CN103509861A - Molecular breeding method for synchronously improving length, strength, and fineness of cotton fiber through polymerizing chromosome segment introgression lines - Google Patents

Molecular breeding method for synchronously improving length, strength, and fineness of cotton fiber through polymerizing chromosome segment introgression lines Download PDF

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CN103509861A
CN103509861A CN201310350208.1A CN201310350208A CN103509861A CN 103509861 A CN103509861 A CN 103509861A CN 201310350208 A CN201310350208 A CN 201310350208A CN 103509861 A CN103509861 A CN 103509861A
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张天真
曹志斌
王鹏
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Abstract

The invention belongs to the field of molecular breeding, and relates to a molecular breeding method for synchronously improving the length, strength, and fineness of cotton fiber through polymerizing chromosome segment introgression lines. The method comprises the following steps: taking two introgression lines, namely IL080-D6-1 and 1047 (IL019-A2-6 chromosome segment introgression line), as the non-recurrent parents, respectively subjecting the non-recurrent parents to carry out hybridization with a recurrent parent (Xinjiang 0768) for one time, backcrossing two generations, in BC2F1 making strains containing different chromosome segments intercross with each other so as to obtain double-segment integrated F2 segregation population, identifying the double-segment homozygous strains through each two molecular labels on two segments, carrying out field tests under four circumstances, and keeping on selfing for two generations so as to obtain a double-segment homozygous novel cotton breed, whose fiber length, fiber strength and fineness have been prominently improved. A high-quality and high-yield novel cotton species, named "Nannong 08-66", has been cultured by utilizing the method provided by the invention.

Description

A kind of molecular breeding method of synchronously improveing cotton fiber length, intensity and fineness by polymerization chromosome segment introgressive line
Technical field
The invention belongs to field of molecular breeding, relate to a kind of molecular breeding method of synchronously improveing cotton fiber length, intensity and fineness by polymerization chromosome segment introgressive line.
Background technology
Cotton is global Important Economic crop.China is the big producing country of cotton, consumption big country and weaving big country.As Cotton Production big country, China's main product cotton region covers 16Ge Sheng, city (autonomous region), plants 7000-8,000 ten thousand mu of cotton areas throughout the year, always produces 1/4th of the total product in the Yue600 Wan Dun，Zhan world.Along with the progress of textile technology, textile product also develops into from coarse yarn the fine sand that high grade cotton yarn even reaches 100, and processing object is long stapled cotton, long stapled cotton and super long stapled cotton in general medium staple cotton is developed into also.Wang Yanqin etc. chose 546 basic points by 2003-2007 years continuous 5 years in three large main product cotton regions, 2201 parts of cotton samples carry out fiber quality and detect the average 28.8mm of staple length that finds China's upland cotton sample, luffing 24.0～34.7mm, main medium staple (28.0～31.0mm) scope in medium staple cotton, in long scope; The average 28.3cN/tex of strength, luffing 19.7-38.3cN/tex, most in medium (26.0～29.0cN/tex) scope, in to strong scope; Mic value is average 4.2, and luffing 2.0～5.2 is most in (3.7～4.9) scope (Wang Yanqin, Yang Wei China, Xu Hongxia, Zhou great Yun, Feng Xinai, Kuang Meng. the subject matter existing in China's Cotton Production and suggestion. Chinese agronomy circular, 2009,25 (14): 86-90).Cotton can meet the needs of textile industry substantially, but lacks length more than 31mm, and strength is more than 34cN/tex, and the extra best best kind of mic value between 3.7～4.2, meets the needs that spin 60 above high grade cotton yarns.
The genetic analysis of general cotton variety fibrous quality proterties all shows typical amounts character inheritance mode, is subject to environmental influence.In different segregating populations, additivity, dominant or epistasis effect all may be occupied an leading position.Utilizing the high-tenacity proterties of wild species and the fine quality gene recombination of sea island cotton gradually to blend transformation, is the main path that contemporary breeding man obtains high tenacity fibre.Sea island cotton is long breeding time, and in ripe evening, bell is little, and output is lower than upland cotton, but fiber finer is strong, is high-count yarn spun raw material; And the output of upland cotton is high, wide adaptability, fibrous quality is medium, but is significantly better than Asiatic cotton and cotton.The F of upland cotton, both hybridization of sea island cotton 1can educate, phenotype is normal, and has the hybrid vigour of obvious output, quality, and India, Israel, China successfully cultivate and goes to sea, the spread utilization on producing of land cross-fertilize seed.But the madness of the typical species hybridization of Posterity phenotype of both hybridization is separated, cotton breeding man is out for cultivating always both has the high yield of upland cotton, has again the new variety of sea island cotton high-quality, never success.The application of molecular marking technique in fibrous quality improvement breeding greatly reduced the blindness of selecting in breeding process, realizes fast external source elite germplasm and permeates to Cultivar, widened the hereditary basis of kind.Both at home and abroad researchist has carried out a large amount of QTL location to fibrous quality, have for marker assisted selection breeding practice.
Chromosome segment introgressive line (Chromosome segment introgression lines, CSIL) be to utilize hybridization, backcross and whole genomic a series of near isogenic lines of nurse crop that molecular marker assisted selection (marker assisted selection, MAS) builds.In its genome, only from a chromosome segment isozygotying of donor parents, and genomic rest part is identical with recurrent parent.It is to carry out genome research, the particularly ideal material of QTL location and Molecular design breeding.Wan Jianmin has utilized the chromosome segment substitution line being built by japonica rice Asominori (recurrent parent) and long-grained nonglutinous rice IR24 (nonrecurrent parent) to carry out the practice of molecular breeding.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, a kind of molecular breeding method of improveing cotton fiber length, fibre strength and mic value is provided.
Object of the present invention can be achieved through the following technical solutions:
A molecular breeding method of synchronously improveing cotton fiber length, intensity and fineness by polymerization chromosome segment introgressive line, comprising:
1) take Xinjiang cotton strain 0768 as recurrent parent (♀), take IL080 D6 1 (ZL201110127252.7) and 1047 (IL019 A2 6 chromosome segment introgressive lines) as nonrecurrent parent (♂), hybridized for 1 generation, in 2 generations that backcrossed, obtain BC 2f 1, per generation is all the mixed miscegenation of receiving;
2) molecule marker is auxiliary selects for the first time: BC 2f 1plantation, application CTAB method is extracted DNA, adopt IL080 D6 molecule marker NAU3677 and NAU1454(table 1 on 1 introgressed segment) and 1047 (IL019 A2 6 chromosome segment introgressive lines) introgressed segment on molecule marker NAU3419 and NAU2277(table 1) carry out pcr amplification, select to contain 2 molecule marker bands simultaneously and be respectively 180bp(NAU3677); Individual plant 300bp(NAU1454) be respectively 400bp(NAU3419 containing 2 molecule marker bands simultaneously); 120bp(NAU2277) individual plant is hybridized, and the heterozygous plant that results cenospecies plantation obtain 2 fragment polymerizations continues selfing;
Table 1 primer characteristic strip size and sequence
Figure BDA0000365265030000021
Figure BDA0000365265030000031
Note: (Han Z G is developed in the primer You Zhe laboratory of NAU numbering from EST-SSR sequence; Guo W Z; Song X L; et al.Genetic mapping of EST-derived microsatellites from the diploid Gossypium arboreum in allotetraploid cotton.Mol Gen Genomics; 2004,272:308-327; Han Z G, Wang C B, Song X L, et al.Characteristics, development and mapping of Gossypium hirsutum derived EST-SSRs in allotetraploid cotton.Theor Appl Genet, 2006,112:430-439; Wangzhen Guo, Caiping Cai, Changbiao Wang, et al.A preliminary analysis of genome structure and composition in Gossypium hirsutum.BMC Genomics, 2008,9:314-331.). all primer information can be from website www.cottonmarkers.orgupper download.
3) molecule marker is auxiliary selects for the second time: the F that plants 2 fragment polymerizations 2segregating population, every strain is extracted after DNA, and recycling molecule marker primer pair NAU3677, NAU1454, NAU3419, NAU2277 determine F 2the genotype of individual plant only has 4 molecule marker bands to be respectively 180bp simultaneously; 300bp; 400bp; The individual plant of 120bp, utilizes 62 polymorphism marks to carry out genetic background selection to these individual plants that isozygoty, and these individual plants that isozygoty are proceeded to field test under the mirror of Fourth Ring 2 generations of selfing to F 2:4.Select F 2:4in a strain self propagated become family, carry out multiple spot test.Its staple length 32.00 33.70mm, fibre strength 34.4 35.7cN/tex, mic value 3.01 3.45, single bell weigh 5.55 6.05g, ginning outturn 36.8 37.2%, therefore it is the improved line that we improve cotton fiber length, fibre strength and mic value, called after " Nan Nong 08 66 ".
Beneficial effect:
A kind of molecular breeding method of synchronously improveing cotton fiber length, intensity and fineness by polymerization chromosome segment introgressive line, overcome and alleviate serious Linkage drag that sea-land hybridization exists and, be conducive to the good genetic resources of fibrous quality in sea island cotton and import in upland cotton.Backcross and can recover the genetic background of recurrent parent, preserved the favourable proterties of nonrecurrent parent simultaneously, binding molecule marker assisted selection objective trait, can rapidly and efficiently cultivate all good New cotton lines of staple length, fibre strength and mic value.
A kind of molecular breeding method of synchronously improveing cotton fiber length, intensity and fineness by polymerization chromosome segment introgressive line provided by the present invention is compared tool with traditional pyramiding breeding technology that backcrosses and is had the following advantages: traditional pyramiding breeding technology that backcrosses exists the defect that crossing work amount is large, select poor reliability, breeding cycle length.By molecular marker assisted selection objective trait, can in 3-4, rapidly and efficiently cultivate the New cotton line of the multiple target characters such as high yield, high-quality.
The improved line of improvement cotton fiber length, fibre strength and the mic value of cultivating by the inventive method " Nan Nong 08-66 " has following major advantage: the sea island cotton chromosome segment introgressive line IL080-D6-1 and 1047 (IL019-A2-6 chromosome segment introgressive line) of take is donor material, take 0768 as recurrent parent, by molecular marker assisted selection, obtained output suitable with recurrent parent 0768, staple length and fibre strength significantly increase, the significantly reduced New cotton line of mic value " Nan Nong 08-66 ".These strain 2012 Nian Xinjiang stone rivers show as staple length 32.00-33.70mm, fibre strength 34.4-35.7cN/tex, mic value 3.01-3.45, ginning outturn 36.8-37.2%, the heavy 5.55-6.10g of single bell is significantly high than the improvement that only imports a segment of IL080-D6-1.
Accompanying drawing explanation
Fig. 1 synchronously improves cotton fiber length, intensity and fineness by polymerization chromosome segment introgressive line
Fig. 2 target interval linked marker NAU1454 is at polymerization biplate section F 2in colony, identify the individual plant that isozygotys
Note: M:DNA marker; P 1: sea 7124; P 2: 0768; F 1: (H7124x TM 1); 1 43:F 2individual plant arrow indication is the amplification difference position of SSR mark
Biological preservation:
IL080 D6 1, Classification And Nomenclature is upland cotton (Gossypium hirsutum.), within on 03 15th, 2011, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, culture presevation number is CGMCC NO.4685.
1047, Classification And Nomenclature is upland cotton (Gossypium hirsutum.), within on 06 17th, 2013, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and culture presevation number is CGMCC NO.7754.
0768, Classification And Nomenclature is upland cotton (Gossypium hirsutum.), within on 05 06th, 2013, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and culture presevation number is CGMCC NO.7559.
South agriculture 08 66, Classification And Nomenclature is upland cotton (Gossypium hirsutum.), within on 05 06th, 2013, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation number is CGMCC NO.7560.
Embodiment:
Embodiment 1
Selection parent: Agricultural University Of Nanjing's crop genetic and germplasm innovation National Key Laboratory Cotton Research Institute take upland cotton Genetic standard line TM 1 be recurrent parent, sea island cotton sea 7124 product disease-resistant, high-quality are donor parents (nonrecurrent parent), at the advanced lines that backcrosses in conjunction with covering complete genomic SSR molecular marker assisted selection, cultivated domestic first cover upland cotton Genetic standard line TM 174 of the sea island cotton chromosome segment introgressive lines of 1 background, its genomic fraction of coverage reaches 83.5%.By the cotton fibre quality that sea island cotton chromosome segment introgressive line is carried out continuous 2 years, detect, filter out staple length be significantly better than contrast introgressive line IL080 D6 1(CGMCC NO.4685) and 1047 (CGMCC NO.7754, for IL019 A2 6 chromosome segment introgressive lines).IL080 D6 1 staple length 31.5 32.5mm, fibre strength 32.7 33.5cN/tex, mic value 4.2 4.3, single bell weigh 6.8 7.2g, ginning outturn 36.8 38.8%, individual plant bell number 15 19, plant height 90 95cm.1047 (IL019 A2 6 chromosome segment introgressive lines) staple length 30.0 30.8mm, fibre strength 30.1 31.4cN/tex, mic value 4.5 4.6, single bell weigh 6.4 7.2g, ginning outturn 36.2 38.4%, individual plant bell number 14 17, plant height 90 95cm
Xinjiang cotton strain 0768(CGMCC NO.7559) be the high yield of suitable Xinjiang plantation, disease-resistant cotton strain.This strain staple length 30.14 30.43mm, fibre strength 35.3 35.7cN/tex, mic value 4.43 4.70, single bell weigh 5.42 5.72g, ginning outturn 38.8 40.5%, individual plant bell number 7 11, plant height 85 90cm.
Breed breeding: winter in 2007 be take Xinjiang strain 0768 as maternal at Sanya, Hainan, IL080 D6 1 and 1047 (IL019 A2 6 chromosome segment introgressive lines) be respectively the hybridization F of 2 combinations of male parent preparation 1.Summer in 2008 is planted respectively a line 0768 and F in testing station, Agricultural University Of Nanjing Jiangpu 1, take 0768 as male parent, the F of 2 combinations 1for maternal preparing hybrid produces BC 1f 1, ripe rear all mixed receipts.Winter in 2008 is planted respectively a line 0768 and BC at Sanya, Hainan 1f 1, take 0768 as male parent, F 1for maternal preparing hybrid produces BC 2f 1, ripe rear all mixed receipts.Field management is carried out according to a conventional method.
Molecular marker assisted selection: summer in 2009 is planted 2 row BC in testing station, Agricultural University Of Nanjing Jiangpu 2f 1, every row 10 12 strains, every strain gather 3 4 blades that do not launch put into the eppendorf pipe of 1.5ml, and add the freshly prepared Extraction buffer 600 μ l of precooling, electric drill grinds, application CTAB method (Paterson AH, Brubaker CL, Wendel JF.A rapid method for extraction of cotton(Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis[J] .Plant Mol Biol Rep, 1993, 11, 2:122 127) extract DNA, adopt IL080 D6 molecule marker NAU3419 and NAU2277 on molecule marker NAU3677 on 1 introgressed segment and NAU1454 and 1047 (IL019 A2 6 chromosome segment introgressive lines) introgressed segment carry out pcr amplification, amplification system 10 μ l, DNA profiling 1 μ l, 94 ℃ of denaturation 5min, 94 ℃ of sex change 0.5min, 57 ℃ of renaturation 0.5min, 72 ℃ are extended 1min, after 30 circulations, 72 ℃ are extended 10min again, amplified production is through native polyacrylamide gel electrophoresis: gel strength is 8%, electrophoretic buffer is 0.5 times of TBE, 170V constant voltage electrophoresis 1.5 hours.The primer of four molecule markers is in Table 1.
Molecule marker is auxiliary to be selected for the first time: the BC that utilizes target area close linkage mark NAU3677, NAU1454, NAU3419 and NAU2277 primer screening tool target fragment 2f 1individual plant, winter in 2009 is handed over F mutually in Sanya plantation 1each 30 strains of colony, selfing results F 2seed.Utilize simultaneously 330 genomic SSR marks of uniform fold to IL080 D6 the donor kind TM-1 and 0768 of 1 and 1047 these two chromosome segment introgressive lines carry out polymorphism screening, obtain altogether the BC of 31 pairs of polymorphism marks to tool target fragment 2f 1individual plant carries out Analysis of Genetic Background, from 2 combinations, picking out respectively more than 80% individual plant A and the B of background recovery rate hybridizes, wherein individual plant A from combination A(0768 * IL080 D6 1) background recovery rate is 80.6%, individual plant B is from combination B(0768 * 1047 (IL019 A2 6 chromosome segment introgressive lines)) background recovery rate is 83.9%.
Molecule marker is auxiliary to be selected for the second time: extract the F of polymerization biplate section in summer in 2010 2200 individual plant DNA of colony, with linked marker NAU3677, NAU1454, NAU3419 and NAU2277(table 1 in two target fragment) analyze the F of biplate section polymerization 2in colony, identify respectively 12 tool single slice A (NAU3677 NAU1454), 10 tool single slice B (NAU3419 NAU2277) and 8 the biplate section target gene types individual plant (NAU3677 NAU1454 and NAU3419 NAU2277) that isozygotys.Land, sea, Yi Zhe laboratory collection of illustrative plates is basis simultaneously, on the former mark density basis that is useful on context analyzer, further increase mark, every 5～10cM, select a SSR mark, selected altogether 630 SSR marks to carry out polymorphism analysis (table 3) to parents, obtain altogether 62 polymorphism marks (table 4), the scope that the genetic background of the individual plant that isozygotys is detected to discovery genetic background recovery rate with these polymorphism marks is between 60%～88.8%, wherein biplate section is isozygotied in the genetic background of polymerization individual plant D1 has 8 SSR marker sites to be shown as TM-1 genotype, background recovery rate is 87.1%.Continue biplate section target gene type to isozygoty F2 individual plant continues in Nanjing and Shihezi of Xinjiang carries out many envrionment tests simultaneously.By to Nanjing (2010), Nanjing (2011), the fiber detected result under borders, Fourth Ring such as Nanjing (2012) and Shihezi (2012) and output analysis of survey results find biplate section isozygoty the extremely significantly super contrast of staple length, fibre strength and fineness improved effect of polymerized strain D1 and yield traits with contrast without significant difference (table 2).We are by this high-quality, high-yield cotton new lines called after " Nan Nong 08-66 " (CGMCC NO.7560).
Field test: 2010, plant respectively F in testing station, Jiangpu, Nanjing summer in 2011 and 2012 2, F 2:3and F 2:4family, adopts the method for nutrition bowl seedling transplanting, and row length is 5 meters, wide 0.8 meter, every row kind 12 strains, randomized complete-block design, single file district, every row 12 15 strains, twice repetition.Summer in 2012 is planted BC in Shihezi of Xinjiang 2f 2:4family is tested, and plot experiment Wei Lianghang presses 0.55(0.4+0.35+0.4 in community) the wide film setting of m, row length is 5 meters, 0.12 meter of spacing in the rows, randomized complete-block design, repeats for 2 times.
After spending by the mixed cotton receiving of row after maturation, every row is got 12 grams of gined cotton samples, repeats 2 Ci，Song Henan cotton quality inspection centers and detects gined cotton quality.Within 2012, under Shihezi of Xinjiang's environment, improve the BC of cotton fiber length, fibre strength and mic value 2f 2:4middle selection plantation staple length 32.00 33.70mm, fibre strength 34.4 35.7cN/tex, mic value 3.01 3.45, single bell weigh 5.55 6.10g, the family of ginning outturn 36.8 37.2%, is improved line " Nan Nong 08-66 " (CGMCC NO.7560).This strain staple length and fibre strength significantly increase compared with 0768, and mic value significantly reduces compared with 0768, and output is suitable with recurrent parent 0768.
The southern agriculture of table 2 08 66 and fibrous quality and the performance of yield factor under 4 environment of contrast
Figure BDA0000365265030000071
# field test: Nanjing (2010), Nanjing (2011), Nanjing (2012) and Shihezi (2012) are named as respectively 1,2,3 and 4*, and * * is illustrated respectively in 0.05,0.01 level and has significant difference
The molecule marker title that table 3 is selected for background and characteristic strip size
Figure BDA0000365265030000072
Figure BDA0000365265030000081
Figure BDA0000365265030000091
Figure BDA0000365265030000101
Figure BDA0000365265030000111
Figure BDA0000365265030000121
Figure BDA0000365265030000131
Figure BDA0000365265030000141
Figure BDA0000365265030000161
Figure BDA0000365265030000171
Figure BDA0000365265030000181
Figure BDA0000365265030000191
Figure BDA0000365265030000201
Figure BDA0000365265030000211
Figure BDA0000365265030000221
Figure BDA0000365265030000231
Figure BDA0000365265030000241
Figure BDA0000365265030000251
Figure BDA0000365265030000261
Figure BDA0000365265030000271
Figure BDA0000365265030000281
Figure BDA0000365265030000291
Figure BDA0000365265030000301
The mark title of tool polymorphism and characteristic strip size between table 40768 and TM 1
Figure BDA0000365265030000302
Figure BDA0000365265030000311
Figure BDA0000365265030000321
Figure IDA0000365265120000011
Figure IDA0000365265120000021
Figure IDA0000365265120000031

Claims (1)

1. by polymerization chromosome segment introgressive line, synchronously improve a molecular breeding method for cotton fiber length, fibre strength and mic value, it is characterized in that comprising:
1) take Xinjiang cotton strain 0768 maternal as recurrent parent, take IL080 D6 1 and 1047 be nonrecurrent parent male parent, hybridize 1 generation hybridization generation BC 1f 1, take 0768 as male parent, BC 1f 1for maternal preparing hybrid produces BC 2f 1, per generation is all the mixed miscegenation of receiving;
2) molecule marker is auxiliary selects for the first time: BC 2f 1plantation, application CTAB method is extracted DNA, adopt IL080 D6 molecule marker NAU3677 on 1 introgressed segment and molecule marker NAU3419 and the NAU2277 on NAU1454 and 1047 introgressed segments carry out pcr amplification, select to contain 2 molecule marker bands simultaneously and be respectively the individual plant of NAU3677 and NAU1454 and containing 2 molecule marker bands, be respectively NAU3419 and NAU2277 individual plant is hybridized simultaneously, the heterozygous plant that results cenospecies plantation obtain 2 fragment polymerizations continues selfing; Wherein said molecule marker NAU3677 forward primer is SEQ ID NO.1, and reverse primer is SEQ ID NO.2, and PCR product size is 180bp; Described molecule marker NAU1454 forward primer is SEQ ID NO.3, and reverse primer is SEQ ID NO.4, and PCR product size is 300bp; Described molecule marker NAU3419 forward primer is SEQ ID NO.5, and reverse primer is SEQ ID NO.6, and PCR product size is 400bp; Described molecule marker NAU2277 forward primer is SEQ ID NO.7, and reverse primer is SEQ ID NO.8, and PCR product size is 120bp;
3) molecule marker is auxiliary selects for the second time: plant 2 fragment polymerization F 2segregating population, every strain is extracted after DNA, and the molecule marker NAU3677 of recycling chromosome segment A and the molecule marker NAU3419 of NAU1454 and chromosome segment B and NAU2277 carry out foreground selection and determine F 2the genotype of individual plant, the individual plant that simultaneously only has 4 molecule marker bands to be respectively 180bp, 300bp, 400bp, 120bp utilizes 62 polymorphism marks to carry out genetic background selection to these individual plants that isozygoty, and these individual plants that isozygoty are proceeded to field test under the mirror of Fourth Ring 2 generations of selfing to F 2:4, at F 2:4in family, select staple length 32.00 33.70mm, fibre strength 34.4 35.7cN/tex, mic value 3.01 3.45, single bell weigh 5.55 6.05g, ginning outturn 36.8 37.2%, background selects the fibrous quality that display background recovery rate is 87.1% significantly to improve the improved line that family D1 is improvement cotton fiber length, fibre strength and mic value.
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