flowers and moderate tree vigor); Huangfeng x Dawuxing-3 (large amount of flowers); 43-15-12 (dwarfed type and large amount of flowers); Baisha x Huangmi 18 (large amount of flowers); Baisha x
Dawuxing-44 and Dawuxing-45 (large amount of flowers); Baisha x
Dawuxing-27 (extremely large amount of flowers); Dawuxing x Bai- sha-3 (extremely large amount of flowers); Baisha * Huangmi-17 (large amount of flowers); Yanhong x Dawuxing-4 (easy flower for- mation, large amount of flowers); Dawuxing x Baisha-7 (effective rate of flowers at almost 100%); and Dawuxing x Baisha-8 (effec- tive rate of flowers at almost 1003).
The present disclosure provides a breeding method of a white- fleshed loquat, including the following steps: (1) using a white- fleshed loquat germplasm resource or a yellow-fleshed loquat germplasm resource with an EjPSY2A° gene as a parent; (2) conducting artificial pollination on collected male par- ent pollen onto an emasculated female parent, followed by fruit bagging; and (3) sowing loquat seeds harvested in the bag the following year, identifying seedlings of white-fleshed loquat germplasms us- ing a molecular marker method after emergence, and conducting nursery establishment and field planting on the seedlings.
In the present disclosure, preferably according to a breeding goal, excellent white-fleshed loquat germplasm resources are se- lected as parents, or excellent yellow-fleshed loquat germplasm resources containing an EjPSY2AY gene are selected as parents using a molecular marker identification technology for loquat flesh col- ors. When conducting molecular marker identification for loquat flesh colors, preferably using a genomic DNA of the loquat germplasm as a template, a PCR reaction system is prepared with a molecular marker primer pair to conduct PCR amplification, and an obtained PCR amplification product is genotyped; when the PCR am- plification product is shown as a band of 300 bp to 400 bp, a sam- ple to be tested is determined as the white-fleshed loquat germplasm; and when the PCR amplification product is shown as a band of 300 bp to 400 bp and a band of greater than 500 bp, the sample to be tested is determined as the yellow-fleshed loquat germplasm containing an EjPSY2AY gene (which can also be used as a parent for hybridization and combination). A reaction system of the PCR amplification is based on 25 |L, preferably including: 1.0
HL of the template, 12.5 pL of a 2xPCR Mix, 1.0 pL of a 10 pM pri- mer F, 1.0 pL of a 10 pM primer R, and ddH:0 as a balance. A pro- gram of the PCR amplification preferably includes: initial dena- turation at 94°C for 4 min; denaturation at 95°C for 1 min, an- nealing at 55°C for 45 sec, and extension at 72°C for 1 min, con- ducting 35 cycles; and extension at 72°C for 10 min. Preferably, the PCR amplification product is genotyped using 8% polyacrylamide gel electrophoresis. The PSY2A-2F has a nucleotide sequence shown in SEQ ID NO: 1: AGCCTTTTCTTGAATGTGGTCG, and the PSY2A-2R has a nucleotide sequence shown in SEQ ID NO: 2: TTGATTGATGTTGGCTAATGA-
GATC. The yellow-fleshed loquat germplasm resources containing an
EjPSsY2A% gene identified by the method preferably include: Eri- obotrya prinoides, Zaohong 89-4, Huangfeng, Xiangzhong No. 11,
Wanzhong 518, Peiyou, Xishu No. 2, Wulila, Xishu No.4, Zaozhong
No. 6, Motegi, Jiefangzhong, Taicheng No.4, Huangjinkuai, Jialing, and Dahongpao.
In the present disclosure, pollen is prepared when loquat flower buds are not blooming. As in an example, non-bloomed loquat flower buds are collected 1 to 2 times in autumn and winter in
Chengdu, all petals and stamens of an upper half of non-bloomed flower buds are collected by clipping, and then filtered through 20-mesh and 35-mesh sieves step by step to obtain pure stamens (a screen aperture can be adjusted according to a size of the stamens of different loquat varieties); the pure stamens are dried in a constant-temperature greenhouse at 25°C to 28°C for 24 h to 36 h to obtain the pollen; and collected pollen are poured into a 50 mL centrifuge tube and added with an appropriate amount of a neutral desiccant (Anhydrous calcium chloride) or a discolored silica gel, and then store in a refrigerator at 4°C. The pollen is preferably derived from all the above parents by hybridization between the parents.
In the present disclosure, the hybridization and combination are conducted from an initial flowering stage to a full flowering stage of the female parent, preferably including pollination at 10:00 to 17:00 in a windless and sunny weather. When conducting the hybridization and combination, flowers that have bloomed or have not whitened are removed, and then artificially emasculated; before pollination, the pollen should be activated in warm sun- light or by body temperature for 0.5 h to 1 h; after pollination, the entire inflorescence is subjected to fruit bagging immediately with a 35 cm x 25 cm large fruit bag, and parental hybridization information is registered on a board.
In the present disclosure, after artificial pollination, the method further includes preferably conducting cold protection and fruit retention, more preferably covering a tree tray with straw to increase a soil temperature before low-temperature freezing in- juries, while covering an entire canopy with a 7.2 m wide cold- proof non-woven fabric (40 g/m’) along a row direction of the field planting.
In the present disclosure, loquat seeds harvested in the bags are sown the following year by a sowing substrate preferably in- cluding a mixture of peat, perlite, and vermiculite with a volume ratio of preferably 3:1:1. In an example, the sowing is preferably conducted in a square tray for seedling cultivation, with a length*width*height of preferably 42 cm*42 cm*5.5 cm, where 25 seeds are evenly sown in each tray, and seedlings are raised in a greenhouse for overwintering.
In the present disclosure, after the seeds germinate, hybrid offspring of the white-fleshed loquat is screened, PCR amplifica- tion is conducted preferably using PSY2A-2F and PSY2A-2R, and a germplasm type is determined according to amplification products; a procedure and a system of the PCR amplification are preferably the same as the above, which are not repeated here. The PCR ampli- fication preferably includes: 45 d to 60 d after sowing, collect- ing tender leaves or shoot tips of the seedlings for molecular marker identification, selecting hybrid offspring of the white- fleshed loquat with a PCR amplification product of a band of 300 bp to 400 bp, and listing information on a board for nursery es- tablishment and centralized field planting.
In the present disclosure, the soil improvement and nursery establishment is conducted preferable by soil ridging, preferably including: before the soil improvement, removing attachments in a garden and conducting land leveling; setting out lines according to a planting row spacing of 4 m x 5 m, and spreading a base fer- tilizer in a ridge area intensively; after ploughing, piling in- ter-row topsoil up in a ploughed area, to form a tile-backed ridge surface with a ridge height of about 50 cm and a bottom width of about 2 m. The base fertilizer includes preferably a decomposed organic fertilizer at a dosage of preferably 1,000 kg/667 m° and superphosphate at a dosage of preferably 100 kg/667 m®; soil with a depth of 30 cm to 40 cm is loosened and mixed with the fertilizers by an excavator, and the inter-row topsoil is piled up in the ploughed area to form the tile-backed ridge surface.
In the present disclosure, before field planting, preferably a field planting map is initially drawn according to different hy-
brid combinations, and seedlings are transplanted to the nursery at a minimum daily temperature in spring of greater than or equal to 10°C (the field planting in Chengdu is preferably conducted from mid-February to mid-March). 3 d to 5 d before transplanting, the leaves are sprayed with a broad-spectrum fungicide; when 1ift- ing, 1/2 to 1/3 of a leaf area is cut off on each leaf to reduce water evaporation. Field planting holes slightly larger than a root width of the hybrid seedling are dug on field planting sites, and the seedlings are gently put into the field planting sites; after righting, mature surface fine soil is filled as field plant- ing soil; and the soil is tread down to make a root system fully contact with the soil, while the root neck is exposed to the ground. After field planting, a watering circle is made with a di- ameter of greater than or equal to 80 cm, enough root-fixing water is applied, the exposed root system is covered with fine soil, and a ridge surface is covered by a mulch with a thickness of greater than or equal to 0.03 mm.
The present disclosure further provides a method for shorten- ing a juvenile phase of a white-fleshed loquat obtained by the breeding method, including the following steps: conducting field planting on seedlings of the white-fleshed loquat on a high ridge with a height of 50 cm and a bottom width of 2 m in a nursery, and covering a mulch on a ridge surface; conducting topdressing by a nitrogen fertilizer at a sprout- ing stage in a first year of the field planting, and then top- dressing by an NPK fertilizer in second and third years after the field planting; in the first year of the field planting, conducting heading trunk at 50 cm to 60 cm, selecting and remaining 3 to 4 main branches, selecting and remaining 2 to 3 lateral branches on each main branch, and conducting bending branch in the second year af- ter the field planting; and in the third year after the field planting, conducting gir- dling on the trunk, and in a fourth year after the field planting, spraying a flower-promoting agent including potassium dihydrogen phosphate, borax, and magnesium sulfate.
In the present disclosure, field planting of seedlings of the white-fleshed loquat is preferably the same as the above, which are not repeated here.
In the present disclosure, after the field planting, seed- lings of the loquat hybrids follow a principle of frequent and thin application, preferably including: in the year of field planting, topdressing can be conducted once every 15 d after each sprouting in late spring, summer, autumn and winter, with 100 g of urea or a high-nitrogen water-soluble fertilizer to each plant in each time; in the second and third years after field planting, a proportion of phosphorus and potassium fertilizers is gradually increased to promote the differentiation of flower buds, with a fertilization time being the same as before, that is, the top- dressing can be conducted once every 15 d after each sprouting in late spring, summer, autumn and winter. For example, in the second year after field planting, 50 g of potassium dihydrogen phosphate is applied each time, which reaches 100 g in the third year.
In the present disclosure, after field planting of the sap- lings, the method further includes sapling shaping, preferably the following steps: in the year after field planting of the hybrid seedlings, wiping off redundant new shoots mainly by a method of wiping off young shoots, and selecting and retaining shoots with suitable directions and positions; and conducting heading trunk at 50 cm to 60 cm, selecting and remaining 3 to 4 main branches, se- lecting and remaining 2 to 3 lateral branches on each main branch, and distribute the branches evenly. The bending branch is conduct- ed in late July of the second year after the field planting, such that the main branch and a central trunk have an open-branching angle of 60° to 80°, and twisting tip to promote flower formation on the lateral branches and auxiliary branches.
In the present disclosure, the loquat trunk is subjected to repeated girdling for 3 circles in June of the third year after the field planting to control tree vigor; from June to July of the fourth year after the field planting, a mixture of "0.3% (m/m) po- tassium dihydrogen phosphate + 0.3% (m/m) borax + 0.1% (m/m) mag- nesium sulfate" is sprayed 3 times at an interval of 10 d. The above measures, combined with ridging, drainage, and ridge surface mulching in the early stage, can reduce a soil moisture (con-
trolled at 15% to 18% on average), making the loquat hybrid proge- nies in a 4th year after sowing have an effective rate of flowers at not less than 77.5%.
In order to further illustrate the present disclosure, the breeding method of a white-fleshed loquat and the method for shortening a juvenile phase provided by the present disclosure are described in detail below in connection with accompanying drawings and examples, but these examples should not be understood as lim- iting the claimed scope of the present disclosure.
Example 1 (1) Parent selection: according to a breeding goal, excellent white-fleshed loquat germplasm resources were selected as parents, or excellent yellow-fleshed loquat germplasm resources containing an EjPSY2A® gene were selected as parents using a molecular marker identification technology (SEQ ID NO: 1 and SEQ ID NO: 2) devel- oped by a project team for loquat flesh colors;
The yellow-fleshed loguat germplasm resources containing an
EjPSy2A% gene included: Eriobotrya prinoides, Zaohong 89-4,
Huangfeng, Xiangzhong No. 11, Wanzhong 518, Peiyou, Xishu No. 2,
Wulila, Xishu No.4, Zaozhong No. 6, Motegi, Jiefangzhong, Taicheng
No.4, Huangjinkuai, Jialing, and Dahongpao.
The white-fleshed logquat germplasm resources included: Ru- antiao Baisha, Xishu No. 3, Xinbai No. 8, Xiangfei, Huangmi, Guan- yu, Ninghaibai, Guifei, Baizaozhong No. 5, Baizaozhong No. 7, Xin- bai No. 7, Dabaili, Qingzhong, Baiyu, Jidanbai, Taipingbai, Huabai
No. 1, Fengyu, Jinyu, Shiro Motegi, Wugongbai, Bingtangzhong, and
Zaojia No. 5.
The genomic DNA was derived from young leaves or shoot tips of the loquat germplasm.
A program of the PCR amplification included: initial denatur- ation at 94°C for 4 min; denaturation at 95°C for 1 min, annealing at 55°C for 45 sec, and extension at 72°C for 1 min, conducting 35 cycles; and extension at 72°C for 10 min.
A reaction system of the PCR amplification was based on 25 uL, including: 1.0 pL of a template, 12.5 pL of a 2xPCR Mix, 1.0 pL of a 10 uM primer F, 1.0 pL of a 10 uM primer R, and ddH:;0 as a balance; the PCR amplification product was genotyped using 8% pol-
yacrylamide gel electrophoresis.
When a PCR amplification product was shown as a band of 300 bp to 400 bp, a sample to be tested was determined as a white- fleshed loquat germplasm; and when the PCR amplification product was shown as a band of 300 bp to 400 bp and a band of greater than 500 bp, the sample to be tested was determined as a yellow-fleshed loguat germplasm containing an EjPSY2A% gene. (2) Preparation of pollen: non-bloomed loquat flower buds were collected 1 to 2 times in autumn and winter in Chengdu, all petals and stamens of an upper half of non-bloomed flower buds were collected by clipping, and then filtered through 20-mesh and 35-mesh sieves step by step to obtain pure stamens; the pure sta- mens were dried in a constant-temperature greenhouse at 25°C to 28°C for 24 h to 36 h to obtain the pollen; and collected pollen were poured into a 50 mL centrifuge tube and added with an appro- priate amount of a neutral desiccant (Anhydrous calcium chloride) or a discolored silica gel, and then store in a refrigerator at 4°C. (3) Hybridization and combination: pollination was conducted from an initial flowering stage to a full flowering stage of the female parent, at 10:00 to 17:00 in a windless and sunny weather.
First, flowers that had bloomed or had not whitened were removed, and then artificially emasculated; before pollination, the pollen should be activated in warm sunlight or by body temperature for 0.5 h to 1 h; after pollination, the entire inflorescence was sub- jected to fruit bagging immediately with a 35 cm x 25 cm large fruit bag. (4) Cold protection and fruit retention: a tree tray was cov- ered with straw to increase a soil temperature before low- temperature freezing injuries, while an entire canopy was covered with a 7.2 m wide cold-proof non-woven fabric (40 g/m*) along a row direction of the field planting. (5) Sowing and seedling raising: in July of the following year, obtained seeds were sown in a square tray for seedling cul- tivation, with a length*width*height of 42 cm*42 cm*5.5 cm, by a sowing substrate including a mixture of peat, perlite, and vermic- ulite with a volume ratio of 3:1:1, where 5*5 seeds were evenly sown in each tray, and seedlings were raised in a greenhouse for overwintering. (6) Screening of hybrid offspring of white-fleshed loquat: by the method in (1), 45 d to 60 d after sowing, tender leaves or shoot tips of the seedlings were collected for molecular marker identification, hybrid offspring of the white-fleshed loquat was selected with a PCR amplification product of a band of 300 bp to 400 bp, and information was listed on a board for nursery estab- lishment and centralized field planting. A total of 674 hybrids were obtained, including selected 451 white-fleshed loquat plants. (7) Nursery establishment: soil improvement and nursery es- tablishment were conducted by soil ridging. Before the soil im- provement, attachments in a garden were removed and land leveling was conducted; lines were set out according to a planting row spacing of 4 m x 5 m, and a base fertilizer containing 1,000 kg/667m? of a decomposed organic fertilizer + 100 kg/é67m° of su- perphosphate was spread in a ridge area intensively; soil with a depth of 30 cm to 40 cm was loosened and mixed with the fertiliz- ers by an excavator, and inter-row topsoil was piled up in a ploughed area to form a tile-backed ridge surface with a ridge height of about 50 cm and a bottom width of about 2 m. (8) Transplanting and field planting: a field planting map was initially drawn according to different hybrid combinations, and seedlings were transplanted to the nursery at a minimum daily temperature in spring of greater than or equal to 10°C (the field planting in Chengdu was preferably conducted from mid-February to mid-March). 3 d to 5 d before transplanting, the leaves were sprayed with a broad-spectrum fungicide; when lifting, 1/2 to 1/3 of a leaf area was cut off on each leaf to reduce water evapora- tion. Field planting holes slightly larger than a root width of the hybrid seedling were dug on field planting sites, and the seedlings were gently put into the field planting sites; after righting, mature surface fine soil was filled as field planting soil; and the soil was tread down to make a root system fully con- tact with the soil, while the root neck was exposed to the ground.
After field planting, a watering circle was made with a diameter of greater than or equal to 80 cm, enough root-fixing water was applied, the exposed root system was covered with fine soil, and a ridge surface was covered by a mulch with a thickness of greater than or equal to 0.03 mm. (8) Sapling fertilization: seedlings of the loquat hybrids followed a principle of frequent and thin application, including: in the year of field planting, topdressing could be conducted once every 15 d after each sprouting in late spring, summer, autumn and winter, with 100 g of urea or a high-nitrogen water-soluble ferti- lizer to each plant in each time; in the second and third years after field planting, a proportion of phosphorus and potassium fertilizers was gradually increased to promote the differentiation of flower buds. (9) Comprehensive juvenile phase shortening technology: in the year after field planting of the hybrid seedlings, redundant new shoots were wiped off mainly by a method of wiping off young shoots, and shoots were selected and retained with suitable direc- tions and positions. The heading trunk was conducted at 50 cm to 60 cm, 3 to 4 main branches were selected and remained, 2 to 3 lateral branches were selected and remained on each main branch, and the branches were distributed evenly. The bending branch was conducted in late July of the second year after the field plant- ing, such that the main branch and a central trunk had an open- branching angle of 60° to 80°, and twisting tip was conducted to promote flower formation on the lateral branches and auxiliary branches (FIG. 2 and FIG. 3).
The loquat trunk was subjected to repeated girdling for 3 circles in June of the third year after the field planting to con- trol tree vigor; from June to July of the fourth year after the field planting, a mixture of "0.3% potassium dihydrogen phosphate + 0.3% borax + 0.1% magnesium sulfate" was sprayed 3 times. The above measures, combined with early sapling fertilization and shaping, ridging, drainage, and ridge surface mulching, could re- duce a soil moisture, making the loquat hybrid progenies in a 4th year after sowing have an effective rate of flowers at not less than 77.5% (FIG. 4).
The method of the present disclosure improves a screening ef- ficiency of hybrid parents of the white-fleshed loquat, improves a preservation time and a pollination efficiency of loguat pollen, and improves a yield of hybrid seeds. The method further avoids influences of grafting or hormones on botanical characters of the logquat hybrid progenies, and shortens the juvenile phase of the loguat hybrid progenies by not less than one year.
Although the above examples have described the present dis- closure in detail, they are only a part of, not all of, the exam- ples of the present disclosure. Other examples may also be ob- tained by persons based on the examples without creative efforts, and all of these examples shall fall within the protection scope of the present disclosure.
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