AU2020268928A1

AU2020268928A1 - Gossypium anomalum-sourced ssr sequence associated with high lint percentage and drought tolerance and application thereof

Info

Publication number: AU2020268928A1
Application number: AU2020268928A
Authority: AU
Inventors: Xinqi FAN; Jin Gao; Qi Guo; Fang Huang; Shan Meng; Wanchao NI; Xinlian SHEN; Peng Xu; Zhenzhen Xu; Caijiao ZHAI; Xianggui ZHANG
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2019-05-09
Filing date: 2020-05-08
Publication date: 2021-01-28
Also published as: WO2020224636A1

Abstract

Provided is a gossypium anomalum-sourced SSR sequence associated with high lint percentage and drought tolerance and an application thereof. Provided is a gossypium anomalum-sourced chromosome segment which is a DNA segment from an SSR molecular marker JAAS6365 to an SSR molecular marker JAAS5604 on chromosome 5 of the gossypium anomalum. The nucleotide sequence of the SSR molecular marker JAAS6365 is shown as SEQ ID No. 1, and the nucleotide sequence of the SSR molecular marker JAAS5604 is shown as SEQ ID No. 2.

Description

G. anomalum-derived SSR sequences related to high lint percentage and drought resistance and application thereof

TECHNICAL FIELD

The present invention relates to the field of cotton breeding, in particular to a G. anomalum-derived SSR sequences related to high lint percentage and drought resistance and application thereof.

BACKGROUND ART

Cotton is an important economic crop, and natural cotton fiber is the main raw material of the world's textile industry. There are four cultivars in Gossypium spp. that can produce natural fibers for textiles, including allotetraploid cotton species Gossypium hirsutum L. and G. barbadense L., and diploid cotton species G. herbaceum L. and G. arboreum L. Among them, G. hirsutum dominates the world's cotton production due to its high yield and wide adaptability. For a long time, increasing yield and improving fiber quality have been two important goals for G. hirsutum variety improvement. With the rapid development of the textile industry, the requirements for fiber quality are getting higher and higher, but while pursuing excellent fiber quality, the lint percentage (one of the factors of cotton yield, the percentage of cotton fiber (lint) obtained after seed cotton ginning to the weight of seed cotton) has been reduced. Therefore, how to coordinate fiber quality and yield in G. hirsutum genetic improvement is an important research topic at present.

In recent years, with the continuous deterioration of the global climate and the destruction of the ecological environment, the area of saline-alkali arid areas has become larger and larger. Due to long-term artificial selection, G. hirsutum has a narrow genetic basis, and its drought resistance is stronger than other crops, but drought and desertification still cause continuous damage to cotton yield and fiber quality. Therefore, it is particularly important to strengthen the research on drought-resistant breeding of cotton and cultivate new drought-tolerant varieties/lines.

The diploid wild species of Gossypium spp. provides abundant genetic resources for cotton breeding. The introgression of foreign genes from wild cotton into G. hirsutum gene pool is an effective method for genetic improvement of G. hirsutum. There were many reports on the appilication of diploid wild cotton for genetic improvement of G. hirsutum, but due to differences in chromosomal ploidy level and genetic structure, the appilication of diploid wild species of Gossypium spp. for genetic improvement of G. hirsutum is still very limited. G. anomalum, a wild species of diploid group B of Gossypium spp., is mainly distributed in the coastal belt in Angola, the South-West and southern borders of the Great Desert Belt in Africa. G. anomalum possesses many excellent characters, such as ultra-strong fiber, strong drought resistance, resistance to insects and immunity to a variety of bacterial diseases. Therefore, it is a valuable genetic resource for genetic improvement of G. hirsutum.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a G. anomalum-derived SSR sequences related to high lint percentage and drought resistance and application thereof.

In the first aspect, the present invention claims a DNA segment.

The DNA segment claimed by the present invention is located on chromosome 5 of G. anomalum and contains at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604.

Wherein, the nucleotide sequence of the SSR molecular marker JAAS6365 is as shown in SEQ ID NO: 1; the nucleotide sequence of the SSR molecular marker JAAS5604 is as shown in SEQ ID NO: 2.

Further, the DNA segment is located on chromosome 5 of G. anomalum and starts from the SSR molecular marker JAAS6365 to the SSR molecular marker JAAS5604.

The DNA segment also contains SSR molecular marker JAAS0803; the nucleotide sequence of the SSR molecular marker JAAS0803 is as shown in SEQ ID NO: 3.

In the second aspect, the present invention claims a vector, expression cassette, recombinant bacteria or transgenic cell line containing the DNA segment in the first aspect.

The vector can be an artificial chromosome, such as a bacterial artificial chromosome or a yeast artificial chromosome. The recombinant bacteria and the transgenic cell line contain the artificial chromosomes.

In the third aspect, the present invention claims use of the DNA segment in the first aspect or the vector, expression cassette, recombinant bacteria or transgenic cell line in the second aspect for improving cotton lint percentage or cultivating cotton varieties or lines with improved lint percentage.

In the fourth aspect, the present invention claims use of the DNA segment in the first aspect or the vector, expression cassette, recombinant bacteria or transgenic cell line in the second aspect for improving drought resistance of cotton or cultivating drought-resistant cotton varieties or lines.

In the fifth aspect, the present invention claims an SSR molecular marker or a set of SSR molecular markers on the DNA segment in the first aspect.

The SSR molecular marker claimed in the present invention is any one of the following (al)-(a3); the set of SSR molecular markers consists of the following (al)-(a3); (al) SSR molecular marker JAAS6365: a DNA molecule having the nucleotide sequence as shown in SEQ ID NO: 1; (a2) SSR molecular marker JAAS5604: a DNA molecule having the nucleotide sequence as shown in SEQ ID NO: 2; (a3) SSR molecular marker JAAS0803: a DNA molecule having the nucleotide sequence as shown in SEQ ID NO: 3.

In the sixth aspect, the present invention claims a primer pair or a set of primer pairs for identifying the SSR molecular marker or the set of SSR molecular markers in the fifth aspect.

The primer pair claimed in the present invention is any one of the following (bl)-(b3); the set of primer pairs consists of the following (bl)-(b3); (b1) primer pair 1 for identifying the SSR molecular marker JAAS6365: designed according to SEQ ID NO: 1; (b2) primer pair 2 for identifying the SSR molecular marker JAAS5604: designed according to SEQ ID NO: 2; (b3) primer pair 3 for identifying the SSR molecular marker JAAS0803: designed according to SEQ ID NO: 3.

In a specific embodiment of the present invention, the primer pair 1 consists of two single-stranded DNA molecules as shown in SEQ ID NO: 4 and SEQ ID NO: 5; the primer pair 2 consists of two single-stranded DNA molecules as shown in SEQ ID NO: 6 and SEQ ID NO: 7; the primer pair 3 consists of two single-stranded DNA molecules as shown in SEQ ID NO: 8 and SEQ ID NO: 9.

In the seventh aspect, the present invention claims a kit containing the primer pair or the set of primer pairs in the sixth aspect.

The kit can also contain one or more of PCR amplification buffer, double-distilled water, DNA polymerase and dNTP.

In the eighth aspect, the present invention claims use of the SSR molecular marker or the set of SSR molecular markers in the fifth aspect or the primer pair or the set of primer pairs in the sixth aspect or the kit in the seventh aspect for screening the DNA segment in the first aspect for cultivating cotton varieties or lines with improved lint percentage.

In the ninth aspect, the present invention claims use of the SSR molecular marker or the set of SSR molecular markers in the fifth aspect or the primer pair or the set of primer pairs in the sixth aspect or the kit in the seventh aspect for screening the DNA segment in the first aspect for cultivating cotton varieties or lines with improved drought resistance.

In the tenth aspect, the present invention claims a method for identifying or assisting in identifying cotton lint percentage traits.

The method for identifying or assisting in identifying cotton lint percentage traits as claimed in the present invention is conducted by using the SSR molecular marker or the set of SSR molecular markers in the fifth aspect or the primer pair or the set of primer pairs in the sixth aspect or the kit in the seventh aspect.

In the eleventh aspect, the present invention claims a method for identifying or assisting in identifying drought resistance of cotton.

The method for identifying or assisting in identifying drought resistance of cotton as claimed in the present invention is conducted by using the SSR molecular marker or the set of SSR molecular markers in the fifth aspect or the primer pair or the set of primer pairs in the sixth aspect or the kit in the seventh aspect.

In the twelfth aspect, the present invention claims a method for improving cotton lint percentage (or a method for cultivating cotton varieties with improved lint percentage).

The method for improving cotton lint percentage (or the method for cultivating cotton varieties with improved lint percentage) as claimed in the present invention can comprises the following step: replacing the original chromosome genome segment of a recipient parent with a DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 (the segment further contains SSR molecular marker JAAS0803; the nucleotide sequence of the SSR molecular marker JAAS0803 is as shown in SEQ ID NO: 3) to obtain a cotton variety with improved lint percentage.

The replacement of the original chromosome genome segment of a recipient parent with a DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 is preferably a homozygous replacement.

Further, the DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 is a DNA segment located on chromosome 5 of G. anomalum and starting from the SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604.

In the thirteenth aspect, the present invention claims a method for improving drought resistance of cotton (or a method for cultivating cotton varieties with improved drought resistance).

The method for improving drought resistance of cotton (or the method for cultivating cotton varieties with improved drought resistance) as claimed in the present invention can comprises the following step: replacing the original chromosome genome segment of a recipient parent with a DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 (the segment further contains SSR molecular marker JAAS0803; the nucleotide sequence of the SSR molecular marker JAAS0803 is as shown in SEQ ID NO: 3) to obtain a cotton variety with improved drought resistance.

Further, the DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 is a DNA segment located on chromosome 5 of G. anomalum and starting from the SSR molecular marker JAAS6365 to the SSR molecular marker JAAS5604.

In the twelfth and thirteenth aspects, the DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 can specifically be a DNA segment located on chromosome 5 of G. anomalum and starting from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604.

In the twelfth and thirteenth aspects, in the methods, the replacement of the original chromosome genome segment of a recipient parent with a DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 is realized by hybridization technology.

In the twelfth and thirteenth aspects, the aforementioned SSR molecular marker or set of SSR molecular markers or the aforementioned primer pair or set of primer pairs or the aforementioned kit can be used to identify whether a cotton to be tested contains the DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604.

In the fourteenth aspect, the present invention claims a cotton or its tissues or organs.

The cotton claimed by the present invention is a cotton variety obtained by replacing the original chromosome genome segment of a recipient parent with a DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 (the segment further contains SSR molecular marker JAAS0803; the nucleotide sequence of the SSR molecular marker JAAS0803 is as shown in SEQ ID NO: 3). Compared with the recipient parent, the cotton variety has improved lint percentage and/or improved drought resistance.

The nucleotide sequence of the SSR molecular marker JAAS6365 is as shown in SEQ ID NO: 1;

the nucleotide sequence of the SSR molecular marker JAAS5604 is as shown in SEQ ID NO: 2.

The tissues and organs can specifically be seeds, cotton bolls and callus. The tissues, organs and plants can be transgenic materials or non-transgenic materials.

In the twelfth aspect to the fourteenth aspect, the recipient parent is a non-G. anomalum variety, preferably G. hirsutum.

The G. anomalum described in the foregoing of the present invention is G. anomalum which is a wild species of diploid group B of Gossypium spp.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the graphic genotypes and distribution of introgression segments of the G. anomalum chromosome segment substitution lines of the present invention. Light gray represents the genotype of the recurrent parent G. hirsutum, Su 8289, black represents the genotype of G. anomalum, and dark gray represents the heterozygous segment. The horizontal axis represents Chr.1-Chr.13 from left to right; the vertical axis represents 74 chromosome segment substitution lines from the top to the bottom, and on the right side of each chromosome segment substitution line is the number and length information of the introgression segments, the proportion of G. anomalum and recurrent parent genome.

FIG. 2 shows the coverage rates of G. anomalum chromosome segments of the introgression lines on 13 chromosomes.

FIG. 3 is a gel electrophoretogram of the BC4F4 generation of the present invention. Panel A shows the detection results of SSR molecular marker JAAS6365; panel B shows the detection results of SSR molecular marker JAAS0803; panel C shows the detection results of SSR molecular marker JAAS5604.

FIG. 4 is a gel electrophoretogram of SSR molecular marker JAAS5604 in BC4F3 generation in the present invention.

FIG. 5 is an analysis diagram showing the correlation between segments on chromosome 5 of G. anomalum and lint percentage in the present invention.

FIG. 6 is a histogram showing the difference in lint percentage between the single segment substitution line CSSL18 with high lint percentage and the recurrent parent Su 8289 in the present invention.

FIG. 7 is an analysis diagram showing the correlation between segments on chromosome 5 of G. anomalum and drought resistance in the present invention.

FIG. 8 shows the identification of drought resistance in the single segment substitution line CSSL18 and recurrent parent Su 8289 in the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were all purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all repeated three times, and the results were averaged.

G. hirsutum var. 86-1, G. hirsutum var. Su8289 and G. anomalum are all recorded in the literature "Caijiao Zhai, Peng Xu, Xia Zhang, et al. Development of Gossypium anomalum derived microsatellite markers and their use for genome-wide identification of recombination between the G. anomalum and G. hirsutum genomes. Theoretical and Applied Genetics, 2015, 128(8): 1531-1540", and are available to the public from the applicant. These materials are only used for repeating the experiments of the present invention, and cannot be used for other purposes.

Example 1. The identification of chromosome segments and development of molecular markers related to high lint percentage and drought resistance traits in cotton

The drought-resistant cotton with high lint percentage in the present invention was cultivated through the following steps:

G. hirsutum 86-1 as the donor was crossed with G. anomalum as the male parent to obtain triploid Fi (AiDiBi), and the tender axillary buds of the triploid seedlings were treated with 0.15% colchicine to obtain fertile hexaploid Fi (A1AiDiDiBiBi);

the hexaploid as the female parent was crossed with G. hirsutum Su 8289 as the parent to obtain pentaploid AiAiDiDiB, all the harvested seeds were planted and 97 individual plants were obtained;

the obtained pentaploid was backcrossed with Su 8289 to obtain BC2F1 seeds, all the harvested seeds were planted and 384 individual plants were obtained, with 50 recombination types, 36 of which were fertile;

all the BC2F1 individuals were backcrossed with Su 8289 and all the harvested seeds were planted, and a total of 4331 BC3F1 individual plants with 40 recombination types were obtained;

the recombinant individuals and the additional line individuals in the BC3F population were continuously backcrossed with Su 8289, and 8540 BC4F1 individual plants with 56 recombinant types were obtained;

the 56 recombination types in the BC4F1 population were self-crossed to obtain self-crossed BC4F2 seeds, the BC4F2 population was planted, and a total of 4543 individual plants were obtained.

The recombination types in the present invention were detected by SSR markers. The SSR markers were obtained as follows: a set of G. anomalum-specific SSR markers (230 markers) that evenly cover the genome was used to detect the G. anomalum introgression segments in different generations by integrating the G. anomalum-specific SSR markers with the published cotton genome-wide SSR physical map.

Starting from BC2F1, the recombinant individuals were selected to continue to be backcrossed twice and self-crossed three times. According to the marker information of the BC2F1 recombination breakpoints, for each generation, 130 key SSR markers that evenly covered the chromosome group were selected from the 230 markers covering the genome. Using PCR technology to detect all recombinant individuals, the size of each generation population is shown in Table 1. Finally, a set of G. anomalum chromosome segment substitution lines (introgression lines) was obtained.

PCR amplification conditions: G. hirsutum Su 8289, G. anomalum, hexaploid Fi and hybrid offspring were used as templates for genotyping. The PCR reaction system was 10 pL: 2 pL of DNA template, IpL of 10xbuffer (Mg2+), 0.2 pL of dNTP, 0.1 pL of Taq DNA polymerase, 0.5 tL of each forward and reverse primers, and 3.7 pL of ddH20.

PCR thermal cycle program: Step 1: pre-denaturation at 95°C for 5 min; 30 cycles of (Step 2: denaturation at 94°C for 30 s; Step 3: annealing at 55°C for 45 s; Step 4: elongation at 72°C for 1 min); Step 5: elongation at 72°C for 10 min; Step 6: storage at 10°C.

Detection of PCR amplification products: Step 1: wash the glass plates, dry them for later use; Step 2: align the set of two glass plates, lay them flat, clamp both sides with clamps, place them on a panel; Step 3: draining with a glass rod and slowly pour 8% polyacrylamide gel (29:1), and insert a comb and leave it for 15 min; Step 4: after the gel has solidified, loosen the clamps, put the glass plates with gel into the electrophoresis tank, pour 1xTBE electrophoresis buffer, and tighten both sides with clamps, and continue to add electrophoresis buffer to cover the gel, pull out the comb; Step 5: load the sample (loading volume is 1 L), and electrophorese the gel for 1.5 h under the condition of constant voltage of 180 V.

All the recombinant individuals in the BC4F1 population obtained in 2015 were self-crossed, BC4F2 seeds were harvested, and the BC4F2 population including 4543 individual plants was planted in the summer of 2016. According to the genetic map and the marker information of the BC2Fi statistical data recombination breakpoints, 130 key SSR primer pairs that evenly cover the chromosome group were selected to detect the genotypes of BC4F2 population, and 51 recombination types were obtained, of which 45 were homozygous.

In December 2016, the BC4F3 population including a total of 1533 individual plants was further planted. According to the genetic map and the marker information of the BC2F1 statistical data recombination breakpoints, 130 key SSR primer pairs that evenly cover the chromosome group were selected to detect the genotypes of the BC4F3 population, and 53 recombination types were obtained, of which 47 recombination types were homozygous.

In 2017, the BC4F4 population including a total of 2225 individual plants was planted. The genotypes of these 2225 plants were detected with 230 SSR primer pairs (including 130 primer pairs) developed by this research group that evenly cover the G. anomalum chromosome group, and a total of 74 recombination types were obtained, of which 71 were homozygous. The population size of each generation is shown in Table 1, and the number of recombination types in each generation is shown in Table 2.

Table 1 Population sizes of BC3F1, BC4F1, BC4F2, BC4F3 and BC4F4 generations for molecular marker-assisted selection BC 3 F1 BC 4 F 1 BC 4 F 2 BC 4 F 3 BC 4F 4 Chromosome (2014, Nanjing)(2014, Nanjing)(2016, Nanjing)(2016, Hainan)(2017, Nanjing) Chr.1 888(86) 774(50) 752 (132) 145 127 Chr.2 495 (77) 464(119) 487(69) 117 155 Chr.3 243 (32) 696(503) 227 (108) 20 55 Chr.4 165(41) 348(123) 240(94) 145 210 Chr.5 170(64) 318(40) 347(48) 113 104 Chr.6 299(228) 869(761) 347 (112) 136 214 Chr.7 121(91) 443 (443) 207(170) 47 98 Chr.8 275 (275) 392(392) 183 (112) 58 121 Chr.9 209(114) 588(410) 235 (108) 66 181 Chr.10 250(201) 550(493) 259(188) 245 293 Chr.11 827 (63) 1377 (57) 706(46) 267 395 Chr.12 128(65) 795(589) 247(69) 73 33 Chr.13 261 (240) 926 (926) 306 (143) 101 239 Total 4331 (1577) 8540 (4906) 4543 (1399) 1533 2225

Note: In the second, third and fourth columns, each number in the parentheses represents the number of individual plants screened in the addition lines.

1. The detection of recombination types in BC4F2, BC4F3 and BC4F4 generations

All the recombinant individuals in the BC4F1 population obtained in 2015 were self-crossed, BC4F2 seeds were harvested, and the BC4F2 population including a total of 4543 individual plants was planted in the summer of 2016. According to the genetic map and the marker information of the BC2F1 recombination breakpoints, 130 key SSR primer pairs that evenly cover the chromosome group were selected for detecting the genotype of these 4543 individual plants, and 51 recombination types were detected, of which 45 were homozygous (Table 2). Compared with BC4F1, 8 new recombination types were detected and the marker intervals of chromosome segments in G. anomalum were located on Chr.6: NAU2714-JAAS1095, NAU2714-JAAS1095 & NAU1272-JAAS2480, JAAS6227-JAAS2480; Chr.9: JAAS1923-JAAS0613; Chr.10: JAAS1256-JAAS3294; Chr.11: JAAS4829-NAU3703, NAU3703-NAU3234 and Chr.13: JAAS4570-JAAS2038, respectively.

In December 2016, the BC4F3 population including a total of 1533 individual plants was planted in Hainan. According to the genetic map and the marker information of the BC2F1 recombination breakpoints, 130 key SSR primer pairs that evenly cover the chromosome group were selected for detecting the genotype of these 1533 plants, and 53 recombination types were obtained, of which 47 were homozygous (Table 2). Compared with BC4F2, two new recombination types were detected in the BC4F3 generation, and the marker intervals of chromosome segments in G. anomalum were located on Chr.5: DC40130-DC40130 and Chr.11: JAAS0280-JAAS3199, respectively.

In 2017, the BC4F4 population including a total of 2225 individual plants was planted. The genotypes of the plants were detected with 230 SSR primer pairs (including 130 primer pairs) developed by this research group that evenly covered the G. anomalum chromosome group, and a total of 74 recombination types were obtained, of which 71 were homozygous. Compared with BC4F3 population, 20 new recombination types were detected in BC4F4, and the marker intervals of chromosome segments in G. anomalum were located on Chr.1: JAAS0826-JAAS1148, NAU3615-JAAS1148, NAU3615-JAAS5817, NAU3615-JAASO392, NAU5100- NAU4045, NAU2083-NAU2083, NAU2083-JAAS2569, NAU4045-NAU4045; Chr.4: JAAS2022-JAAS2076; Chr.8: NAU1037-JAAS6420; Chr.10: JAAS3294-JAAS3294, JAAS1256-JAAS1256; Chr.11: JAAS4829-JAAS4829, JAAS3088-JAAS5224, JAAS4259-JAAS4259, respectively. Since genome-wide marker scanning was performed on all individuals, some new detected recombinant individuals contained different chromosomal introgression segments.

A summary of the recombination types for six consecutive generations during the construction of G. anomalum chromosome segment substitution lines is shown in Table 2. Each generation from BC2Fi to BC4F4 had new recombination types. At the same time, due to the poor fertility and low seed vigor of the offspring plants, some recombination types were lost in each generation. In order to make up for the loss of recombination types, in the BC3F1, BC4F1 and BC4F2 generations, the recombination individuals were backcrossed with the recurrent parents, and at the same time the addition line was backcrossed with the recurrent parent, so that some lost recombination types were detected again and more new recombination types derived from the lost types were produced. The number of recombination types increased from the 50 (BC2F1) to 74 (BC4F4). Finally, 71 G. anomalum chromosome segment substitution lines (homozygous genotype) with stable inheritance capacities were obtained. Specifically, the number of recombination types identified in different populations is shown in Table 2.

Table 2 Number of recombination types identified in BC2F1, BC3F1, BC4Fi, BC4F2, BC4F3 and BC4F4 generations Chromosome BC 2F 1 BC 3F1 BC 4F1 BC4F 2 BC 4F 3 BC 4F 4

Chr.1 3 4 9 8 8 13

Chr.2 4 5 6 3 (2) 3 (2) 3

Chr.3 1 1 3 1 (1) 1 (1) 1 (1)

Chr.4 1 5 3 2(1) 2(1) 3

Chr.5 4 6 6 6 7 7

Chr.6 2 2 3 5 5 6(1)

Chr.7 1 0 1 1 1 1

Chr.8 1 0 3 1 1 2

Chr.9 3 2 2 3 (1) 3 (1) 3 (1)

Chr.10 1 2 2 1 1 3

Chr.11 11 11 11 13 (1) 14(1) 17

Chr.12 3 2 3 3 3 3

Chr.13 1 0 4 4 4 4

Total 36 40 56 51(6) 53 (6) 74*(3)

Note: The number in parentheses represents the number of heterozygous recombination types. *Because recombination types containing more than two chromosome segments were detected in the BC4F4 generation, the total number is greater than the total number of recombination types accumulated in chromosomes.

2. The identification of G. anomalum chromosome segment substitution lines

In the BC4F4 generation, 230 specific SSR primer pairs evenly covering the G. anomalum genome were used to identify the genome-wide foregrounds and backgrounds of all recombinant individuals. Finally, 74 stable inherited chromosome segment substitution lines were identified and numbered CSSL1-CSSL74 in turn. Among them, there were 43 single segment substitution lines, 24 double segment substitution lines and 7 triple segment substitution lines; there were one single segment substitution line and two double segment substitution lines with introgression segments that remain heterozygous, and the remaining 71 chromosome segment substitution lines were homozygous. The chromosome segments of the double segment substitution lines and triple segment substitution lines mainly contained the overlapping introgression segments on Chr.1, Chr.2, Chr.5, Chr.6, Chr.9, Chr.10, Chr.11, Chr.12 and Chr.13. The types of chromosome segment substitution lines and the corresponding types of introgression segments are shown in Table 3.

Table 3 G. anomalum chromosome introgression segments and chromosome segment substitution lines detected in BC4F4 Length of

Chromosome Introgression segment introgression Chromosome segment substitution line segment

(cM) JAAS0826-JAAS1148 (1-12) 89.50 CSSL69# JAAS3039-JAAS2569 (6-20) 134.20 CSSL1 NAU3615-JAAS1148 (10-12) 16.10 CSSL2,CSSL44+ NAU3615-JAAS5817 (10-17) 60.35 CSSL3 NAU3615-JAAS0392 (10-18) 84.40 CSSL4 NAU3615-JAAS2569 (10-20) 102.55 CSSL5 Chr.1 NAU5100-NAU3714 (13-14) 12.65 CSSL6 NAU5100-NAU4045 (13-16) 26.30 CSSL7 NAU5100-JAAS2569 (13-20) 86.45 CSSL8 NAU2083-NAU2083 (15-15) 8.90 CSSL45*, CSSL48+, CSSL68#, CSSL71 NAU2083-NAU4045 (15-16) 13.65 CSSL46* NAU2083-JAAS2569 (15-20) 73.80 CSSL9 NAU4045-NAU4045 (16-16) 4.75 CSSL1O, CSSL47*, CSSL70#, CSSL72# NAU2929-JAAS1857 (21-27) 75.55 CSSL11 Chr.2 NAU2929-JAASO426 (21-29) 100.80 CSSL49* JAAS2050-HAU1219 (32-36) 55.34 CSSL12 Chr.3 JAAS4512-JAAS4003 (37-43)* 71.10 CSSL13 Chr.4 JAAS2662-JAAS2977 (50-54) 44.00 CSSL14

Length of

Chromosome Introgression segment introgression Chromosome segment substitution line segment (cM) JAAS2022-JAAS2076 (55-57) 24.25 CSSL15 JAAS2022-JAAS5943 (55-61) 57.25 CSSL16 JAAS6365-JAAS0803 (62-63) 12.65 CSSL17* JAAS6365-JAAS5604 (62-64) 25.10 CSSL18 JAAS6365-JAAS1953 (62-67) 51.60 CSSL51+ Chr.5 JAAS6365-DC40130 (62-69) 80.40 CSSL50+ CSSL52*, CSSL53*, CSSL54*, CSSL55*, DC40130-DC40130 (69-69) 16.0552+, 73#,CSSL74#,CSSL68# CSSL734, CSSL744, CSSL684 HAU1248-NAU911 (76-77) 36.50 CSSL19, CSSL17, CSSL52 CSSL20, CSSL56*, CSSL57+, CSSL58*, NAU2714-JAAS1095 (86-87) 25.55CSL0 CSSL704 NAU4969-JAAS2480 120.55 CSSL59* Chr.6 (90-103)* NAU1272-JAAS2480 (95-103) 92.70 CSSL21,CSSL56* JAAS6227-JAAS2480 40.45 CSSL22, CSSL57*, CSSL60*, CSSL69 (101-103) NAU3678-NAU1305 Chr.7 119.60 CSSL23 (104-114) NAU1037-JAAS6420 121.90 CSSL24 (129-140) Chr.8 NAU2914-JAAS6420 84.95 CSSL25 (132-140) JAAS3113-JAAS4094 81.00 CSSL26 (141-147) NAU3100-JAAS4094 68.30 CSSL61* (143-147)* Chr.9 NAU3358-JAAS0163 29.70 CSSL61* (149-150)* JAASO163-NBRIO961 62.00 CSSL27, CSSL53* (150-155) JAAS3294-JAAS3294 12.20 CSSL50*, CSSL73# (156-156) Chr.10 JAAS3294-JAAS1256 CSSL44+, CSSL45+, CSSL51*, CSSL54*, 29.60 (156-157) CSSL68#

Length of

Chromosome Introgression segment introgression Chromosome segment substitution line segment (cM) JAAS1256-JAAS1256 17.40 CSSL28,CSSL49' (157-157) JAAS4829-JAAS4829 CSSL29, CSSL46', CSSL55', CSSL62', 17.50 (172-172) CSSL72" JAAS4829-JAAS4769 17.50 CSSL30, CSSL63+, CSSL64+, CSSL71" (172-174) JAAS4829-NAU3703 77.95 CSSL31 (172-177) JAAS4829-NAU3234 145.60 CSSL32, CSSL65+, CSSL74# (172-184) JAAS0280-JAAS3199 113.40 CSSL33 (176-189) JAASO280-NAU980 (176-198) 146.90 CSSL59+ Chr.ll NAU3703-NAU3234 71.30 CSSL34, CSSL66* (177-184) JAAS3088-JAAS5224 19.30 CSSL63* (185-187) JAAS3088-JAAS3199 CSSL35, CSSL58*, CSSL60*, CSSL64+, 29.75 (185-189) CSSL69", CSSL70", CSSL71" JAAS3088-NAU980 (185-198) 63.25 CSSL36, CSSL67+ CSSL37, CSSL65*, CSSL66*, CSSL73", JAAS3310-NAU980O(190-198) 33.50CSL4 CSSL74" JAAS4259-JAAS4259 0.35 CSSL47*, CSSL72" (194-194) JAAS4259-NAU980 (194-198) 18.15 CSSL38 JAAS6317-JAAS3735 27.20 CSSL39 (199-201) JAAS6317-JAAS6372 Chr.12 53.25 CSSL40, CSSL67* (199-203) JAAS3735-JAAS6372 40.85 CSSL41 (201-203) JAAS4570-NAU2038 24.00 CSSL48* (214-217) Chr.13 JAAS4570-JAAS0784 55.70 CSSL42 (214-219)

Length of

(cM) JAAS6264-JAAS0784 31.70 CSSL62+ (218-219) NAU2697-JAAS0049 85.10 CSSL43 (224-230)

Note: *The introgression segment is heterozygous. The underlined one means that it is obtained by backcrossing with addition lines. The ones marked with+ are double segment substitution lines, those marked with # are triple segment substitution lines, and those not marked are single segment substitution lines.

GGT software (Van Berloo R. GGT 2.0: Versatile software for visualization and analysis of genetic data. Journal of Heredity, 2008, 99(2): 232-236. https://doi.org/i0.1093/jhered/esm109) was used to infer the chromosomal composition of the lines and defines the positions and sizes of the donor segments according to Young and Tanksley (Young ND, Tanksley SD. Restriction fragment length polymorphism maps and the concept of graphical genotypes. Theoretical and Applied Genetics, 1989, 77(1): 95-101. https://doi.org/10.1007/bf00292322) method. The distribution of chromosome segments of 74 chromosome segment substitution lines was mapped (FIG. 1), it can be found that the G. anomalum segments (shown in black) on the diagonal line covered most of the genome, and most chromosome segment substitution lines contained overlapped segments. The length of the G. anomalum segment in the chromosome segment substitution line ranged from 4.75 cM (CSSL10) to 267.45 cM (CSSL59), with an average length of 68.91 cM and a cumulative length of 4972.79 cM, which was equivalent to 2.15 times the total length of the G. anomalum genome. Due to the overlap between the introgression segments, the total length of the G. anomalum chromosomes covered by all the introgression segments was 1607.1 cM, and the coverage rate was about 70% (FIG. 2). The numbers of chromosome segment substitution lines containing Chr.1 or Chr.11 chromosome segments were larger, 20 and 27 respectively, and the coverage of these two G. anomalum chromosomes had reached 100%. The numbers of chromosome segment substitution lines containing Chr.3 or Chr.7 chromosome segments were the smallest, only one respectively. However, due to the longer segment length, the chromosome coverage rate was not low (59.9% and 61.2%, respectively). The chromosome which had the lowest coverage rate was Chr.10, only 17.9%. It can also be found from FIG. 1 that there were not many black and dark gray segments on the light gray area outside the diagonal line, indicating that the genetic background recovery rate of G. hirsutum Su 8289 of this set of chromosome segment substitution line materials was relatively high, varying from 88.43% (CSSL59) to 99.79% (CSSL1), with an average recovery rate of 97.09%.

The gel electrophoretogram of SSR detection in BC4F4 strains is shown in FIG. 3.

In FIG. 3, panel A shows the detection results of SSR molecular marker JAAS6365; Lane 1: Marker, Lane 2: genotype of G. hirsutum Su 8289, Lane 3: genotype of G. hirsutum 86-1, Lane 4: genotype of G. anomalum, Lane 5: genotype of hexaploid Fi, Lane 6: genotype of CSSL18, Lane 7: genotype of CSSL50, Lane 8: genotype of CSSL51, Lane 9: genotype of CSSL19, Lane 10: genotype of CSSL52, Lane 11: genotype of CSSL53.

In FIG. 3, panel B shows the detection results of SSR molecular marker JAAS0803; Lane 1: Marker, Lane 2: genotype of Su 8289, Lane 3: genotype of 86-1, Lane 4: genotype of G. anomalum, Lane 5: genotype of hexaploid Fi, Lane 6: genotype of CSSL18, Lane 7: genotype of CSSL50, Lane 8: genotype of CSSL51, Lane 9: genotype of CSSL19, Lane 10: genotype of CSSL52, Lane 11: genotype of CSSL53.

In FIG. 3, panel C shows the detection results of SSR molecular marker JAAS5604; Lane 1: Marker, Lane 2: genotype of Su 8289, Lane 3: genotype of 86-1, Lane 4: genotype of G. anomalum, Lane 5: genotype of hexaploid Fi, Lane 6: genotype of CSSL18, Lane 7: genotype of CSSL50, Lane 8: genotype of CSSL51, Lane 9: genotype of CSSL19, Lane 10: genotype of CSSL52, Lane 11: genotype of CSSL53. The genotypes of CSSL18, CSSL50 and CSSL51 were the same as the genotype of hexaploid Fi. The reasons are as follows: G. anomalum B 1 and At subgenome of G. hirsutum are prone to recombination. In general, the Dt subgenome of G. hirsutum further has an unrecombined SSR site that is homologous to the At subgenome. During PCR amplification, the primers bind to the SSR sites of the B 1 genome of G. anomalum and also bind to the SSR sites of the Dt subgenome. Therefore, the genotypes of CSSL18, CSSL50 and CSSL51 are the same as that of hexaploid Fi. In order to verify this conclusion, in the BC4F3 population of CSSL18, 20 self-crossed plants were selected for genotyping. The results showed that their genotypes did not segregate, indicating that the three chromosome segment substitution lines had been stably inherited and were homozygous chromosome segment substitution lines (FIG. 4).

FIG. 4 shows the detection results of SSR molecular marker JAAS5604 in BC4F3 generation of CSSL18. Lane 1: marker, Lane 2: genotype of Su 8289, Lane 3: genotype of 86-1, Lane 4: genotype of G. anomalum, Lane 5: genotype of hexaploid Fi, Lane 6-Lane 25: genotypes of 20 individual plants in the BC4F3 population.

SSR marker analysis: in the collection of genotype data, the band pattern that was the same as that of Su 8289 or 86-1 was marked as "1", the band pattern that was the same as that of G. anomalum was marked as "2", and the band pattern that was the same as that of Fi was marked as "3",missing or ambiguous band pattern was marked as

. From the gel electrophoretogram of SSR detection in BC4F4 strain shown in FIG. 3, it can be seen that in the detection results of SSR molecular markers JAAS6365 and JAAS0803, the band patterns of the three chromosome segment substitution lines CSSL50, CSSL51 and CSSL18 were consistent with the G. anomalum band patterns ("2"). The SSR molecular marker JAAS5604 in the three chromosome segment substitution lines CSSL50, CSSL51 and CSSL18 had the same band patterns as that of hexaploid Fi ("3"), but did not segregate in the BC4F3 generation of CSSL50, CSSL51 and CSSL18 (FIG. 4), indicating that the three chromosome segment substitution lines had been stably inherited and were homozygous chromosome segment substitution lines. Therefore, CSSL50, CSSL51 and CSSL18 all contained SSR molecular markers JAAS6365, JAAS5604 and JAAS0803 located on chromosome 5 of G. anomalum.

Statistical analysis showed that the chromosome segment substitution lines with high lint percentage and strong drought resistance had introgression segments on chromosome 5 from G. anomalum.

A. Identification of high lint percentage traits

In order to verify the genetic contribution of the G. anomalum segment introgressed into Chr.5 of G. hirsutum to lint percentage, the genotypes and lint percentages of the chromosome segment substitution lines of chromosome 5 were analyzed. The results are shown in FIG. 5. In FIG. 5, the left part is the genetic background analysis of the chromosome 5 chromosome segment substitution lines on chromosome 5. Light gray represents the gene segment of Su 8289, and black represents the gene segment of G. anomalum. The right part shows the phenotypic statistics of lint percentage of different chromosome segment substitution lines and recurrent parent Su 8289. *Indicates a significant difference.

It can be seen from FIG. 5 that the chromosome segment substitution lines of chromosome 5 had different substitution segments. Among them, three chromosome segment substitution lines CSSL50, CSSL51 and CSSL18 contained specific G. anomalum SSR molecular markers JAAS6365, JAAS0803 and JAAS5604 (i.e., the three chromosome segment substitution lines CSSL50, CSSL51 and CSSL18 contained a segment located on chromosome 5 of G. anomalum and starting from SSR molecular marker JAAS6365 to JAAS5604), and compared with the recurrent parent Su 8289, their lint percentages have different degrees of improvement. The other three chromosome segment substitution lines did not contain a G. anomalum chromosome segment containing the three SSR molecular markers, and their lint percentages were not significantly different from that of the recurrent parent Su 8289. These results partly suggested that the G. anomalum chromosome segment that containing the three SSR molecular markers JAAS6365, JAAS0803 and JAAS5604 are related to high lint percentage. Using the single-marker analysis method of WinQTLCart 2.5 software, the correlation between all 23 markers on chromosome 5 and lint percentage traits was analyzed and the results showed that markers JAAS6365, JAAS0803 and JAAS5604 were significantly correlated with lint percentage, while the other markers were not (Table 4).

Table 4 Correlation analysis between markers JAAS6365, JAAS0803, JAAS5604 and lint percentage Trait Chromosome Marker P value R2 Lint percentage Chr.5 JAAS6365 0.00832195 0.111 Lint percentage Chr.5 JAAS0803 0.00832195 0.111 Lint percentage Chr.5 JAAS5604 0.00832195 0.111

The CSSL18 was planted in different environments. Environment 1: in 2017, Nanjing, Jiangsu province, Jiangsu Academy of Agricultural Sciences, Lishui Plant Science Base; Environment 2: in 2018, Nanjing, Jiangsu province, Jiangsu Academy of Agricultural Sciences, Lishui Plant Science Base; Environment 3: in 2018, Korla, Xinjiang Autonomous Region. Each test site was arranged in random blocks, repeated 3-4 times, and the planting density and production management were the same as the local field production of the test sites.

The statistical data of lint percentage is shown in Table 5.

Table 5 Phenotypic statistics of lint percentage in CSSL18 and recurrent parent Su 8289 Material Lint percentage (%) Mean SD Environment name Parallel test 1 Parallel test 2 Parallel test 3 Parallel test 4 (%) 1 Su 8289 40.10 38.94 40.57 - 39.87±0.68 1 CSSL18 40.00 42.70 43.21 - 41.97±1.40 2 Su 8289 40.12 40.48 41.19 - 40.60±0.44 2 CSSL18 45.00 44.70 50.00 - 46.57±2.43 3 Su 8289 42.98 42.78 43.19 43.56 43.13±0.29 3 CSSL18 45.60 43.33 43.87 43.33 44.03±0.92

Note: "-" means not detected.

It can be seen from Table 5 that a single segment substitution line of chromosome 5, CSSL18, (in the process of constructing a set of G. anomalum chromosome segment substitution line populations, each generation was identified with SSR molecular marker-assisted selection; compared with the recurrent parent Su 8289, the only difference of CSSL18 was that the corresponding segment on chromosome 5 was replaced by "the segment located on chromosome 5 of G. anomalum and starting from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604" and the rest of the genome was completely the same as the recurrent parent Su 8289; CSSL18 was a homozygous tetraploid) had a lint percentage of 41.97-46.57 in different environments, which was significantly higher than that of the recurrent parent Su 8289 (39.87-43.13). It can be used as an important material for high yield breeding and lint percentage molecular genetic mechanism research of cotton.

The values in Table 5 were also reflected in FIG. 6.

In FIG. 6, it is shown that there was a significant difference in lint percentage between CSSL18 and Su8289.

From the above experimental results, it can be seen that the chromosome segment located on chromosome 5 of G. anomalum and starting from SSR molecular marker JAAS6365 to the SSR molecular marker JAAS5604 can improve the cotton lint percentage. The three SSR molecular markers (JAAS6365, JAAS0803 and JAAS5604) on this chromosome segment and the developed primer pairs can be used to screen target chromosome segments to develop cotton varieties with improved lint percentage.

B. Identification of drought resistance

In order to verify the genetic contribution of the G. anomalum segment introgressed into Chr.5 of G. hirsutum to drought resistance, the genotypes and drought resistance indicators of the chromosome segment substitution lines of chromosome 5 were analyzed. CSSL50, CSSL51, CSSL18, CSSL52, CSSL53, CSSL19 and the recurrent parent Su 8289 were planted in an incubator. The culture conditions were as follows: temperature: 28°C, photoperiod: 16h/8h light/dark, humidity: 60%. Using the method of PEG treatment to simulate drought stress, the drought resistance of 6 segment substitution lines and recurrent parent Su 8289 were evaluated. At the two-euphylla and one-bud seeding stage, the 6 substitution lines and recurrent parent Su 8289 were treated with 20% PEG6000, and after 72 hours, the drought resistance was systematically evaluated by observing the wilting degree of the plants. The identification of drought resistance refered to the grading standard of verticillium wilt of cotton: Grade 0: no symptom; Grade 1: one euphylla wilts; Grade 2: less than 70% of the leaves of plants with more than two euphylla wilt or fall off; Grade 3: no less than 70% of the leaves of plants wilt but the plants are not dead; Grade 4: a large number of leaves fall off to a bare trunk and the plants are dead. Wilting rate: after PEG treatment, the percentage of wilting cotton plants planted in the incubator in the total number of cotton plants was investigated. Drought index: the product of the number of wilting plants at each grade and the corresponding grade value divided by the product of the total number of plants investigated and the highest wilting Grade 4, and then multiplied by 100%. The results are shown in FIG. 7. In FIG. 7, the left part is the genetic background analysis of chromosome 5 chromosome segment substitution lines on chromosome 5. Light gray represents the gene segment of Su 8289, and black represents the gene segment of G. anomalum. The right part is the drought resistance statistics of the 6 chromosome segment substitution lines and recurrent parent Su 8289.

Number of wilting plants Wilting rate = >100% Total number of plants investigated

Number of wilting plants at each grade x Corresponding grade value Disease index= x100%

Total number of plants investigated x 4

It can be seen from FIG. 7 that the chromosome segment substitution lines of chromosome 5 had different substitution segments. Among them, three chromosome segment substitution lines CSSL50, CSSL51 and CSSL18 contained specific G. anomalum SSR molecular markers JAAS6365, JAAS0803 and JAAS5604 (i.e., the three chromosome segment substitution lines CSSL50, CSSL51 and CSSL18 all contained a segment located on chromosome 5 of G. anomalum and starting from SSR molecular marker JAAS6365 to JAAS5604), and their wilting rates and disease indexes were significantly lower than those of the recurrent parent Su 8289. The other three chromosome segment substitution lines did not contain G. anomalum chromosome segments containing the three SSR molecular markers, and their wilting rates and disease indexes were not significantly different from those of the recurrent parent Su 8289. These results partly suggested that the G. anomalum chromosome segment that containing the three SSR molecular markers JAAS6365, JAAS0803 and JAAS5604 are related to drought resistance.

Using the single-marker analysis method of WinQTLCart 2.5 software, the correlation between all 23 markers of chromosome 5 and drought resistance-related traits, including wilting rate and drought index was analyzed and the results showed that markers JAAS6365, JAAS0803 and JAAS5604 were significantly correlated with wilting rate and drought index, while the other markers were not (Table 6).

Table 6 Correlation analysis between markers JAAS6365, JAAS0803, JAAS5604 and drought resistance related traits Trait Chromosome Marker P value R2 Chr.5 JAAS6365 3.97E-54 0.1920 Wilting rate Chr.5 JAAS0803 3.97E-54 0.1920 Chr.5 JAAS5604 3.97E-54 0.1920 Chr.5 JAAS6365 4.54E-31 0.1802 Drought index Chr.5 JAAS0803 4.54E-31 0.1802 Chr.5 JAAS5604 4.54E-31 0.1802

The CSSL18 and the recurrent parent were planted in an incubator. The culture conditions were as follows: temperature: 28C, photoperiod: 16h/8h light/dark, humidity: 60%. Using the method of PEG treatment to simulate drought stress, the drought resistance of single segment substitution line CSSL18 and the parent Su 8289 was evaluated. At the two-euphylla and one-bud seeding stage, CSSL18 and recurrent parent Su 8289 were treated with 20% PEG6000, and after 72 hours, the drought resistance was systematically evaluated by observing the wilting degree of the plants (FIG. 8, Table 7).

Table 7 Phenotypic statistics of drought resistance related traits of CSSL18 and recipient Su 8289 Material Parallel test Parallel test Parallel test Trait name 1 2 3 Mean SD(%)

Su 8289 82.86 73.33 81.82 79.34 5.22 Wilting rate (%) CSSL18 33.33 39.13 35.71 36.06 ±2.91 Droughtindex Su 8289 65.00 66.67 61.36 64.34± 2.71 (%) CSSL18 16.67 15.22 18.45 16.78 ± 1.62

It can be seen from Table 7 that the wilting rate and drought index of a single segment substitution line of chromosome 5, CSSL18, (in the process of constructing a set of G. anomalum chromosome segment substitution line populations, each generation was identified with SSR molecular marker-assisted selection; compared with the recurrent parent Su 8289, the only difference of CSSL18 was that the corresponding segment on chromosome 5 was replaced by "the segment located on chromosome 5 of G. anomalum and starting from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604" and the rest of the genome was completely the same as the recurrent parent Su 8289; CSSL18 was a homozygous tetraploid) are significantly lower than those of recurrent parent Su 8289. It can be used as an important material for drought resistance breeding and drought resistance genetic mechanism research of cotton (FIG. 8, Table 7).

From the above experimental results, it can be seen that the chromosome segment located on chromosome 5 of G. anomalum and containing the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 can improve the drought resistance of cotton. The three SSR molecular markers (JAAS6365, JAAS0803 and JAAS5604) on this chromosome segment and the developed primer pairs can be used to screen target chromosome segments to develop cotton varieties with improved drought resistance.

The specific information of each SSR molecular marker and its detection primers are as follows:

the nucleotide sequence of SSR molecular marker JAAS6365 is as shown in SEQ ID NO: 1;

the nucleotide sequence of SSR molecular marker JAAS5604 is as shown in SEQ ID NO:

2;

the nucleotide sequence of SSR molecular marker JAAS0803 is as shown in SEQ ID NO: 3.

The primers used to detect the SSR molecular marker JAAS6365 (i.e., the primer pair used for the detection of panel A in FIG. 3) are as follows:

JAAS6365-F: 5'-AGCATCCAAAACCCATTTGCT-3'(SEQ ID NO: 4);

JAAS6365-R: 5'-ACCGCATCCTAAGGAAAGCT-3'(SEQ ID NO: 5).

The primers used to detect the SSR molecular marker JAAS5604 (i.e., the primer pair used for the detection of panel C in FIG. 3 and FIG. 4) are as follows:

JAAS5604-F: 5'-TGACGTCGTTGATCCACCTC-3' (SEQ ID NO: 6);

JAAS5604-R: 5'- TCCCATGGGTGTGGTAAAACC-3' (SEQ ID NO: 7).

The primers used to detect the SSR molecular marker JAAS0803 (i.e., the primer pair used for the detection of panel B in FIG. 3) are as follows:

JAAS0803-F: 5'-ACTTTTTGCATTATCTAAGGTTCTGT-3'(SEQ ID NO: 8);

JAAS0803-R: 5'-ACCGATACTCTTTTTCCCTGCA-3' (SEQ ID NO: 9).

Although specific embodiments have been used to illustrate and describe the present invention, it should be appreciated that many other changes and modifications can be made without departing from the spirit and scope of the present invention. Therefore, this means that all these changes and modifications that fall within the scope of the present invention are included in the appended claims.

Industrial application

In this present invention, the cotton chromosome segment substitution lines with high-lint percentage and drought resistance were developed by replacing the genetic background of G. hirsutum Su 8289 with the segment between SSR molecular markers JAAS6365 and JAAS5604 located on chromosome 5 of G. anomalum based on integrating distant hybridization and molecular marker-assisted selection technologies. Therefore, the chromosome segment developed by the present invention (the segment between the SSR molecular markers JAAS6365 and JAAS5604 located on chromosome 5 of G. anomalum) has an important application value in developing drought-resistant cotton with high lint percentage in the future. In the present invention, the molecular markers were used to detect the target segment with high accuracy and stability, and this facilitated the early and rapid identification. Therefore, the three SSR molecular markers and their primer pairs provided by the present invention are expected to greatly improve the selection efficiency and cotton breeding speed of lint percentage and drought resistance traits and are of great significance for accelerating the breeding process of new cotton varieties with high lint percentage and drought resistance. The drought-resistant cotton line with high lint percentage provided by the present invention can be used for fine mapping, cloning and functional analysis of high lint percentage and drought-resistant related genes through SSR molecular markers and their primer pairs, which can not only clarify the molecular genetic mechanism of cotton high lint percentage and drought resistant traits, but also provides the material and molecular basis for breeding varieties with high lint percentage and drought resistance that can be used in production.

Claims

1. A DNA segment, wherein the DNA segment is located on chromosome 5 of G. anomalum and contains at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604;

2. The DNA segment according to claim 1, wherein the DNA segment is located on chromosome 5 of G. anomalum and starts from the SSR molecular marker JAAS6365 to the SSR molecular marker JAAS5604.

3. A vector, expression cassette, recombinant bacteria or transgenic cell line containing the DNA segment according to claim 1 or 2.

4. The vector, expression cassette, recombinant bacteria or transgenic cell line according to claim 3, wherein the vector is an artificial chromosome;

the artificial chromosome is a bacterial artificial chromosome or a yeast artificial chromosome;

the recombinant bacteria and the transgenic cell line contain the artificial chromosomes.

5. Use of the DNA segment according to claim 1 or 2 or the vector, expression cassette, recombinant bacteria or transgenic cell line according to claim 3 or 4 for improving cotton lint percentage or cultivating cotton varieties or lines with improved lint percentage.

6. Use of the DNA segment according to claim 1 or 2 or the vector, expression cassette, recombinant bacteria or transgenic cell line according to claim 3 or 4 for improving drought resistance of cotton or cultivating drought-resistant cotton varieties or lines.

7. An SSR molecular marker or a set of SSR molecular markers on the DNA segment according to claim 1 or 2, wherein the SSR molecular marker is any one of the following (al)-(a3); the set of SSR molecular markers consists of the following (al)-(a3);

(al) SSR molecular marker JAAS6365: a DNA molecule having the nucleotide sequence as shown in SEQ ID NO: 1; (a2) SSR molecular marker JAAS5604: a DNA molecule having the nucleotide sequence as shown in SEQ ID NO: 2; (a3) SSR molecular marker JAAS0803: a DNA molecule having the nucleotide sequence as shown in SEQ ID NO: 3.

8. A primer pair or a set of primer pairs for identifying the SSR molecular marker or the set of SSR molecular markers according to claim 7, wherein the primer pair is any one of the following (bl)-(b3); the set of primer pairs consists of the following (bl)-(b3);

(bl) primer pair 1 for identifying the SSR molecular marker JAAS6365: designed according to SEQ ID NO: 1; (b2) primer pair 2 for identifying the SSR molecular marker JAAS5604: designed according to SEQ ID NO: 2; (b3) primer pair 3 for identifying the SSR molecular marker JAAS0803: designed according to SEQ ID NO: 3.

9. The primer pair or the set of primer pairs according to claim 8, wherein the primer pair 1 consists of two single-stranded DNA molecules as shown in SEQ ID NO: 4 and SEQ ID NO: 5; the primer pair 2 consists of two single-stranded DNA molecules as shown in SEQ ID NO: 6 and SEQ ID NO: 7; the primer pair 3 consists of two single-stranded DNA molecules as shown in SEQ ID NO: 8 and SEQ ID NO: 9.

10. A kit containing the primer pair or the set of primer pairs according to claim 8 or 9.

11. The kit according to claim 10, wherein the kit further contains one or more of PCR amplification buffer, double-distilled water, DNA polymerase and dNTP.

12. Use of the SSR molecular marker or the set of SSR molecular markers according to claim 7 or the primer pair or the set of primer pairs according to claim 8 or 9 or the kit according to claim 10 or 11 for screening the DNA segment according to claim 1 for cultivating cotton varieties or lines with improved lint percentage.

13. Use of the SSR molecular marker or the set of SSR molecular markers according to claim 7 or the primer pair or the set of primer pairs according to claim 8 or 9 or the kit according to claim 10 or 11 for screening the DNA segment according to claim 1 for cultivating cotton varieties or lines with improved drought resistance.

14. A method for identifying or assisting in identifying cotton lint percentage traits, which is conducted by using the SSR molecular marker or the set of SSR molecular markers according to claim 7 or the primer pair or the set of primer pairs according to claim 8 or 9 or the kit according to claim 10 or 11.

15. A method for identifying or assisting in identifying drought resistance of cotton, which is conducted by using the SSR molecular marker or the set of SSR molecular markers according to claim 7 or the primer pair or the set of primer pairs according to claim 8 or 9 or the kit according to claim 10 or 11.

16. A method for improving cotton lint percentage, comprising the following step: replacing the original chromosome genome segment of a recipient parent with a DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 to obtain a cotton variety with improved lint percentage;

17. A method for improving drought resistance of cotton, comprising the following step: replacing the original chromosome genome segment of a recipient parent with a DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 to obtain a cotton variety with improved drought resistance;

18. The method according to claim 16 or 17, wherein the DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 is a DNA segment located on chromosome 5 of G. anomalum and starting from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604.

19. The method according to any one of claims 16-18, wherein in the method, the replacement of the original chromosome genome segment of a recipient parent with a DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604 is realized by hybridization technology.

20. The method according to any one of claims 16-19, wherein in the method, the SSR molecular marker or the set of SSR molecular markers according to claim 7 or the primer pair or the set of primer pairs according to claim 8 or 9 or the kit according to claim 10 or 11 is used to identify whether a cotton to be tested contains the DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604.

21. A cotton or its tissues or organs, wherein the cotton is a cotton variety obtained by replacing the original chromosome genome segment of a recipient parent with a DNA segment located on chromosome 5 of G. anomalum and containing at least the segment from SSR molecular marker JAAS6365 to SSR molecular marker JAAS5604;